And Chemical-Activated Nucleosides and Unnatural Amino Acids. (Under the Direction of Dr

ABSTRACT

LIU, QINGYANG. Synthesis of Photo- and Chemical-Activated Nucleosides and Unnatural Amino Acids. (Under the direction of Dr. Alexander Deiters).

Synthetic oligonucleotides coupled with photolabile caging groups have been developed to regulate a variety of biological processes in a spatial and temporal fashion. A

UV-cleavable caging group was installed on deoxyadenosine and two morpholino oligonucleotide (MO) monomers of which the morpholino core synthesis was also investigated. The synthesis of a two-photon caging group was optimized and two chromophores with > 400 nm absorption maximum were applied to cage thymidine. These caged monomers can serve as light-triggers of oligonucleotide function upon incorporation.

Two phosphine-labile azido thymidine derivatives were synthesized as orthogonal small molecule-triggers to the above light-triggers.

Additionally, two coumarin linkers were synthesized, which can cyclize a linear MO so as to inactivate MO activity until > 400 nm light irradiation. These two linkers have been applied to the wavelength-selective regulation of zebrafish embryo development. An azide linker was also synthesized to control MOs using phosphines, as well as a UV-cleavable phosphoramidite to regulate DNA oligonucleotide activities.

On the regulation of proteins, a two-photon caged lysine, four azido lysines and an azido tyrosine were synthesized to control protein function with either light or small molecules. The phosphine-induced cleavage of the azido groups were investigated on a coumarin reporter. A fluorescent lysine and an isotope labeled lysine were also synthesized as additional biophysical probes to label protein. These unnatural amino acids have been or will be incorporated into proteins through exogenous tRNA-aaRSs pairs.

Synthesis of Photo- and Chemical-Activated Nucleosides and Unnatural Amino Acids

by Qingyang Liu

A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Chemistry

Raleigh, North Carolina

2014

APPROVED BY:

______Dr. Alexander Deiters Dr. Christian Melander Committee Co-chair Committee Co-chair

______Dr. Daniel L. Comins Dr. Gavin Williams

DEDICATION

谨以此文致家严家慈，感谢他们的理解，鼓励和支持。

谨以此文悼念先师黄宪教授，感谢他教导我以务实、非功利的态度面对科研。

I dedicate this work to my parents for their understanding, encouragement and support.

I dedicate this work in memory of Prof. Xian Huang for showing me the ethic of work.

BIOGRAPHY

Qingyang Liu was born on March 7th, 1986 in Hebei, China and grown up to be a big sports fan. She enjoys watching any kind of sports and ran track and played soccer till high school. After graduation from high school, she went to Zhejiang University in Hangzhou,

China where she received her BS degree in Chemistry in 2009 and experienced a different culture in southern China. This developed her interest in organic chemistry working on the methodology of vinylidenecyclopropane under Prof. Xian Huang’s mentorship. In the summer of 2008, she came to North Carolina State University as an exchange student for two months and worked on the development of microwave assisted cyclotrimerization towards natural product synthesis. This experience motivated her to pursue a graduate degree in the

US. She started her PhD training under the supervision of Dr. Alexander Deiters at NCSU where she enjoyed the fusion of western and eastern cultures. This fusion was well demonstrated in her wedding ceremony to Thomas K. Chen on June 15, 2013. With curiosity and excitement, she looks forward to explore wherever and whatever the Spirit will lead her.

iii

ACKNOWLEDGMENTS

First of all, I want to thank my advisor, Dr. Alexander Deiters for his guidance in the past five years. He taught me to be confident as a non-native English speaker and encouraged me to practice my English. He also trained me to maintain good time management and to develop a good work ethic which will be beneficial to me even after graduation.

I would like to thank Yan who has been such a great friend and mentor to me for all these years. Thanks to Doug, Jesse, and Harry for their kindness during my visit in 2008 which played an important part in my decision to join this lab. Thanks to Jeane, Meryl, Alex

P., James, and Andrew for making our office such an enjoyable place and for helping me both in research and in improving my English; Laura and Kalyn for teaching me protein expression and about American culture; Rajendra, Matt and Subhas for sharing their expertise in chemistry. Thanks to Colleen, Jessica, Ji, and Jihe for staying in Raleigh with me after the lab moved and to Taylor for making the boring moments of life more lively. I also want to thank Robin for her help outside the lab and Dr. Melander for supervising me in the past year.

I would like to thank my parents for their love and support. Knowing that I have a place to go back to no matter what happens has always been a comfort during hard times. I thank my husband and my in-law families who have treated me as a member of their own ever since we first met and give me a home in this unfamiliar country. Finally I want to thank the Shaws for being my family in Raleigh, and my church family at St. Giles who gives me lots of help and comfort.

TABLE OF CONTENTS

LIST OF FIGURES ...... vii LIST OF SCHEMES ...... ix LIST OF ABBREVIATIONS ...... xiii

CHAPTER 1: Regulation of Biological Activity with Oligonucleotides ...... 1 1.1 Synthetic oligonucleotides in the regulation of biological processes ...... 1 1.1.1 Transcriptional level of regulation ...... 2 1.1.2 Translational level of regulation ...... 3 1.1.3 Protein level of regulation ...... 7 1.2 Oligonucleotide improvement through chemical modification ...... 8 1.3 Regulation of synthetic oligonucleotide activites with light ...... 11 1.3.1 Evolution of caging groups ...... 11 1.3.2 Caging approaches to regulate oligonucleotide function ...... 18

CHAPTER 2: Synthesis of Photocaged Nucleoside Phosphoramidites ...... 25 2.1 Synthesis of 6-nitropiperonylmethyl-caged deoxyadenosine phosphoramidite ..25 2.2 Development of caged nucleosides with red-shifted cleavage wavelengths ...... 28 2.2.1 Synthesis and photolysis of coumarin caged nucleosides ...... 29 2.2.2 Synthesis and photolysis of 3-(2-propyl)-4-dimethylamino-4-nitro- biphenyl nucleosides ...... 32 2.3 Development of small molecule-triggered nucleosides ...... 39 2.4 Conclusion ...... 46 2.5 Experimental ...... 47

CHAPTER 3: Synthesis of Photocaged Morpholino Oligonucleotide Subunits ...... 82 3.1 Synthesis of NPOM-caged MO subunit ...... 84 3.1.1 Optimization of the NPOM-caged thymine-MO subunit synthesis ...... 84 3.1.2 Synthesis of the NPOM-caged guanine-MO subunit ...... 90 3.2 Synthesis of two-photon caged thymine-MO subunit ...... 92 3.3 Synthesis of the PNVOM MO-T ...... 95 3.4 Conclusion ...... 96 3.5 Experimental ...... 97

CHAPTER 4: Synthesis of Photocleavable Oligonucleotide Linkers ...... 120 4.1 Synthesis of o-nitrobenzyl linker for DNA oligonucleotides ...... 120 4.2 Synthesis of coumarin-based linkers for MOs ...... 121 4.2.1 Synthesis of 7-diethylaminocoumarin linkers ...... 122 4.2.2 Development of coumarin linkers with red-shifted absorption maximums ...... 128 4.2.3 Synthesis of azidobenzyl MO linker ...... 136 4.3 Conclusion ...... 138

4.4 Experimental ...... 139

CHAPTER 5: Engineering Protein Function through Unnatural Amino Acid Mutagenesis ...... 168 5.1 Post transcriptional modification of protein ...... 168 5.2 Unnatural amino acid mutagenesis ...... 169

CHAPTER 6: Synthesis of Unnatural Amino Acids ...... 174 6.1 Synthsis of two-photon caged lysine ...... 174 6.2 Synthesis of azido lysine and tyrosine analogs ...... 177 6.2.1 Development of azidomethylene and the azidoethyl lysine and tyrosine analogs ...... 177 6.2.2 Synthesis of ortho-azidobenzyl and para-azidobenzyl lysine analogs ...... 187 6.3 Synthesis of nitrobenzofuran lysine ...... 200 6.4 Synthesis of p-nitrobenzyl ε-15N lysine ...... 205 6.5 Conclusion ...... 209 6.6 Experimental ...... 210

REFERENCES ...... 260

APPENDICES ...... 291 Appendix A: Additional Information on CHAPTER 3 ...... 292

LIST OF FIGURES

Figure 1.1 Regulation of biological molecules with oligonucleotides ...... 1

Figure 1.2 Three generations of synthetic oligonucleotides ...... 10

Figure 1.3 UV cleavable caging groups ...... 13

Figure 1.4 The improved three-dimensional focus of two-photon excitation...... 16

Figure 1.5 Two-photon caging groups ...... 17

Figure 1.6 Chemical structures of caged nucleobases ...... 23

Figure 2.1 Photocaged deoxynucleoside phosphoramidites ...... 25

Figure 2.2 Absorption spectra of 76-79 (50 µM in MeOH)...... 33

Figure 2.3 LC-MS analysis of the decaging of the N-ANBP-caged thymidine 83 ...... 35

Figure 2.4 HPLC analysis of the O-ANBP-caged thymidine 94 decaging ...... 38

Figure 2.5 Structure of azide protected fluorophores and prodrugs ...... 40

Figure 2.6 Photo- and chemically triggered nucleosides ...... 47

Figure 3.1 Gene regulation with an NPOM-caged MO in zebrafish embryos ...... 84

Figure 3.2 Photocaged MO subunits ...... 97

Figure 4.1 Wavelength-selective regulation of zebrafish embryo development ...... 127

Figure 4.2 Absorption spectra of the DEACM 196, the thiocoumarin (TC) 197 and the MNCM 198 in MeOH at a 0.1 mM concentration...... 132

Figure 4.3 Comparison of the DEACM- and the MNCM-cyclized ntla MO at different wavelengths (360 nm, 405 nm, and 470 nm) ...... 136

Figure 4.4 Photo/chemical-cleavable oligonucleotide linkers ...... 138

Figure 5.1 Incorporation of UAA through an orthogonal tRNA-aaRSs pair ...... 171

Figure 5.2 Chemical structures of selected encoded unnatural lysine amino acids by

vii

tRNApyl-PylRS pairs...... 172

Figure 5.3 Photo-regulation over protein function...... 173

Figure 6.1 Incorporation of the NDBF lysine 231 in mammalian cells ...... 175

Figure 6.2 Proposed structures of the AzM- and the AzE-modified lysines 234 and 235 ....178

Figure 6.3 The SDS-PAGE of sfGFP bearing the AzM lysine 246 incorporated by the wild- type tRNApyl-PylRS pair in E. coli...... 181

Figure 6.4 The SDS-PAGE of sfGFP bearing the AzE lysine 265 incorporated by the wild- type PylRS in E. coli...... 184

Figure 6.5 PPh3 reduction of the AzM coumarin 271 (10 mM) in a 9:1 mixture of MeOH/PBS buffer (pH = 7.4) ...... 186

Figure 6.6 The SDS-PAGE of sfGFP bearing the AzM tyrosine 276 incorporated by the EV- 16 PylRS in E. coli...... 187

Figure 6.7 HPLC analysis of oAzBn naphthylmethylamine 280 cleavage upon TPPTS reduction...... 189

Figure 6.8 Fluorescence (ex 495 nm / em 520 nm) of oAzBn rhodamine 287 reduction with different phosphines ...... 192

Figure 6.9 Fluorescence (360 nm / 480 nm) of oAzBn coumarin 290 reduction through different phosphines...... 194

Figure 6.10 Fluorescence (360 nm/ 480 nm) of pAzBn coumarin 291 reduction in different solvents ...... 194

Figure 6.11 Incorporation of the oAzBn lysine 299 in mammalian cells ...... 198

Figure 6.12 Small molecule activation of firefly luciferase ...... 199

Figure 6.13 Unnatural amino acid analogs ...... 210

Figure A.1 Photolysis instrument setup...... 276

Figure A.2 Size of the cuvette used in photolysis experiment ...... 277

viii

LIST OF SCHEMES

Scheme 1.1 Transcriptional regulation over gene expression ...... 3

Scheme 1.2 Antisense agent induced gene silencing through different mechanisms ...... 4

Scheme 1.3 DNAzyme induced gene silencing. DNAzyme binds to mRNA and catalyzes the cleavage of mRNA so as to stop mRNA translation ...... 5

Scheme 1.4 siRNA induced gene silencing ...... 5

Sheme 1.5 miRNA regulation over gene expression...... 7

Scheme 1.6 Light activation of caged molecule ...... 12

Scheme 1.7 Photolysis mechanism of different caging groups ...... 15

Scheme 1.8 Photo-induced cis-trans isomerization of azobenzene group ...... 18

Scheme 1.9 Different caging approaches to regulate oligonucleotide hybridization with light ...... 20

Scheme 2.1 Synthesis of the NPE-NH2 (47) caging precursor ...... 26

Scheme 2.2 Synthesis of the NPM-dA (41) ...... 28

Scheme 2.3 Synthesis of the DEACM caging group 56 ...... 29

Scheme 2.4 Synthesis of the O-DEACM-caged nucleosides 66-68 ...... 30

Scheme 2.5 Synthsis of the N-DEACM-caged nucleosides 72-74...... 31

Scheme 2.6 Synthesis of the 4-nitro-biphenyl derivatives 76-79 ...... 32

Scheme 2.7 Synthesis of the N-ANBP-caged thymidine 83 ...... 34

Scheme 2.8 Photolysis of the N-ANBP-caged thymidine 83 ...... 34

Scheme 2.9 Synthesis of the secondary ANBP alcohol 90 ...... 36

Scheme 2.10 Synthesis of the O-ANBP-caged thymidine 94 ...... 37

Scheme 2.11 Photolysis of the O-ANBP-caged thymidine 94 ...... 38

Scheme 2.12 Mechanism of the Staudinger reduction or organic azides to amines ...... 40

Scheme 2.13 Synthesis of the AzM thymidine 102 ...... 41

Scheme 2.14 Reduction of the AzM thymidine 100 with PPh3 (A) which was followed by 1H NMR (B) ...... 42

Scheme 2.15 Synthesis of the pAzBn thymidine 105 and the pAzBnCH2 thymidine 108 ...43

Scheme 2.16 Reduction of the AzBnCH2 thymidine 108 with PPh3 (A) which was followed by 1H NMR (B) ...... 44

Scheme 2.17 Reduction of the AzBn thymidine 108 with PPh3 (A), which was followed by 1H NMR (B) ...... 46

Scheme 3.1 Synthesis of the thymine-MO subunit 112 from 5-methyluridine 110 ...... 85

Scheme 3.2 Synthesis of the uracil-MO subunit 115 from the TIPS protected uridine 113 ...... 85

Scheme 3.3 Attempts to Synthesize the NPOM thymine-MO subunit from caged 5-methyluridine 122 ...... 87

Scheme 3.4 Synthesis of the NPOM-caged thymine-MO chlorophosphoramidate 131 ...... 88

Scheme 3.5 Synthesis of the NPOM thymine-MO subunit 130 from 5-methyluridine 110 ...... 89

Scheme 3.6 Synthesis of the guanine-MO subunit 136 ...... 90

Scheme 3.7 Synthesis of the NPOM guanine-MO chlorophosphoramidate 141 ...... 92

Scheme 3.8 Optimized synthesis of the NDBF alcohol ...... 94

Scheme 3.9 Synthesis of the NDBF-caged thymine-MO subunit 152 ...... 95

Scheme 3.10 Synthesis of the PNVOM MO-T chlorophosphoramidate 155 ...... 96

Scheme 4.1 Improved synthesis of the ONB linker 160 ...... 121

Scheme 4.2 Structure of the DMNB linker 161 (A) and its cyclization of MO (B) ...... 122

Scheme 4.3 Synthesis of the maleimide DEACM linker 170 ...... 123

Scheme 4.4 Synthesis of the chloroacetamide DEACM linker 176 ...... 124

Scheme 4.5 Synthesis of non-cleavable linker 181 ...... 125

Scheme 4.6 Multi-wavelength regulations over MO activities ...... 126

Scheme 4.7 Attempts to form the thiocoumarin linker 186 ...... 129

Scheme 4.8 Attempts to synthesize thiocoumarin linker through amine 191 ...... 130

Scheme 4.9 Attempts to synthesize the thiocoumarin linker 195 through the chloroacetamide 193 ...... 131

Scheme 4.10 Synthesis of dicyanocoumarin amine 199...... 132

Scheme 4.11 Synthesis of the acid 203, containing a chloroacetamide group...... 133

Scheme 4.12 Attempts to couple the amine 199 with the activated acids 204 and 205...... 134

Scheme 4.13 Synthesis of the MNCM linker 208...... 135

Scheme 4.14 Synthesis of the pAzBn linker 215...... 137

Scheme 6.1 Synthesis of the NDBF lysine 231...... 174

Scheme 6.2 Synthesis of photo-cleavable hydroxyl acid 232...... 176

Scheme 6.3 Synthesis and cleavage of the AzM naphthylmethylamine 238...... 179

Scheme 6.4 Attempts to make the AzM lysine 234 from Boc Lys 239...... 179

Scheme 6.5 Synthesis of the AzM lysine 246 from Fmoc Lys 242...... 180

Scheme 6.6 Synthesis and cleavage of the AzE naphthylmethylamine 249...... 181

Scheme 6.7 Attempts to synthesize the AzEK 235 from Boc Lys OMe 239...... 182

Scheme 6.8 Attempts to synthesize AzE lysine 235 with via different esters...... 184

Scheme 6.9 Synthesis of AzM coumarin 271...... 186

Scheme 6.10 Synthesis of AzMY 276...... 187

Scheme 6.11 Synthesis and decaging of oAzBn naphthylmethyl amine 280...... 189

Scheme 6.12 Synthesis of the oAzBn rhodamine 287...... 191

Scheme 6.13 Synthesis of AzBn coumarins 290-291...... 193

Scheme 6.14 Synthesis of the Boc protected AzBn lysine 296-297...... 196

Scheme 6.15 Synthesis of the TFA salt 298 and the HCl salt 299 of oAzBn lysine...... 196

Scheme 6.16 Synthesis of the HCl salt of oAzBn lysine 299 via succinimidyl carbonate. ..197

Scheme 6.17 Synthesis of the HCl salt of pAzBn lysine 302...... 200

Scheme 6.18 Attempts to synthesize and activate the hydroxylethyl NBDs 304-306...... 202

Scheme 6.19 Attempts to synthesize the aminoethyl lysine 311...... 203

Scheme 6.20 Synthesis of the urea NBD lysine 315...... 204

Scheme 6.21 Synthesis of the carbamate NBD lysine 316...... 204

Scheme 6.22 Synthesis of alcohol 319-321...... 206

Scheme 6.23 Synthesis of Lys 324 as a model study for 15N Lys...... 207

Scheme 6.24 Attempts to synthesize pNBn lysine 328 via a copper complex...... 207

Scheme 6.25 Synthesis of pNBn lysine 328 via 9-BBN protected Lys...... 208

Scheme 6.26 Synthesis of the pNBn15N lysine 332...... 209

xii

LIST OF ABBREVIATIONS

μL micro liter

μM micromolar aaRs aminoacyl tRNA synthetase

Ac acetyl

AcOH acetic acid

ATP adenosine triphosphate

AMP adenosine monophosphate

AMNB 5-aminomehthyl-2-nitrobenzyl

ANBP 4-dimethylamino-4-nitro-biphenyl

AzM azidomethylene

AzE azidoethyl

9-BBN 9-borabicyclo(3,3,1)nonane

Bhc 6-bromo-7-hydroxycoumarin-4-methyl

BHQ 8-bromo-7-hydroxyquinoline

Bn benzyl

Boc t-butoxycarbonyl

BPO benzoyl peroxide

CbzCl benzyl chloroformate

CHCl3 chloroform

CDCl3 deuterated chloroform

13C NMR carbon nuclear magnetic resonance

xiii

CPP cell penetrating peptides

DBU 1,8-diazabicycloundec-7-ene

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

DEACM (7-diethylaminocoumarin-4-yl)methyl

DEAD diethyl azodicarboxylate dA 2ʹ-deoxyadenosine

DIAD diisopropyl azodicarboxylate

DIBALH diisobutylaluminium hydride

DIPEA diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMNB 4,5-dimethoxy-2-nitrobenzyl

DMSO dimethyl sulfoxide

DMF dimethyl formamide

DMTr dimethoxytrityl

DMA dimethylacetamide

DNA deoxyribonucleic acid

2-DPBA 2-(diphenylphosphino)benzoic acid

4-DPBA 4-(diphenylphosphino)benzoic acid

DSC disuccinimidyl carbonate

EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

xiv

EDTA ethylenediaminetetraacetic acid

EGFP enhanced green fluorescent protein

ESI electrospray ionization

Et ethyl

Et2O diethylether

EtOAc ethyl acetate

EtOH ethanol

Fm 9-fluorenemethyl

Fmoc fluorenylmethoxycarbonyl

GFP green fluorescent protein

HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate

HBTU O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium

hexafluorophosphate

HMDS hexamethyldisilizane

1H NMR proton nuclear magnetic resonance

HPLC high performance liquid chromatography

HRMS high resolution mass spectrometry

IBX 2-iodoxybenzoic acid i-Pr isopropyl

IR infrared

LG leaving group

LiHMDS lithium hexamethyldisiliazide

LNA locked nucleic acid

M molar mc dicyanocoumarin m-CPBA meta-chloroperoxybenzoic acid

Me methyl

MeOH methanol miRNA microRNA mg milligram

MHz megahertz mL milliliter mM millimolar mmol millimole mol mole mRNA messenger RNA

MO morpholino oligonucleotide

MsCl methanesulfonyl chloride

MW microwave

NBD nitrobenzofuran

NCS N-chlorosuccinamide

NPM ortho-nitropiperonyl methyl

NPOM nitropiperonyloxymethyl

xvi

NDBF 3-nitro-2-ethyl-dibenzofuran

NHS N-hydroxysuccinimide nm nanometer

NMO N-methylmorpholine-N-oxide

NPE 2-(o-nitrophenyl)ethyl

NPP 2-(o-nitrophenyl)propyl oAzBn ortho-azidobenzyl

ONB ortho-nitrobenzyl pAzBn para-azidobenzyl

PBS phosphate buffered saline

PCC pyridinium chlorochromate

PDC pyridinium dichromate

Ph phenyl

PMB para-methoxybenzyl

PNA peptide nucleic acid

PPh3 triphenylphosphine

PyBop benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

PPi pyrophosphate inorganic ppm parts per million p-TsOH p-toluenesulfonic acid rt room temperature

RISC RNA-induced silencing complex

xvii

RT-PCR real-time polymerase chain reaction siRNA small interfere RNA sfGFP superfold green fluorescent protein

SPPS stepwise solid-phase peptide synthesis

TBAF tetrabutylammonium fluoride

TBAI tetrabutylammonium iodide

TBDMS t-butyldimethylsilyl t-BuOK potassium tert-butoxide tc thiocoumarin

TEA triethylamine

TFA trifluoroacetic acid

Tf trifluoromethyl

TFO triplex-forming oligonucleotide

THF tetrahydrofuran

THP tris(hydroxypropyl)phosphine

TIPS triisopropylsilyl

TLC thin layer chromatography

TMS trimethylsilyl

TPPTS triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt tRNA transfer ribonucleic acid

UAA unnatural amino acid

UV ultra violet

xviii

CHAPTER 1 Regulation of Biological Activity with Oligonucleotides

1.1 Synthetic oligonucleotides in the regulation of biological processes

The ability to react to changes in the environment is considered an important characteristic of life. In nature, these accommodations are achieved via complex biological processes.1 In order to study various mechanisms of these biological systems as well as to regulate detrimental defects in cells, artificial approaches to control biological functions were developed. Synthetic oligonucleotides, as one of these approaches, have been extensively applied in the control of a wide range of biological molecules including DNA, RNA and protein (Figure 1.1). For instance, triplex-foming oligonucleotides (TFO) and DNA decoys regulate DNA function through transcriptional control. Antisense agents, DNAzymes and small interfering RNAs (siRNAs) directly target mRNA translation; meanwhile antagomirs control mRNA function through the regulation of miRNAs. Aptamers can bind to protein after translation and control protein function. The predictability in sequence design coupled with the well-established polymerization chemistry allows for both high flexibility and specificity in this approach, making it a powerful class of tools in the study and regulation of cellular processes.

Figure 1.1 Regulation of biological molecules with oligonucleotides.

1.1.1 Transcriptional level of regulation

In general, synthetic oligonucleotides have been developed to regulate biological activities at three different levels: tanscription, translation and protein function. Normally, gene is transcripted into mRNA by transcription factor and RNA polymerase, and the subsequent mRNA translation afford protein of certain function (Scheme 1.1A). At transcrptional level, triplex-forming oligonucleotides (TFOs) can site-specifically bind to the promoter region in double-stranded DNA forming a triplex helix which prevents transcription factors from binding and thus induce gene silencing (Scheme 1.1B). Apart from controlling gene expression, TFOs have also been successfully applied as tools in gene repair,2 gene modification,3 inhibition of DNA replication,4 and double-strand DNA detection.5

DNA decoys are another way of transcriptional regulation over DNA expression

(Scheme 1.1C). A DNA decoy is a short double-stranded DNA molecule that contains the binding sequence for a transcription factor. The decoy sequesters the transcription factor and prevents it from binding to its genomic promoter region, thereby inhibiting transcription.6

Since their introduction in 1990, several DNA decoys have been reported as versatile antigene reagents to deactivate gene function.6, 7 Targeting transcription, instead of translation, holds promise as the catalytic formation of mRNA is inhibited, in contrast to targeting mRNA function directly. However, the delivery of oligonucleotides to the nucleus remains challenging, potentially limiting their efficiency.6

target gene

A B C TFO DNA transcription decoy factor transcription transcription factor factor

transcription mRNA

DNA triplex translation

protein

Scheme 1.1 Transcriptional regulation over gene expression. (A) Normally, transcription factor binds to the promoter sequence in DNA duplex and start DNA transcription followed by translation to give protein. (B) TFO prevents the transcription factor from binding to target gene through the formation of a DNA triplex, thus blocks gene transcription. (C) In presence of DNA decoy, transcription factor binds to the decoy instead of target gene, and DNA transcription is prevented.

1.1.2 Translational level of regulation

Various synthetic reagents have been developed to target mRNA in order to inhibit translation and thus, the corresponding gene expression. Antisense agents are among the most widely used tools. Antisense agents are oligonucleotides that site-specifically target mRNA and block subsequent protein expression, through either ribonuclease H (RNase H) mediated mRNA cleavage (Scheme 1.2A) or steric blocking of the ribosome (Scheme 1.2B).8

Both mechanisms lead to an efficient inhibition of protein expression and gene silencing.

Thus, antisense agents have been proven to be powerful tools for the study of gene function in cells and multicellular model organisms, especially zebrafish embryos.9

target gene

DNA

transcription target antisense mRNA agent mRNA mRNA C

ribosome A B

RNase H ribosome

translation

no protein no protein protein

Scheme 1.2 Antisense agent induced gene silencing through different mechanisms. An antisense agent selectively binds to target mRNA (green) and stops mRNA translation through (A) RNase H-mediated mRNA cleavage or (B) blocking of mRNA processing by the ribosome. Meanwhile, (C) other non-targeted mRNAs (blue) are translated into proteins normally.

Alternatively, catalytic deoxyribozymes (DNAzymes) can also block gene expression at translational level through induced cleavage of mRNA (Scheme 1.3). DNAzymes were first discovered in 1994 through the evolution of an artificial DNA oligonucleotide that can cleave RNA in a sequence-specific manner.10 Since then, various DNAzymes with different functions have been engineered and applied as biological sensors, DNA computation devices, ligases, and potential gene silencing agents.11-15

target gene DNAzyme transcription

mRNA

translation DNAzyme

protein

Scheme 1.3 DNAzyme induced gene silencing. DNAzyme binds to mRNA and catalizes the cleavage of mRNA so as to stop mRNA translation. In absence of DNAzyme, protein is expressed normally.

Small interfering RNA (siRNA) as well as its genetically encodable precusors, small hairpin RNA (shRNA),16 is another class of powerful gene silencing tools targeting at mRNA. As short noncoding RNA duplexes, siRNAs are processed through the RNA interference pathway and inhibit the expression of its complement gene (Scheme 1.4).17 siRNAs have been widely applied to the study of gene function and gene regulation.18, 19

DNA shRNA siRNA RISC

transcription

mRNA translation

protein

Scheme 1.4 siRNA induced gene silencing. A siRNA, generated from shRNA or directly transfected into cell, selectively binds to mRNA and interfere mRNA translation through RNA-induce silencing complex (RISC). In absence of siRNA, encoded protein is successfully expressed.

Recently, microRNAs (miRNAs) are evaluated as regulators of biological function and have been found to be involved in a wide range of cellular processes.20 The overexpression of miRNAs is related to several human diseases, including many cancers.21 miRNAs are short non-coding RNAs that are produced from the genome. The transcription of miRNA genes delivers pri-miRNAs, which are cleaved into pre-miRNA hairpin. The pre- miRNA hairpin is then exported from nucleus into the cytosol, where the pre-miRNA cleavage results in a miRNA duplex and subsequently a mature miRNA is formed (Scheme

1.5). The miRNA can bind to its complementary mRNA and thereby inhibit mRNA translation (Scheme 1.5A).22 In an effort to control miRNA activity, oligomers called antagomirs are designed to inhibit miRNA function by forming miRNA:antagomir duplexes

(Scheme 1.5B).23 Different from all above approaches, antagomirs recover the protein expression silenced by the miRNA. Small molecules known as miRNA inhibitors can also regulate miRNA level to trigger gene expression.24

pri-miRNA pre-miRNA miRNA RISC duplex miRNA A

transcription

antagomir

B transcription mRNA

trnaslation

mRNA trnaslation

C DNA protein

Sheme 1.5 miRNA regulation over gene expression. Apart from been transcripted to mRNA and then translated into protein (C), DNA also encodes pri-miRNA which delivers matural miRNA in three steps through the formation of pre-miRNA and mRNA duplex. (A) Mature miRNA selectively binds to mRNA and induces gene silencing. (B) in presence of antagomir, miRNA forms a duplex with antagomir rather than binding to mRNA and the coressponding protein expression is recovered.

1.1.3 Protein level of regulation

Both transcriptional and translational regulation results in the control over protein expression. Nucleic acid aptamers, however, can trigger protein activity after translation.

Nucleic acid aptamers are single-strand oligonucleotides folded into certain three dimentional configurations that can recognize and bind to a variety of targets including small molecules, nucleic acids, and proteins with high specificity. Upon binding to protein or other targets, nucleic acid aptamers act through different mechanisms to control the function of their targets.25 Recently, aptamers have been applied to biosensors,26 drug delivery,27 nanotechnology,28, 29 and molecular imaging.30 Post-transcriptional modification is another

design aspect that can be exploited to control protein function, which will be discussed in

CHAPTER 5.

In addition to above regulatory applications, oligonucleotides have been programed into DNA logic gates which produce controlled outputs in response to biological or enternal inputs.31 DNA computation based on the assembly of complex circuits of DNA logic gates can possibly perform complex algorithms while interacting with biological and chemical environments.

1.2 Oligonucleotide improvement through chemical modification

As discussed above, oligonucleotides have been applied to control a wide range of processes. The chronicle of oligonucleotide regulation starts with the introduction of the first antisense agent by Stephenson and Zamecnik in 1978. They applied a 13 nucleotide single- stranded DNA (ssDNA) to chick embryo fibroblast tissue cultures in order to inhibit the Rous sarcoma virus.32 ssDNA and their modifications including phosphorothioate DNA (PS-

DNA), and methylphosphonate DNA are considered to be the first generation of antisense oligonucleotides (Figure 1.2A). These oligonucleotides are able to trigger a response by the innate immune system and have been applied to all approaches listed in Chapter 1.1. 33

Meanwhile, these antisense agents are subjected to nuclease cleavage and suffer from potential cellular toxicity. 34, 35

Second generation of antisense oligonucleotides (Figure 1.2B) refers to 2 modified

DNA nucleotides and the most common modifications are 2ʹ-O-alkyl RNA oligonucleotides.8, 33 Compared to DNA oligonucleotides, 2ʹ-O-alkyl RNA agents are

relatively resistant to nucleases, less toxic, and show better affinity to the complementary mRNA. 2ʹ-O-alkyl RNA agents, especially 2ʹ-OMe RNA agents are still one of the most popular gene silencing reagents today.36, 37

Oligomers with significantly different structures from the last two generations of regulatory oligonucleotides are classified as third generation oligonucleotides. The widely used third generation oligonucleotides include peptide nucleic acids (PNAs), locked nucleic acids (LNAs) and morpholino oligonucleotides (MOs) (Figure 1.2C). Because of the structure modification, third generation antisense agents exhibit good biological stability, low off-target effects, increased binding affinity, and low toxicity among other desirable properties.38, 39 PNAs have an achiral, neutral polyamide backbone, thus are resistant to normal nucleases as well as peptidases and are capable to bind to target sequence with enhanced affinity.40 Taking advantage of their unique properties, PNAs have been applied to a variety of hybridization-based applications including DNA decoys, TFOs, antisense agents, antagomirs, PCR primers, and biosensors.41-43 LNAs provide a substantial increase in binding affinity as well as an improved cellular uptake compared to the last two generations of antisense agents.44 The high binding affinity makes LNAs a promising class of regulatory oligonucleotides, which have also been applied to all approaches introduced in Chapter

1.1.45-47 MOs are designed to have low production cost and have been proven to be one of the most promising gene knockdown tools in developmental biology.48 Also, MOs have been applied to treat RNA virus infection, regulate RNA splicing, and block microRNA function as antagomirs both in vitro and in vivo.43, 49-52

Figure 1.2 Three generations of synthetic oligonucleotides. A) First generation. B) Second generation. C) Third generation antisense agent. B = A, T, G, C. R= alkyl group, e.g., Me.

The improvement of synthetic oligonucleotides through chemical modifications are still ongoing to seek desired properties including low toxicity, high binding affinity, good cellular stability, efficient cellular uptake, and biophysical probe labelling.53, 54 Meanwhile another problem with the application of oligonucleotides in complex biological systems is the lack of spatial and temperal control. To solve this problem, our developed caging technology is introduced to the oligonucleotides.

1.3 Regulation of synthetic oligonucleotide activites with light

In order to elucidate the underlying mechanisms that maintain and control biological systems, synthetic approaches have been developed for conditional control of cellular processes. Light is a versatile regulatory element, as it is fully orthogonal to most cellular components, is non-invasive, and can be easily controlled in timing and localization to certain tissues, cells, and even sub-cellular compartments.55 Coupled with gene regulatory methods introduced in chapter 1.1, light has been used to control biological activities with high spatial and temporal resolution. This precise control enables, for example, the study of genes essential for embryonic development by deactivating them at specific time points and locations thus minimizing embryonic lethality.56 The light regulation of biological processes is often based on photo-labile protecting groups, so called ‘caging groups’,57, 58 that are installed on small molecules, proteins, or oligonucleotides, thereby serving as light-triggered switches for cellular mechanisms.59-65

1.3.1 Evolution of caging groups

The term ‘caging’ was coined by J. F. Hoffman in 1978 to describe the application of photo-labile protecting groups in a biological setting with the aim to achieve spatial and temporal control over the protected substrate.57 These caging groups should: (1) render the substrate biologically inactive, (2) be readily cleaved by non-damaging light, and (3) the substrate should fully regain its biological activity after photolysis, while (4) the caging group should not produce highly reactive or toxic byproducts (Scheme 1.6).66

Scheme 1.6 Light activation of a caged molecule. The red ball represents a biomolecule and the blue frame represents a photo-caging group. The biomolecule is inactive with the caging group and active after light irradiation cleaves the caging group.

Most caging groups are derivatives of the ortho-nitrobenzyl (ONB) moiety (1, Figure

1.3) as they are easy to synthesize and give high yields in both the caging and decaging processes. Based on the ONB group, a number of caging groups with different alkyl chain were synthesized to improve decaging rate including 1-(o-nitrophenyl)-ethyl (NPE) group,67 o-nitromandelyloxycarbonyl (Nmoc) group,68 and 1-(o-nitrophenyl)-2,2,2-trifluoroethyl

(NPT) group.69 The benzyl ring of ONB can also bear a wide range of functional groups such as hydroxyl, amino, and carboxyl groups. It is found that the installations of electro-donating groups on benzyl ring can red-shift the decaging wavelength and reduce the possibility of photo-damage. Thus nitroveratryl (NV) group,70 4,5-dimethoxy-2-nitrobenzyl

(DMNB) group,71 (1-(4,5-dimethoxy-2-nitrophenyl)-2,2,2-trifluoroethyl (DMNPT) group,69

6-nitropiperonyloxy-methyl (NPOM) group,72 and 5-aminomehthyl-2-nitrobenzyl (AMNB) group73 were developed. Recently, the propargyl-6-nitroveratryloxymethyl (PNVOM) group was reported, which provides the added benefit of conjugations via Click reaction with the ability for light regulation.74 The decaging of these ONB groups is initiated via a one-proton absorption process with light irradiation of UV or near-UV region (between 200nm and

370nm).75 After initiation, the decaging of ONB group (4) proceeds through a Norrish type II

mechanism,66 affording a free substrate and a nitrosobenzocarbonyl molecule (5) (Scheme

1.7A). The nitroso byproduct (5), however, can potentially react with nucleophiles in living system and be toxic. Pentadienylnitrobenzyl (PeNB) group can trap the nitroso group through an internal hetero Diels-Alder reaction.76

Figure 1.3 UV cleavable caging groups.57, 67-74, 76-84 LG: leaving group.

As an alternative to ONB groups, nitrophenylpropyl (NPP) group (2, Figure 1.3) was also evaluated for photo-cleavage and exhibited an enhanced decaging rate. Furthermore, the

NPP group (6) is cleaved through β-elimination to give a nitrovinyl byproduct (7) rather than a nitroso after light irradiation (Scheme 1.7B).85 These two advantages make NPP derivatives an attractive class of caging groups. The commonly used NPP caging groups include 2-(4,5- dimethoxy-2-nitrophenyl)propyl (DMNPP) group,78 3-(4,5-dimethoxy-2-nitrophenyl)-2-butyl

(DMNBP) group,78 and 2-(3,4-methylenedioxy-6-nitrophenyl)-propyl (MNPP) group.79

Coumarin groups (3, Figure1.3) are another popular class of caging groups because of their longer-wavelength absorptions (400-500 nm).86 Different modifications have been made

on coumarin backbone especially at the 3- and 7- positions to improve its photo properties.

The early reported coumarin caging groups are 7-hydroxylcoumarin (HCM) and 7- methoxylcoumarin (MCM) derivatives,81 followed by the introduction of 7- dimethylaminocoumarin (DMACM) group and 7-diethylaminocoumarin (DEACM) group, which show more red-shifted absorption maximum (385-400 nm).82 Recently, the absorption maximum of coumarins have been shifted to 400-500 nm region through the following modifications: (1) the installation of a strong electron-donating group on 3-position such as

3-cyano-7-diethylaminocouamrin (NdiEt-3CN-c),83 (2) thionation of coumarin backbone affording 7-methoxythiocoumarin (OMe-tc) and 7-diethylaminothiocoumarin (NdiEt-tc),84, 87 and (3) malononitrile modification on coumarin backbone to give 7-Diethylamino-4-methyl- dicyanocoumarin (NdiEt-mc).83 The application of coumarin caging groups (8), however, are limited to leaving groups with a low pKa due to the formation of an ion pair in the photolysis mechanism of coumarins (Scheme 1.7C).88

Scheme 1.7 Photolysis mechanism of different caging groups: (A) Photolysis mechanism of ONB groups (B) Photolysis mechanism of NPP groups (C) Photolysis mechanism of coumarin groups.

Although coumarin caging groups exhibit a significant red-shift absorption maximum, in order to further shift the photo-cleavage wavelength to minimize photo- damage, two-photon caging groups have been introduced. Rather than absorbing one photon at high energy, a two-photon caging group absorbs two photons simultaneously in a single quantized event, and the energy of each photon absorbed is only half of the energy needed for excitation.89 Thus the irradiation wavelength of a two-photon excitation is about 700 nm

(near-IR or IR region). Furthermore, two-photon excitation ensures its better spatial selectivity through better three-dimensional focus and higher photon density requirement compared to one-photon activation (Figure 3).89-93

caged active remnant of compound compound caging group

Figure 1.4 The improved three-dimensional focus of two-photon excitation. In two-photon excitation, only one molecule at a focused position regains its activity while molecules all through the depth of tissue are activated in one photon-decaging.6

The ONB, NPP, and coumarin caging groups discussed above are designed for one- photon absorption and bear low decaging efficiency in two-photon excitation. Efforts have been made to improve their two-photon properties through extension of conjugation and installation of donor/acceptor substituents.94, 95 The first two-photon caging group reported is

6-bromo-7-hydroxycoumarin-4-methyl (Bhc) group (13, Figure 1.5),92 which also set the core structure for future coumarin two-photon caging groups.96 The 8-bromo-7-hydroxyquinoline

(BHQ) group (14) was introduced with an improved two-photon effect.98 However, it is difficult to optimize the photophysical properties of the BHQ group.97 ONB group, on the contrary, can tolerate a variety of modifications and yield at several two-photon caging

groups. Popular ONB two-photon caging groups include 3-nitro-2-ethyldibenzofuran

(NDBF) group (10),98 4-nitro-biphenyl derivatives (11) such as 3-(2-propyl)-4-methoxy-4- nitro-biphenyl (PMNB) group 99 and 3-(2-propyl)-4-dimethylamino-4-nitro-biphenyl

(ANBP) group,100 4,4’-bis-(8-(4-nitro-3-(2-propyl)-styryl))-3,3’-dimethoxybiphenyl

(BNSMB) group (12),94 and 2,7-bis-(4-nitro-8-(3-(2-propyl)-styryl))-9,9-bis-(1-(3,6- dioxaheptyl))-fluorene (BNSF) group (13).94

Figure 1.5 Two-photon caging groups.

Photo-switchable chromophores are among the earliest reported photo-triggered regulators of biological function although they are not exactly ‘caging’ groups.101 Several series of photo-switches based on azobenzene,102 spiropyrans,103 and diarylethenes,104 fulgides,105 and overcrowded alkenes106 have been developed. The azobenzene group (16) is the most commonly used photo-switch, which undergoes a cis-trans isomerization upon UV or visible light irradiation and regulates their target activity in a reversible manner (Scheme

1.8).107

Scheme 1.8 Photo-induced cis-trans isomerization of azobenzene group

1.3.2 Caging approaches to regulate oligonucleotide function

In order to accomplish the photo-regulation of oligonucleotide function, several different approaches have been developed that all take advantage of the photo-labile caging groups as well as the high modularity and synthetic accessibility of oligonucleotides (Scheme

1.9). One of the early and most widely used strategies involves the insertion of a photo-labile moiety within the oligonucleotide backbone.108 Upon irradiation, the oligomer is cleaved, thereby inactivating its function (Scheme 1.9A). To optically activate oligonucleotide function, a complementary inhibitor strand is tethered to a functional strand through a photo- cleavable linker (Scheme 1.9B). In the absence of light irradiation the caged duplex is inactive; after light irradiation, the linker is cleaved and the active, functional strand is released. Most photo-cleavable linkers are based on the ONB moieties,109-111 but the BHQ group has also been used in this approach as well.112 Since its introduction in 1995,108 light- cleavable linkers have been applied to DNA,110 2´-O-methyl RNA,113 PNA,114 and morpholino oligonucleotides.56, 109, 111 However, three synthetic components are needed for this strategy (two oligonucleotide strands and the photo-cleavable linker) and the design of the short inhibitor strand is not trivial.112, 115 If the inhibitor strand is too short, its binding energy is not sufficient to prevent the mRNA from hybridizing to the oligonucleotide. On the

other hand, if its binding affinity to the oligonucleotide is too high, it will not be efficiently released after linker photolysis. For MOs specifically, the Chen lab have derived an equation

(1) as a general guide for inhibitor design based on the melting temperature and biological

112 studies. The equation shows that the melting temperature of the MO duplex (Tm) is related to the number of A/T and G/C base pairs. An efficient inhibitor strand will be generated by designing a hairpin MO with a melting temperature between 41 ˗ 44 C.

Tm = 1.9  (A + T) + 5.7  (G + C) (1)

Recently, light-activated circular oligonucleotides, generated by linking both ends of a linear oligonucleotide through photo-cleavable moieties, were introduced as a modification of the above approach. Compared to linear oligonucleotides, circular ones show reduced structural flexibility in binding to their complementary target sequences due to the induced curvature. Thus, antisense agents can be rendered inactive through circularization and are readily activated through photolysis of a linker, enabling duplex formation with target strands

(Scheme 1.8C).73, 116-118

photo-cleavable linkers reversible photo-switches A deactivation D

B activation photo-caged nucleobases E activation

C activation F deactivation

Scheme 1.9 Different caging approaches to regulate oligonucleotide hybridization with light. A) Photo-deactivation via light-induced strand breakage. B) Photo-activation via light-induced release of an inhibitor strand. C) Photo-activation via linearization of a light-cleavable circular oligonucleotide. D) Reversible control over oligomer hybridization via diazobenzene incorporation. E) Photo-activation via photolysis of caged nucleobases. F) Photo-deactivation via removal of nucleobase-caging groups from an inhibitor strand. Target strands, typically mRNA, are shown in green; functional oligonucleotides, e.g., an antisense agent, are shown in blue; inhibitor strands are colored dark red; nucleobase-caging groups and light-cleavable linkers are indicated by a red circle.

The groups of Asanuma and Komiyama introduced an photo-switchible azobenzene group into oligonucleotides and accomplished the regulation of duplex formation between modified oligonucleotides and their target in a reversible manner (Scheme 1D).119-121

However, the limited dehybridization at room temperature and the incomplete trans to cis switching of most azobenzenes currently limits intracellular applications of this approach.122

However, no reversible photo-switches with improved stability and photoisomerization efficiency have been installed on oligonucleotides, which shows the potential of this approach.123-125

A versatile approach to optically trigger a wide range of oligonucleotide function involves the installation of caging groups onto oligomers at various positions, including backbone phosphates,126-128 2´-OH groups,129, 130 and nucleobases. Among these approaches, caged nucleobases have demonstrated the broadest applicability in our lab. With caging groups blocking Watson-Crick hydrogen bonding, the oligonucleotide is inactive until photolysis of the caging groups restores oligonucleotide function (Scheme 1D). The corresponding light-deactivation of function is accomplished via a caged self-complementary strand (Scheme 1E). The functional arm can still bind to its target sequence before light irradiation, but a hairpin structure is formed and the activity of the oligonucleotide is inhibited after photolysis.

Since the introduction of the caged nucleobase 17 in 2004,131 several other caging groups have been installed on specific nucleobases (Figure 1.6). For example, the NPE group has been applied to five nucleobases 18, 21, 25, 32, and 36.132-134 In order to improve decaging rates, NPP caged nucleotides 19, 22, 26, and 33 were developed.133, 135, 136 To red- shift the absorption maximum and thereby the decaging wavelength and to increase the stability of the caging group during oligonucleotide synthesis, our lab installed the 6- nitropiperonyl methyl group (NPM) on cytosine (34) and its corresponding hydroxymethylene analog (NPOM) on guanine (23), thymine (27), and uracil (37).137-140 The decaging wavelength of all these groups are within the UV range, typically 360-366 nm, and thus is orthogonal to all commonly used fluorescent proteins. This enables the direct interfacing with many reporter systems,58 while maintaining low cellular and toxicity due to light exposure.141, 142 Furthermore, these caging groups are stable under ambient light and no

additional precautions are required during synthesis and handling. The application of coumarin caging groups and p-hydroxyphenacyl (pHP) group allows for multi-wavelength control of different biological processes.80, 87, 143, 144 Recently, the DEACM caged guanine 24 and thymine 29 was reported,136, 145 enabling photolysis at >405 nm and pHP caged thymine

28 can be cleaved at 313 nm.136 In addition, two-photon caging groups have been introduced, which enable activation at 720-800 nm. For example, the NDBF group was employed in the caging of 20, 30, and 35; however, its application in optochemical oligonucleotide control has yet to be demonstrated in biological systems.134, 146 Our group recently accomplished the conjugation of multiple HIV TAT peptides to an antisense agent through a PNVOM caged thymine (31) which aids the cellular delivery of oligonucleotides.74

Perturbation effects of nucleobase caging groups vary, based on the oligonucleotide function, the number of caged nucleobases, and their position.136 In certain cases, especially the regulation of DNA:protein interactions and catalytic DNAzyme activity, a single caging group located at a crucial site is sufficient to block activity until light exposure.137, 138, 147, 148

However, multiple caging groups are usually required to regulate base-pairing interactions between oligonucleotides such as TFOs, DNA decoys, antisense agents, siRNAs, and antagomirs. Generally, one caging group every 5-6 bases evenly distributed throughout an oligonucleotide fully abrogates hybridization to its complement, until the caging groups are removed through irradiation.133, 149, 150

Figure 1.6 Chemical structures of caged nucleobases, grouped by letter code, that have been incorporated into oligonucleotides.131-139, 145, 146, 151, 152 The light-removable caging groups are shown in red.

This nucleobase-caging approach can be readily applied to a wide-range of oligomers with different sequences, structures, and functions by simply incorporating caged monomers into the oligonucleotide through standard solid-phase synthesis methods. Thus, nucleobase-

caged oligonucleotides provide the programmability and flexibility to target any selected sequence or gene with high specificity, and depending on the design, either allow for light- induced activation or deactivation of biological processes.153 Additionally, recent reports show the potential of nucleobase-caging in emerging oligonucleotide technologies, including

DNA computation, DNA/RNA sensing, and DNA nanotechnology.154-156

Meanwhile, cyclic oligonucleotide strategy is a relatively new caging approach that combines several advantages of the above three approaches: 1) the oligonucleotides can be purchased readily; 2) one caging group linker is sufficient to induce deactivation; 3) no byproduct oligomers are generated after photolysis; 4) different caging groups, even coumarins, which require a low pKa of the caged target, are tolerated.

This dissertation presents the development of new caged nucleotides in CHAPTER 2 and caged MO-monomers in CHAPTER 3 to further broaden the application of caged oligonucleotides. In CHAPTER 4, the synthesis of novel photo-cleavable linkers for cyclic oligonucleotides will be discussed, which provide multi-wavelength regulations.

CHAPTER 2: Synthesis of Photocaged Nucleoside Phosphoramidites

2.1 Synthesis of 6-nitropiperonylmethyl-caged deoxyadenosine phosphoramidite

Previously, Dr. Lusic and Dr. Uprety in the Deiters group synthesized 6- nitropiperonyloxymethyl (NPOM)-caged thymidine phosphoramidite (38), deoxyguanosine phosphoramidite (39), and 6-nitropiperonylmethyl (NPM)-caged deoxycytosine phosphoramidite (40) (Figure 2.1). These phosphoramidites have been successfully incorporated into TFOs, DNA decoys, antisense agents and DNAzymes to trigger biological activities.153 To complete our tool box of caged deoxynucleoside phosphoramidites as well as to provide more flexibility in caged oligomer design, the NPM-caged deoxyadenosine phosphoramidite (NPM-dA) 41 was synthesized.

Figure 2.1 Photocaged deoxynucleoside phosphoramidites.

The caging of the adenine residue was reported recently, including 1-(2-nitrophenyl) ethyl (NPE)-caged adenosine phosphoramidite (42) and 2-ethyldibenzofuran (NDBF)-caged deoxyadenosine phosphoramidite (43) (Figure 2.1).134, 157 Based on the reported approaches,

1-(6-nitropiperonyl) ethylamine (NPM-NH2) (47) was constructed as a caging precursor in three steps from commercially available 6-nitropiperonal (44) (Scheme 2.1). Methylation of the aldehyde of 6-nitropiperonal (44) afforded the secondary alcohol 45 in 97% yield

72 (AlMe3, DCM). The alcohol 45 underwent a Mitsunobu reaction to deliver the phthalimide

157 46 (isoindole-1,3-dione, PPh3, DIAD in THF, 84% yield). Cleavage of the phthalimide in

46 was achieved through treatment with hydrazine in ethanol, giving the caging precursor

157 NPM-NH2 (47) in 68% yield.

Scheme 2.1 Synthesis of the NPE-NH2 (47) caging precursor.

The nucleoside segment was prepared from commercially available 2-deoxyinosine

(48), which was first treated with TBDMSCl and imidazole in DMF to give the protected 2- deoxyinosine 49 in quantitative yield (Scheme 2.2).134 A 2,4,6-triisopropylbenzenesulfonyl group was installed onto 49 converting the amide oxygen into a good leaving group (2,4,6- triisopropylbenzenesulfonylchloride, DIPEA and DMAP in DCM, 50% yield).134 The formation of 1-N sulfonated byproduct limited the yield of the sulfonation which is consistent

157 with previous report. Thus different solvents (DMF, CH3CN, and THF) and bases (TEA,

Na2CO3, and DBU) were screened in search for optimized conditions. Then the caging precursor NPM-NH2 (47) reacted with 50, and the TBDMS-protected NPM-caged deoxyadenosine (51) was obtained in 83% yield (DIPEA, DMAP, DMF, 90oC).157 Through treatment with TBAF, the silyl ether in 51 was cleaved in quantitative yield.134 A subsequent

DMTr protection of the 5-hydroxyl group of 52 delivered the DMTr-protected caged deoxyadenosine (53) in 63% yield (DMTr-Cl, pyridine).134 Compound 53 can be stored for a prolonged period of time without any significant decomposition. The NPM-dA (41) was made freshly from 53 by introducing the phosphoramidite onto the 3-hyroxyl group (2- cyanoethyl-N,N-diisopropyl-chlorophosphoramidite and DIPEA in DCM, 61% yield).134

Scheme 2.2 Synthesis of the NPM-dA (41).

2.2 Development of caged nucleosides with red-shifted cleavage wavelengths

The NPE- and NPOM-caged nucleosides (38-41) shown above are cleaved by 365 nm light. Although they have been successfully applied in regulating oligonucleotide functions,

UV irradiation has limited penetration depth and may cause potential photo-damage in biological systems.158 Thus, the development of caged nucleosides that can be activated through light of red-shifted wavelengths is an area of active research as discussed in

CHAPTER 1.3.2.134, 145, 146, 159, 160

2.2.1 Synthesis and photolysis of coumarin caged nucleosides

Coumarin groups (3, Figure 1.3) can be cleaved with 400-500 nm light,86 but are only suitable caging groups for acidic substrates because of their photo-cleavage mechanism

(Scheme 1.7C).88 Recently, however, Heckel et al. introduced a diethylaminocoumarin

(DEACM) group into guanine (24, Figure 1.6), and successfully cleaved the DEACM group with 405 nm light.145 Here the DEACM group was installed either on the 4-O position or the

3-N position of pyrimidine bases and subjected to photolysis.

A two-step conversion was employed to transform commercially available 7- diethylamino-4-methylcoumarin (54) into the DEACM alcohol (55) as reported previously with minor modification (Scheme 2.3).161 The usage of dioxane as a solvent instead of xylene for SeO2 oxidation improved the yield to 66% for the synthesis of 55. The alcohol 55 then underwent Appel reaction delivering DEACM-Br 56 in 76% yield.162

Scheme 2.3 Synthesis of the DEACM caging group 56.

To investigate the behavior of DEACM groups on pyrimidine bases, three commercially available nucleosides were subjected to caging, including 2ʹ-deoxyuridine (57),

5-fluoro-2ʹ-deoxyuridine (58), and thymidine (59) (Scheme 2.5). These three bases lack an exo-amine thus are easier to work with. Furthermore, the pKas of the amides in these bases

are comparable to if not lower than the pKa of the guanosine amide,145 thereby favoring

DEACM decaging.163 Also these caged-pyrimidine bases can interrupt A-T pairing and supplement the DEACM-guanine that controls G-C pairing.

The synthesis starts with the TBDMS protection of the 3ʹ- and 5ʹ-hydroxyl groups on the nucleosides 57-59 in high yields (Scheme 2.4).164 The obtained TBDMS-protected nucleosides 60-62 were reacted with the DEACM alcohol 55 via a Mitsunobu reaction to give the caged nucleosides 63-65 in 63-86% yield.145 Upon the cleavage of the silyl ethers with TBAF, the O-DEACM-caged nucleosides 66-68 were obtained in 77-93% yields.165

Scheme 2.4 Synthesis of the O-DEACM-caged nucleosides 66-68.

For the synthesis of N-caged nucleosides, the DEACM-Br (56) was used to alkylate

138, 166 the protected nucleosides (60-62) in the presence of DBU (Scheme 2.5). A SN2 substitution followed by a TBAF deprotection delivered the N-caged nucleosides 72-74 in medium to high yields.

Scheme 2.5 Synthesis of the N-DEACM-caged nucleosides 72-74.

The six nucleosides 66-68 and 72-74 (20 µM in PBS buffer pH 7.4) were irradiated with a Xe/Hg lamp equipped with a 405 nm filter and analyzed by LC-MS. Unfortunately no nucleoside cleavage was observed in our study. Meanwhile, the successful cleavage of the O-

DEACM-caged thymidine 67 was reported by Heckel et al..159 This confirmed that light is capable to cleave DEACM group on pyrimidine bases and our failure in the photolysis is probably due to instrumental limitations (see Appendix A).

2.2.2 Synthesis and photolysis of 3-(2-propyl)-4-dimethylamino-4-nitro-biphenyl nucleosides

Caging groups based on the 4-nitro-biphenyl group (11, Figure 1.5) were developed as two-photon caging groups.100 Meanwhile, these groups can absorb strongly around 400 nm, which makes them attractive even as one-photon caging groups.

The synthesis of 4-nitro-biphenyl derivatives began with the alcohol 75 which was made in four steps following literature.167 A set of phenylboronic acids, containing hydrogen, trimethylsilyl, methoxyl, and dimethylamino groups at the para position was employed to react with the alcohol 75 (Scheme 2.6). The Suzuki coupling proceeded in moderate to good yields (57-98%) when conducted with Pd(OAc)2 in EtOH/H2O under microwave irradiation

(200 W, 60 ˚C, 10 minutes), affording the 4-nitrobiphenyl derivatives 76-79. On the absorption spectrum of these 4-nitrobiphenyl groups, 3-(2-propyl)-4-dimethylamino-4-nitro- biphenyl (ANBP) group showed the most red-shifted absorption maximum (Figure 2.2), thus the ANBP group was selected to caged thymidine.

Scheme 2.6 Synthesis of the 4-nitro-biphenyl derivatives 76-79.

2 1.8 1.6 1.4 1.2 NEt2 1 Abs 0.8 H 0.6 OMe 0.4 SiMe3 0.2 0 300 400 500 wavelength/nm

Figure 2.2 Absorption spectra of 76-79 (50 µM in MeOH).

Following a similar route as the synthesis of the NPOM-caging precursor developed by Dr. Lusic in the Deiters group,72 the ANBP alcohol 79 was converted to the thioether 80 using BPO and Me2S in 41% yield (Scheme 2.7). Subsequent treatment with sulfuryl chloride delivered the ANBP-Cl (81) which was confirmed by NMR, and was reacted with the TBDMS-protected thymidine 61 without further purification to afford the protected N-

ANBP-caged thymidine 82 in 14% yield (DBU, DMF). Efforts to optimize the caging step, including a screen of different bases (Cs2CO3 and DIPEA) and solvents (THF, DCM, and

CH3CN), were not successful due to decomposition of the starting materials. The instability is possibly caused by the presence of both a nucleophilic amino and an electrophilic chloro group in 81. Upon removal of the silyl groups, the N-ANBP-caged thymidine 83 was obtained.

Scheme 2.7 Synthesis of the N-ANBP-caged thymidine 83.

The N-ANBP-caged thymidine 83 was then subjected to light irradiation at a 0.1 mM concentration in PBS buffer at pH 7.4 (Scheme 2.8) and was analyzed by LC-MS (Figure

2.3). After 10 minutes of irradiation (405 nm, 0.5 mW), the ANBP caging group was completely cleaved, delivering the free thymidine 58.

Scheme 2.8 Photolysis of the N-ANBP-caged thymidine 83.

A. N-ANBP T N-ANBP T

B. T

thymidine

C. N-ANBP T+UV thymidine

Figure 2.3 LC-MS analysis of the decaging of the N-ANBP-caged thymidine 83.

In hope of improvement on caging yields, a secondary ANBP alcohol 87 was synthesized. This secondary alcohol is a closer mimic to the NPOM group and would undergo photolysis through a Norrish type II mechanism which is different from the decaging of 79 as discussed in CHAPTER 1.3.1.66 Starting from 5-bromo-2-nitrobenzoic acid

(84), the carboxylic acid underwent an activation followed by a nucleophilic acyl substitution affording the amide 85 in 64% yield (Scheme 2.9).168 The DIBAL-H reduction of the amide

85 gave the aldehyde 86 in 75% yield,169 which was methylated to the secondary alcohol 87

72 in 84% yield (AlMe3 in DCM). A primary alcohol 88 was also obtained from the reduction of the acid 84 after thionyl chloride activation in 56% yield (over two steps).170 The two alcohols 87 and 88 was then reacted with 4-(dimethylamino)phenylboronic acid 89 via a

Suzuki coupling, among which only the secondary alcohol 87 gave the desired product 90.

However, the conversion of the secondary ANBP alcohol 90 into the thioether 91 suffered from low yield (20%) due to the formation of uncharacterized byproducts.171

Scheme 2.9 Synthesis of the secondary ANBP alcohol 90.

In addition to the N-caging approach, the primary ANBP alcohol 79 was installed on the 4-O position of thymidine 61 through a Mitsunobu reaction (PPh3 and DIAD in THF,

85% yield) (Scheme 2.10).145 This was followed by the cleavage of the silyl protecting groups by TBAF to afford the O-ANBP-caged thymine 93 (O-ANBP-T) in 60% yield. The 5- hydroxyl group of 93 was protected through treatment with DMTrCl in pyridine delivering the protected O-ANBP-T 94 in 76% yield.

Scheme 2.10 Synthesis of the O-ANBP-caged thymidine 94.

The photolysis of O-ANBP-caged thymidine 94 was tested at 0.1 mM concentration in pH 7.4 PBS buffer (Scheme 2.11) and was analyzed by HPLC (Figure 2.4). For recording thymidine 58 spectrum shown in Figure 2.7B, a 100 mM solution of thymidine 58 in PBS buffer (pH 7) was measured and the high concentration is owing to the low fluorescence of

58 compared to the caged thymidine 94. After 10 minutes of irradiation (405 nm, 0.5 W), the

O-ANBP-caged thymidine peak disappeared and the free thymidine 58 was obtained.

Scheme 2.11 Photolysis of the O-ANBP-caged thymidine 94.

A. O-ANBP T O-ANBP T

B. T thymidine

C. O-ANBP T+UV thymidine

Figure 2.4 HPLC analysis of the O-ANBP-caged thymidine 94 decaging.

The caged nucleoside 94 was subjected to standard oligonucleotide polymerization conditions in order to test the stability of the caging group: 1) con. NH4OH, 60 ˚C, 16 hrs; 2)

5% TFA r.t. 5 hrs; 3) 40% MeNH2 in MeOH, 60 ˚C, 16 hrs; and 4) 1:1 mixture of con.

NH4OH and 40% MeNH2 in MeOH, 60 ˚C, 2 hrs. No decomposition was observed both by

TLC and 1H NMR, indicating that the nucleoside 94 is stable for oligonucleotide synthesis.

2.3 Development of small molecule-triggered nucleosides

With the approaches reported so far to optochemically regulate oligonucleotide activities (CHAPTER 1.3), technology developments that allow for selective and step-wise regulation are still ongoing. For instance, Heckel et al. accomplished four levels of wavelength-selective decaging of nucleotides through the installation of different chromophores on nucleobases.159 To further extend conditional control of DNA and RNA function, chemical triggered nucleosides which are stable to light irradiation and thus orthogonal to photocaged nucleosides, can be an asset.

Organic azides were first introduced in 1864,172 and in the last two centuries these energy-rich intermediates serve as useful building blocks in organic synthesis, peptide synthesis, and the synthesis of other biological active compounds. As an important property of organic azides, the azido group can be reduced to an amine by phosphines via a Staudinger reduction (Scheme 2.12).172 The electron lone pair on phosphorus nucleophilically attacks the terminal nitrogen atom of azide giving a phosphazine intermediate, which results in the release of nitrogen through a four-member transition state to afford an iminophosphoranes intermediate. In the presence of water, the iminophosphorane intermediate is hydrolyzed to a

primary amine product and phosphine oxide. The Staudinger reduction has been proved to be an efficient method to cleave organic azides that were used as protecting groups on primary amines and hydroxyl groups.173, 174 Moreover, the mild condition of the Staudinger reduction may enable regulation in vivo. Thus, the azidomethylene (AzM) protected fluorophores (95 and 96 in Figure 2.5) have been used to detect oligonucleotide hybridization and the p- azidobenzyl (pAzBn) group has been applied to control prodrug activities (97 and 98).175-178

+ PR R' N3 R' N N N R' N N N 3 R' N N N PR3

N N R R H2O R-NH2 + O PR3 R P N R' R P N R' R N R 2

Scheme 2.12 Mechanism of the Staudinger reduction or organic azides to amines.179

Figure 2.5 Structure of azide protected fluorophores and prodrugs.

The AzM and pAzBn group were tested as phosphine-labile protecting groups on nucleosides. The synthesis of AzM thymidine 102 commenced with the TBDMS-protected thymidine 61 where the methylthiomethyl group was installed delivering the thioether 99 in

93% yield (Scheme 2.13). This was followed by a three-step transformation to give the protected AzM thymidine 100 (94% yield in three steps).180 Surprisingly, the deprotection of the TBDMS groups with TBAF resulted in the cleavage of azide at the same time. Thus, the silyl groups were removed by acetyl chloride in MeOH (78% yield)181 to furnish the AzM thymidine 101 which was readily reacted with DMTrCl to afford DMTr-protected AzM thymidine 102 in 75% yield.

Scheme 2.13 Synthesis of the AzM thymidine 102.

The reduction of the protected AzM thymidine 100 was carried out in CD3OD at a 10 mM concentration with 5 eq. of PPh3 and was followed by NMR (Scheme 2.14A). As shown in Scheme 2.14B, the AzM group gave a singlet peak (a in Scheme 2.14B, δ = 5.03 ppm) that

decreased upon the addition of PPh3 and completely disappeared after 80 minutes. This indicates the successful cleavage of the AzM group with PPh3. Furthermore, a double doublet signal (δ = 6.11 ppm) appeared and changed inverse to the signal of 1-H on the sugar (b/c in

Scheme 2.14B, δ = 6.27 ppm). This might be the phosphazine or iminophosphoranes intermediate peak. The reaction mixture was purified by column chromatography, affording the thymidine 61 in 78% yield.

B b/c TBDMS T a

80 mins

70 mins

50 mins

40 mins

30 mins

20 mins

10 mins

5 mins

AzM T

1 Scheme 2.14 Reduction of the AzM thymidine 100 with PPh3 (A) which was followed by H NMR (B).

With the AzM thymidine 100 investigated, we moved onto the pAzBn-modified nucleosides (105 and 108). The pAzBn alcohol 104 was made in 54% yield via a diazotization followed by a nucleophilic aromatic substitution of p-aminobenzyl alcohol 103

182 (Scheme 2.15). The alcohol 104 was exposed to methanesulfonyl chloride (MeSO2Cl) in the presence of DIPEA,183 followed by the TBDMS-protected thymidine 61 and DBU, affording the pAzBn thymidine 105 (68% yield through two steps). Meanwhile, treatment of the pAzBn alcohol 104 with BPO and Me2S generated the thioether 106 in 87% yield, which was further converted to the chloromethyl ether 107 by sulfonyl chloride in DCM. Reaction of the TBDMS-protected thymine 61 and the chloromethyl ether 107 gave the pAzBnCH2 thymidine 108 in 51% yield (DBU, THF).

Scheme 2.15 Synthesis of the pAzBn thymidine 105 and the pAzBnCH2 thymidine 108.

Similarly, the reduction of the pAzBn-modified nucleosides (105 and 108) was tested in CD3OD and was followed by NMR. The AzBnCH2 thymidine 108 was treated with 5 eq. of PPh3 at a 10 mM concentration (Scheme 2.16A). Within 60 minutes, the two methylene peaks (a and b in Scheme 2.16B, δ = 4.64 and 5.48 ppm) of 108 disappeared affording the 4- aminobenzyl alcohol (NH2BnOH) peaks (e and f in Scheme 2.16B, δ = 4.45 and 6.72 ppm).

After column purification, the thymidine 61 was recovered in 72% yield.

B c/d e TBDMS T f NH2BnOH a b 60 mins

50 mins

40 mins

30 mins

20 mins

10 mins

5 mins

AzBnCH2 T

Scheme 2.16 Reduction of the AzBnCH2 thymidine 108 with PPh3 (A) which was followed by 1H NMR (B).

The AzBn thymidine 105 treated under the same condition as the AzBnCH2 thymidine 108 failed to give the free thymidine 61 (Scheme 2.17A). This is possibly due to the lack of the oxymethylene spacer thus less entropy increase can be generated upon fragmentation. The azide was successfully reduced to an amine, as indicated by the disappearance of the methylene peak from 105 (a in Scheme 2.17B, δ = 5.06 ppm) and the appearance of the benzene peak of 103 (d/e in Scheme 2.17B, δ = 6.68 ppm). But the methylene peak of the NH2BnOH 103 (f in Scheme 2.17B, δ = 6.72 ppm) and the 1ʹ-H peak of the TBDMS-protected thymidine 61 (f in Scheme 2.17B, δ = 6.28 ppm) were absent after reduction. Overall, the aminobenzyl thymidine 109 was obtained in 86% yield.

The stability of the AzM thymidine 100 and the AzBnCH2 thymidine 108 were tested under the following conditions: 1) conc. NH4OH, 60 ˚C, 16 hrs; 2) 5% TFA r.t. 5 hrs; 3) 40%

MeNH2 in MeOH, 60 ˚C, 16 hrs; and 4) 1:1 mixture of conc. NH4OH and 40% MeNH2 in

MeOH, 60 ˚C, 2 hrs. No decomposition was observed by TLC or NMR. However, a possible problem of these azido-nucleosides is that they cannot be incorporated under standard oligonucleotide synthesis condition owing to the reduction of the azide by phosphoramidites.

Recently, an 2´-azido-modified RNA monomer was coupled to an oligomer on solid support resulting in an siRNA with a site-selective azido modification.184 This provides a possible approach for the incorporation of the AzM (102) and the AzBnCH2 thymidine (108).

B g d/e TBDMS T

f NH2BnOH

b/c a 40 mins

30 mins

20 mins

10 mins

5 mins

AzBnCH2 T

Scheme 2.17 Reduction of the AzBn thymidine 108 with PPh3 (A), which was followed by 1H NMR (B).

2.4 Conclusion

In summary, five photo- and chemically triggered nucleosides were synthesized and successfully cleaved with either light irradiation or through phosphine reduction (Figure 2.6).

These nucleosides will enable the regulation over gene functions upon introduction into oligonucleotides. The NPE-caged dA 41 can be cleaved by UV irradiation (365 nm), while the N- and O-ANBP-caged thymidine (83 and 94) can allow regulation of oligonucleotide activity with >400 nm light. Although the photolysis of the DEACM-caged nucleosides gain

no success so far, these DEACM-caged nucleosides have been cleaved by Heckel’s group thus still have the potential to trigger oligonucleotide function with improved light source and instrument setting. Two reduction-labile azido thymidines (102 and 108) were also developed, which are orthogonal to photo-triggers and may benefit the selective activation of one oligomer over another. The biological application of these photo- and chemically triggered nucleosides are being pursued in the Deiters lab.

Figure 2.6 Photo- and chemically triggered nucleosides.

2.5 Experimental

All reactions were performed in flame-dried glassware under a nitrogen atmosphere and stirred magnetically. Reactions were followed by thin layer chromatography (TLC) using glass-back silica gel plates (Sorbent technologies, 250 µm thickness). Tetrahydrofuran was

distilled from sodium/benzophenone ketyl prior to use. DCM, DMF, MeCN, EtOH, MeOH and pyridine were distilled from calcium hydride and stored over 4 Ǻ molecular sieves. Other reagents and solvents were obtained from commercial sources were stored under nitrogen and used directly without further purification. Yields refer to chromatographically and spectroscopically pure compounds unless otherwise stated. Flash chromatography was performed on silica gel (60 Å, 40-63 μm (230 × 400 mesh), Sorbtech) as a stationary phase.

High resolution mass spectral analysis (HRMS) was performed at the University of

Pittsburgh. The 1H NMR and 13C NMR spectra were recorded on a 300 MHz or a 400 MHz

Varian NMR spectrometer. Chemical shifts are given in δ units (ppm) for 1H NMR spectra and 13C NMR spectra.

1-(6-Nitrobenzo[d][1,3]dioxol-5-yl)ethan-1-ol (45). A 2 M solution of AlMe3 (1.5 eq., 15 ml, 30 mmol) was added to the aldehyde 44 (1 eq., 3.9 g, 20 mmol) in DCM (60 ml) at –78

˚C. The mixture was stirred at –78 ˚C for 1.5 hours. Ice was added until the generation of methane seized, followed by the addition of aq. NaOH (1 M, 80 ml). The mixture was stirred till clear and the aqueous layer was extracted with DCM (3 × 50 ml). The combined organic layer was washed with brine (50 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 45 as a white solid in 90% yield (3.8 g, 18 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.50-1.52 (d, J = 6.3 Hz, 3H), 5.39-5.48 (m, 1H), 6.09 (s, 2H), 7.24 (s, 1H), 7.43

(s, 1H). The analytical data matched reported results.139

2-(1-(6-Nitrobenzo[d][1,3]dioxol-5-yl)ethyl)isoindoline-1,3-dione (46). Isoindole-1,3-dione

(1 eq., 1.4 g, 9.5 mmol) was added to a solution of the alcohol 45 (1 eq., 2 g, 9.5 mmol) and

PPh3 (1.2 eq., 2.98 g, 11.4 mmol) in THF (28 ml). The mixture was cooled to 0 ˚C and DIAD

(1.2 eq., 2.3 g, 11.4 mmol) was added. The resulting solution was stirred at room temperature for 3 hours and was concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 46 as a yellow

1 solid in 84% yield (2.7 g, 7.9 mmol). H NMR (300 MHz, CDCl3): δ = 1.91-1.93 (d, J = 7.2

Hz, 3H), 4.91-4.99 (m, 1H), 6.05-6.09 (m, 2H), 7.32 (s, 1H), 7.35 (s, 1H), 7.67-7.70 (m, 2H),

7.77-7.79 (m, 2H). The analytical data matched reported results.139

1-(6-Nitrobenzo[d][1,3]dioxol-5-yl)ethan-1-amine (47). Hydrazine (2.5 eq., 0.64 ml, 20 mmol) was added to a solution of the phthalimide 46 (1 eq., 2.7 g, 8 mmol) in EtOH (37 ml) at 0 ˚C and the mixture was heated to reflux for 1 hour. The solution was cooled to 0 ˚C, diluted with Et2O (40 ml), and the resulting precipitate was removed through filtration and washed with Et2O (2 × 20 ml). The combined filtrate was washed with water (50 ml) and brine (50 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 47 as a yellow

1 solid in 68% yield (1.14 g, 5.44 mmol). H NMR (300 MHz, CDCl3): δ = 1.38-1.40 (d, J =

6.6 Hz, 3H), 4.62-4.69 (q, J = 6.6 Hz, 1H), 6.07 (s, 2H), 7.25 (s, 1H), 7.34 (s, 1H). The analytical data matched reported results.139

4,5-Di-tert-Butyldimethylsilyl-2-deoxylinosine (49). TBDMSCl (4 eq., 1.2 g, 7.9 mmol) and imidazole (7 eq., 944 mg, 13.9 mmol) were added to a solution of 2’-deoxyinosine 48 (1 eq., 0.5 g, 1.98 mmol) in DMF (25 ml). The reaction mixture was stirred for 20 hours at room temperature and quenched with EtOH (2 ml). The volatiles were removed and the residue was dissolved in DCM (20 ml). The organic solution was washed with 1 M HCl (15 ml), sat.

NaHCO3 (15 ml), water (15 ml) and brine (10 ml), dried over Na2SO4, filtered, and concentrated affording 49 as a colorless foam in quantitative yield (952 g, 1.98 mmol). 1H

NMR (300 MHz, CDCl3): δ = 0.06-0.08 (m, 12H), 0.89 (s, 18H), 2.38-2.59 (m, 2H), 3.76-

3.85 (m, 2H), 3.98-4.01 (q, J = 3.6 Hz, 1H), 4.57-4.60 (q, J = 5.7 Hz, 1H), 6.37-6.41 (t, J =

6.3 Hz, 1H), 8.13-8.14 (m, 2H). The analytical data matched reported results.134

9-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-9H-purin-6-yl 2,4,6-triisopropylbenzenesulfonate (50). DIPEA (5 eq., 1.77 ml, 10.2 mmol) followed by 2,4,6-triisopropylbenzenesulfonyl chloride (1.8 eq., 1.1 g, 3.65 mmol) was added to a solution of the silyl-protected deoxylinosine 49 (1 eq., 977 mg,

2.03 mmol) and DMAP (0.1 eq., 25 mg, 0.2 mmol) in DCM (10 mL) at 0 ºC. The reaction mixture was stirred at 0 ºC for 30 minutes and concentrated. The crude product was purified by column chromatography on silica gel with hexanes/EtOAc (9:1 to 2:1) as the eluent,

1 delivering 50 as a colorless foam (756 mg, 50%). H NMR (300 MHz, CDCl3): δ = 0.04-0.07

(m, 12H), 0.86-0.88 (m, 18H), 1.21-1.26 (m, 18 H), 2.40-2.69 (m, 1H), 2.56-2.64 (m, 1H),

2.85-2.93 (m, 1H), 3.71-3.87 (m, 2H), 3.98-4.02 (q, J = 3.6 Hz, 1H), 4.28-4.36 (m, 2H), 4.56-

4.61 (q, J = 5.7 Hz, 1H), 6.45-6.49 (t, J = 6.3 Hz, 1H), 7.18 (s, 2H), 8.36 (s, 1H), 8.53 (s,

1H). The analytical data matched reported results.134

9-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-N-(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)-9H-purin-6-amine

(51). DIPEA (3 eq., 0.32 ml, 1.86 mmol) was added to a solution of the deoxylinosine 50 (1 eq., 463 mg, 0.62 mmol) and the amine 47 (1.3 eq., 169 mg, 0.81 mmol) in DMF (5 mL) at 0

ºC. The reaction mixture was heated to 90 ºC for 48 hours, diluted with EtOAc (10 ml), and washed with 1 M citric acid (5 ml) and brine (5 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 51 as a slightly yellow foam in 83% yield (343

1 mg, 0.51 mmol). H NMR (400 MHz, CDCl3): δ = 0.06 (s, 12H), 0.87-0.89 (m, 18H), 1.64-

1.66 (d, J = 6.4 Hz, 3H), 2.32-2.40 (m, 1H), 2.53-2.61 (m, 1H), 3.71-3.85 (m, 2H), 3.94-

43.97 (m, 1H), 4.53-4.58 (m, 1H), 5.98-6.02 (m, 2H), 6.36-6.40 (m, 2H), 7.02 (s, 1H), 7.24

+ (s, 1H),7.42 (s, 1H), 8.20 (s, 1H). HRMS-LC: m/z calcd for C31H48N6O7Si2 [M+H] :

673.3201; found: 673.3220.

(2R,3S,5R)-2-(Hydroxymethyl)-5-(6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl)amino)-

9H-purin-9-yl)tetrahydrofuran-3-ol (52). TBAF in THF (3 eq., 1 M, 1.53 ml, 1.53 mmol) was added to a solution of the silyl ether 51 (1 eq., 343 mg, 0.51 mmol) in THF (15 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH

(9:1) as the eluent, affording 52 as a slightly yellow foam in 99% yield (224 mg, 0.50 mmol).

1 H NMR (300 MHz, CDCl3): δ = 1.66-1.68 (d, J = 6.6 Hz, 3H), 2.25-2.41 (m, 2H), 2.85-2.97

(m, 1H), 3.72-3.89 (m, 2H), 4.17 (br, 1H), 4.75 (br, 1H), 5.99-6.03 (m, 2H), 6.36-6.40 (t, J =

6.3 Hz, 1H), 6.99 (s, 1H), 7.24 (s, 1H),7.41 (s, 1H), 8.14 (s, 1H). LRMS-LC: m/z calcd for

+ C19H20N6O7 [M+H] : 445.1; found: 455.7.

(2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-((1-(6- nitrobenzo[d][1,3]dioxol-5-yl)ethyl)amino)-9H-purin-9-yl)tetrahydrofuran-3-ol (53).

DMTCl (1.3 eq., 296 mg, 0.87 mmol) was added to a solution of the mucleotide 52 (1 eq.,

298 mg, 0.67 mmol) in pyridine (21 ml). The reaction mixture was stirred overnight at room temperature. The reaction was quenched with MeOH (1 ml), and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography with

DCM/acetone (2:1) and 1% TEA as the eluent, affording 53 as a yellow foam in 63% yield

1 (315 mg, 0.42 mmol). H NMR (300 MHz, CDCl3): δ = 1.65-1.67 (d, J = 6.6 Hz, 3H), 2.41-

2.62 (m, 2H), 3.34-3.42 (m, 2H), 3.77 (s, 6H), 4.17 (m, 1H), 4.64 (m, 1H), 5.97-6.02 (m,

3H), 6.33-6.37 (t, J = 6.3 Hz, 1H), 6.77-6.80 (m, 5H), 6.03 (s, 1H), 7.21-7.27 (m, 7H), 7.35-

13 7.42 (m, 3H), 8.14-8.15 (m, 1H). C NMR (400 MHz, CDCl3): δ = 10.7, 40.5, 46.0, 55.3,

63.8, 72.2, 84.3, 86.3, 86.6, 102.8, 105.3, 106.1, 113.2, 119.9, 126.9, 127.9, 128.1, 130.6,

135.6, 135.6, 135.7, 138.1, 138.4, 138.5, 144.5, 146.6, 152.1, 153.0, 158.5. HRMS-LC: m/z

+ calcd for C40H38N6O9 [M+H] : 747.2779; found: 747.2721.

(2R,3S,5R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-((1-(6-nitrobenzo-

[d][1,3]dioxol-5-yl)ethyl)amino)-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (41). DIPEA (10 eq., 035 ml, 2 mmol) was added to a solution of the alcohol 53 (1 eq., 150 mg, 0.2 mmol) in DCM (3.7 ml) at 0 ˚C and the mixture was stirred for 10 minutes at 0 ˚C. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (2 eq.,

0.09 ml, 0.4 mmol) was added and the reaction mixture was allowed to warm to room temperature over 3 hours. The solvent was removed under reduced pressure and the crude product was purified by silica gel chromatography with hexane/acetone (2:1) and 1% TEA as the eluent, affording 41 as a light yellow foam in 61% yield (115.5 mg, 0.12 mmol). 1H NMR

(400 MHz, CDCl3): δ = 1.12-1.18 (m, 12H), 1.71-1.73 (d, J = 6.6 Hz, 3H), 2.41-2.61 (m,

2H), 3.20-3.40 (m, 2H), 3.34-3.72 (m, 4H), 3.77 (s, 6H), 4.20-4.30 (m, 1H), 4.62-4.75 (m,

1H), 5.96-6.02 (m, 2H), 6.35-6.41 (t, J = 6.3 Hz, 1H), 6.78-6.81 (m, 4H), 6.99 (m, 1H), 7.19-

7.28 (m, 8H), 7.35-7.37 (m, 2H), 7.43 (s, 1H), 8.14-8.15 (m, 1H). 31P NMR (300 MHz,

+ CDCl3): δ = 149.06. HRMS-LC: m/z calcd for C49H55N8O10P [M+H] : 947.3857; found:

947.3871

7-(Diethylamino)-4-(hydroxymethyl)-2H-chromen-2-one (55). Selenium oxide (1.5 eq.,

1.66 g, 15 mmol) was added to a solution of 7-diethylamino-4-methyl coumarin (1 eq., 2.31 g, 10 mmol) in dioxane (60 ml). The reaction mixture was heated to reflux overnight and concentrated. The crude product was confirmed by NMR and dissolved in EtOH (65 ml).

Sodium borohydride (0.5 eq., 189 mg, 5 mmol) was added and the mixture was stirred overnight at room temperature. The solution was concentrated under reduced pressure and

the reaction was quenched with 1 M HCl (10 ml), followed by the addition of water (20 ml).

The mixture was extracted with DCM (3 × 20 ml). The combined organic layer was washed with water (30 ml) and brine (30 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with DCM/acetone (5:1) as the eluent, affording 55 as a yellow solid in 66% yield (1.6 g, 6.6 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.09-1.14 (t, J = 7.2 Hz, 6H), 3.27-3.34 (q, J = 7.2 Hz, 4H), 4.77 (s, 2H), 6.25 (s,

1H), 6.33-6.34 (d, J = 2.7 Hz, 1H), 6.45-6.49 (dd, Ja = 8.7 Hz, Jb = 2.7 Hz, 1H), 7.21-7.24 (d,

J = 8.7 Hz, 1H). The analytical data matched reported results.185

4-(Bromomethyl)-7-(diethylamino)-2H-chromen-2-one (56). Triphenylphosphine (2 eq.,

212 mg, 0.81 mmol) was added to a mixture of the alcohol 55 (1 eq., 100 mg, 0.40 mmol) and CBr4 (2 eq., 268 mg, 0.81 mmol) in THF (1.7 ml). The resulting mixture was stirred for 1 hour at room temperature and concentrated under reduced pressure. The residue was taken up in water (1 ml) and the aqueous layer was extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated.

The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 56 as a yellow solid in 76% yield (96 mg, 0.31 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.19-1.24 (t, J = 7.2 Hz, 6H), 3.39-3.46 (q, J = 7.2 Hz, 4H), 4.40 (s, 2H), 6.14 (s,

1H), 6.52-6.53 (d, J = 2.7 Hz, 1H), 6.62-6.66 (dd, Ja = 8.7 Hz, Jb = 2.7 Hz, 1H), 7.48-7.51 (d,

J = 9.0 Hz, 1H). The analytical data matched reported results.186

1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (60). TBDMSCl (4 eq., 490 mg, 4 mmol) and imidazole (7 eq., 387 mg, 7 mmol) were added to a solution of 2’-deoxyluridine

57 (1 eq., 228 mg, 1 mmol) in DMF (1.2 ml). The mixture was stirred for 20 hours at room temperature and the reaction was quenched with EtOH (1 ml). The mixture was concentrated and the residue was taken up in DCM (10 ml). The solution was washed with 1 M HCl (10 ml), sat. NaHCO3 (10 ml), water (10 ml) and brine (10 ml), dried over Na2SO4, filtered, and concentrated, affording 60 as a colorless foam in 98% yield (448 g, 0.98 mmol). 1H NMR

(300 MHz, CDCl3): δ = 0.07-0.10 (m, 12H), 0.89-0.92 (m, 18H), 2.04-2.09 (m, 1H), 2.29-

2.31 (m, 1H), 3.74-3.77 (m, 1H), 3.88-3.92 (m, 2H), 4.39-4.43 (m, 1H), 5.66-5.69 (dd, Ja =

8.0 Hz, Jb = 2.4 Hz, 1H), 6.27-6.30 (t, J = 6.4 Hz, 1H), 7.89-7.91 (d, J = 8.4 Hz, 1H). The analytical data matched reported results.187

1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (61). TBDMSCl (4 eq., 1.24 g, 8.3 mmol) and imidazole (7 eq., 983 mg, 14.4 mmol) were added to a solution of thymidine 58 (1 eq., 500 mg, 2.06 mmol) in DMF (2.5 ml). The mixture was stirred for 20 hours at room temperature and the reaction was quenched with EtOH (2 ml). The mixture was concentrated and the residue was taken up in DCM (20 ml). The solution was washed with 1 M HCl (15 ml), sat. NaHCO3 (15 ml), water (15 ml) and brine (10 ml), dried over

Na2SO4, filtered, and concentrated affording 61 as a colorless foam in 97% yield (942 g, 2

1 mmol). H NMR (300 MHz, CDCl3): δ = 0.07-0.11 (m, 12H), 0.89-0.93 (m, 18H), 1,91 (s,

3H), 1.92-2.04 (m, 1H), 2.21-2.30 (m, 1H), 3.71-3.96 (m, 3H), 4.38-4.42 (m, 1H), 6.24-6.37

(m, 1H), 7.49 (s, 1H), 8.46 (br, 1H). The analytical data matched reported results.188

1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione (62). TBDMSCl (4 eq., 490 mg, 3.2 mmol) and imidazole (7 eq., 387 mg, 5.7 mmol) were added to a solution of 2’- deoxy-5-fluorounridine 59 (1 eq., 200 mg, 0.81 mmol) in DMF (1 ml). The mixture was stirred for 20 hours at room temperature and the reaction was quenched with EtOH (1 ml).

The mixture was concentrated and the residue was taken up in DCM (10 ml). The solution was washed with 1 M HCl (7 ml), sat. NaHCO3 (7 ml), water (7 ml) and brine (7 ml), dried over Na2SO4, filtered, and concentrated affording 62 as a colorless foam in 97% yield (373 g,

1 0.79 mmol). H NMR (300 MHz, CDCl3): δ = -0.02-0.13 (m, 12H), 0.82-0.93 (m, 18H),

2.02-2.08 (m, 1H), 2.19-2.32 (m, 1H), 3.76-3.78 (m, 1H), 3.88-3.94 (m, 2H), 4.40 (br, 1H),

6.27-6.30 (m, 1H), 8.02-8.06 (m, 2H). The analytical data matched reported results.189

1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-4-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)pyrimidin-

2(1H)-one (63). The alcohol 55 (1.2 eq., 33 mg, 0.13 mmol), the nucleotide 60 (1 eq., 50 mg,

0.11 mmol), triphenylphosphine (1.8 eq., 52 mg, 0.2 mmol), and diisopropyl azodicarboxylate (1.8 eq., 25.5 µl, 0.2 mmol) were dissolved in THF (1.3 ml) and stirred for

16 hours at room temperature. The solution was diluted with water (2 ml) and the aqueous layer was extracted with EtOAc (3 × 1 ml). The organic layers were combined, washed with

brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (3:1) as the eluent, affording 63 as a yellow

1 foam in 63% yield (48 mg, 0.07 mmol). H NMR (300 MHz, CDCl3): δ = 0.08-0.12 (m,

12H), 0.88-0.89 (m, 18H), 1.15-1.20 (t, J = 6.9 Hz, 6H), 2.07-2.16 (m, 1H), 2.31-2.40 (m,

1H), 3.38-3.45 (q, J = 7.2 Hz, 4H), 3.78 (m, 1H), 3.90-3.94 (m, 2H), 4.43-4.46 (m, 1H), 5.22

(s, 2H), 5.66 (s, 1H), 5.80-5.83 (d, J = 8.4 Hz, 1H), 6.27-6.32 (m, 1H), 6.50-6.51 (d, J = 2.4

Hz, 1H), 6.59-6.65 (dd, Ja = 9.0 Hz, Jb = 2.4 Hz, 1H), 7.46-7.49 (d, J = 9.0 Hz, 1H), 7.98-

+ 8.00 (d, J = 8.1 Hz, 1H). LRMS-LC: m/z calcd for C35H55N3O7Si2 [M+H] : 686.4; found:

686.5.

1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-4-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)-5- methylpyrimidin-2(1H)-one (64). The alcohol 55 (1.2 eq., 150 mg, 0.61 mmol), the thymidine 61 (1 eq., 238 mg, 0.51 mmol), triphenylphosphine (1.8 eq., 239 mg, 0.91 mmol), and diisopropyl azodicarboxylate (1.8 eq., 0.11 ml, 0.91 mmol) were dissolved in THF (6 ml) and stirred for 16 hours at room temperature. The solution was diluted with water (10 ml) and extracted with EtOAc (3 × 5 ml). The organic layers were combined, washed with brine

(10 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (3:1) as the eluent, affording 64 as a yellow

1 foam in 86% yield (304 mg, 0.43 mmol). H NMR (400 MHz, CDCl3): δ = 0.05-0.11 (m,

12H), 0.85-0.92 (m, 18H), 1.15-1.19 (t, J = 6.9 Hz, 6H), 1.94 (s, 3H), 2.20-2.30 (m, 2H),

3.34-3.42 (q, J = 7.2 Hz, 4H), 3.72-3.94 (m, 3H), 4.36-4.41 (m, 1H), 5.21 (s, 2H), 5.60 (s,

1H), 6.30-6.36 (m, 1H), 6.46-6.47 (d, J = 2.4 Hz, 1H), 6.54-6.60 (dd, Ja = 9.0 Hz, Jb = 2.4

Hz, 1H), 7.44-7.47 (d, J = 9.0 Hz, 1H), 7.55-7.56 (d, J = 1.2 Hz, 1H). LRMS-LC: m/z calcd

+ for C36H57N3O7Si2 [M+H] : 700.4; found: 700.7. The analytical data matched reported results.159

1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-4-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)-5- fluoropyrimidin-2(1H)-one (65). The alcohol 55 (1.2 eq., 31 mg, 0.13 mmol), the nucleotide 62 (1 eq., 50 mg, 0.1 mmol), triphenylphosphine (1.8 eq., 47.2 mg, 0.18 mmol), and diisopropyl azodicarboxylate (1.8 eq., 21 µl, 0.18 mmol) were dissolved in THF (1.2 ml) and the mixture was stirred for 16 hours at room temperature. The solution was diluted with water (2 ml) and the aqueous layer was extracted with EtOAc (3 × 2 ml). The organic layers were combined, washed with brine (3 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (3:1) as the eluent, affording 65 as a yellow foam in 83% yield (58 mg, 0.08 mmol). 1H NMR (400 MHz,

CDCl3): δ = 0.07-0.15 (m, 12H), 0.88 (s, 18H), 1.18-1.23 (t, J = 6.9 Hz, 6H), 2.32-2.39 (m,

2H), 3.37-3.44 (q, J = 7.2 Hz, 4H), 3.77-3.81 (m, 1H), 3.94-3.97 (m, 2H), 4.39-4.43 (m, 1H),

5.22 (s, 2H), 5.65 (s, 1H), 6.23-6.33 (m, 1H), 6.50-6.51 (d, J = 2.4 Hz, 1H), 6.50-6.63 (dd, Ja

= 9.0 Hz, Jb = 2.4 Hz, 1H), 7.46-7.49 (d, J = 9.0 Hz, 1H), 8.15-7.17 (d, J = 1.2 Hz, 1H).

+ LRMS-LC: m/z calcd for C35H54FN3O7Si2 [M+H] : 704.4; found: 704.4.

4-((7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)-1-((2R,4S,5R)-4-hydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (66). TBAF in THF (3 eq., 1

M, 0.21 ml, 0.21 mmol) was added to a solution of the silyl ether 63 (1 eq., 48 mg, 0.07 mmol) in THF (1.1 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 66 as a yellow solid with

1 87% yield (28 mg, 0.061 mmol). H NMR (300 MHz, CDCl3): δ = 1.15-1.20 (t, J = 6.9 Hz,

6H), 2.07-2.16 (m, 1H), 2.31-2.40 (m, 1H), 3.38-3.45 (q, J = 7.2 Hz, 4H), 3.78 (m, 1H), 3.90-

3.94 (m, 2H), 4.43-4.46 (m, 1H), 5.22 (s, 2H), 5.66 (s, 1H), 5.80-5.83 (d, J = 8.4 Hz, 1H),

6.27-6.32 (m, 1H), 6.50-6.51 (d, J = 2.4 Hz, 1H), 6.59-6.65 (dd, Ja = 9.0 Hz, Jb = 2.4 Hz,

1H), 7.46-7.49 (d, J = 9.0 Hz, 1H), 7.98-8.00 (d, J = 8.1 Hz, 1H). LRMS-LC: m/z calcd for

+ C23H27N3O7 [M+H] : 458.2; found: 458.5.

4-((7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)-1-((2R,4S,5R)-4-hydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidin-2(1H)-one (67). TBAF in

THF (3 eq., 1 M, 1.31 ml, 1.31 mmol) was added to a solution of the silyl ether 64 (1 eq., 304 mg, 0.43 mmol) in THF (10 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 67 as a yellow solid in 93% yield (188 mg, 0.41 mmol). 1H NMR (400 MHz, DMSO): δ = 1.11-1.14 (t, J = 6.8 Hz, 6H),

1.88 (s, 3H), 2.15-2.20 (m, 2H), 3.41-3.46 (q, J = 6.4 Hz, 4H), 3.58-3.63 (m, 2H), 3.79-3.80

(d, J = 2.8 Hz, 1H), 4.27 (s, 1H), 5.10-5.12 (m, 3H), 5.27-5.28 (d, J = 4.0 Hz, 1H), 5.44 (s,

1H), 6.20-6.23 (t, J = 6.4 Hz, 1H), 6.55 (s, 1H), 6.72-6.74 (d, J = 9.2 Hz, 1H), 7.66-7.68 (d, J

= 9.2 Hz, 1H), 7.92 (s, 1H). The analytical data matched reported results.159

4-((7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methoxy)-5-fluoro-1-((2R,4S,5R)-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (68). TBAF in

THF (3 eq., 1 M, 0.25 ml, 0.25 mmol) was added to a solution of the silyl ether 65 (1 eq., 58 mg, 0.083 mmol) in THF (1.9 ml). The reaction mixture was stirred at room temperature for

1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 68 as a yellow solid in 77% yield (30.5 mg, 0.64 mmol). 1H NMR (400 MHz, DMSO): δ = 1.11-1.14 (t, J = 6.8 Hz, 6H),

2.17-2.25 (m, 2H), 3.41-3.47 (q, J = 6.8 Hz, 4H), 3.59-3.69 (m, 2H), 3.81-3.84 (dd, Ja = 6.8

Hz, Jb = 3.6 Hz, 1H), 4.28-4.29 (m, 1H), 5.11 (s, 2H), 5.24-5.26 (t, J = 4.8 Hz, 1H), 5.29-

5.30 (d, J = 4.4 Hz, 1H), 5.72 (s, 1H), 6.15-6.18 (t, J = 4.8 Hz, 1H), 6.55-6.56 (d, J = 2.4 Hz,

1H), 6.72-6.75 (dd, Ja = 9.2 Hz, Jb = 2.4 Hz, 1H), 7.64-7.66 (d, J = 7.2 Hz, 1H), 8.43-8.45

+ (dd, Ja = 6.8 Hz, Jb = 4.0 Hz, 1H). LRMS-LC: m/z calcd for C23H26FN3O7 [M+H] : 476.2; found: 476.6.

1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-3-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl)pyrimidine-

2,4(1H,3H)-dione (69). DBU (1.5 eq., 10 µl, 0.066 mmol) was added to a solution of the nucleotide 60 (1 eq., 20 mg, 0.44 mmol) in DMF (0.4 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of the coumarin 56 (1.2 eq., 16 mg, 0.05 mmol) in

DMF (0.2 ml) was added drop-wise and the resulting mixture was stirred overnight, quenched with water (1 ml), extracted with EtOAc (3 × 1 ml). The organic layers were combined, washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (3:1) as the eluent, affording 69 as a yellow foam in 56% yield (17 mg, 0.025 mmol). 1H NMR (400

MHz, CDCl3): δ = 0.08-0.12 (m, 12H), 0.88-0.94 (m, 18H), 1.18-1.23 (t, J = 7.2 Hz, 6H),

2.31-2.40 (m, 2H), 3.38-3.45 (q, J = 7.2 Hz, 4H), 3.74-3.79 (m, 1H), 3.90-3.94 (m, 2H), 4.40-

4.48 (m, 1H), 5.22 (s, 2H), 5.66 (s, 1H), 5.80-5.83 (d, J = 8.4 Hz, 1H), 6.24-6.31 (m, 1H),

6.50-6.51 (d, J = 2.7 Hz, 1H), 6.59-6.64 (dd, Ja = 9.0 Hz, Jb = 2.4 Hz, 1H), 7.46-7.49 (d, J =

+ 9.0 Hz, 1H), 7.98-8.00 (d, J = 8.1 Hz, 1H). LRMS-LC: m/z calcd for C35H55N3O7Si2 [M+H] :

686.4; found: 686.5.

1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-3-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl)-5- methylpyrimidine-2,4(1H,3H)-dione (70). DBU (1.5 eq., 9 µl, 0.06 mmol) was added to a solution of the nucleotide 61 (1 eq., 20 mg, 0.04 mmol) in DMF (0.4 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of the coumarin 56 (1.2 eq., 16 mg,

0.05 mmol) in DMF (0.2 ml) was added drop-wise and the resulting mixture was stirred overnight. The reaction was quenched with water (1 ml), and the aqueous layer was extracted with EtOAc (3 × 1 ml). The organic layers were combined, washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (3:1) as the eluent, affording 70 as a yellow foam in

1 95% yield (26 mg, 0.037 mmol). H NMR (400 MHz, CDCl3): δ = 0.07-0.13 (m, 12H), 0.88-

0.94 (m, 18H), 1.17-1.28 (t, J = 7.2 Hz, 6H), 2.23-2.33 (m, 2H), 3.37-3.44 (q, J = 7.2 Hz,

4H), 3.74-3.79 (dd, Ja = 11.4 Hz, Jb = 2.7 Hz, 1H), 3.86-3.93 (m, 2H), 4.39-4.42 (m, 1H),

5.23 (s, 2H), 5.62 (s, 1H), 6.32-6.37 (m, 1H), 6.49-6.50 (d, J = 2.4 Hz, 1H), 6.59-6.63 (dd, Ja

= 9.0 Hz, Jb = 2.4 Hz, 1H), 7.47-7.50 (d, J = 9.3 Hz, 1H), 7.57-7.58 (d, J = 1.2 Hz, 1H).

+ LRMS-LC: m/z calcd for C36H57N3O7Si2 [M+H] : 700.4; found: 700.7.

1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-3-((7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl)-5- fluoropyrimidine-2,4(1H,3H)-dione (71). DBU (1.5 eq., 9.4 µl, 0.06 mmol) was added to a solution of the nucleotide 62 (1 eq., 20 mg, 0.04 mmol) in DMF (0.4 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of the coumarin 56 (1.2 eq., 15.7 mg,

1 77% yield (23 mg, 0.032 mmol). H NMR (400 MHz, CDCl3): δ = = 0.07-0.15 (m, 12H),

0.88-0.95 (m, 18H), 1.18-1.23 (t, J = 7.2 Hz, 6H), 2.27-2.39 (m, 2H), 3.38-3.45 (q, J = 7.2

Hz, 4H), 3.77-3.81 (m, 1H), 3.93-3.97 (m, 2H), 4.38-4.44 (m, 1H), 5.22 (s, 2H), 5.66 (s, 1H),

6.26-6.33 (m, 1H), 6.50-6.51 (d, J = 2.4 Hz, 1H), 6.60-6.63 (dd, Ja = 8.7 Hz, Jb = 2.4 Hz,

1H), 7.46-7.49 (d, J = 9.0 Hz, 1H), 8.15-8.17 (d, J = 6.0 Hz, 1H). HRMS-LC: m/z calcd for

+ LRMS-LC: m/z calcd for C35H54FN3O7Si2 [M+H] : 704.4; found: 704.5.

3-((7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methyl)-1-((2R,4S,5R)-4-hydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (72). TBAF in THF

(3 eq., 1 M, 0.06 ml, 0.06 mmol) was added to a solution of the silyl ether 69 (1 eq., 14 mg,

0.02 mmol) in THF (0.5 ml). The reaction mixture was stirred at room temperature for 1 hour and was concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 72 as a yellow solid in 58%

1 yield (5.3 mg, 0.01 mmol). H NMR (400 MHz, CDCl3): δ = 1.18-1.23 (t, J = 7.2 Hz, 6H),

2.31-2.40 (m, 2H), 3.38-3.45 (q, J = 7.2 Hz, 4H), 3.74-3.79 (m, 1H), 3.90-3.94 (m, 2H), 4.40-

4.48 (m, 1H), 5.22 (s, 2H), 5.66 (s, 1H), 5.80-5.83 (d, J = 8.4 Hz, 1H), 6.24-6.31 (m, 1H),

6.50-6.51 (d, J = 2.7 Hz, 1H), 6.59-6.64 (dd, Ja = 9.0 Hz, Jb = 2.4 Hz, 1H), 7.46-7.49 (d, J =

+ 9.0 Hz, 1H), 7.98-8.00 (d, J = 8.1 Hz, 1H). LRMS-LC: m/z calcd for C23H27N3O7 [M+H] :

458.2; found: 458.4.

3-((7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methyl)-1-((2R,4S,5R)-4-hydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (73).

TBAF in THF (3 eq., 1 M, 0.24 ml, 0.24 mmol) was added to a solution of the silyl ether 70

(1 eq., 54 mg, 0.08 mmol) in THF (1.8 ml). The reaction mixture was stirred at room temperature for 1 hour and was concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 73 as a

1 yellow solid in 82% yield (31 mg, 0.065 mmol). H NMR (400 MHz, CDCl3): δ = 1.11-1.19

(t, J = 6.8 Hz, 6H), 1.88 (s, 3H), 2.10-2.22 (m, 2H), 3.42-3.47 (q, J = 7.2 Hz, 4H), 3.56-3.65

(m, 2H), 3.79-3.80 (d, J = 2.4 Hz, 1H), 4.27 (s, 1H), 5.10-5.12 (m, 3H), 5.27-5.28 (d, J = 3.6

Hz, 1H), 5.43 (s, 1H), 6.19-6.23 (t, J = 6.8 Hz, 1H), 6.55-6.56 (d, J = 1.2 Hz, 1H), 6.72-6.74

(d, J = 8.4 Hz, 1H), 7.66-7.68 (d, J = 9.2 Hz, 1H), 7.92 (s, 1H). LRMS-LC: m/z calcd for

+ C24H29N3O7 [M+H] : 472.2; found: 472.0.

3-((7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methyl)-5-fluoro-1-((2R,4S,5R)-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (74).

TBAF solution in THF (3 eq., 1 M, 0.23 ml, 0.23 mmol) was added to a solution of the silyl ether 71 (1 eq., 54 mg, 0.08 mmol) in THF (1.7 ml). The reaction mixture was stirred at room temperature for 1 hour and was concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 74 as a

1 yellow solid in 55% yield (20 mg, 0.042 mmol). H NMR (400 MHz, CD3CN): δ = 1.15-1.19

(t, J = 6.8 Hz, 6H), 2.09-2.23 (m, 2H), 3.42-3.47 (q, J = 7.2 Hz, 4H), 3.70--3.77 (m, 2H),

3.80-3.88 (t, J = 2.8 Hz, 1H), 4.36 (m, 1H), 5.14 (s, 2H), 5.64 (s, 1H), 6.18-6.23 (t, J = 6.8

Hz, 1H), 6.53-6.54 (d, J = 2.8 Hz, 1H), 6.70-6.72 (dd, Ja = 8.8 Hz, Jb = 2.4 Hz, 1H), 7.57-

7.59 (d, J = 8.8 Hz, 1H), 8.24-8.26 (d, J = 7.2 Hz, 1H). LRMS-LC: m/z calcd for

+ C23H26FN3O7 [M+H] : 476.2; found: 476.4.

2-(4-Nitro-[1,1'-biphenyl]-3-yl)propan-1-ol (76). The alcohol 75 (1 eq., 26 mg, 0.1 mmol), phenylboronic acid (1.2 eq., 15 mg, 0.12 mmol), potassium carbonate (2.7 eq., 37 mg, 0.27

mmol), tetrabutylammonium bromide (1 eq., 32 mg, 0.1 mmol) and palladium acetate (0.1 eq., 2 mg, 0.01 mmol) were dissolved in EtOH/H2O (2:1, 0.75 ml) and heated under microwave irradiation in a CEM Discover in standard mode (200 W) for 1 minute. The solution was diluted with water (1 ml) and the aqueous layer was extracted with EtOAc (3 ×

1 ml). The organic layers were combined, washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 76 as a colorless oil in 57% yield (15 mg, 0.057

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.38-1.40 (d, J = 6.9 Hz, 3H), 1.74 (br, 1H), 3.62-

3.69 (q, J = 6.9 Hz, 1H), 3.80-3.86 (m, 2H), 7.43-7.61 (m, 6H), 7.66-7.67 (d, J = 1.8 Hz, 1H),

7.86-7.89 (d, J = 8.4 Hz, 1H). The analytical data matched reported results.190

2-(4-Nitro-4'-(trimethylsilyl)-[1,1'-biphenyl]-3-yl)propan-1-ol (77). The alcohol 75 (1 eq.,

26 mg, 0.1 mmol), 4-(trimethylsilyl)phenyl boronic acid (1.2 eq., 23 mg, 0.12 mmol), potassium carbonate (2.7 eq., 37 mg, 0.27 mmol), tetrabutylammonium bromide (1 eq., 32 mg, 0.1 mmol) and palladium acetate (0.1 eq., 2 mg, 0.01 mmol) were dissolved in

EtOH/H2O (2:1. 0.75 ml) and heated under microwave irradiation in a CEM Discover in standard mode (200 W) for 1 minute. The solution was diluted with water (1 ml) and extracted with EtOAc (3 × 1 ml). The organic layers were combined, washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 77 as a light yellow oil

1 in 75% yield (25 mg, 0.075 mmol). H NMR (400 MHz, CDCl3): δ = 1.38-1.40 (d, J = 6.8

Hz, 3H), 3.60-3.70 (m, 1H), 3.83-3.86 (m, 2H), 7.54-7.57 (m, 2H), 7.58-7.59 (m, 1H), 7.64-

7.65 (m, 1H), 7.66-7.68 (m, 2H), 7.86-7.88 (d, J = 8.4 Hz, 1H). 13C NMR (400 MHz,

CDCl3): δ = -1.4, 17.4, 36.2, 67.8, 124.8, 125.6, 126.5, 126.8, 133.9, 138.7, 139.2, 141.1,

+ 145.6, 148.8. HRMS-LC: m/z calcd for C18H23NO3Si [M+H˗H2O] : 312.1391; found:

312.1420.

2-(4'-Methoxy-4-nitro-[1,1'-biphenyl]-3-yl)propan-1-ol (78). The alcohol 75 (1 eq., 26 mg,

0.1 mmol), 4-methoxylbenzene boronic acid (1.2 eq., 18 mg, 0.12 mmol), potassium carbonate (2.7 eq., 37 mg, 0.27 mmol), tetrabutylammonium bromide (1 eq., 32 mg, 0.1 mmol) and palladium acetate (0.1 eq., 2 mg, 0.01 mmol) were dissolved in EtOH/H2O (2:1.

0.75 ml) and heated under microwave irradiation in a CEM Discover in standard mode (200

W) for 1 min. The solution was diluted with water (1 ml) and extracted with EtOAc (3 × 1 ml). The organic layers were combined, washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 78 as a red solid in 98% yield (32 mg, 0.098

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.36-1.39 (d, J = 6.9 Hz, 3H), 1.86 (br, 1H), 3.62-

3.69 (q, J = 6.6 Hz, 1H), 3.80-3.87 (m, 5H), 6.99-7.02 (d, J = 8.4 Hz, 2H), 7.48-7.55 (m, 3H),

13 7.61-7.62 (d, J = 2.1 Hz, 1H), 7.83-7.86 (d, J = 8.7 Hz, 1H).. C NMR (400 MHz, CDCl3): δ

= -1.4, 17.4, 36.2, 67.8, 124.8, 125.6, 126.5, 126.8, 133.9, 138.7, 139.2, 141.1, 145.6, 148.8.

The analytical data matched reported results.99

2-(4'-(Dimethylamino)-4-nitro-[1,1'-biphenyl]-3-yl)propan-1-ol (79). The alcohol 75 (1 eq., 595 mg, 2.3 mmol), 4-(dimethylamino)phenyl boronic acid (1.2 eq., 452 mg, 2.7 mmol),

potassium carbonate (2.7 eq., 851 mg, 6.2 mmol), tetrabutylammonium bromide (1 eq., 737 mg, 2.3 mmol) and palladium acetate (0.1 eq., 52 mg, 0.23 mmol) were dissolved in

EtOH/H2O (2:1, 18 ml) and heated under microwave irradiation in a CEM Discover in standard open vessel mode (200 W) for 10 mins. The solution was diluted with water (10 ml) and extracted with EtOAc (3 × 10 ml). The organic layers were combined, washed with brine

(15 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 79 as a red solid

1 in 97% yield (666 mg, 2.2 mmol). H NMR (300 MHz, CDCl3): δ = 1.35-1.37 (d, J = 6.9 Hz,

3H), 3.01 (s, 6H), 3.60-3.70 (m, 1H), 3.82-3.84 (d, J = 6.6 Hz, 2H), 6.77-6.80 (d, J = 8.1 Hz,

2H), 7.46-7.51 (m, 3H), 7.58-7.59 (d, J = 1.8 Hz, 1H), 7.83-7.86 (d, J = 8.7 Hz, 2H). The analytical data matched the reported results.100

N,N-Dimethyl-3'-(1-((methylthio)methoxy)propan-2-yl)-4'-nitro-[1,1'-biphenyl]-4-amine

(80). Dimethylsulfide (8 eq., 59 µl, 0.8 mmol) was added to a solution of the alcohol 79 (1 eq., 30 mg, 0.1 mmol) in CH3CN (0.5 ml) at 0 ˚C, followed by benzoyl peroxide (4 eq., 4 ×

31 mg, 0.4 mmol) in four portions over 30 minutes. The mixture was stirred for 2 hours at 0

˚C and the reaction was quenched with 1 M NaOH (1 ml). The mixture was stirred overnight at room temperature and the aqueous layer was extracted with Et2O (3 × 1 ml). The organic layers were combined, washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 80 as a red solid in 41% yield (14 mg, 0.04

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.38-1.40 (d, J = 6.6 Hz, 3H), 1.96 (s, 3H), 3.03 (s,

6H), 3.60-3.70 (m, 1H), 3.79-3.84 (m, 2H), 4.55- 4.63 (m, 2H), 6.79-6.82 (d, J = 8.7 Hz, 2H),

7.47-7.55 (m, 3H), 7.65-7.66 (d, J = 2.4 Hz, 1H), 7.84-7.87 (d, J = 8.4 Hz, 2H). ). 13C NMR

(400 MHz, CDCl3): δ = 15.1, 18.2, 34.2, 40.3, 63.2, 72.3, 112.5, 124.2, 125.1, 125.4, 126.3,

+ 128.0, 139.4, 145.7, 147.9, 150.7. HRMS-LC: m/z calcd for C19H24N2O3S [M+H] : 361.1586; found: 361.1564.

3'-(1-(Chloromethoxy)propan-2-yl)-N,N-dimethyl-4'-nitro-[1,1'-biphenyl]-4-amine (81).

Sulfuryl chloride (1.2 eq., 7.3 µl, 0.09 mmol) was added to a solution of the thioether 80 (1 eq., 27 mg, 0.075 mmol) in DCM (0.2 ml) at 0 ˚C. The mixture was stirred for 30 mins at 0

˚C and dried, delivering the crude product as red oil which was confirmed by 1H NMR and

1 used without purification. H NMR (300 MHz, CDCl3): δ = 1.38-1.40 (d, J = 6.6 Hz, 3H),

3.03 (s, 6H), 3.60-3.70 (m, 1H), 3.94-4.02 (m, 2H), 5.46 (s, 2H), 6.79-6.82 (d, J = 8.7 Hz,

2H), 7.47-7.55 (m, 3H), 7.65-7.66 (d, J = 2.4 Hz, 1H), 7.84-7.87 (d, J = 8.4 Hz, 2H).

1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-3-((2-(4'-(dimethylamino)-4-nitro-[1,1'-biphenyl]-3-yl)propoxy)- methyl)-5-methylpyrimidine-2,4(1H,3H)-dione (82). DBU (2.5 eq., 28 µl, 0.19 mmol) was added to a solution of the nucleoside 61 (1 eq., 32 mg, 0.07 mmol) in DMF (0.45 ml), and the mixture was stirred for 30 minutes at room temperature. A solution of the chloromethyl ether

81 (1.1 eq., 0.075 mmol) in DMF (0.15 ml) was added drop-wise and the resulting mixture was stirred overnight. The reaction was quenched with water (1 ml), and the aqueous layer was extracted with EtOAc (3 × 1 ml). The organic layers were combined, washed with brine

(2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 82 as a red solid

1 in 14% yield (7.6 mg, 0.01 mmol). H NMR (400 MHz, CDCl3): δ = 0.07-0.11 (m, 6H),

0.88-0.92 (m, 18H), 1.35-1.38 (m, 3H), 1.85-1.89 (m, 5H), 2.88 (s, 6H), 2.96 (m, 1H), 3.71-

3.93 (m, 5H), 4.38-4.41 (m, 1H), 5.36-5.42 (s, 2H), 6.32 (m, 1H),7.13-7.15 (d, J = 8.4 Hz,

13 1H), 7.44-7.48 (m, 4H), 7.54-7.60 (m, 2H), 7.81-7.84 (m, 1H). C NMR (400 MHz, CDCl3):

δ = -1.4, 17.4, 36.2, 67.8, 124.8, 125.6, 126.5, 126.8, 133.9, 138.7, 139.2, 141.1, 145.6,

+ 148.8. LRMS-LC: m/z calcd for C40H62N4O8Si2 [M+H] : 783.4; found: 783.4.

4-(2-(4'-(Dimethylamino)-4-nitro-[1,1'-biphenyl]-3-yl)propoxy)-1-((2R,4S,5R)-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidin-2(1H)-one (83).

TBAF in THF (3 eq., 1 M, 30 µl, 0.03 mmol) was added to a solution of the silyl ether 82 (1 eq., 7 mg, 0.01 mmol) in THF (0.2 ml). The reaction mixture was stirred at room temperature for 1 hour and was concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (1:1) as the eluent, affording 83 as a red foam

1 in 43% yield (2.4 mg, 0.004 mmol). H NMR (300 MHz, CDCl3): δ = 1.33-1.36 (d, J = 7.2

Hz, 3H), 1.85-1.86 (m, 3H), 2.20-2.33 (m, 2H), 2.88 (s, 6H), 3.69-4.00 (m, 6H), 4.47-4.75

(m, 1H), 5.39 (s, 2H), 6.10-6.15 (m, 1H), 7.14-7.17 (d, J = 8.1 Hz, 1H), 7.42-7.45 (m, 1H),

7.43-7.45 (m, 2H), 7.58-7.60 (m, 2H), 7.79-7.83 (dd, Ja = 8.4 Hz, Jb = 1.8 Hz, 1H). LRMS-

+ LC: m/z calcd for C28H34N4O8 [M+H] : 555.2; found: 555.3.

5-Bromo-2-nitrobenzoic acid (84). 3-Bromobenzoic acid (1 eq., 4 g, 20 mmol) in con.

H2SO4 (6.8 ml) was added to a mixture of conc. H2SO4 (6.8 ml) and conc. HNO3 (13.6 ml) at

0 ˚C. The mixture was warm to room temperature overnight and the reaction was quenched with water (50 ml). The mixture was filtered and the precipitate was washed with (2 × 10 ml), affording 84 as white solid in quantitative yield (5 g, 20 mmol). 1H NMR (300 MHz,

191 CDCl3): δ = 7.81-7.82 (m, 2H), 7.99 (s, 1H). The analytical data matched reported results.

5-Bromo-N-methoxy-N-methyl-2-nitrobenzamide (85). Oxalyl chloride (1.3 eq., 0.22 ml,

2.6 mmol) was added to a solution of the acid 84 (1 eq., 500 mg, 2 mmol) in DCM (4 ml), followed by DMF (2 drops). The mixture was stirred for 4 hours at room temperature.

DIPEA (3.5 eq., 0.61 ml, 3.5 mmol) and N,O-dimethylhydrozylamine hydrochloride (1.3 eq.,

127 mg, 1.3 mmol) were added and stiring was continued overnight at room temperature.

The solution was diluted with DCM (3 ml), and was washed with sat. NaHCO3 (2 ml), 1 M

HCl (2 ml) and brine (2 ml). The organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 85 as a white solid in 64% yield (367 mg, 1.28

1 mmol). H NMR (300 MHz, CDCl3): δ = 3.37 (s, 3H), 3.40 (s, 3H), 7.68-7.74 (m, 2H), 8.02-

+ 8.05 (d, J = 8.7 Hz, 1H). LRMS-LC: m/z calcd for C9H9BrN2O4 [M+H] : 290.0; found:

288.9.

5-Bromo-2-nitrobenzaldehyde (86). DIBAl-H (1 eq., 1 M, 1.3 ml, 1.3 mmol) was added to a solution of the amide 85 (1 eq., 368 mg, 1.3 mmol) in toluene (1.3 ml) at –78 ˚C. The

mixture was stirred for 1.5 hours at –78 ˚C before warmed to 0 ˚C, and 1 M HCl (1 ml) was added. The mixture was diluted with water (2 ml) and the aqueous layer was extracted with

Et2O (3 × 2 ml). The organic layers were combined, washed with brine (2 ml), dried over

Na2SO4, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 86 as a colorless oil in 75% yield (220 mg, 0.95

1 mmol). H NMR (300 MHz, CDCl3): δ = 7.59-7.62 (dd, Ja = 8.7 Hz, Jb = 2.1 Hz, 1H), 8.02-

8.04 (d, J = 8.4 Hz, 1H), 8.06-8.07 (d, J = 2.1 Hz, 1H), 10.41 (s, 1H). The analytical data matched reported results.192

1-(5-Bromo-2-nitrophenyl)ethan-1-ol (87). AlMe3 in hexane (1.5 eq., 2 M, 0.72 ml, 1.43 mmol) was added to a solution of the aldehyde 86 (1 eq., 220 mg, 0.96 mmol) in DCM (4.3 ml) at –78 ˚C. The mixture was stirred for 1.5 hours at –78 ˚C and slowly warmed to -20 ˚C for one hour. Ice was added until the gernation of methane seized, followed by the addition of 1 M NaOH (10 ml). The mixture was stirred till clear and extracted with DCM (3 × 10 ml). The organic layers were combined, washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 87 as a colorless oil in 84% yield (198 mg, 0.81

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.56-1.58 (d, J = 6.3 Hz, 3H), 5.44-5.50 (q, J = 6.3

Hz, 1H) 7.53-7.57 (dd, Ja = 8.7 Hz, Jb = 2.4 Hz, 1H), 7.81-7.84 (d, J = 8.4 Hz, 1H), 8.02-8.03

(d, J = 2.1 Hz, 1H). The analytical data mathed reported results.193

(5-Bromo-2-nitrophenyl)methanol (88). Thionyl chloride (3 eq., 0.22 ml, 3 mmol) was added to a solution of the acid 84 (1 eq., 250 mg, 1 mmol) in toluene (2 ml) at room temperature. The mixture was heated to reflux for 3 hours and concentrated under reduced pressure. The crude product was confirmed by 1H NMR and used in the next step without further purification. NaBH4 (1 eq., 38 mg, 1 mmol) was added to the acid chloride generated in the previous step in THF/DMF (3:1, 2 ml) at 0 ˚C and the resulting mixture was stirred overnight from 0 ˚C to room temperature. The reaction was quenched by the addition of 1 M

HCl (3 ml), and the aqueous layer was extracted with EtOAc (3 × 2 ml). The organic layers were combined washed with sat. NaHCO3 (2 ml), water (2 ml) and brine (2 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 88 as a colorless oil in

1 56% yield (129 mg, 0.56 mmol). H NMR (300 MHz, CDCl3): δ = 5.02 (s, 2H), 7.59-7.62

(dd, Ja = 8.7 Hz, Jb = 2.1 Hz, 1H), 7.98-8.02 (m, 2H). The analytical data matched reported results.194

1-(4'-(Dimethylamino)-4-nitro-[1,1'-biphenyl]-3-yl)ethan-1-ol (90). The alcohol 88 (1 eq.,

198 mg, 0.8 mmol), 4-(dimethylamino)phenyl boronic acid (89) (1.2 eq., 158 mg, 0.96 mmol), potassium carbonate (2.7 eq., 298 mg, 2.16 mmol), tetrabutylammonium bromide (1 eq., 258 mg, 0.8 mmol) and palladium acetate (0.1 eq., 18 mg, 0.08 mmol) were dissolved in

EtOH/H2O (2:1, 12 ml) and heated under microwave irradiation in a CEM Discover in standard open vessel mode (200 W) for 10 min. The solution was diluted with water (10 ml) and extracted with EtOAc (3 × 10 ml). The combined organic layers were washed with brine

(15 ml), dried over Na2SO4, filtered, and concentrated. The crude the product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 90 as a red solid

1 in 93% yield (179 mg, 0.74 mmol). H NMR (400 MHz, CDCl3): δ = 1.61-1.63 (d, J = 6.4

Hz, 3H), 3.03 (s, 6H), 5.48-5.52 (q, J = 6.4 Hz, 1H), 6.75-6.77 (d, J = 8.8 Hz, 2H), 7.50-7.56

13 (m, 3H), 7.95-8.00 (m, 2H). C NMR (400 MHz, CDCl3): δ = 24.4, 40.4, 65.9, 112.5, 124.3,

124.8, 125.5, 125.9, 128.1, 142.4, 144.9, 146.7, 150.9. HRMS-LC: m/z calcd for C16H18N2O3

[M+H]+: 287.1398; found: 287.1399.

N,N-Dimethyl-3'-(1-((methylthio)methoxy)ethyl)-4'-nitro-[1,1'-biphenyl]-4-amine (91).

NaH (2 eq., 5.2 mg, 0.2 mmol) followed by chloromethyl methyl sulfide (5 eq., 41 µl, 0.5 mmol) was added to a solution of the alcohol 90 (1 eq., 28 mg, 0.1 mmol) in THF (1 ml) at 0

˚C. The mixture was stirred overnight from 0 ˚C to room temperature and the reaction was quenched with water (2 ml). The aqueous solution was extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude the product was purified by silica gel chromatography with hexane/EtOAc (9:1) as the eluent, affording 91 as an orange solid in 20% yield (6.9 mg, 0.02

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.61-1.63 (d, J = 6.4 Hz, 3H), 1.93 (s, 3H), 3.03 (s,

6H), 4.56-4.57 (m, 2H), 5.48-5.52 (q, J = 6.4 Hz, 1H), 6.75-6.77 (d, J = 8.8 Hz, 2H), 7.50-

7.56 (m, 3H), 7.95-8.00 (m, 2H).

1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-4-(2-(4'-(dimethylamino)-4-nitro-[1,1'-biphenyl]-3-yl)propoxy)-5-

methylpyrimidin-2(1H)-one (92). The alcohol 79 (1 eq., 30 mg, 0.1 mmol), the thymidine

61 (1 eq., 47 mg, 0.1 mmol), triphenylphosphine (1.8 eq., 47 mg, 0.18 mmol), and diisopropyl azodicarboxylate (1.8 eq., 36 µl, 0.18 mmol) were dissolved in THF (0.6 ml) and the mixture was stirred for 16 hours at room temperature. The solution was diluted with water (1 ml) and the aqueous layer was extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated.

The crude the product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 92 as a red foam in 85% yield (64 mg, 0.085 mmol). 1H NMR (300

MHz, CDCl3): δ = 0.08-0.09 (m, 6H), 0.87-0.91 (m, 18H), 1.35-1.37 (d, J = 6.9 Hz, 3H),

1.83-1.87 (m, 3H), 2.20-22.32 (m, 2H), 3.02 (s, 6H), 3.69-3.93 (m, 4H), 4.18-4.23 (m, 1H),

4.31-4.40 (m, 1H), 5.02-5.11 (m, 1H), 6.22-6.31 (m, 1H), 6.80-6.82 (d, J = 7.5 Hz, 2H), 7.40-

7.45 (m, 2H), 7.56-7.59 (m, 2H), 7.69-7.73 (m, 2H). LRMS-LC: m/z calcd for C39H60N4O7Si2

[M+H]+: 753.4; found: 753.8.

4-(2-(4'-(Dimethylamino)-4-nitro-[1,1'-biphenyl]-3-yl)propoxy)-1-((2R,4S,5R)-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidin-2(1H)-one (93).

TBAF in THF (3 eq., 1 M, 0.26 ml, 0.26 mmol) was added to a solution of the silyl ether 92

(1 eq., 64 mg, 0.085 mmol) in THF (1.9 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (1:1) as the eluent, affording 93 as

1 a red foam in 60% yield (27 mg, 0.05 mmol). H NMR (400 MHz, CDCl3): δ = 1.36-1.38 (d,

J = 7.2 Hz, 3H), 1.82-1.84 (m, 3H), 2.22-2.38 (m, 2H), 3.03 (s, 6H), 3.79-3.09 (m, 4H),

4.12-4.17 (m, 1H), 4.33-4.42 (m, 1H), 4.50-4.52 (m, 1H), 6.01-6.08 (m, 1H), 6.83-6.85 (d, J

= 7.6 Hz, 2H), 7.29-7.31 (d, J = 4.0 Hz, 1H), 7.42-7.45 (m, 1H), 7.56-7.59 (d, J = 8.8 Hz,

13 2H), 7.67-7.72 (m, 2H). C NMR (400 MHz, CDCl3): δ = 13.4, 19.6, 32.0, 40.0, 40.2, 40.7,

46.5, 45.6, 62.4, 71.3, 71.5, 87.0, 87.7, 88.1, 110.1, 112.9, 124.6, 126.5, 128.3, 135.0, 135.3,

+ 138.6, 138.8, 145.5, 148.2, 151.0, 163.4. LRMS-LC: m/z calcd for C27H32N4O7 [M+H] :

525.2; found: 525.9.

4-(2-(4'-(Dimethylamino)-4-nitro-[1,1'-biphenyl]-3-yl)propoxy)-1-((2R,4S,5R)-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidin-2(1H)-one (94).

DMTCl (1.5 eq., 157 mg, 0.46 mmol) was added to a solution of the nucleoside 93 (1 eq.,

161 mg, 0.31 mmol) in pyridine (4.6 ml). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:3) as the eluent, affording 94 as a red foam

1 in 75% yield (195 mg, 0.24 mmol). H NMR (400 MHz, CDCl3): δ = 1.36-1.38 (d, J = 7.2

Hz, 3H), 1.82-1.84 (m, 3H), 2.22-2.38 (m, 2H), 3.03 (s, 6H), 3.79-3.09 (m, 10H), 4.12-4.17

(m, 1H), 4.33-4.42 (m, 1H), 4.50-4.52 (m, 1H), 6.01-6.08 (m, 1H), 6.83-6.85 (m, 6H), 7.24-

7.31 (m, 8H), 7.42-7.45 (m, 2H), 7.56-7.59 (d, J = 8.8 Hz, 2H), 7.67-7.72 (m, 2H). LRMS-

+ LC: m/z calcd for C48H51N4O9 [M+H] : 827.4; found: 827.4.

1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)- tetrahydrofuran-2-yl)-5-methyl-3-((methylthio)methyl)pyrimidine-2,4(1H,3H)-dione

(99). DBU (2 eq., 33 ml, 2.2 mmol) was added to a solution of the thymidine 61 (1 eq., 520

mg, 1.1 mmol) in DMF (2.5 ml) and the resulting mixture was stirred for 20 minutes at room temperature. Chloromethyl menthylsulfide (5 eq., 0.46 ml, 5.5 mmol) was added and the reaction mixture was stirred overnight before been quenched by sat. NaHCO3 (3 ml). The solution was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with brine (3 ml), dried over Na2SO4, filtered, and concentrated. The crude the product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 99 as

1 a clear foam in 93% yield (555 mg, 1 mmol). H NMR (300 MHz, CDCl3): δ = 0.03-0.07 (m,

12 H), 0.85-0.88 (m, 18 H), 1.89 (s, 3H), 2.23 (s, 3H), 2.33-2.46 (m, 2H), 3.68-3.94 (m, 3

H), 4.35-4.37 (m, 1 H), 4.99 (s, 1 H), 6.33-6.34 (m, 1H), 7.44 (s, 1 H). 13C NMR (300 MHz,

CDCl3): δ = -5.0, -4.9, -4.4, -4.2, 13.7, 26.9, 41.9, 45.1, 63.4, 72.7, 86.0, 88.3, 110.4, 134.2,

+ 151.0, 163.5. HRMS-LC: m/z calcd for C24H46N2O5SSi2 [M+H] : 531.2744; found: 531.2712.

3-(Azidomethyl)-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (100).

NCS (2 eq., 98 mg, 0.73 mmol) was added to a solution of the thioether 99 (1 eq., 200 mg,

0.37 mmol) in DCM (6 ml) and the reaction mixture was stirred at room temperature for 2 hours. TMSCl (2 eq., 93 µl, 0.73 mmol) was added and the mixture was stirred for 3 hours.

The reaction was quenched with sat. NaHCO3 (2 ml) and the aqueous layer was extracted with DCM (3 × 2 ml). The combined organic layer was washed with water (2 ml), and brine

(2 ml), and concentrated under reduced pressure. The crude the product was confirmed by 1H

NMR and used in next step without further purification. The chloride was dissolved in DMF

(1.9 ml) and sodium azide (3 eq., 68.8 mg, 1.11 mmol) was added. The resulting mixture was

stirred overnight at room temperature, and the reaction was quenched with sat. NaHCO3 (2 ml). The aqueous layer was extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude the product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 100 as a clear foam in 94% yield (183 mg, 0.35 mmol). 1H NMR (300 MHz,

CDCl3): δ = 0.06-0.11 (m, 12 H), 0.88-0.92 (m, 18 H), 1.94 (s, 3 H), 1.98-2.04(m, 1 H), 2.24-

2.31 (m, 1 H), 3.73-3.90 (m, 2 H), 3.94-3.96 (m, 1 H), 4.37-4.41 (m, 1 H), 4.50-4.52 (m, 0.5

H), 5.17-5.18 (m, 0.5 H), 5.32-5.34 (m, 2 H), 6.33-6.37 (m, 1H), 7.52 (s, 1 H). 13C NMR

(300 MHz, CDCl3): δ = ˗4.9, ˗4.8, ˗4.3, ˗4.1, 13.7, 26.9, 42.0, 56.2, 63.5, 72.8, 86.2, 88.5,

+ 110.7, 135.0, 151.1, 163.6. HRMS-LC: m/z calcd for C23H43N5O5Si2 [M+H] : 526.2881; found: 526.2853.

3-(Azidomethyl)-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione (101). Acetic chloride (0.5 eq., 6 µl, 0.08 mmol) was added to a solution of the silyl ether 100 (1 eq., 85 mg, 0.16 mmol) in MeOH (3.3 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with EtOAc as the eluent, affording 101 as a clear foam in 78% yield (37 mg, 0.08 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.94 (s, 3 H), 2.34-2.37 (m, 2 H), 3.83-3.94 (m, 2 H), 4.01-4.03 (m, 1 H), 4.49

(s, 0.5 H), 4.55-4.59 (m, 1 H), 5.16 (s, 1 H), 5.32 (s, 2 H), 6.20-6.24 (t, J = 4.8 Hz, 1 H), 7.51

13 (s, 1 H). C NMR (300 MHz, CDCl3): δ = 13.4, 40.5, 55.9, 62.6, 71.7, 87.3, 87.4, 110.7,

+ 136.0, 151.0, 163.2. HRMS-LC: m/z calcd for C11H15N5O5 [M+H] : 298.1151; found:

298.1133.

3-(Azidomethyl)-1-((2R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (102). DMTCl (1.5 eq., 19.3 mg, 0.06 mmol) was added to a solution of the nucleoside 101 (1 eq., 11.3 mg,

0.038 mmol) in pyridine (0.6 ml). The reaction mixture was stirred overnight at room temperature and then concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:3) as the eluent, affording 102 as a

1 colorless foam in 75% yield (18 mg, 0.028 mmol). H NMR (300 MHz, CDCl3): δ = 1.48 (s,

3 H), 2.32-2.46 (m, 2 H), 3.36-3.51 (m, 2 H), 4.05-4.06 (d, J = 2.4 Hz, 1 H), 4.50-4.51 (m,

0.5 H), 4.56 (br, 1 H), 5.17-5.19 (m, 1 H), 5.33 (s, 2 H), 6.41-6.44 (t, J = 4.8 Hz, 1 H), 6.83-

6.85 (m, 4H), 7.27-7.30 (m, 7H), 7.38-7.40 (m, 2H), 7.64 (s, 1H). 13C NMR (300 MHz,

CDCl3): δ = 13.9, 41.5, 55.7, 56.2, 63.8, 72.8, 85.8, 86.6, 87.4, 110.9, 113.7, 127.6, 128.5,

130.5, 135.1, 135.7, 144.7, 151.1, 159.2, 163.4. LRMS-LC: m/z calcd for C32H33N5O7

[M+H]+: 600.2; found: 600.7.

(4-Azidophenyl)methanol (104). Sodium nitrite (1.125 eq., 1.26 g, 18.3 mmol) in water (10 ml) was added to a solution of 4-aminobenzyl alcohol (1 eq., 2 g, 16.2 mmol) in 5 M HCl (32 ml) at 0 ˚C. The mixture was stirred for 30 minutes and sodium azide (4 eq., 4 × 1.05 g) was added in four portion over 1 hour with vigrous effervescence. The resulting mixture was stirred at 0 ˚C for 2 hours before been poured into ice-cold water (100 ml). The solution was

basified to pH 8 by adding solid NaHCO3 and was extracted with EtOAc (3 × 50 ml). The combined organic layer was washed with brine (100 ml), dried over Na2SO4, filtered, and concentrated. The crude the product was purified by recrystallization in EtOAc/hexane affording 104 as a needle crystal in 54% yield (1.3 g, 8.7 mmol). 1H NMR (300 MHz,

CDCl3): δ = 4.64 (s, 2H), 6.98-7.01 (d, J = 8.4 Hz, 2H), 7.31-7.34 (d, J = 8.4, 2H). The analytical data matched reported results.182

3-(4-Azidobenzyl)-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (105).

TEA (1.25 eq., 44 µl, 0.34 mmol) followed by methanesulfonyl chloride (1.25 eq., 26 µl,

0.34 mmol) was added to a solution of the alcohol 104 (1.25 eq., 50 mg, 0.34 mmol) in DCM

(2 ml). The mixture was stirred for 2 hours and concentrated under reduced pressure. The crude the product was confirmed by 1H NMR and used in the next step without further purification. DBU (2 eq., 80 µl, 0.54 mmol) was added to a solution of the TBDMS protected thymidine 61 (1, 127 mg, 0.27 mmol) in DMF (1.5 ml) and the mixture was stirred for 30 minutes. A solution of the azide generated in the precious step in DMF (0.5 ml) was added.

The reaction was stirred overnight and quenched by sat. NaHCO3 (2 ml). The solution was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with sat. NaHCO3

(2 ml), water (2 ml) and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 105 as a colorless oil in 68% yield (109 mg, 0.23 mmol). 1H NMR (300

MHz, CDCl3): δ = 0.06-0.10 (m, 12H), 0.88-0.91 (m, 18H), 1.92 (s, 3H), 2.21-2.26 (m, 2H),

3.72-3.73 (m, 1H), 3.76-3.86 (m, 1H), 3.92-3.93 (m, 1H), 4.37-4.40 (m, 1H), 5.02-5.11 (m,

2H), 6.35-6.38 (t, J = 4.8 Hz, 1H), 6.92-6.95 (d, J = 8.4 Hz, 2H), 7.46-7.50 (m, 3H). 13C

NMR (300 MHz, CDCl3): δ = ˗5.1, ˗4.5, ˗4.2, 13.7, 18.4, 18.7, 26.1, 26.3, 41.8, 44.3, 63.4,

72.7, 85.9, 88.2, 100.5, 119.3, 131.3, 134.0, 134.2, 139.6, 151.3, 163.8. HRMS-LC: m/z calcd

+ for C29H47N5O5Si2 [M+H] : 602.3194; found: 602.3155.

(((4-Azidobenzyl)oxy)methyl)(methyl)sulfane (106). Dimethylsulfide (8 eq., 200 µl, 2.64 mmol) was added to a solution of the alcohol 104 (1 eq., 50 mg, 0.33 mmol) in CH3CN (1.5 ml) at 0 ˚C, followed by benzoyl peroxide (4 eq., 4 × 100 mg, 1.34 mmol) in four portions over 30 minutes. The mixture was stirred for 2 hours at 0 ˚C, quenched with 1 M NaOH (1 ml), and stirred overnight at room temperature. The aqueous layer was extracted with Et2O (3

× 1 ml) and the combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 106 as a yellow oil in 87% yield (60 mg, 0.29

1 mmol). H NMR (300 MHz, CDCl3): δ = 2.15 (s, 3H), 4.55 (s, 2H), 4.65 (s, 2H), 6.97-6.99

13 (d, J = 8.4 Hz, 2H), 7.30-7.32 (d, J = 8.4, 2H). C NMR (300 MHz, CDCl3): δ = 13.6, 68.4,

+ 74.0, 118.7, 129.3, 133.9, 139.1. LRMS-LC: m/z calcd for C9H11N3OS [M+H] : 210.1; found: 210.4.

1-Azido-4-((chloromethoxy)methyl)benzene (107). Sulfuryl chloride (1.5 eq., 12 µl, 0.15 mmol) was added to a solution of the thioether 106 (1 eq., 21 mg, 0.1 mmol) in DCM (0.2 ml) at 0 ˚C. The mixture was stirred for 30 minutes at 0 ˚C and dried, delivering the crude

1 product 107 as a red oil which was used without purification. H NMR (300 MHz, CDCl3): δ

= 2.15 (s, 3H), 4.67 (s, 2H), 5.62 (s, 2H), 6.97-6.99 (d, J = 8.4 Hz, 2H), 7.30-7.32 (d, J = 8.4,

2H).

3-(((4-Azidobenzyl)oxy)methyl)-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert- butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)- dione (108). DBU (1.5 eq., 8 µl, 0.12 mmol) was added to a solution of the nucleoside 61 (1 eq., 39 mg, 0.08 mmol) in DMF (0.3 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of chloromethyl ether 107 (1.2 eq., 0.1 mmol) in DMF (0.2 ml) was added drop-wise and the resulting mixture was stirred overnight, quenched with water (1 ml), extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 108 as a yellow oil in 11%

1 yield (5.8 mg, 0.009 mmol). H NMR (300 MHz, CD3OD): δ = 0.13-0.14 (m, 12H), 0.90-

0.93 (m, 18H), 1.29 (s, 3H), 2.07-2.20 (m, 2H), 3.82-3.93 (m, 3H), 4.40-4.47 (m, 1H), 4.54

(s, 2H), 5.48 (s, 2H), 6.18-6.25 (m, 1H), 6.98-7.00 (d, J = 8.4 Hz, 2H), 7.32-7.35 (m, 2H),

+ 7.65 (s, 1H). LRMS-LC: m/z calcd for C30H49N5O6Si2 [M+H] : 632.3; found: 632.0.

CHAPTER 3: Synthesis of Photocaged Morpholino Oligonucleotide Subunits

Morpholino oligomers (MOs) are the most commonly used gene-silencing reagents applied in zebrafish embryos.9 Three strategies targeting different RNA molecules have been reported to trigger gene expression using MOs in zebrafish embryos. The earliest strategy is the use of translation-silencing MOs to target the initiation codon of mature mRNA, thus blocking translation as shown in Scheme 1.2. Recently, MOs have been designed to bind to pre-mRNA and inhibit correct pre-mRNA splicing.195 This strategy requires additional information on intron and exon structure compared to the first strategy, but allows the quantitative measurement of MO efficiency by RT-PCR. MOs targeting miRNAs have also been reported, thereby expanding the application of MOs in zebrafish.196

Generally, to block translation directly, MOs of 25 base pairs are synthesized and microinjected into embryos at the 1-2 cell stage, followed by distribution throughout the developing embryo and sustained blocking of target gene expression for several days.48

Usually within three to five days the embryos injected with antisense MOs show abnormal development and the observed phenotype can be used to elucidate the function of the targeted gene. To further confirm that the phenotype is induced by target gene-silencing rather than off-target effects, mRNA encoding target protein but without sequence overlap with antisense MOs can be co-injected with MOs. If the phenotype is recovered, the corresponding gene is responsible for the mutant phenotype. The usage of two different sets of MOs targeting the same gene can also eliminate off-target effects, especially when one translation-silencing MO and one splicing-inhibiting MO are used in combination.

A problem with the MO gene-silencing methodology is a general lack of conditional control. As mentioned above, after being injected, MOs are distributed evenly throughout the embryo which leads to gene knock down throughout the whole embryo right from the injection time point onward. To accurately control embryo development and assign gene function, selective gene perturbation within a certain region and at a specific stage of development can potentially reveal the mechanisms behind embryonic development that cannot be observed otherwise.

Approach A-C and E in Scheme 1.9 have been applied to accomplish the spatial and temporal control over MO activity,109, 111, 116, 118, 197 among which the regulation through caged nucleobases was reported by our group.197 Dr. Lusic in the Deiters lab synthesized a 6- nitropiperonyloxymethyl (NPOM)-caged thymine-MO subunit that was readily incorporated into MOs targeting a variety of genes. The efficiency of this approach in vivo was demonstrated by the successful regulation of the chordin gene, which is expressed in early embryonic development and triggers dorsal-ventral axis formation and brain formation.

Embryos injected with a chordin MO with four caging groups were either kept in the dark or exposed to UV light (Figure 3.1), and as show in Figure 3.1D, irradiation resulted in 90% chordin mutant phenotype, indicating successful light-induced MO activation and gene- silencing. By activating the nucleobase cMO at different time points, it was found that irradiation before 10 hpf mostly led to a severe chordin mutant phenotype, while this phenotype was not observed at later irradiations. The importance of chordin activity before

10 hpf is consistent with known chordin function and proves the applicability of NPOM- caged MO reagents in temporal gene control.198 Apart from optimizing the synthesis of

NPOM thymine-MO subunit (131), the synthesis of an NPOM-caged guanine-MO subunit

(141), a propargyl-6-nitroveratryloxymethyl (PNVOM)-caged thymine-MO subunit (155), and a 3-nitro-2-ethyldibenzofuran (NDBF)-caged thymine-MO subunit (152) will be discussed below.

A B

wild type chordin MO C D

nucleobase cMO nucleobase cMO + UV

Figure 3.1 Gene regulation with an NPOM-caged MO in zebrafish embryos. MOs were injected at the one to four cell stage and phenotypes were assessed at 24-28 hpf. A. Wild-type (WT) embryo showing a normal phenotype. B. Embryos injected with a chordin MO show a distinct phenotype, including an abnormal tail fin and a reduced head. C. Embryos injected with the NPOM-caged MO and shielded from light show a normal phenotype. D. Embryos injected with the NPOM-caged MO and exposed to 365 nm light after injection show the chordin phenotype. Adapted from J. Am. Chem. Soc. 2010, 132, 15644.

3.1 Synthesis of NPOM-caged MO subunit

3.1.1 Optimization of the NPOM-caged thymine-MO subunit synthesis

The morpholino ring structure is usually synthesized from a nucleoside starting material. 109, 199 Taking the thymine-MO subunit (112) as an example, the synthesis started

from 5-methyluridine 110 in two steps (Scheme 3.1). The yield however was only 13% and the purification was troublesome required more efficient ways to make the morpholino ring.

Scheme 3.1 Synthesis of the thymine-MO subunit 112 from 5-methyluridine 110.

Dr. Lusic in the Deiters lab developed a route in which the uracil-MO subunit 115 was synthesized from the triisopropylsilyl (TIPS) protected uridine 113 via a dialdehyde 114 in 50% overall yield (Scheme 3.2).166 Apart from the yield improvement, the introduction of the TIPS group allows the MO subunit to be purified through column chromatography. Thus, this route was adapted to the synthesis of thymine-MO subunits.

Scheme 3.2 Synthesis of the uracil-MO subunit 115 from the TIPS protected uridine 113.

Owing to the high price of 5-methyluridine (110) at the time, the 5-methyluridine derivatives were made in house. The first is to synthesize the benzoyl protected 5- methyluridine 119 from thymine (116) which was silylated in 1,2-dichloroethane with

TMSCl and then reacted with 1-o-acetyl-2,3,5-tribenzoylribofuranose (118) (Scheme 3.3).200

The reaction of the benzoyl protected 5-methyluridine 119 with the NPOM-Cl 120, which was synthesized from 6-nitropiperinal (44) following previous report,72 yielded the caged product 121 in 93% yield. The subsequent removal of the benzoyl groups to give the

200 unprotected sugar moiety 122 proceeded readily with 7 N NH3 in methanol (90% yield).

The unprotected sugar moiety 122 then underwent a TBDMS protection and the 5-protected caged 5-methyluridine 123 was obtained in 73% yield.201 However, we were not able to convert neither 122 nor 123 into the morpholino subunits efficiently. The highest yield was

40% in case of the 5-protected caged 5-methyluridine 125.

Scheme 3.3 Attempts to Synthesize the NPOM thymine-MO subunit from caged 5- methyluridine 122.

In an alternate route, the benzoyl groups in compound 119 were cleaved by NH3 affording 5-methyluridine (110) in 93% yield (Scheme 3.4). The 5ʹ-TBDMS protected 5- methyluridine 126 was obtained through treatment of 5-methyluridine (110) with 1.1 equivalents of TBDMSCl and 1.2 equivalents of imidazole in DMF in 67% yield. Increasing the amount of TBDMSCl or imidazole resulted in more di-protected 5-methyluridine and decreasing their amount lead to an incomplete reaction, both of which lowered the yield. The resulting protected 5-methyluridine 126 underwent oxidation followed by (NH4)2B4O7 and

NaCNBH3 in methanol to give the thymine-MO subunit 127 (69% yield). Protection of the

amine in 127 with trityl chloride and triethylamine in DCM delivered the trityl protected subunit 128 in 89% yield.109 The NPOM group was installed on the 3-N position of thymine in 128 using NPOM-Cl (120) and base. Different bases were tested and DBU gave the best yield of 74%. The removal of the silyl ether was accomplished with TBAF, affording the 5’- hydroxyl thymine-MO subunit 130 in high yield (94% yield). The resulting caged thymine-

MO subunit 130 was activated by treatment with Me2NPOCl2 and LiHMDS, and the NPOM- caged thymine morpholino chlorophosphoramidate 131 was obtained in 80% yield.109 The chlorophosphoramidate 131 was sent to Syntrix Biosystems, Inc. where it was successfully incorporated into MOs in a yield comparable to uncaged MO-subunits.

Scheme 3.4 Synthesis of the NPOM-caged thymine-MO chlorophosphoramidate 131.

Although the morpholino ring was assembled in good yield (69%) in the above route, two additional steps, TBDMS protection and its deprotection, were involved which required column purification. This limits the above synthesis to a relatively small scale and is unfavorable for the commercialization of caged MO reagents. Thus, a more direct route to the NPOM-caged thymine-MO subunit 130 was developed (Scheme 3.5). Similarly to the synthesis shown in Scheme 3.1, 5-methyluridine 110 was oxidized and cyclized to give the intermediate 110. The subsequent reduction was conducted using borane triethylamine complex, which is less toxic compared to sodium cyanoborohydride and thus superior for a large-scale synthesis.202 Furthermore, the treatment of the crude product generated above with p-toluenesulfonic acid allowed the secondary amine 112 to be purified by recrystallization as a p-toluenesulfonic acid salt 132 (47% yield in 4 steps). Trityl protection of the amine group on 132 was achieved in 94% yield furnishing the protected monomer 133, which underwent a caging reaction with NPOM-Cl (120), generating the NPOM-caged thymine-MO alcohol 130 (DBU, DMF, 74%). This provides a shorter synthesis of the caged thymine-MO subunit 130 with simplified purification, reduced toxicity, and comparable yield.

Scheme 3.5 Synthesis of the NPOM thymine-MO subunit 130 from 5-methyluridine 110.

3.1.2 Synthesis of the NPOM-caged guanine-MO subunit

With the NPOM-caged thymine-MO subunit in hand, the synthesis of an NPOM- caged guanine-MO subunit was pursued to provide more flexibility in the photochemical control of MO activity.

The synthesis of trityl protected guanine-MO subunit 136 was very similar to the synthesis of the thymine-MO subunit 132, commencing with the phenylacetyl protection of guanosine (134) to give the protected guanosine 135 in 53% yield (Scheme 3.6).203 The remainder of the synthesis was identical to MO-T 132, staring with the oxidation of protected guanosine 135 with NaIO4 in 40% water/methanol. After the removal of the sodium iodate byproduct by filtration, the dialdehyde intermediate was obtained and directly treated with

(NH4)2B4O7 followed by borane-triethylamine complex affording the crude guanine-MO amine. The pure product was isolated as a p-toluenesulfonic acid salt (136) (20% yield in 4 steps) by acidifying the mixture to pH 3-4 with p-toluenesulfonic acid. A diisopropylacetyl group was also tested as the protecting group on the primary amine instead of phenylacetyl group,204 but the diisopropylacetyl protected guanosine failed to give the desired product.

Scheme 3.6 Synthesis of the guanine-MO subunit 136.

Meanwhile, the trityl protected guanine-MO subunit 137 was produced in large scale by our collaborators at Syntrix Biosystems Inc. and the synthesis was continued with their material (Scheme 3.7). The caging of amide on 137 with NPOM-Cl (120) in the presence of

DBU was tested in DMF. However, the selectivity between the two amide groups was very low and a 1:1 mixture of 1-N-NPOM- and exocyclic-NPOM-caged MO subunits was obtained. With the hope of selectively masking the exocyclic amide, several protection groups were analyzed, but none provided the desired selectivity. Therefore, to prevent the exocyclic amine from reacting, a dimethylformamidine group was installed on the exocyclic amine to replace the phenylacetyl group in two steps from compound 137.205 The free amine

138 was obtained in 85% yield by treatment of the phenylacteyl protected monomer 137 with

2 M NH3 in methanol. The amine of 138 was then reacted with N,N-dimethylformamide dimethyl acetal in THF/methanol at 60 oC giving the amidine protected amine 139 in 92% yield. The subsequent caging of 139 with the NPOM-Cl 120 was achieved in 94% yield using DBU in DMF. The resulting NPOM-caged guanine-MO subunit 140 was activated by

Me2NPOCl2 to give the NPOM-caged guanine-MO chlorophosphoramidate 141 (67% yield), which was also sent to Syntrix Biosystems, Inc. to get incorporated into oligonucleotides.

Scheme 3.7 Synthesis of the NPOM guanine-MO chlorophosphoramidate 141.

3.2 Synthesis of two-photon caged thymine-MO subunit

Due to the drawbacks of one-photon activation mentioned in CHAPTER 1.3.1, two- photon caging groups are introduced into biomolecules to trigger activity with IR light with enhanced spatial resolution. A recently developed two-photon caging group is the 3-nitro-2- ethyldibenzofuran (NDBF) group, however, the application of NDBF group is limited by the low yields in its reported synthesis.98, 146 Efforts have been made to optimize the synthesis of the NDBF caging group and to install the NDBF group on the thymine-MO subunit 152.

The synthesis of NDBF group started from commercially available 4-fluoro-2- nitrotoluene (142) following previous report (Scheme 3.8).146 Through treatment with N,N- dimethylformamide dimethyl acetal followed by NaIO4, 4-fluoro-2-nitrotoluene (142) was oxidized into the aldehyde 143 in 86% yield. A Cu-catalyzed Ullmann coupling of the resulting aldehyde 143 and ortho-iodophenol provided the diphenyl ether 144 (CuBr, K2CO3,

pyridine, 60oC, 67% yield). The alcohol 145 was obtained in high yield (98%) by methylation of the diphenyl ester 144 with AlMe3 in DCM. The subsequent Heck reaction is the key step in the formation of the dibenzofuran ring. In a previous synthesis, the Heck

o reaction was carried out on the alcohol 144 with low yield (Pd(OAc)2, Cs2CO3, DMA, 80 C,

12 hours, 38% yield).146 The attempts to improve the yield by modifying the reaction conditions, including different solvents, palladium catalysts, bases, temperatures and reaction times all failed (7-18% yield). It is noteworthy that the intramolecular Heck reaction in the presence of a free hydroxyl has rarely been reported while the Heck arylation has been well investigated.206-208 The hydroxyl group is likely to interfere with the reaction and lead to byproducts. Therefore, the aldehyde 144 and the thioether 146, which was synthesized from the alcohol 145 in 75% yield, were tested as substrates for the Heck reaction with the hope of improving the yield without elongating the synthesis. However, the yields remained to be very low (13-39% for the aldehyde 144 and 33-43% for the thioether 146). Silyl groups which are shown to be tolerable in Heck reaction,209 were introduced on the alcohol 145 with high yields (90-98%) and the resulting TMS and TBDMS protected alcohols (147 and 148) were used in Heck reaction. Both TMS and TBDMS protected alcohol gave promising yields. The TMS group could be removed during work up, which eliminated an additional deprotection step.

Scheme 3.8 Optimized synthesis of the NDBF alcohol.

Thus, the synthesis of NDBF alcohol 149 was accomplished in 81% yield from the

TMS protected alcohol 147 (Scheme 3.9). The resulting alcohol 149 was treated with BPO and Me2S affording the methylthiomethyl ether derivative 150 (74% yield) which was further transformed into the caging precursor NDBF-Cl (151) by employing sulfuryl chloride in

DCM. The NDBF-caged thymine-MO subunit 152 was obtained through reacting the NDBF-

Cl (151) with the trityl protected thymine-MO subunit 133 in the presence of DBU (86% yield).

Scheme 3.9 Synthesis of the NDBF-caged thymine-MO subunit 152.

3.3 Synthesis of the PNVOM MO-T

Recently, the PNVOM group was reported by our lab, which bears an alkyne group and thus is available for modifications through Click chemistry. Dr. Uprety accomplished the synthesis of PNVOM-caged thymidine, which was incorporated into DNA antisense agents and conjugated to cell penetrating peptides (CPP). The CPP facilitated the delivery of oligonucleotide without any transfection reagents while the o-nitrobenzyl core enabled spatial and temporal control over gene silencing.74

This PNVOM group was also introduced to thymine MO subunit (Scheme 3.10).

Following the reported protocol, the PNVOM-Cl 153 was synthesized from commercially available vanillin in 5 steps.74 This caging precursor 153 was reacted with the amide on 133 in the presence of DBU, delivering the PNVOM thymine-MO 154 in 86% yield. Activation

of the 5ʹ- hydroxyl group on 154 with Me2NPOCl2 completed the synthesis of the chlorophosphoramidate 155 in 73% yield.

Scheme 3.10 Synthesis of the PNVOM MO-T chlorophosphoramidate 155.

3.4 Conclusion

In summary, photocaged MO subunits have been synthesized which can provide spatial and temporal control over MO activity and expand the toolbox of reagents available to investigate zebrafish development (Figure 3.2). The synthesis of NPOM-caged thymine-MO subunit (131) was optimized to three steps before activation, and NPOM-caged guanine-MO subunit (141) was synthesized following a similar route. To improve the spatial resolution in the photoregulation as well as to minimize the possible photo-damage, an NDBF group as a two-photon caging group was installed on the thymine-MO subunit affording the IR- cleavable subunit 152. Furthermore, the PNVOM-caged thymine-MO subunit 155 was

developed, which enables the modification of MOs through Click reaction while simultaneously maintaining the photoregulatory ability.

Figure 3.2 Photocaged MO subunits.

3.5 Experimental

MeOH and pyridine were distilled from calcium hydride and stored over 4 Ǻ molecular sieves. Other reagents and solvents were obtained from commercial sources were stored under nitrogen and used directly without further purification. Yields refer to

chromatographically and spectroscopically pure compounds unless otherwise stated. Flash chromatography was performed on silica gel (60 Å, 40-63 μm (230 × 400 mesh), Sorbtech) as a stationary phase. High resolution mass spectral analysis (HRMS) was performed at the

University of Pittsburgh. The 1H NMR and 13C NMR spectra were recorded on a 300 MHz or a 400 MHz Varian NMR spectrometer. Chemical shifts are given in δ units (ppm) for 1H

NMR spectra and 13C NMR spectra.

(2R,3R,4R,5R)-2-((Benzoyloxy)methyl)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-

1(2H)-yl)tetrahydrofuran-3,4-diyl dibenzoate (119). TMSCl (0.8 eq., 0.4 ml, 3.2 mmol) followed by HMDS (0.8 eq., 0.7 ml, 3.2 mmol) was added to a solution of thymine (1 eq.,

504 mg, 4 mmol) in DCE (15 ml). The reaction mixture was heated to 80 ˚C for 3 hours and cooled to room temperature. A solution of β-D-ribofuranose 1-acetate 2,3,5-tribenzoate (1 eq., 2 g, 4 mmol) in DCE (15 ml) was added, followed by SnCl4 (2 eq., 0.94 ml, 8 mmol).

The resulting mixture was stirred for 2 hours, quenched with sat. NaHCO3 (20 ml), and extracted with DCM (3 × 10 ml). The combined organic layer was washed with water (30 ml) and brine (30 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with DCM and 2% TEA as the eluent, affording 119 as

1 a colorless foam in 98% yield (2.25 g, 3.9 mmol). H NMR (300 MHz, CDCl3): δ = 1.56 (s,

3H), 4.60-4.72 (m, 2H), 4.84-4.92 (m, 1H), 5.71-5.75 (m, 1H), 5.87-5.92 (m, 1H), 6.40-6.42

(d, J= 6.3 Hz, 1H), 7.14 (s, 1H), 7.35-7.41 (m, 4H), 7.49-7.59 (m, 5H), 7.92-7.96 (m, 4H),

8.11-8.14 (m, 2H).The analytical data matched reported results.200

(2R,3R,4R,5R)-2-((Benzoyloxy)methyl)-5-(5-methyl-3-((1-(6-nitrobenzo[d][1,3]dioxol-5- yl)ethoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3,4-diyl dibenzoate (121). DBU (1.5 eq., 19 µl, 0.13 mmol) was added to a solution of the nucleoside

119 (1 eq., 50 mg, 0.088 mmol) in DMF (1 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of chloromethyl ether 120 (1.5 eq., 0.13 mmol) in DMF (0.5 ml) was added drop-wise and the resulting mixture was stirred overnight, quenched with sat.

NaHCO3 (2 ml), and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording

1 121 as a yellow foam in 93% yield (65 mg, 0.082 mmol). H NMR (300 MHz, D2O): δ =

1.43-1.45 (m, 3H), 1.53-1.54 (m, 3H), 4.58-4.70 (m, 2H), 4.85-4.93 (m, 1H), 5.11-5.39 (m,

3H), 5.60-5.71 (m, 1H), 5.86-5.92 (m, 1H), 5.98-6.07 (m, 2H), 6.27-6.38 (m, 1H), 6.99-7.01

(m, 1H), 7.20 (s, 1H), 7.29-7.7.40 (m, 5H), 7.52-7.61 (m, 5H), 7.90-8.00 (m, 4H), 8.09-8.18

(m, 2H).

1-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-methyl-3-

((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione (122).

The nucleoside 121 (1 eq., 67 mg, 0.085 mmol) was dissolved in a 2 M solution of NH3 in

MeOH (150 eq., 6.35 ml, 12.7 mmol). The reaction mixture was stirred at room temperature for 2 days and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 122 as a light yellow

1 foam with 90% yield (36.7 mg, 0.05 mmol). H NMR (300 MHz, CDCl3): δ = 1.50-1.52 (d,

J= 6.3 Hz, 3H), 1.73-1.74 (d, J= 3.3 Hz, 3H), 3.74-3.96 (m, 2H), 4.10-4.17 (m, 1H), 4.20-

4.40 (m, 2H), 5.19-5.30 (m, 2H), 5.36-5.59 (m, 1H), 5.46-5.59 (m, 1H), 6.07-6.09 (m, 2H),

7.16 (s, 1H), 7.24 (s, 1H) 7.47 (s, 1H).

1-((2R,3R,4S,5R)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-3,4-dihydroxytetrhydrofuran-

2-yl)-5-methyl-3-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)methyl)pyrimidine-2,4

(1H,3H)-dione (123). The alcohol 122 (1 eq, 50 mg, 0.1 mmol) was dissolved in DMF (0.5 ml) and TBDMSCl (1.1 eq, 17 mg, 0.11 mmol) was added, followed by imidazole (1.1 eq,

7.8 mg, 0.11 mmol). The mixture was stirred at room temperature overnight, quenched with sat. NaHCO3 (2 ml) and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over NaSO4, filtered and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOA (2:1) as the eluent, yielding 123 as an off-white solid in 73% yield (45 mg, 0.076 mmol). 1H NMR (300

MHz, CDCl3): δ = 0.030.07 (m, 6H), 0.84-0.89 (m, 9H), 1.45-1.48 (d, J= 6.3 Hz, 3H), 1.81-

1.85 (m, 3H), 3.71-3.99 (m, 2H), 4.00-4.25 (m, 3H), 5.07-5.26 (m, 2H), 5.30-5.44 (m, 1H),

5.63-5.75 (m, 1H), 6.05-6.07 (m, 2H), 7.14 (s, 1H), 7.39-7.48 (m, 2H).

1-((2R,6S)-6-(hydroxymethyl)morpholin-2-yl)-5-methyl-3-((1-(6-nitrobenzo[d][1,3] dioxol-5-yl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione (124). Sodium periodate (1.1 eq.,

12 mg, 0.055 mmol) was added to a solution of the nucleotide 122 (1 eq., 24 mg, 0.05 mmol) in MeOH/water (3:1, 0.6 ml). The reaction mixture was stirred at room temperature for 24 hours and the solvent was removed under reduced pressure. The residue was taken up in

100

acetone (2 ml) and filtered through celite. The filtrate was concentrated under reduced pressure, and the crude product was confirmed by NMR and used in next step without further purification. The crude dialdehyde was dissolved in a pH 7 solution of MeOH buffered with

TEA and AcOH (1 ml). Ammonium biborate (1 eq., 13.2 mg, 0.05mmol) was added and the resulting mixture was stirred for one hour at room temperature. The mixture was cooled to 0

˚C and sodium cyanoborohydride (1 eq., 3.1 mg, 0.05 mmol) was added. The reaction was stirred at room temperature overnight and concentrated. The residue was dissolved in EtOAc

(2 ml) and washed with sat. NaHCO3 (2 ml), water and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 124 as a yellow foam in 20% yield (5 mg, 0.01

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.24-1.38 (m, 2H), 1.51-1.53 (d, J= 6.3 Hz, 3H),

1.89 (s, 3H), 2.47-2.69 (m, 2H), 3.00-3.18 (m, 2H), 3.24-3.39 (m, 1H), 3.59-3.3.79 (m, 1H),

5.13-5.41 (m, 2H), 5.60-5.67 (m, 1H), 6.08-6.10 (m, 2H), 7.13-7.16 (m, 1H), 7.29 (s, 1H),

7.45 (s, 1H).

1-((2R,6S)-6-(((tert-Butyldimethylsilyl)oxy)methyl)morpholin-2-yl)-5-methyl-3-((1-(6- nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione (125). Sodium periodate (1.1 eq., 12 mg, 0.055 mmol) was added to a solution of the nucleotide 123 (1 eq.,

30 mg, 0.05 mmol) in MeOH/water (3:1, 0.6 ml). The reaction mixture was stirred at room temperature for 24 hours and the solvent was removed under reduced pressure. The residue was taken up in acetone (2 ml) and filtered through celite. The filtrate was concentrated under reduced pressure, and the crude product was confirmed by NMR and dissolved in a pH

101

7 solution of MeOH buffered with TEA and AcOH (1 ml). Ammonium biborate (1 eq., 13.2 mg, 0.05 mmol) was added and the resulting mixture was stirred for one hour at room temperature. The mixture was cooled to 0 ˚C and sodium cyanoborohydride (1 eq., 3.1 mg,

0.05 mmol) was added. The reaction was stirred at room temperature overnight and then concentrated. The residue was dissolved in EtOAc (2 ml), washed with sat. NaHCO3 (2 ml), water and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 125

1 as a yellow foam in 40% yield (11.5 mg, 0.02 mmol). H NMR (300 MHz, CDCl3): δ = 0.08

(s, 6H), 0.92 (s, 9H), 1.24-1.38 (m, 2H), 1.51-1.53 (d, J= 6.3 Hz, 3H), 1.89 (s, 3H), 2.47-2.69

(m, 2H), 3.00-3.18 (m, 2H), 3.24-3.39 (m, 1H), 3.59-3.3.79 (m, 1H), 5.13-5.41 (m, 2H), 5.60-

5.67 (m, 1H), 6.08-6.10 (m, 2H), 7.13-7.16 (m, 1H), 7.29 (s, 1H), 7.45 (s, 1H).

5-Methyluridine (110). The nucleoside 119 (1 eq., 500 mg, 0.88 mmol) was dissolved in a 2

M solution of NH3 in MeOH (50 eq., 22 ml, 44 mmol). The reaction mixture was stirred at room temperature for 2 days and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording

1 110 as a light yellow foam in 93% yield (211 mg, 0.82 mmol). H NMR (300 MHz, D2O): δ

= 1.91 (s, 3H), 3.74-3.90 (m, 2H), 4.08-4.09 (m, 1H), 4.18-4.22 (m, 1H), 4.27-4.30 (m, 1H),

5.88-5.89 (d, J= 4.5 Hz, 1H), 7.67 (s, 1H). The analytical data matched reported results.200

1-((2R,3R,4S,5R)-5-(((tert-Butyldimethylsilyl)oxy)methyl)-3,4-dihydroxytetrahydro- furan-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (126). The alcohol 110 (1 eq, 50 mg,

102

0.19 mmol) was dissolved in DMF (0.5 ml) and TBDMSCl (1.1 eq, 32 mg, 0.21 mmol) was added, followed by imidazole (1.1 eq, 14.5 mg, 0.21 mmol). The mixture was stirred at room temperature overnight, quenched with sat. NaHCO3 (2 ml) and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over

NaSO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with EtOA as the eluent, yielding 126 as a white solid in 67%

1 yield (48 mg, 0.13 mmol). H NMR (300 MHz, CDCl3): δ = 0.06 (s, 6H), 0.87 (s, 9H), 1.79

(s, 3H), 3.72-3.88 (m, 2H), 3.81-3.83 (m, 1H), 4.02-4.04 (m, 2H), 5.84-5.86 (d, J= 4.5 Hz,

1H), 7.48 (s, 1H). The analytical data matched reported results.210

1-((2R,6S)-6-(((tert-Butyldimethylsilyl)oxy)methyl)morpholin-2-yl)-5-methylpyrimidine-

2,4(1H,3H)-dione (127). Sodium periodate (1.1 eq., 25 mg, 0.12 mmol) was added to a solution of the nucleotide 126 (1 eq., 40 mg, 0.11 mmol) in MeOH/water (3:1, 0.6 ml). The reaction mixture was stirred at room temperature for 24 hours and the solvent was removed under reduced pressure. The residue was taken up in acetone (2 ml) and filtered through celite. The filtrate was concentrated under reduced pressure. The crude product was confirmed by NMR and dissolved in a pH 7 solution of MeOH buffered with TEA and

AcOH (1 ml). Ammonium biborate (1 eq., 28 mg, 0.11 mmol) was added and the resulting mixture was stirred for one hour at room temperature. The mixture was cooled to 0 ˚C and sodium cyanoborohydride (1 eq., 6.7 mg, 0.11 mmol) was added. The reaction was stirred at room temperature overnight and concentrated under reduced pressure. The residue was dissolved in EtOAc (2 ml), washed with sat. NaHCO3 (2 ml), water and brine (2 ml), dried

103

over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with chloroform/MeOH (9:1) as the eluent, affording 127 as a colorless

1 foam in 69% yield (26 mg, 0.074 mmol). H NMR (300 MHz, CDCl3): δ = 0.03-0.04 (m,

6H), 0.88 (s, 9H), 1.90 (s, 3H), 2.58-2.70 (m, 2H), 2.99-3.19 (m, 2H), 3.58-3.75 (m, 2H),

3.79-3.83 (m, 1H), 5.66-5.73 (m, 1H), 7.24 (s, 1H). LRMS-LC: m/z calcd for C16H29N3O4Si

[M+H]+: 356.2; found: 356.9.

1-((2R,6S)-6-(((tert-Butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-5- methylpyrimidine-2,4(1H,3H)-dione (128). TrCl (1.5 eq., 30 mg, 0.11 mmol) followed by

TEA (3 eq., 30 µl, 0.21 mmol) was added to a solution of the alcohol 127 (1 eq., 25 mg, 0.07 mmol) in DCM (1 ml). The reaction mixture was stirred overnight at room temperature and diluted with DCM (2 ml). The solution was washed with sat. NaHCO3 (2 ml), water and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) and 1% TEA as the eluent, affording

1 128 as an off-white solid in 66% yield (28 mg, 0.047 mmol). H NMR (300 MHz, CDCl3): δ

= 0.02-0.04 (m, 6H), 0.83 (s, 9H), 1.86 (s, 3H), 3.21-3.32 (m, 2H), 3.52-3.60 (m, 1H), 3.67-

3.74 (m, 1H), 4.19-4.28 (m, 1H), 6.13-6.17 (m, 1H), 7.01 (s, 1H), 7.14-7.18 (m, 3H), 7.23-

+ 7.30 (m, 7H), 7.43-7.46 (m, 5H). LRMS-LC: m/z calcd for C35H43N3O4Si [M+H] : 598.3; found: 598.8.

1-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-5-methyl-3-

((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione (129).

104

Cs2CO3 (5.7 eq., 63 mg, 0.19 mmol) was added to a solution of the morpholino subunit 128

(1 eq., 20 mg, 0.03 mmol) in DMF (0.5 ml) and the mixture was stirred for one hour at room temperature. A solution of the chloromethyl ether 120 (2.8 eq., 0.096 mmol) in DMF (0.25 ml) was added drop-wise. The resulting mixture was stirred overnight, quenched with water

(2 ml), and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 129 as a

1 yellow foam in 84% yield (23 mg, 0.028 mmol). H NMR (300 MHz, CDCl3): δ = -0.04-0.01

(m, 6H), 0.79 (s, 9H), 1.24-1.35 (m, 2H), 1.46-1.48 (d, J= 5.4 Hz, 3H), 1.74 (s, 3H), 3.21-

3.32 (m, 2H), 3.50-3.62 (m, 1H), 3.69-3.80 (m, 1H), 4.13-4.22 (m, 1H), 5.10-5.22 (m, 1H),

5.26-5.37 (m, 2H), 5.97-6.12 (m, 3H), 6.83-6.90 (m, 1H), 7.09-7.20 (m, 4H), 7.24-7.39(m,

+ 8H), 7.44-7.50 (m, 5H). LRMS-LC: m/z calcd for C45H52N4O9Si [M+H] : 821.4; found:

821.8.

1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-5-methyl-3-((1-(6-nitrobenzo[d]

[1,3]dioxol-5-yl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione (130). Method A: DBU (1.5 eq., 1.16 ml, 7.75 mmol) was added to a solution of the morpholino subunit 133 (1 eq., 2.5 g,

5.2 mmol) in DMF (25 ml) and the mixture was stirred for 30 minutes at room temperature.

A solution of the chloromethyl ether 120 (1.5 eq., 7.75 mmol) in DMF (15 ml) was added drop-wise. The resulting mixture was stirred overnight, quenched with water (30 ml), and extracted with EtOAc (3 × 15 ml). The combined organic layer was washed with brine (20 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica

105

gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 130 as a yellow foam in 67% yield (2.45 g, 3.47 mmol). Method B: TBAF in THF (1.5 eq., 1 M, 0.04 ml, 0.042 mmol) was added to a solution of the silyl ether 129 (1 eq., 23 mg, 0.028 mmol) in THF (0.5 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 130 as a yellow foam in 94% yield (19 mg,

1 0.026 mmol). H NMR (300 MHz, CDCl3): δ = 1.50-1.53 (m, 2H), 1.60 (s, 3H), 1.79-1.80 (d,

J= 3.6 Hz, 3H), 3.03-3.12 (m, 1H), 3.28-3.35(m, 1H), 3.51-3.62 (m, 2H), 4.21-4.34 (m, 1H),

5.30-5.42 (m, 3H), 5.96-6.09 (m, 3H), 6.80-6.86 (m, 1H), 7.13-7.19 (m, 4H), 7.24-7.32 (m,

+ 8H), 7.42-7.44 (m, 5H). LRMS-LC: m/z calcd for C39H38N4O9 [M+H] : 707.3; found: 707.1.

The analytical data matched reported results.197

((2S,6R)-6-(5-Methyl-3-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)methyl)-2,4-dioxo-

3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl dimethylphosphoramidochloridate (131). LiHMDS in hexane (2.2 eq., 1 M, 7.5 ml, 7.5 mmol) was added to a solution of the caged morpholino subunit 130 (1 eq., 2.4 g, 3.4 mmol) in THF (25 ml) at –78

˚C and the mixture was stirred for 20 minutes at room temperature. A solution of

Me2NPOCl2 (1.1 eq., 0.46 ml, 3.74 mmol) in DMF (10 ml) was added drop-wise at –78 ˚C.

The resulting mixture was stirred for 2 hours at –78 ˚C, quenched with sat. NH4Cl (30 ml) and extracted with EtOAc (3 × 15 ml). The combined organic layer was washed with water

(20 ml) and brine (20 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with EtOAc as the eluent, affording 131 as a

106

1 yellow foam in 80% yield (2.25 g, 2.71 mmol). H NMR (400 MHz, CDCl3): δ = 1.32-1.43

(m, 2H), 1.51-1.52 (m, 3H), 1.77-1.52 (m, 3H), 2.60-2.65 (m, 6H), 3.09-3.20 (m, 1H), 3.27-

3.36 (m, 1H), 3.55-3.63 (m, 1H), 4.09-4..13 (m, 1H), 4.25-4.45 (m, 1H), 5.26- 5.41 (m, 3H),

5.96-6.16 (m, 3H), 6.87-7.00 (m, 1H), 7.16-7.22 (m, 4H), 7.28-7.40 (m, 8H), 7.46-7.50 (m,

13 5H). C NMR (400 MHz, CDCl3): δ = 13.4, 14.6, 21.5, 24.1, 30.8, 37.1, 49.3, 52.0, 52.2,

60.8, 64.1, 67.7, 70.0, 70.2, 73.5, 73.9, 75.0, 81.4, 103.2, 103.3, 105.2, 105.5, 106.8, 106.9,

110.3, 127.0, 128.4, 129.6, 134.5, 138.1, 138.2, 142.6, 147.3, 150.7, 150.8, 152.6, 163.3. 31P

+ NMR (400 MHz, CDCl3): δ = 19.98. HRMS-LC: m/z calcd for C41H43ClN5O10P [M+Na] :

854.2334; found: 854.5000.

1-((2R,6S)-6-(Hydroxymethyl)morpholin-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione p- toluenesulfonic acid salt (132). The nucleotide 110 (1 eq., 5 g, 20 mmol) in MeOH (50 ml) was added to a solution of NaIO4 (1.05 eq., 4.5 g, 21 mmol) in MeOH (10 ml) at 60 °C. The reaction mixture was stirred at room temperature for 2 hrs and filtered. The solid was washed with a 3:1 mixture of MeOH/water (20 ml). Ammonium biborate (1.6 eq., 8.4 g, 32 mmol) was added to the combined filtrate and the mixture was stirred for 1 hr at room temperature before cooled to 0 °C. The borane triethylamine complex (2.3 eq., 7.1 ml, 46 mmol) was added and the pH was adjusted to 3-4 with p-TsOH (1 g/ml in MeOH). The reaction was stirred at 4 °C for 12 hrs and then taken up in a 6:1 mixture of MeOH/water. After filtration, the precipitation was dried, affording the morpholino 132 as a white solid in 47% yield (4.0

1 g, 9.4 mmol). H NMR (300 MHz, CDCl3): δ = 1.97 (s, 3H), 2.71-2.72 (m, 2H), 2.99-3.19

(m, 2H), 3.58-3.75 (m, 2H), 3.71-3.79 (m, 1H), 5.22-5.29 (m, 1H), 6.10-6.21 (m, 1H), 7.24

107

(s, 1H), 7.32-7.35 (d, J = 8.1 Hz, 2H), 7.68-7.71 (d, J = 8.1 Hz, 2H). LRMS-LC: m/z calcd

+ for C10H16N3O4 [M] : 242.1; found: 242.3.

1-((2R,6S)-6-(Hydroxymethyl)-4-tritylmorpholin-2-yl)-5-methylpyrimidine-2,4(1H,3H)- dione (133). TrCl (1.2 eq., 1 g, 3.6 mmol) followed by TEA (3 eq., 1.3 ml, 9 mmol) was added to a solution of the amine 132 (1 eq., 1.28 g, 3 mmol) in DCM (15 ml). The reaction mixture was stirred overnight at room temperature and poured into water (7 ml). The mixture was kept at 4 °C for 20 hours and the precipitate was isolated through filtration affording the trityl protected morhpholino 133 as a white solid in 94% yield (1.36 g, 2.8 mmol). 1H NMR

(300 MHz, CDCl3): δ = 1.86 (s, 3H), 3.21-3.32 (m, 2H), 3.52-3.60 (m, 1H), 3.67-3.74 (m,

1H), 4.19-4.28 (m, 1H), 6.13-6.17 (m, 1H), 7.01 (s, 1H), 7.14-7.18 (m, 3H), 7.23-7.30 (m,

7H), 7.43-7.46 (m, 5H). The analytical data matched reported results.211

N-(9-((2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9- dihydro-1H-purin-2-yl)-2-phenylacetamide (135). Guanosine 134 (1 eq., 566 mg, 2 mmol) was co-evaporated with pyridine (5 ml × 3) and dissolved in pyridine (10 ml). TMSCl (7.5 eq., 1.9 ml, 15 mmol) was added and the mixture was stirred for 8 hrs before PhCH2COCl

(1.5 eq., 0.4 ml, 3 mmol) was added followed by HOBt (1.6 eq., 432 mg, 3.2 mmol). The reaction mixture was stirred at room temperature overnight and cooled to 0 ˚C. The reaction was quenched with 25% NH4OH (6 ml) in water and the mixture was stirred at 0 ˚C for 30 minutes. The solvent was removed under reduced pressure, and the residue was dissolved in water (10 ml). The aqueous layer was washed with EtOAc (3 × 10 ml) and cooled to 4 ˚C

108

overnight. The suspension was filtered and the solid was collected and dried, affording the protected guanosine 135 as a white solid in 53% yield (425 mg, 1.1 mmol). 1H NMR (300

MHz, DMSO): δ = 3.51-3.64 (m, 2H), 3.81 (s, 2H), 3.87-3.94 (m, 1H), 4.10-4.16 (m, 1H),

4.39-4.43 (m, 1H), 5.01-5.06 (m, 1H), 5.18-5.20 (m, 1H), 5.47-5.50 (m, 1H), 5,80-5.82 (d, J=

6.0 Hz, 1H), 7.34-7.35 (m, 5H), 8.27 (s, 1H). The analytical data matched reported results.211

N-(9-((2R,6S)-6-(Hydroxymethyl)morpholin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-2- phenylacetamide p-toluenesulfonic acid salt (136). The nucleoside 135 (1 eq., 200 mg, 0.5 mmol) in MeOH (2 ml) was added to a solution of NaIO4 (1.05 eq., 112 mg, 0.52 mmol) in

MeOH (1 ml) at 60 °C. The reaction was stirred at room temperature for 2 hrs and filtered.

The solid was collected and washed with a 3:1 mixture of MeOH/water (2 ml). Ammonium biborate (4 eq., 527 mg, 2 mmol) was added to the combinded filtrate and the mixture was stirred for 1 hour at room temperature before cooled to 0 °C. Borane triethylamine complex

(2 eq., 0.15 ml, 1 mmol) was added and the pH was adjusted to 3-4 with p-TsOH (1 g/ml in

MeOH). The reaction was stirred at 4 °C for 12 hrs and taken up in a 6:1 mixture of

MeOH/water. The precipitation was filtered and dried, affording the morpholino 136 as a

1 white solid in 20% yield (55 mg, 9.4 mmol). H NMR (300 MHz, CDCl3): δ = 1.39-1.50 (m,

1H), 1.80-1.91 (m, 1H), 3.17-3.25 (m, 3H), 3.68 (s, 2H), 4.17-4.23 (m, 2H), 4.78-4.84 (br,

1H), 5.99-6.04 (m, 1H), 6.63 (s, 2H), 7.23-7.35 (m, 7H), 7.63 (s, 1H), 10.60 (m, 3H). The analytical data matched reported results.202

109

2-Amino-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-1,9-dihydro-6H-purin-

6-one (138). NH3 in MeOH (20 eq., 7 M, 7 ml, 47.9 mmol) was added to a solution of the morpholino subunit 137 (1 eq., 1.5 g, 2.39 mmol) in MeOH (17 ml). The reaction mixture was stirred at room temperature overnight and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) and 1%

TEA as the eluent, affording 138 as a colorless foam in 85% yield (1.03 g, 2.02 mmol). 1H

NMR (300 MHz, CDCl3): δ = 1.39-1.50 (m, 1H), 1.80-1.91 (m, 1H), 3.17-3.25 (m, 3H),

4.17-4.23 (m, 2H), 4.78-4.84 (br, 1H), 5.99-6.04 (m, 1H), 6.63 (s, 2H), 7.23-7.35 (m, 4H),

7.40-7.45 (m, 6H), 7.46-7.57 (m, 5H), 7.63 (s, 1H), 10.60 (br, 1H). LRMS-LC: m/z calcd for

+ 212 C29H28N5O3 [M+Na] : 531.2; found: 531.2. The analytical data matched reported results.

(E)-N'-(9-((2R,6S)-6-(Hydroxymethyl)-4-tritylmorpholin-2-yl)-6-oxo-6,9-dihydro-1H- purin-2-yl)-N,N-dimethylformimidamide (139). N,N-Dimethylformamide dimethyl acetal

(4 eq., 1.05 ml, 7.87 mmol) was added to a solution of the morpholino subunit 138 (1 eq., 1 g, 1.97 mmol) in THF/MeOH (1:1, 13 ml). The mixture was heated to 60 ˚C overnight and concentrated. The crude product was purified by silica gel chromatography with

DCM/MeOH (9:1) and 1% TEA as the eluent, affording 139 as a colorless foam in 92% yield

(1.03 mg, 1.82 mmol). 1H NMR (300 MHz, DMSO): δ = 1.37-1.42 (m, 1H), 1.77-1.82 (m,

1H), 3.08 (s, 3H), 3.15-3.18 (m, 2H), 3.25 (s, 3H), 3.32-3.35 (m, 2H), 4.17-4.23 (m, 1H),

4.72-4.79 (m, 1H), 6.08-6.14 (m, 1H), 7.09-7.58 (m, 15H), 7.72 (s, 1H), 8.55 (s, 1H), 11.30

+ (s, 1H). LRMS-LC: m/z calcd for C32H33N7O3 [M+H] : 564.27; found: 564.21.

110

(E)-N'-(9-((2R,6S)-6-(Hydroxymethyl)-4-tritylmorpholin-2-yl)-1-((1-(6-nitrobenzo[d]

[1,3]-dioxol-5-yl)ethoxy)methyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide (140). DBU (1.5 eq., 0.41 ml, 2.73 mmol) was added to a solution of the morpholino subunit 139 (1 eq., 1.02 g, 1.82 mmol) in DMF (16 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of the chloromethyl ether 120 (1.2 eq.,

2.184 mmol) in DMF (8 ml) was added drop-wise, and the resulting mixture was stirred overnight, quenched with water (20 ml), and extracted with EtOAc (3 × 15 ml). The combined organic layer was washed with brine (20 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with

DCM/MeOH (19:1) and 1% TEA as the eluent, affording 140 as a yellow foam in 94% yield

1 (1.34 g, 1.71 mmol). H NMR (300 MHz, CDCl3): δ = 1.43-1.45 (d, J= 6.0 Hz, 3H), 1.45-

1.67 (m, 2H), 2.84-2.91 (m, 6H), 3.01-3.11 (m, 2H), 3.42-3.61 (m, 2H), 4.20-4.33 (m, 1H),

5.33-5.62 (m, 2H), 5.73-5.63 (m, 4H), 7.13-7.52 (m, 17H), 7.97 (s, 1H), 8.35 (s, 1H). 13C

NMR (400 MHz, CDCl3): δ = 24.0, 24.1, 30.3, 35.4, 35.5, 36.7, 41.5, 41.8, 46.2, 48.9, 52.9,

63.7, 79.4, 79.6, 79.8, 102.5, 102.9, 107.2, 107.3, 126.8, 128.1, 129.3, 135.4, 135.6, 157.3,

+ 157.7, 157.9. LRMS-LC: m/z calcd for C42H42N8O8 [M+H] : 787.3204; found: 787.3.

((2S,6R)-6-(2-(((E)-(Dimethylamino)methylene)amino)-1-((1-(6-nitrobenzo[d][1,3] dioxol-5-yl)ethoxy)methyl)-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2- yl)methyl dimethylphosphoramidochloridate (141). LiHMDS in hexane (3 eq., 1 M, 0.19 ml, 0.19 mmol) was added to a solution of the caged morpholino subunit 140 (1 eq., 50 mg,

0.064 mmol) in THF (1 ml) at –78 ˚C and the mixture was stirred for 20 minutes at room

111

temperature. A solution of Me2NPOCl2 (1.5 eq., 11 µl, 0.1 mmol) in DMF (0.4 ml) was added drop-wise, and the resulting mixture was stirred for 2 hours at –78 ˚C. The reaction was quenched with sat. NH4Cl (1.5 ml) and the aqueous layer was extracted with EtOAc (3 ×

1 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with EtOAc as the eluent, affording 141 as a yellow foam in 67% yield (39

1 mg, 0.04 mmol). H NMR (300 MHz, CDCl3): δ = 1.44-1.46 (d, J= 6.3 Hz, 3H), 1.46-1.80

(m, 2H), 2.57-2.63 (m, 6H), 3.07-3.11 (m, 3H), 3.15-3.25 (m, 2H), 3.28-3.32 (m, 3H), 4.03-

4.15 (m, 2H), 4.40-4.51 (m, 1H), 5.31-5.40 (m, 2H), 5.67-5.78 (m, 1H), 5.85-6.02 (m, 3H),

+ 7.13-7.52 (m, 18H), 8.43 (s, 1H). HRMS-LC: m/z calcd for C44H47ClN9O9P [M+H] :

912.3001; found: 912.3067.

4-Fluoro-2-nitrobenzaldehyde (143). N,N-Dimethylformamide dimethyl acetal (3 eq, 10.3 ml, 77.4 mmol) was added to a stirring solution of 4-fluoro-2-nitrotoluene (142) (1 eq, 4g,

25.8 mmol) in DMF (40 ml), and the mixture was refluxed for 12 hours. The solution was cooled in an ice-bath and slowly poured into an ice-cold water/DMF (4:1, 200 ml) solution containing NaIO4 (3 eq, 16.8 g). The reaction was stirred for 16 hours, and filtered. The filtrate was extracted with toluene (3 × 100 ml), and the combined organic layer was washed with water (50 ml) and brine (50 ml), dried over NaSO4. After filtration, the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography with hexanes/EtOAc (2:1) as the eluent, affording 143 as a yellow solid in

1 87% yield (3.8g, 22 mmol). H NMR (300 MHz, CDCl3): δ = 7.44-7.45 (m, 1H), 7.77-7.81

112

(dd, Ja = 8.3 Hz, Jb = 2.4 Hz, 1H), 7.99-8.04 (dd, Ja = 8.3 Hz, Jb = 5.7 Hz, 1H), 10.35 (s, 1H).

The analytical data matched reported results.146

4-(2-Iodophenoxy)-2-nitrobenzaldehyde (144). The aldehyde 143 (1 eq, 3.8 g, 22 mmol) and 2-iodophenol (1 eq, 2.5 ml, 22 mmol) were dissolved in pyridine (76 ml). CuBr (1 eq,

3.1g, 22 mmol) and K2CO3 (2 eq, 6.1 g, 44 mmol) were added. The mixture was heated to

60 C for 16 hours, cooled to room temperature and filtered. The filtrate was diluted with diethyl ether (100 ml), washed with 1 M NaOH (50 ml), water (50 ml) and brine (50 ml), dried over NaSO4 and filtered. The crude product was purified by silica gel chromatography with hexanes/EtOAc (9:1) as the eluent, affording 144 as a yellow solid in 67% yield (5.4 g,

1 15 mmol). H NMR (300 MHz, CDCl3): δ = 7.05-7.12 (m, 2H), 7.17-7.20 (m, 1H), 7.41-

7.48 (m, 2H), 7.91-7.98 (m, 2H), 10.30 (s, 1H). The analytical data matched reported results.146

1-[4-(2-Iodophenoxy)-2-nitrophenyl]ethanol (145). AlMe3 in hexane (1.2 eq., 2 M, 13.6 ml, 27.3 mmol) was added to a solution of the aldehyde 144 (1 eq., 8.4 g, 22.7 mmol) in

DCM (92 ml) at 0 ˚C. The mixture was stirred for 1.5 hours at 0 ˚C. Ice was added until the generation of methane seized, followed by the addition of 1 M NaOH (200 ml). The mixture was stirred till clear and extracted with DCM (3 × 100 ml). The combined organic layer was washed with brine (100 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 145 as a yellow solid in 98% yield (8.6 mg, 22.4 mmol). 1H NMR (300 MHz,

113

CDCl3): δ = 1.54-1.56 (d, J = 6.3 Hz, 3H), 5.32-5.38 (q, J = 6.3 Hz, 1H), 6.97-7.01 (m, 2H),

7.18-7.24 (m, 1H), 7.34-7.40 (m, 2H), 7.75-7.78 (d, J = 8.4 Hz, 1H), 7.87-7.90 (dd, Ja = 7.8

146 Hz, Jb = 1.2 Hz, 1H). The analytical data matched reported results.

((1-(4-(2-Iodophenoxy)-2-nitrophenyl)ethoxy)methyl)(methyl)sulfane (146).

Dimethylsulfide (8 eq., 213 µl, 2.9 mmol) was added to a solution of the alcohol 145 (1 eq.,

139 mg, 0.36 mmol) in CH3CN (2 ml) at 0 ˚C, followed by benzoyl peroxide (4 eq., 4 ×

113.2 mg, 1.45 mmol) in four portions over 30 mins. The mixture was stirred for 2 hours at 0

˚C and the reaction was quenched with 1 M NaOH (5 ml). The mixture was stirred overnight at room temperature and extracted with Et2O (3 × 2 ml). The combined organic layer was washed with brine (3 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording

1 146 as a yellow solid in 75% yield (121 mg, 0.27 mmol). H NMR (300 MHz, CDCl3): δ =

1.51-1.53 (d, J = 6.3 Hz, 3H), 2.11 (s, 3H), 4.28-4.32 (d, J = 11.4 Hz, 1H), 4.58-4.61 (d, J =

11.4 Hz, 1H), 5.30-5.40 (q, J = 6.3 Hz, 1H), 6.96-7.02 (m, 2H), 7.17-7.22 (dd, Ja = 4.3 Hz, Jb

= 2.5 Hz, 1H), 7.35-7.40 (m, 2H), 7.84-7.87 (d, J = 8.7 Hz, 1H), 8.16-8.19 (dd, Ja = 9.0 Hz,

+ Jb = 1.5 Hz, 1H). LRMS-LC: m/z calcd for C16H16INO4S [M+H] : 446.0; found: 446.0.

(1-(4-(2-iodophenoxy)-2-nitrophenyl)ethoxy)trimethylsilane (147). The alcohol 145 (1 eq,

1.4 g, 3.7 mmol) was dissolved in DCM (7.5 ml) and TMSCl (2 eq, 0.9 ml, 7.4 mmol) was added, followed by TEA (3 eq, 1.55 ml, 11 mmol) and DMAP (0.5 eq, 226 mg, 1.85 mmol).

The mixture was stirred at room temperature overnight and diluted with DCM (10 ml). The

114

solution was washed with sat. NH4Cl (10 ml), water (10 ml) and brine (10 ml) and dried over

NaSO4, filtered and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, yielding 147 as a yellow solid in

1 90% yield (1.5 g, 3.3 mmol). H NMR (300 MHz, CDCl3): δ = 0.07 (s, 9H), 1.45-1.47 (d, J =

6.3 Hz, 3H), 5.36-5.42 (q, J = 6.3 Hz, 1H), 6.95-7.00 (m, 2H), 7.17-7.20 (dd, Ja = 4.5 Hz, Jb

= 2.7 Hz, 1H), 7.33-7.38 (m, 2H), 7.78-7.81 (d, J = 9.0 Hz, 1H), 7.87-7.90 (dd, Ja = 7.8 Hz,

+ Jb = 1.5 Hz, 1H). LRMS-LC: m/z calcd for C17H20INO4Si [M+H] : 458.0; found: 458.3.

tert-Butyl(1-(4-(2-iodophenoxy)-2-nitrophenyl)ethoxy)dimethylsilane (148). The alcohol

145 (1 eq, 2.1 g, 5.6 mmol) was dissolved in DMF (16 ml) and TBDMSCl (2 eq, 1.7 g, 11.2 mmol) was added, followed by imidazole (3 eq, 1.1 g, 16.8 mmol). The mixture was stirred at room temperature for 12 hours, quenched with sat. NaHCO3 (20 ml) and extracted with

EtOAc (3 × 10 ml). The combined organic layer was washed with water (20 ml) and brine

(20 ml), dried over NaSO4, filtered and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, yielding 148 as a yellow

1 solid in 98% yield (2.7 g, 5.46 mmol). H NMR (300 MHz, CDCl3): δ = -0.07-0.03 (m, 6H),

0.86 (s, 9H), 1.42-1.45 (d, J = 6.3 Hz, 3H), 5.36-5.42 (q, J = 6.3 Hz, 1H), 6.97-7.02 (m, 2H),

7.16-7.23 (dd, Ja = 4.5 Hz, Jb = 2.7 Hz, 1H), 7.34-7.42 (m, 2H), 7.70-7.83 (d, J = 8.7 Hz,

1H), 7.87-7.90 (dd, Ja = 7.8 Hz, Jb = 1.5 Hz, 1H). LRMS-LC: m/z calcd for C20H26INO4Si

[M+H]+: 500.1; found: 500.2.

115

1-(3-Nitrodibenzofuran-2-yl)-ethanol (149). The silyl ether 147 (1 eq, 1.52 g, 3.32 mmol) was dissolved in DMA (30 ml) and Pd(OAc)2 (0.1 eq, 74.5 mg, 0.33 mmol) was added, followed by Cs2CO3 (2 eq, 2.2 g, 6.65 mmol). The mixture was heated to 80 ˚C for 16 hours.

The reaction mixture was diluted with EtOAc (30 ml), washed with water (20 ml) and brine

(20 ml), and dried over NaSO4. After filtration, the solvent was evaporated and the crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, yielding 149 as a yellow solid in 81% yield (677 mg, 2.64 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.63-1.65 (d, J = 6.3 Hz, 3H), 5.52-5.58 (q, J = 6.3 Hz, 1H), 7.40-7.42 (t, J = 7.5

Hz, 1H), 7.57-7.59 (m, 2H), 7.98-8.00 (d, J = 8.1 Hz, 1H), 8.09 (s, 1H), 8.36 (s, 1H). The analytical data matched reported results.146

Methyl ((1-(3-nitrodibenzofuran-2-yl)ethoxy)methyl)sulfane (150). Dimethylsulfide (8 eq., 1 ml, 14.1 mmol) was added to a solution of the alcohol 149 (1 eq., 454 mg, 1.77 mmol) in CH3CN (10 ml) at 0 ˚C, followed by benzoyl peroxide (4 eq., 4 × 552.5 mg, 7.1 mmol) in four portions over 30 mins. The mixture was stirred for 2 hours at 0 ˚C and quenched with 1

M NaOH (20 ml). The mixture was stirred overnight at room temperature and extracted with

Et2O (3 × 20 ml). The combined organic layer was washed with brine (30 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 150 as a yellow solid in

1 74% yield (413 mg, 1.3 mmol). H NMR (300 MHz, CDCl3): δ = 1.62-1.64 (d, J = 6.3 Hz,

3H), 2.14(s, 3H), 4.34-4.38 (d, J = 11.4 Hz, 1H), 4.65-4.68 (d, J = 11.4 Hz, 1H) 5.51-5.57 (q,

116

J = 6.3 Hz, 1H), 7.39-7.41 (t, J= 7.5 Hz, 1H), 7.55-7.57 (m, 2H), 7.00-8.02 (d, J= 8.1 Hz,

1H), 8.17 (s, 1H), 8.30 (s, 1H). The analytical data matched reported results.146

2-(1-(Chloromethoxy)ethyl)-3-nitrodibenzo[b,d]furan (151). Sulfuryl chloride (1.2 eq.,

126.7 µl, 1.56 mmol) was added to a solution of the thioether 150 (1 eq., 354 mg, 1.3 mmol) in DCM (1 ml) at 0 ˚C. The mixture was stirred for 30 minutes at 0 ˚C and dried, delivering the crude product as red oil which was used without purification.146

1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-5-methyl-3-((1-(3-nitrodibenzo

[b,d]-furan-2-yl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione (152). DBU (1.5 eq., 194 µl,

1.3 mmol) was added to a solution of the morpholino subunit 133 (1 eq., 419 mg, 0.87 mmol) in DMF (4 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of the chloromethyl ether 151 (1.5 eq., 1.3 mmol) in DMF (4 ml) was added drop-wise. The resulting mixture was stirred overnight, quenched with water (10 ml), and extracted with

EtOAc (3 × 5 ml). The combined organic layer was washed with brine (10 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 152 as a yellow foam in

1 86% yield (562 mg, 0.75 mmol). H NMR (400 MHz, CDCl3): δ = 1.23-1.34 (m, 2H), 1.46

(s, 3H), 1.64-1.66 (d, J = 6.3 Hz, 3H), 3.02-3.18 (m, 2H), 4.52-4.64 (m, 2H), 4.28-4.37 (m,

1H), 3.35-3.43 (m, 3H), 5.93-5.99 (m, 1H), 6.58 (s, 1H), 7.10-7.20 (m, 3H), 7.29-7.30 (m,

6H), 7.32-7.51 (m, 6H), 7.55-7.60 (m, 3H), 7.96-7.99 (d, J= 8.1 Hz, 1H), 8.21 (s, 1H), 8.29

13 (s, 1H). C NMR (400 MHz, CDCl3): δ = 12.8, 13.1, 14.4, 21.3, 24.7, 49.0, 51.7, 60.7, 63.8,

117

64.0, 70.2, 70.6, 73.7, 74.5, 81.0, 108.1, 108.4, 110.0, 112.2, 112.4, 119.9, 122.1, 122.7,

122.9, 124.0, 126.7, 128.2, 129.0, 129.1, 129.6, 134.0, 134.2, 135.9, 136.3, 146.9, 150.5,

+ 153.8, 154.0, 158.3, 158.5, 163.0, 163.2. LRMS-LC: m/z calcd for C44H40N4O8 [M+Na] :

775.2744; found: 755.37.

1-((2R,6S)-6-(Hydroxymethyl)-4-tritylmorpholin-2-yl)-3-((1-(5-methoxy-2-nitro-4-

(prop-2-yn-1-yloxy)phenyl)ethoxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione (154).

DBU (2 eq., 0.3 ml, 2.06 mmol) was added to a solution of the morpholino subunit 133 (1 eq., 500 mg, 1.03 mmol) in DMF (7 ml) and the mixture was stirred for 30 minutes at room temperature. A solution of the chloromethyl ether 153 (1.1 eq., 1.14 mmol) in DMF (2 ml) was added drop-wise to above solution. The resulting mixture was stirred overnight, quenched with water (10 ml), and extracted with EtOAc (3 × 10 ml). The combined organic layer was washed with brine (20 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 154 as a yellow foam in 86% yield (661 mg, 0.89 mmol). 1H NMR (300

MHz, CDCl3): δ = 1.56-1.58 (d, J = 6.3 Hz, 3H) 1.88 (s, 3H), 2.60-2.63 (m, 1H), 3.18-3.32

(m, 2H), 3.42-3.57 (m, 1H), 3.65-3.71 (m, 1H), 4.01 (s, 3H), 4.19-4.35 (m, 2H), 4.57-4.61

(m, 1H), 4.85-4.86 (d, J = 2.1 Hz, 2H), 5.55-5.62 (m, 3H), 6.11-6.15 (m, 1H), 7.02 (s, 1H),

7.12-7.18 (m, 3H), 7.25-7.33 (m, 8H), 7.44-7.50 (m, 5H), 7.71 (s, 1H). HRMS-LC: m/z calcd

+ for C42H42N4O9 [M+Na] : 769.2849; found: 769.2844.

118

((2S,6R)-6-(3-((1-(5-methoxy-2-nitro-4-(prop-2-yn-1-yloxy)phenyl)ethoxy)methyl)-5- methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl dimethylphosphoramidochloridate (155). LiHMDS in hexane (2.2 eq., 1.6 M, 0.24 ml,

0.38 mmol) was added to a solution of the alcohol 154 (1 eq., 130 mg, 0.17 mmol) in THF

(1.7 ml) at –78 ˚C. The mixture was stirred for 10 minutes and Me2NPOCl2 (1.1 eq., 23 µl,

0.19 mmol) in THF (0.6 ml) was slowly added. The reaction mixture was stirred at –78 ˚C for one hour, quenched with sat. NH4Cl (3 ml), and extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with EtOAc as the eluent, affording 155 as a yellow foam in 73% yield (108 mg, 0.124 mmol). 1H NMR

(300 MHz, CDCl3): δ = 1.56-1.58 (d, J = 6.3 Hz, 3H) 1.88 (s, 3H), 2.60-2.63 (m, 7H), 3.18-

3.32 (m, 2H), 3.42-3.57 (m, 1H), 3.65-3.71 (m, 1H), 4.01 (s, 3H), 4.19-4.35 (m, 2H), 4.57-

4.61 (m, 1H), 4.85-4.86 (d, J = 2.1 Hz, 2H), 5.55-5.62 (m, 3H), 6.11-6.15 (m, 1H), 7.02 (s,

1H), 7.12-7.18 (m, 3H), 7.25-7.33 (m, 8H), 7.44-7.50 (m, 5H), 7.90 (s, 1H). LRMS-LC: m/z

+ calcd for C44H47ClN5O10P [M+Na] : 872.3; found: 872.4.

119

CHAPTER 4: Synthesis of Photocleavable Oligonucleotide Linkers

Photocleavable linkers, which can tether two oligonucleotides or cyclize the two ends of a linear oligonucleotide, have been used to regulate biological activities (see CHAPTER

1.3.2). In this chapter, the synthesis of light-sensitive linkers for both DNA and morpholino oligonucleotides based on different chromophores are presented.

4.1 Synthesis of o-nitrobenzyl linker for DNA oligonucleotides

The synthesis of the o-nitrobenzyl (ONB) linker 160 was reported in 1995 by Taylor et al. with a 40% yield over four steps.108 Following the same route, improvements were made through the application of new conditions (Scheme 4.1). The initial step was an allylic stannane addition onto the commercially available 2-nitrobenzaldehyde 156 using allyl

213 tributyl tin in the presence of ZnCl2 in a 4:1 mixture of CH3CN/water (97% yield). When the alkene 157 was treated with OsO4 and 4-methyl morpholine oxide (NMO), a triol

214 intermediate was formed and further oxidized into an aldehyde by NaIO4. A subsequent

215 reduction with NaBH4 afforded the diol 158 in 74% yield over three steps. After the protection of the primary hydroxyl group in 158 with a DMTr group (DMTrCl, pyridine,

98% yield),216 the secondary alcohol 159 was reacted with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of DIPEA in DCM to furnish the linker 160 with a

61% overall yield.217 This linker 160 can be incorporated into DNA oligonucleotides through standard automated oligonucleotide synthesis methodology.108 Taking advantage of this ONB linker (160) and a 3-nitro-2-ethyldibenzofuran (NDBF) linker made by Dr. McIver, the wavelength-selective activations of oligonucleotides are being pursued in the Deiters group.

120

Scheme 4.1 Improved synthesis of the ONB linker 160.

4.2 Synthesis of coumarin-based linkers for MOs

Cyclic, caged MOs (cyclic cMOs) are a recently developed class of MO reagents that can be regulated with light. A photocleavable linker with either a dimethoxynitrobenzyl

(DMNB) 116 or an 5-aminomehthyl-2-nitrobenzyl (AMNB) group 118 has been used to connect the two ends of a linear MO forming a cyclic MO. Taking advantage of the commercially available 5ʹ-amine and 3ʹ-disulfide MOs, the DMNB-containing cyclic cMO was developed by Chen et al. The synthesis commenced with the coupling of the 5ʹ-amine group on the MO to the succinimidyl ester at one end of the DMNB linker (161), which was followed by the reduction of the disulfide bond (Scheme 4.2). The released free thiol group at the 3ʹ-terminus of the MO reacted spontaneously with the chloroacetamide functionality at the other end of the DMNB linker, thereby delivering the light-activatable cyclic MO. This approach was tested through no tail a (ntla) and pancreas transcription factor 1 alpha (ptf1α) gene-silencing in zebrafish.116 In collaboration with the Chen lab, MO linkers based on

121

coumarin caging groups were synthesized to achieve multi-wavelength regulation of gene function during zebrafish embryo development.

Scheme 4.2 Structure of the DMNB linker 161 (A) and its cyclization of MO (B). The succinimidyl ester of 161 was reacted with the 5ʹ-NH2 of the MO (blue) and the 3ʹ-disulfide is reduced to a thio intermediate which cyclized to give the cyclic MO.

4.2.1 Synthesis of 7-diethylaminocoumarin linkers

Two 7-diethylaminocoumarin (DEACM) linkers were designed to bear an amine- reactive succinimidyl carbonate on one end and a thio-reactive group on the other end. Two thio-specific groups, a maleimide and a chloroacetamide, were applied resulting in two

DEACM linkers (170 and 176).218, 219

The synthesis of the maleimide linker 170 commenced with the aldehyde 166 which underwent a Henry reaction delivering the alcohol 167 in 69% yield (Scheme 4.3).220

122

Reduction of the nitro group in 167 was accomplished through the treatment with zinc in acetic acid (55% yield).221 The obtained β-amino alcohol 168 was coupled to the succinimide ester 165, which was formed in two steps from β-alanine (162) and maleic anhydride (163), to afford the amide 169.222 After a DSC activation in the presence of DMAP in DCM,223 the linker 170 was obtained in 33% yield. This linker was tested by Dr. Govan in our lab and Dr.

Yamazoe in the Chen lab, but unfortunately it failed to cyclize the MOs.

Scheme 4.3 Synthesis of the maleimide DEACM linker 170.

A chloroacetamide linker 176 mimicking the DMNB linker 161 was synthesized from the aldehyde 166 to produce the alcohol 171 (allyl tributyl tin, ZnCl2, CH3CN/water, 74% yield),213 which was protected by a TBDMS group, providing the alkene 172 in 92% yield

(Scheme 4.4).224 The alkene 172 was subjected to a hydroboration with borane dimethyl

123

sulfide complex to give the alcohol 173.225 The installation of the chloroacetamide group was achieved in three steps with a 49% yield following a previous report.116 This was followed by the removal of the silyl group with TBAF in 44% yield, delivering the secondary alcohol

175.226 Finally, the hydroxyl group of 175 was activated by DSC and the linker 176 was delivered in 87% yield (DSC, DMAP, DCM).227

Scheme 4.4 Synthesis of the chloroacetamide DEACM linker 176.

Meanwhile, a non-cleavable linker 181 was synthesized as a control to the DEACM linker 176 (Scheme 4.5). The synthesis began with the protection of the commercially available 1,4-butandiol 177 to form the mono-protected product 178 in 97% yield

(TBDMSCl, imidazole, THF).228 The alcohol 178 was then activated by CDI in DCM, and

124

treated with ethylenediamine followed by chloroacetyl chloride resulting in the chloroacetamide 179 (69% yield over three steps).116 The deprotection of the silyl group gave the free alcohol 180 (TBAF, THF, 76% yield), which was activated by DSC to form the succinimidyl carbonate 181 (DSC and DMAP in DCM, 86% yield). The non-cleavable linker

181 together with the chloroacetamide DEACM linker 176 was sent to the Chen lab where their abilities to control MO activity were studied.

Scheme 4.5 Synthesis of non-cleavable linker 181.

Taking advantage of the DEACM linker 176 and an ONB linker synthesized by Dr.

Yamazoe in Chen’s lab, the following approach to control MO activities in a wavelength- selective manner was developed (Scheme 4.6). Two MOs targeting different genes are cyclized with different caging groups. Upon 405 nm light irradiation, only the DEACM cyclized MO can be cleaved, releasing the active MO (blue) to bind to its target mRNA (light green). After a subsequent 365 nm light irradiation, both MOs (blue and red) are active which result in the silencing of both genes.

125

active MO active MO

mRNA mRNA

cMOs 405 nm 365 nm active MO

mRNA

cMO

= =

DEACM ONB

Scheme 4.6 Multi-wavelength regulations over MO activities. Two different MOs (blue and red) are cyclized by different linkers (pink and yellow). Upon 405 nm irradiation, the DEACM linker (pink) is cleaved selectively and the translation of the mRNA in light green is blocked. Upon a subsequent 365 nm irradiation, the ONB linker (yellow) is also cleaved, and the translation of both mRNAs are blocked.

Specifically, the selected zebrafish genes were flatting head (flh) and spadetail (spt).

The silence of flh with MO results in the ectopic expression of myoD (Figure 4.1B), which is a class of proteins that regulate myogenesis and participate in somite development.229 On the other hand, the knockdown of both flh and spt genes blocks the expression of myoD completely (Figure 4.1C). The 405 nm cleavable DEACM linker 176 was applied to an flh

MO to produce the cyclic flh MO (DEACM flh cMO) while the ONB linker was introduced into a spt MO generating the cyclic spt MO (ONB spt cMO). Zebrafish embryos co-injected with these two cMOs were split into three groups: 1) shielded from light, 2) exposed to 405 nm light, and 3) exposed to both 365 nm and 405 nm light. As shown in Figure 4.1D, embryos kept in the dark showed a 100% normal phenotype, which suggested that both

126

linkers successfully inactivated the MOs. The embryos exposed to 405 nm light mostly demonstrated the abnormal phenotype (B) indicating that the DEACM linker was selectively cleaved. In the embryos exposed to both 405 nm and 365 nm irradiation, both DEACM and

ONB linkers were cleaved and lead to the silencing of both genes and the severe abnormal phenotype (C).

A B C

D 100 A B C

50 % ofembryos

0 WT flh MO spt MO spt cMO + flh cMO ˗light +405 nm +405 nm +365 nm

Figure 4.1 Wavelength-selective regulation of zebrafish embryo development. (A) Wild-type zebrafish embryo shows normal phenotype. (B) Flh-silenced zebrafish embryo shows abnormal somite development. (C) Flh- and spt-silenced zebrafish embryo shows severe abnormal somite. (D) Zebrafish embryos were injected with the DEACM cyclized flh MO (flh cMO) and the ONB cyclized spt MO (spt cMO). Embryos that were protected from light demonstrated normal phenotype indicating both DEACM and ONB linkers successfully blocked MO activities upon cyclization. Most of the embryos exposed to 405 nm light demonstrated the flh-silenced phenotype indicating flh cMO was selectively activated in the presence of spt cMO. Embryos exposed to both 365 nm and 405 nm irradiations demonstrates severe abnormal somite indicating both cMOs were active.

127

4.2.2 Development of coumarin linkers with red-shifted absorption maximums

Recently, modification of the coumarin backbone through thionation was reported to red-shift the absorption maximum due to the empty 3d orbital of the sulfur atom which is a feasible electron acceptor thus can enhance the intramolecular charge transfer.84, 87, 230 In hope of better wavelength-selectivity, efforts have been made to synthesize a thiocoumarin

MO linker.

The developed route to make the DEACM linker 176 was adapted to the synthesis of the thiocoumarin linker 186 (Scheme 4.7). Beginning with the alkene 172, the carbonyl group was converted into a thiocarbonyl group in 78% yield (Lawesson’s reagent, toluene).230 This was followed by a hydroboration with borane dimethylsulfide complex to afford the alcohol 183 in 42% yield. The activation of alcohol 183 as the imidazole carbamate 184 was confirmed by 1H NMR. A subsequent treatment with either ethylenediamine or N-Boc-ethylenediamine failed to give desired product (185), probably due to the poor stability of the thiocarbonyl group in the presence of nucleophiles. Thionation on the chloroacetamide 174 was also unsuccessful and lead to the decomposition of starting material.

128

Scheme 4.7 Attempts to form the thiocoumarin linker 186.

Alternatively, the thiocoumarin 191 was designed and synthesized, starting with the tosylation of N-Boc-ethylenediamine (187) in 97% yield,231 however, the Mitsunobu reaction of the tosyl amine 188 and the thiocoumarin alcohol 183 only gave starting material (Scheme

4.8). Thus, the coumarin alcohol 173 was used instead, affording 189 in 89% yield (N-Boc-

232 ethylenediamine, PPh3, DIAD, THF). This was followed by a thionation to deliver the thiocoumarin 190 in 37% yield. The thiocoumarin 190 was subjected to a variety of conditions to remove the Boc group and only decomposition of thiocoumarin 190 was observed.

129

Scheme 4.8 Attempts to synthesize thiocoumarin linker through amine 191.

The route was then changed to avoid the exposure of thiocoumarin to free amines

(Scheme 4.9). The amine 192 was generated through a Boc deprotection from 188 in

233 quantitative yield (TFA, Et3SiH, DCM), which was reacted with chloroacetyl chloride in the presence of DIPEA to furnish the chloroacetamide 193 in 72% yield.116 Using this chloroacetamide 193, a Mitsunobu reaction was attempted on both the coumarin 173 and the thiocoumarin 183 with no success in either case.

130

Scheme 4.9 Attempts to synthesize the thiocoumarin linker 195 through the chloroacetamide 193.

Overall, the instability of the thiocoumarin limited the design and synthesis of thiocoumarin linkers. Meanwhile, a 7-diethylamino-4-methyl-dicyanocoumarin was reported by Jullien et al. which gave a comparable red-shifted absorption maximum as 7- diethylamino-4-methyl-thiocoumarin (3, Figure 1.3).83 Thus, a MO linker 208 bearing a malononitrilecoumarin (MNCM) core was designed. The synthesis commenced with the alcohol 167 where a TBDMS group was installed to obtain the coumarin 196 in 84% yield

(Scheme 4.10). This was followed by a thionation with Lawesson’s reagent, affording the thiocoumarin 197. Treatment of the thiocoumarin 197 with malononitrile in the present of silver nitrate and TEA resulted in the malononitrilecoumarin 198 in 89% yield.234 The absorption spectrum of 196-198 confirmed that the malononitrile modification demonstrated a more significant red-shifted effect and lower absorption at 365 nm compared to 196 (Figure

4.2). The reduction of the nitro group on 198 was achieved in 70% yield to furnish the amine

199 (Zn, HOAc).

131

Scheme 4.10 Synthesis of dicyanocoumarin amine 199.

1 0.9 0.8 0.7 0.6 0.5 DEACM

0.4 TC absorbance 0.3 MNCM 0.2 0.1 0 300 400 500 600 wavelength/nm

Figure 4.2 Absorption spectra of the DEACM 196, the thiocoumarin (TC) 197 and the MNCM 198 in MeOH at a 0.1 mM concentration.

Meanwhile, the commercially available 6-aminohexanoic acid (200) was esterified to the methyl ester 201 with thionyl chloride in MeOH (96% yield) (Scheme 4.11).235 The amine of 201 was then reacted with chloroacetyl chloride to deliver the chloroacetamide 202 in 96% yield (DIPEA, DCM). The hydrolysis of the methyl ester was accomplished with 1 M

LiOH in a 1:1 mixture of MeOH/water, affording the acid 203 in 84% yield.236

132

Scheme 4.11 Synthesis of the acid 203, containing a chloroacetamide group.

The initial attempts to couple the amine 199 with the acid 203 started with the activation of the acid 203 in two ways (Scheme 4.12). The first was the generation of the acid chloride 204 through treatment with oxalyl chloride in the present of DMF (catalytic amount).237 A second was the coupling of the acid 203 with N-hydroxysuccinimide (NHS) affording the NHS ester 205 in 55% yield. Several solvents and bases were screened for the reaction of the amine 199 with the activated acids (204 and 205) with no success.

133

Scheme 4.12 Attempts to couple the amine 199 with the activated acids 204 and 205.

The acid 203 and the amine 199 were then subjected to a screening of different coupling reagents in search for the best condition, and the highest yield was 44% obtained with HATU in the present of DMAP in THF (Scheme 4.13). The subsequent TBAF deprotection afforded the alcohol 207 (69%), which reacted with DSC in the presence of

TEA in CH3CN as the solvent, completing the synthesis of the MNCM linker 208 in 47% yield.

134

Scheme 4.13 Synthesis of the MNCM linker 208.

This MNCM linker 208 was sent to Chen’s lab and tested to regulate the ntla MO which has previously been used to test the light-activation of MOs due to the significant abnormal phenotypes generated from ntla gene-silencing (Figure 4.3A-D). Zebrafish embryos ejected with either the DEACM cylized ntla MO or the MNCM cylized ntla MO were kept in the dark or irradiated with light at one of the following wavelengths: 360 nm,

405 nm, or 470 nm. The phenotypic distribution of these embryos was shown in Figure 4.3E.

Surprisingly, the MNCM group demonstrated no improvement on wavelength-selectivity compared to the DEACM group. This is potential the result of not strictly monochromatic light sources used for decaging.

135

A B C D

class 1 class 2 class 3 class 4

E class 1 class 2 class 3 class 4

DEACM cyclic ntla cMO MNCM cyclic ntla cMO 100 100

80 80

60 60

40 40

% of embryos% of % of embryos% of

20 20

0 0 ˗ UV 470 nm 405 nm 360 nm ˗ UV 470 nm 405 nm 360 nm

Figure 4.3 Comparison of the DEACM- and the MNCM-cyclized ntla MO at different wavelengths (360 nm, 405 nm, and 470 nm). Four classes of phenotypes (A-D) were generated due to the ntla gene-silencing at 24 hours post fertilization (hpf). Embryos injected with the indicated cyclic MOs were either kept in the dark or irradiated with 360 nm, 405 nm, or 470 nm light at 3.5 hpf. (D) The phenotypic distributions for treated embryos are shown.

4.2.3 Synthesis of azidobenzyl MO linker

As discussed in CHAPTER 2.3, organic azides have been used as protecting groups which can be cleaved by phosphines via Staudinger reduction, and the development of chemical triggers to bioactive molecules is of interest. To this end, a pAzBn MO linker (215) was synthesized which can be a reductive regulator over MO activity that is orthogonal to the light regulators discussed above.

The synthesis of the pAzBn MO linker 215 was very similar to the synthesis of

DEACM linker 176, but starts with 4-azidobenzyl alcohol 104 instead (Scheme 4.14). The

136

alcohol 104 was oxidized to aldehyde 209 using PCC in 85% yield,238 followed by an allylic stannane addition with allyl tributyl tinn to afford the alkene 210 in 97% yield (Scheme

4.12). After a TBDMS protection to 211, the alkene was exposed to borane dimethylsulfide, and the reaction was quenched by hydrogen peroxide and NaOH to obtain the alcohol 212 in

46% yield through two steps. The subsequent three-step reaction starting with a CDI activation followed by the treatment of ethylenediamine and acylation with chloroacetyl chloride was completed in 77% yield for the TBDMS protected linker 213. Upon cleavage of the silyl group, the free alcohol of 214 was activated by DSC, affording the pAzBn linker

215 in 71% yield. This linker is under investigation on its regulatory ability over MOs in the

Chen lab.

Scheme 4.14 Synthesis of the pAzBn linker 215.

137

4.3 Conclusion

In summary, five different oligonucleotide linkers were synthesized (Figure 4.4). An

ONB phosphoramidite 160 can link two DNA oligomers and enable the light regulation of their function. Two DEACM MO linkers (170 and 176) bearing two different thiol-specific groups were designed to cyclize MOs, in which the chloroacetyl chloride one (176) has been applied to the wavelength-selective regulation of MO function in zebrafish embryos. A MO linker based on the novel MNCM chromophore (208) was also developed which has an absorption maximum at 490 nm. As a phosphine-labile linker, the pAzBn linker (215) may add another level of regulation over cMOs. The biological applications of these linkers are being pursued in the Deiters lab and the Chen lab at Stanford University.

Figure 4.4 Photo/chemical-cleavable oligonucleotide linkers.

138

4.4 Experimental

MeOH, EtOH, and pyridine were distilled from calcium hydride and stored over 4 Ǻ molecular sieves. Other reagents and solvents were obtained from commercial sources were stored under nitrogen and used directly without further purification. Yields refer to chromatographically and spectroscopically pure compounds unless otherwise stated. Flash chromatography was performed on silica gel (60 Å, 40-63 μm (230 × 400 mesh), Sorbtech) as a stationary phase. High resolution mass spectral analysis (HRMS) was performed at the

University of Pittsburgh. The 1H NMR and 13C NMR spectra were recorded on a 300 MHz or a 400 MHz Varian NMR spectrometer. Chemical shifts are given in δ units (ppm) for 1H

NMR spectra and 13C NMR spectra.

1-(2-Nitrophenyl)but-3-en-1-ol (157). Allyltri-n-butyl tin (1.5 eq., 1.63 ml, 4.9 mmol) and zinc chloride (1.5 eq., 676 mg, 4.96 mmol) were added to a solution of the 2- nitrobenzaldehyde 156 (1 eq., 500 mg, 3.31 mmol) in CH3CN/water (4:1, 16 ml). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The residue was taken up in water (10 ml) and extracted with DCM (3 × 20ml). The combined organic layer was washed with brine (30 ml), dried over Na2SO4, and concentrated. The crude product was purified by silica gel chromatography with

139

hexane/EtOAc (1:1) as the eluent, affording 157 as a white solid in 97% yield (620 mg, 3.11

1 mmol). H NMR (400 MHz, CDCl3): δ = 2.37-2.45 (m, 1H), 2.53 (br, 1H), 2.66-2.71 (m,

1H), 5.17-5.21 (m, 2H), 5.28-5.31 (dd, Ja = 8.4 Hz, Jb = 3.6 Hz, 1H), 5.83-5.93 (m, 1H),

7.39-7.44 (m, 1H), 7.62-7.66 (m, 1H ), 7.81-7.83 (d, J = 8.0 Hz, 1H), 7.92-7.93 (d, J = 8.4

+ Hz, 1H). HRMS-LC: m/z calcd for C10H11NO3 [M+H˗H2O] : 176.0712; found: 176.0716.

The analytical data matched reported results.108

1-(2-Nitrophenyl)propane-1,3-diol (158). 4-Methyl morpholine oxide (1.13 eq., 440 mg,

3.76 mmol) was dissolved in water (17 ml) and the alkene 157 (1 eq., 639 mg, 3.31 mmol) in acetone (9 ml) was added, followed by the addition of a catalytic amount of osmium tetroxide. The reaction mixture was stirred at room temperature overnight, concentrated under reduced pressure, neutralized to pH 7 through the addition of 1 N HCl, and extracted with EtOAc (3 × 20 ml). The combined organic layer was concentrated under reduced pressure. The crude product was confirmed by 1H NMR and used in the next step without further purification. The crude alcohol was dissolved in acetone/water (1:1, 160 ml) and sodium periodate (3 eq., 2.14 g, 10 mmol) was added. The resulting mixture was stirred for 2 hours at room temperature, concentrated under reduced pressure and extracted with EtOAc (3

× 20 ml). The combined organic layer was dried over Na2SO4, filtered and concentrated. The yielding aldehyde was confirmed by crude NMR and subjected to next step without further purification. NaBH4 (0.5 eq., 64 mg, 1.7 mmol) was added to a solution of the crude aldehyde in EtOH (22 ml), and the reaction mixture was stirred at room temperature for 2 hours. The reaction was quenched by the addition of water (10 ml), and the mixture was

140

concentrated under reduced pressure. The aqueous layer was extracted with EtOAc (3 × 20 ml). The combined organic layer was washed with brine (20 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1 to 1:2) as the eluent, affording 158 as an off-white solid in 36% yield

1 (549 mg, 2.78 mmol). H NMR (400 MHz, CDCl3): δ = 1.90-1.98 (m, 1H), 2.05-2.11 (m,

1H), 3.88-3.99 (m, 2H), 5.46-5.49 (dd, Ja = 9.2 Hz, Jb = 2.8 Hz, 1H), 7.39-7.43 (m, 1H),

+ 7.63-7.67 (m, 1H), 7.87-7.92 (m, 2H). HRMS-LC: m/z calcd for C9H11NO4 [M+H˗H2O] :

180.0661; found: 180.0667. The analytical data matched reported results.108

3-(Bis(4-methoxyphenyl)(phenyl)methoxy)-1-(2-nitrophenyl)propan-1-ol (159). DMTCl

(1.5 eq., 526 mg, 1.55 mmol) was added to a solution of the alcohol 158 (1 eq., 204 mg, 1.03 mmol) in pyridine (15 ml) and the mixture was stirred overnight at room temperature. The reaction was quenched with MeOH (1 ml), and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc

(2:1) and 1% TEA as the eluent, affording 159 as a white solid in 98% yield (441 mg, 0.98

1 mmol). H NMR (400 MHz, CDCl3): δ = 1.93-2.02 (m, 1H), 2.16-2.21 (m, 1H), 3.40-3.47

(m, 2H), 3.79 (s, 6H), 5.43-5.46 (dd, Ja = 8.4 Hz, Jb = 2.0 Hz, 1H), 6.84-6.86 (m, 4H), 7.23-

7.37 (m, 10 H), 7.44-7.46 (d, J = 8.8 Hz 1H), 7.57-7.60 (t, J = 7.2 Hz, 1H), 7.83-7.85 (d, J =

+ 8.0 Hz, 1H), 7.88-7.90 (d, J = 8.0 Hz, 1H). HRMS-LC: m/z calcd for C30H29NO6 [M+H] :

522.1893; found: 521.8489. The analytical data matched reported results.108

141

3-(Bis(4-methoxyphenyl)(phenyl)methoxy)-1-(2-nitrophenyl)propyl (2-cyanoethyl) diisopropylphosphoramidite (160). DIPEA (4 eq., 0.7 ml, 3.92 mmol) was added to a solution of the alcohol 159 (1 eq., 441 mg, 0.98 mmol) in DCM (18 ml) at 0 ˚C and the mixture was stirred for 10 minutes. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (2 eq., 0.4 ml, 1.96 mmol) was added and the reaction mixture was warm to room temperature over 2 hours. The solvent was removed under reduced pressure and the crude product was purified by silica gel chromatography with hexane/EtOAc (3:1) and 1% TEA as the eluent, affording 160 as a clear foam in 86% yield (592 mg, 0.84 mmol). 1H NMR (300 MHz,

CDCl3): δ = 0.75-0.77 (d, J = 6.6 Hz, 3H), 1.00-1.08 (m, 6H), 1.17-1.21 (dd, Ja = 6.6 Hz, Jb

= 1.8 Hz, 3H), 1.96-2.04 (m, 1H), 2.24-2.34 (m, 1.5H), 2.38-2.42 (m, 1H), 2.63-2.69 (m, 0.5

H), 3.24-3.38 (m, 3H), 3.46-3.64 (m, 3H), 3.79 (s, 6H), 5.42-5.62 (m, 1H), 6.78-6.84 (m,

4H), 7.15-7.24 (m, 2H), 7.28-7.43 (m, 8 H), 7.45-7.61 (m, 1H), 7.61-7.76 (m, 1H), 7.85-7.91

31 (m, 1H). P NMR (300 MHz, CDCl3): δ = 149.6, 151.2. The analytical data matched reported results.108

3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid (164). Maleic anhydrate 163 (1 eq., 490 mg, 5 mmol) was added to a solution of β-alanine 162 (1 eq., 475 mg, 5 mmol) in

DMF (5 ml). The reaction mixture was stirred for 6 hours at room temperature. Partial of the solution (1ml) was diluted with water (2 ml), extracted with EtOAc (3 × 2 ml) and concentrated under reduced pressure, affording the crude product 164 as a white solid in 83% yield (140 mg, 0.83 mmol). The rest was subjected to next step in situ. 1H NMR (300 MHz,

DMSO): δ = 2.54-2.59 (t, J = 6.9 Hz, 2H), 2.97-3.02 (t, J = 6.9 Hz, 2H), 6.02 (s, 2H).

142

- HRMS-LC: m/z calcd for C7H7NO5 [M-H] : 168.0291; found: 168.0295. The analytical data matched reported results.222

2,5-Dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (165). N-

Hydroxylsuccinimide (1.2 eq., 552 mg, 4.8 mmol) was added to a solution of the acid 164 (1 eq., 676 mg, 4 mmol) in DMF (4 ml) at 0 ˚C, followed by DCC (2.1 eq., 1.7 g, 8.4 mmol).

The reaction mixture was stirred overnight from 0 ˚C to room temperature and filtered. The solid was washed with cold DMF (2 ml). The combined filtrate was poured onto ice, and extracted with DCM (10 ml × 3). The organic layer was combined dried over Na2SO4 and concentrated, affording the product 165 as a white solid in 72% yield (766 mg, 2.88 mmol).

1 H NMR (300 MHz, CDCl3): δ = 2.98-3.02 (t, J = 6.9 Hz, 2H), 2.87 (s, 4H), 3.89-3.93 (t, J =

6.9 Hz, 2H), 6.72 (s, 2H). The analytical data matched reported results.222

7-(Diethylamino)-2-oxo-2H-chromene-4-carbaldehyde (166). Selenium oxide (1.5 eq.,

3.32 g, 30 mmol) was added to a solution of 7-diethylamino-4-methylcoumarin 54 (1 eq.,

4.64 g, 20 mmol) in dioxane (120 ml). The reaction mixture was heated to reflux overnight and filtered through celite. The filtrate was concentrated under reduced pressure and the crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 166 as a red solid in 34% yield (1.68 g, 6.8 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.12-1.20 (t, J = 7.2 Hz, 6H), 3.33-3.40 (q, J = 7.2 Hz, 4H), 6.36 (s, 1H), 6.42-

6.43 (d, J = 2.7 Hz, 1H), 6.53-6.57 (dd, Ja = 9.0 Hz, Jb = 2.7 Hz, 1H), 8.19-8.22 (d, J = 2.7

13 Hz, 1H), 10.95 (s, 1H). C NMR (400 MHz, CDCl3): δ = 12.4, 44.7, 97.4, 103.6, 109.4,

143

117.1, 126.9, 143.7, 150.9, 157.3, 161.7, 192.5. HRMS-LC: m/z calcd for C14H15NO3

[M+H]+: 246.1130; found: 246.1130. The analytical data matched reported results.161

7-(Diethylamino)-4-(1-hydroxy-2-nitroethyl)-2H-chromen-2-one (167). Nitromethane (10 eq., 0.54 ml, 10 mmol) was added to a solution of the aldehyde 166 (1 eq., 245 mg, 1 mmol) in THF (2 ml), followed by N,N,Nʹ,Nʹ,-tetramethylethylene diamine (0.3 eq., 45 µl, 0.3 mmol). The reaction mixture was stirred overnight at room temperature, quenched by water

(3 ml) and extracted with EtOAc (3 × 3 ml). The combined organic layer was washed with brine (5 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (3:2) as the eluent, affording 167 as an off-white

1 solid in 69% yield (211 mg, 0.69 mmol). H NMR (300 MHz, CDCl3): δ = 1.18-1.25 (t, J =

7.2, 6H), 3.37-3.44 (q, J = 7.2 Hz, 4H), 3.88 (s, 1H), 4.51-4.64 (m, 2H), 5.78-5.81 (m, 1H),

6.35 (s, 1H), 6.48-6.49 (d, J = 2.7 Hz, 1H), 6.58-6.62 (dd, Ja = 9.0 Hz, Jb = 2.7 Hz, 1H), 7.40-

13 7.43 (d, J = 2.7 Hz, 1H). C NMR (400 MHz, CDCl3): δ = 12.6, 45.0, 62.3, 80.0, 98.2,

105.2, 106.7, 109.4, 124.2, 151.0, 152.5, 156.7, 162.5. HRMS-LC: m/z calcd for C15H18N2O5

[M+H]+: 307.1294; found: 260.1298 (nitro group fell off).

4-(2-Amino-1-hydroxyethyl)-7-(diethylamino)-2H-chromen-2-one (168). Zinc powder (40 eq., 4.2 g, 64 mmol) was added to a solution of the coumarin 167 (1 eq., 500 mg, 1.6 mmol) in acetic acid (20 ml). The reaction mixture was stirred at room temperature for 16 hours and filtered through celite. The mixture was neutralized to pH 7.0 by aq. NH4OH and extracted with EtOAc (3 × 30 ml). The combined organic layer was washed with water (50 ml) and

144

brine (50 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) and 1% TEA as the eluent, affording 168 as a

1 yellow solid in 55% yield (243 mg, 0.88 mmol). H NMR (300 MHz, CDCl3): δ = 1.14-1.18

(t, J = 7.2, 6H), 3.10-3.22 (m, 2H), 3.33-3.39 (q, J = 7.2 Hz, 4H), 4.93-5.01 (m, 1H), 6.26 (s,

1H), 6.46-6.60 (m, 2H), 7.30-7.33 (d, J = 2.7 Hz, 1H). HRMS-LC: m/z calcd for C15H18N2O3

[M+H]+: 277.1552; found: 277.1546.

N-(2-(7-(Diethylamino)-2-oxo-2H-chromen-4-yl)-2-hydroxyethyl)-3-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)propanamide (169). 4-Methylmorpholine (0.5 eq., 88 µl, 0.8 mmol) was added to a solution of the amine 168 (1 eq., 442 mg, 1.6 mmol) and the succinimide ester 165 (3.1 eq., 1.3 g, 5 mmol) in DMF (10 ml). The reaction mixture was stirred overnight at room temperature and quenched with water (10 ml). The mixture was acidified to pH 3-4 with 1 N HCl and extracted with DCM (3 × 10 ml). The combined organic layer was washed with water (10 ml) and brine (10 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with

DCM/acetone (2:1) as the eluent, affording 169 as a yellow solid in 50% yield (211 mg, 0.8

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.12-1.17 (t, J = 6.9, 6H), 2.52-2.56 (t, J = 6.9, 2H),

2.85-2.94 (m, 2H), 3.31-3.38 (q, J = 6.9 Hz, 4H), 3.82-3.86 (t, J = 6.9, 2H), 5.14-5.17 (m,

1H), 6.27 (s, 1H), 6.41 (s, 1H), 6.59-6.63 (d, J = 9.0 Hz, 1H), 6.67 (s, 2H), 7.62-7.65 (d, J =

+ 9.0 Hz, 1H). HRMS-LC: m/z calcd for C22H25N3O6 [M+H] : 428.1822; found: 428.1833.

145

1-(7-(Diethylamino)-2-oxo-2H-chromen-4-yl)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)propanamido)ethyl (2,5-dioxopyrrolidin-1-yl) carbonate (170). N,Nʹ-Disuccinimidyl carbonate (5 eq., 384 mg, 1.5 mmol) followed by a catalytic amount of DMAP was added to a solution of the alcohol 169 (1 eq., 128 mg, 0.3 mmol) in DCM (6 ml). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (2:1) as the eluent, affording 170 as a yellow solid in 33% yield (56 mg, 0.1 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.15-1.19 (t, J = 6.9, 6H), 2.52-2.56 (t, J = 6.9, 2H), 2.66 (s, 4H), 2.85-2.94 (m,

2H), 3.35-3.42 (q, J = 6.9 Hz, 4H), 3.76-3.92 (m, 2H), 5.72-5.81 (m, 1H), 6.12 (s, 1H), 6.19-

6.22 (d, J = 9.0 Hz, 1H), 6.46 (s, 1H), 6.67 (s, 2H), 7.78-7.81 (d, J = 9.0 Hz, 1H). HRMS-LC:

+ m/z calcd for C27H28N4O10 [M+H] : 569.1884; found: 569.1906.

7-(Diethylamino)-4-(1-hydroxybut-3-en-1-yl)-2H-chromen-2-one (171). Allyltri-n-butyl tin (0.99 ml, 3 mmol) and zinc chloride (409 mg, 3 mmol) were added to a solution of the aldehyde 165 (500 mg, 2 mmol) in 10 ml of CH3CN/water (4:1). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The residue was taken up in water (10 ml), and was extracted with DCM (3 × 10 ml). The combined organic layer was washed with brine (10 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 171 as a green solid in 83% yield (477 mg, 1.66 mmol). 1H NMR (400 MHz,

CDCl3): δ = 1.14-1.18 (t, J = 7.2, 6H), 2.38-2.45 (m, 1H), 2.57-2.63 (m, 1H), 3.33-3.38 (q, J

= 7.2 Hz, 4H), 4.97-5.00 (dd, Ja = 8.0 Hz, Jb = 4.0 Hz, 1H), 5.10-5.17 (m, 2H), 5.81-5.90 (m,

146

1H), 6.22 (s, 1H), 6.40-6.41 (d, J = 2.4 Hz, 1H), 6.52-6.55 (dd, Ja = 9.2 Hz, Jb = 2.4 Hz, 1H),

13 7.36-7.38 (d, J = 9.2 Hz, 1H). C NMR (400 MHz, CDCl3): δ = 12.5, 41.6, 44.7, 68.7, 97.8,

105.1, 106.2, 108.6, 118.8, 124.9, 133.6, 150.3, 156.4, 158.2, 163.0. HRMS-LC: m/z calcd

+ for C17H21NO3 [M+H] : 288.1660; found: 288.1602.

4-(1-((tert-Butyldimethylsilyl)oxy)but-3-en-1-yl)-7-(diethylamino)-2H-chromen-2-one

(172). TBDMSCl (904 mg, 6 mmol) and imidazole (612 mg, 9 mmol) were added to a solution of the alcohol 171 (862 mg, 3 mmol) in DMF (3.4 ml). The reaction mixture was stirred overnight at room temperature and quenched with sat. NaHCO3 (15 ml). The aqueous phase was extracted with EtOAc (3 × 10 ml). The combined organic layer was washed with water (15 ml) and brine (10 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 172

1 as a yellow solid in 97% yield (1.16 g, 2.9 mmol). H NMR (400 MHz, CDCl3): δ = 0.03 (s,

6H), 0.86 (s, 9H), 1.13-1.16 (t, J = 7.2, 6H), 2.38-2.48 (m, 2H), 3.32-3.37 (q, J = 7.2 Hz, 4H),

4.85-4.88 (m, 1H), 4.97-5.02 (m, 2H), 6.13 (s, 1H), 6.13-6.14 (s, 1H), 6.45-6.46 (d, J = 2.8

13 Hz, 1H), 6.52-6.55 (dd, Ja = 9.2 Hz, Jb = 2.8 Hz, 1H), 7.43-7.45 (d, J = 9.2 Hz, 1H). C

NMR (400 MHz, CDCl3): δ = -5.1, -4.8, -3.6, 12.4, 18.0, 25.7, 42.9, 44.6, 71.0, 97.8, 105.7,

105.9, 108.4, 117.8, 125.2, 133.8, 150.2, 156.5, 158.3, 162.7. HRMS-LC: m/z calcd for

+ C23H35NO3Si [M+H] : 402.2464; found: 402.2468.

4-(1-((tert-Butyldimethylsilyl)oxy)-4-hydroxybutyl)-7-(diethylamino)-2H-chromen-2-one

(173). A 2 M solution of BH3•Me2S complex in THF (0.5 ml, 1 mmol) was added to a

147

solution of the alkene 172 (80 mg, 0.2 mmol) in THF (0.7 ml) at 0 ˚C and the mixture was stirred for 3 hours at 0 ˚C. A 3 M aq. NaOH (0.5 ml) and 30% hydrogen peroxide in water

(0.4 ml) were added. The mixture was allowed to warm to room temperature over 2 hours and was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over Na2SO4, and concentrated under reduced pressure.

The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 173 as a yellow solid in 49% yield (41 mg, 0.098 mmol). 1H NMR (400

MHz, CDCl3): δ = -0.04 (s, 3H) 0.08 (s, 3H), 0.91 (s, 9H), 1.18-1.21 (t, J = 7.2, 6H), 1.62-

1.90 (m, 5H), 3.37-3.42 (q, J = 7.2 Hz, 3H), 3.62-3.67 (m, 2H), 4.91-4.93 (dd, Ja = 6.4 Hz, Jb

= 4.0 Hz, 1H), 6.17 (s, 1H), 6.49-6.50 (d, J = 2.8 Hz, 1H), 6.54-6.57 (dd, Ja = 9.2 Hz, Jb = 2.8

13 Hz, 1H), 7.47-7.49 (d, J = 9.2 Hz, 1H). C NMR (400 MHz, CDCl3): δ = -5.0, -4.6, 12.6,

18.3, 25.9, 28.5, 34.5, 44.8, 62.7, 71.0, 97.9, 105.9, 106.1, 108.5, 125.4, 150.3, 156.6, 158.7,

+ 162.7. HRMS-LC: m/z calcd for C23H37NO4Si [M+H] : 420.2570; found: 420.4193.

4-((tert-Butyldimethylsilyl)oxy)-4-(7-(diethylamino)-2-oxo-2H-chromen-4-yl)butyl (2-(2- chloroacetamido)ethyl)carbamate (174). 1,1ʹ-Carbonyldiimidazole (45 mg, 0.28 mmol) was added to a solution of the alcohol 173 (47 mg, 0.11 mmol) in DCM (3 ml). The reaction mixture was stirred at room temperature for 3 hours and was diluted with DCM (2 ml). The solution was washed with water (5 ml) and brine (5 ml), dried over Na2SO4, and concentrated under reduced pressure. The crude product was confirmed by 1H NMR and used in next step without further purification. The activated alcohol was dissolved in DCM (2.5 ml) and ethylene diamine (16 µl, 0.25 mmol) was added. The resulting mixture was stirred for 2

148

hours at room temperature. The solvent was removed under reduced pressure and the crude product was confirmed by 1H NMR and subjected to next step without further purification.

The amine residue was redissoled in DCM (2 ml), and DIPEA (8 µl, 0.05 mmol) was added.

The mixture was stirred at room temperature for 10 minutes and cooled to 0˚C. Chloroacetyl chloride (16 µl, 0.2 mmol) was added and the solution was stirred at 0˚C for 10 minutes. The reaction was quenched with sat. NaHCO3 (2 ml) and the aqueous layer was extracted with

DCM (3 × 2 ml). The combined organic layer was washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 174 as a yellow foam in

1 65% yield (42 mg, 0.07 mmol). H NMR (400 MHz, CDCl3): δ = -0.04 (s, 3H) 0.08 (s, 3H),

0.91 (s, 9H), 1.18-1.22 (t, J = 7.2, 6H), 1.71-1.80 (m, 4H), 3.35-3.43 (m, 8H), 4.03 (s, 2H),

4.05-4.06 (m, 2H), 4.89 (m, 1H), 5.04 (m, 1H), 6.17 (s, 1H), 6.50-6.51 (d, J = 2.8 Hz, 1H),

13 6.54-6.57 (dd, Ja = 8.8 Hz, Jb = 2.4 Hz, 1H), 7.13 (br, 1H), 7.44-7.47 (d, J = 8.8 Hz, 1H). C

NMR (400 MHz, CDCl3): δ = -5.0, -4.5, 12.6, 18.3, 24.9, 25.9, 29.8, 34.6, 40.5, 40.8, 42.6,

44.8, 64.9, 70.8, 97.9, 105.9, 106.1, 108.4, 125.3, 150.4, 156.7, 157.4, 158.4, 162.7, 166.9.

+ HRMS-LC: m/z calcd for C28H44ClN3O6 Si[M+H] : 582.2766; found: 582.2761.

4-(7-(Diethylamino)-2-oxo-2H-chromen-4-yl)-4-hydroxybutyl (2-(2-chloroacetamido)- ethyl)-carbamate (175). TBAF in THF (1.5 eq., 1 M, 0.1 ml, 0.1 mmol) was added to a solution of the silyl ether 174 (38 mg, 0.06 mmol) in THF (1.5 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with chloroform/acetone (1:1) as the

149

eluent, affording 175 as a yellow foam in 44% yield (13 mg, 0.03 mmol). 1H NMR (400

MHz, CDCl3): δ = 1.17-1.21 (t, J = 7.2, 6H), 1.80-1.90 (m, 4H), 3.34-3.45 (m, 9H), 4.03 (s,

2H), 4.10-4.13 (m, 2H), 5.01 (m, 1H), 5.44 (m, 1H), 6.25 (s, 1H), 6.45-6.46 (d, J = 2.0 Hz,

1H), 6.54-6.57 (dd, Ja = 9.2 Hz, Jb = 2.4 Hz, 1H), 7.21 (br, 1H), 7.37-7.39 (d, J = 9.2 Hz,

13 1H). C NMR (400 MHz, CDCl3): δ = 12.6, 25.3, 33.7, 40.6, 40.7, 42.7, 44.8, 64.9, 69.3,

97.9, 105.1, 106.2, 108.7, 125.1, 150.5, 156.5, 157.5, 158.9, 163.1, 167.2. HRMS-LC: m/z

+ calcd for C22H30ClN3O6 [M+H] : 468.1901; found: 468.1870.

4-(7-(Diethylamino)-2-oxo-2H-chromen-4-yl)-4-((((2,5-dioxopyrrolidin-1-yl)oxy)- carbonyl)oxy)butyl (2-(2-chloroacetamido)ethyl)carbamate (176). N,Nʹ-Disuccinimidyl carbonate (36 mg, 0.14 mmol) and a catalytic amount of DMAP were added to a solution of the alcohol 175 (13 mg, 0.03 mmol) in DCM (0.5 ml). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (2:1) as the eluent, affording

1 176 as a yellow solid in 82% yield (15 mg, 0.025 mmol). H NMR (400 MHz, CDCl3): δ =

1.19-1.22 (t, J = 7.2, 6H), 1.72-1.86 (m, 2H), 2.10-2.14 (m, 2H), 2.84 (s, 4H), 3.35-3.42 (m,

8H), 4.03 (s, 2H), 4.15-4.18 (t, J = 7.2, 2H), 5.43 (m, 1H), 5.96-5.99 (t, J = 6.0, 1H), 6.15 (s,

1H), 6.51-6.52 (d, J = 2.8, 1H), 6.59-6.63 (dd, Ja = 9.2 Hz, Jb = 2.8 Hz, 1H), 7.18 (br, 1H),

13 7.33-7.35 (d, J = 9.2 Hz, 1H). C NMR (400 MHz, CDCl3): δ = 12.5, 14.3, 24.3, 25.5, 25.6,

31.9, 40.3, 42.6, 44.9, 64.5, 78.3, 98.2, 105.1, 109.4, 124.6, 150.9, 151.2, 151.6, 156.7, 157.4,

+ 161.8, 167.3, 172.2. HRMS-LC: m/z calcd for C27H33ClN4O10 [M+H] : 609.1963; found:

609.1990.

150

4-((tert-Butyldimethylsilyl)oxy)butan-1-ol (178) was prepared according to a previously reported procedure.239 TBDMSCl (301 mg, 2 mmol) in THF (0.9 ml) was added drop-wise to a solution of 1,4-butanediol 177 (0.75 ml, 8.5 mmol) and imidazole (168 mg, 2.5 mmol) in

THF (3.5 ml) during 20 minutes. The reaction mixture was stirred at 0 ˚C for 1 hour, warmed to room temperature and diluted with Et2O (9 ml). The solution was washed with sat. NH4Cl

(10 ml) and brine (10 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 178

1 as a colorless foam in 97% yield (396 mg, 1.9 mmol). H NMR (300 MHz, CDCl3): δ = 0.06

(s, 6H), 0.89 (s, 9H), 1.62-1.65 (m, 4H), 3.63-3.68 (m, 4H). The analytical data matched reported results.239

4-((tert-Butyldimethylsilyl)oxy)butyl (2-(2-chloroacetamido)ethyl)carbamate (179). 1,1ʹ-

Carbonyldiimidazole (2.5 eq., 203 mg, 1.25 mmol) was added to a solution of the alcohol

178 (1 eq., 102 mg, 0.5 mmol) in DCM (13 ml). The reaction mixture was stirred at room temperature for 3 hours and diluted with DCM (10 ml). The solution was washed with water

(15 ml) and brine (15 ml), dried over Na2SO4 and concentrated. The crude product was confirmed by 1H NMR and used in next step without further purification. The activated alcohol was dissolved in DCM (12.5 ml) and ethylene diamine (2.5 eq., 84 µl, 1.25 mmol) was added. The resulting mixture was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure and the crude product was confirmed by 1H NMR before being subjected to next step without further purification. The crude amine and DIPEA

(0.5 eq., 44 µl, 0.25 mmol) were dissolved in DCM (10 ml). The mixture was stirred at room

151

temperature for 10 minutes and cooled to 0˚C. Chloroacetyl chloride (3 eq., 119 µl, 1.5 mmol) was added and the resulting mixture was stirred at 0˚C for 10 minutes. The reaction was quenched with sat. NaHCO3 (10 ml), and the aqueous layer was extracted with DCM (3

× 10 ml). The combined organic layer was washed with brine (20 ml), dried over Na2SO4, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 179 as a colorless foam in 69% yield (127 mg,

1 0.34 mmol). H NMR (400 MHz, CDCl3): δ = 0.03-0.07 (m, 6H), 0.87-0.89 (m, 9H), 1.52-

1.67 (m, 4H), 3.33-3.45 (m, 4H), 3.59-3.63 (t, J = 6.4, 2H), 4.03-4.11 (m, 4H), 5.20 (br, 1H),

13 7.22 (br, 1H). C NMR (400 MHz, CDCl3): δ = -5.2, -3.5, 18.4, 25.7, 26.0, 29.2, 40.4, 40.8,

+ 42.6, 62.7, 65.3, 157.6, 167.1. HRMS-LC: m/z calcd for C15H31ClN2O4Si [M+Na] :

389.1639; found: 389.1643.

4-Hydroxybutyl (2-(2-chloroacetamido)ethyl)carbamate (180). TBAF in THF (1.5 eq., 1

M, 0.23 ml, 0.23 mmol) was added to a solution of the silyl ether 179 (1 eq., 55 mg, 0.15 mmol) in THF (3.4 ml). The reaction mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:3) as the eluent, affording 180 as a colorless foam in

1 76% yield (29 mg, 0.11 mmol). H NMR (400 MHz, (CD3)2CO): δ = 1.52-1.68 (m, 4H),

3.23-3.28 (m, 2H), 3.33-3.37 (m, 2H), 3.53-3.57 (m, 3H), 3.99-4.02 (t, J = 6.4, 2H), 4.06 (s,

13 1H), 6.37 (br, 1H), 7.67 (br, 1H). C NMR (400 MHz, (CD3)2CO): δ = 26.5, 29.2, 40.7,

+ 40.9, 43.3, 62.0, 65.0, 157.8, 167.1. HRMS-LC: m/z calcd for C9H17ClN2O4 [M+Na] :

275.0775; found: 275.0775.

152

4-(((2-(2-Chloroacetamido)ethyl)carbamoyl)oxy)butyl 2-(2,5-dioxopyrrolidin-1-yl)- acetate (181). N,Nʹ-Disuccinimidyl carbonate (5 eq., 71 mg, 0.28 mmol) and a catalytic amount of DMAP were added to a solution of alcohol 180 (1 eq., 14 mg, 0.055 mmol) in

DCM (1 ml). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (2:1) as the eluent, affording 181 as a white solid in 86%

1 yield (21 mg, 0.047 mmol). H NMR (300 MHz, CDCl3): δ = 1.73-1.87 (m, 4H), 2.84 (s,

4H), 3.33-3.46 (m, 4H), 4.03 (s, 2H), 4.09-4.4.13 (t, J = 6.0, 2H), 4.34-4.38 (t, J = 6.4, 2H),

13 5.35 (br, 1H), 7.11 (br, 1H). C NMR (400 MHz, CDCl3): δ = 25.8, 25.9, 30.1, 40.5, 41.1,

42.9, 65.0, 71.4, 151.9, 157.9, 169.5, 172.6.

4-(1-((tert-Butyldimethylsilyl)oxy)but-3-en-1-yl)-7-(diethylamino)-2H-chromene-2- thione (182). Lawesson’s reagent (2.5 eq., 504 mg, 1.25 mmol) was added to a solution of the coumarin 172 (1 eq., 200 mg, 0.5 mmol) in dry toluene (5 ml). The reaction mixture was heated to 90 ˚C for 24 hours and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 182 as an orange solid in

1 78% yield (163 mg, 0.39 mmol). H NMR (300 MHz, CDCl3): δ = -0.04 (s, 3H), 0.07 (s,

3H), 0.89 (s, 9H), 1.19-1.24 (t, J = 7.2 Hz, 6H), 2.44-2.51 (m, 2H), 3.39-3.46 (q, J = 6.9 Hz,

4H), 4.82-4.85 (t, J = 5.7 Hz, 1H), 5.00-5.09 (m, 2H), 5.75-5.84 (m, 1H), 6.70-6.73 (m, 2H),

+ 7.09 (s, 1H), 7.59-7.63 (d, J = 9.6 Hz, 1H). LRMS-LC: m/z calcd for C25H35NO2SSi [M+H] :

418.2; found: 418.2.

153

4-(1-((tert-Butyldimethylsilyl)oxy)-4-hydroxybutyl)-7-(diethylamino)-2H-chromene-2- thione (183). A 2 M solution of BH3•Me2S complex in THF (0.85 ml, 1.7 mmol) was added to a solution of the alkene 182 (140 mg, 0.34 mmol) in THF (1.2 ml) at 0 ˚C and the mixture was stirred for 3 hours at 0 ˚C. A 3 M aq. NaOH in water (0.7 ml) and 30% hydrogen peroxide in water (0.6 ml) were added. The mixture was allowed to warm to room temperature over 2 hours and extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 183 as an orange solid in 42% yield (62 mg, 0.14

1 mmol). H NMR (400 MHz, CDCl3): δ = -0.04 (s, 3H) 0.09 (s, 3H), 0.91 (s, 9H), 1.21-1.26

(t, J = 7.2, 6H), 1.59-1.95 (m, 5H), 3.40-3.45 (q, J = 7.2 Hz, 3H), 3.63-3.71 (m, 2H), 4.87-

4.90 (m, 1H), 6.87-6.92 (m, 2H), 7.12 (s, 1H), 7.62-7.65 (d, J = 7.2 Hz, 1H). LRMS-LC: m/z

+ calcd for C23H37NO3SSi [M+H] : 436.2; found: 436.5.

tert-Butyl (4-((4-methylphenyl)sulfonamido)butyl)carbamate (188). TEA (1.5 eq., 110 µl,

0.75 mmol) was added to a solution of the amine 187 (1 eq., 95 µl, 0.5 mmol) in DCM (2.5 ml) at ˗10 ˚C, followed by a solution of p-toluenesulfonyl chloride (1.1 eq., 105 mg, 0.55 mmol) in DCM (0.5 ml). The mixture was stirred overnight and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/EtOAc

(9:1) as the eluent, affording 188 as a white solid in 97% yield (166 mg, 0.48 mmol). 1H

NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.42-1.58 (m, 4H), 2.38 (s, 3H), 2.85-3.10 (m,

154

4H), 7.28-7.31 (d, J = 7.8 Hz, 2H), 7.67-7.70 (d, J = 7.8 Hz, 2H). The analytical data matched reported results.240

tert-Butyl (4-((N-(4-((tert-butyldimethylsilyl)oxy)-4-(7-(diethylamino)-2-oxo-2H- chromen-4-yl)butyl)-4-methylphenyl)sulfonamido)butyl)carbamate (189). The carbamate

188 was added to a solution of the alcohol 173 (1 eq., 10 mg, 0.025 mmol) and PPh3 (3 eq.,

19.7 mg, 0.075 mmol) in THF (0.3 ml). The mixture was cooled to 0 ˚C and DIAD (2.5 eq.,

12 µl, 0.063 mmol) was added. The resulting solution was stirred at room temperature for 5 hours and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 189 as a yellow solid in

1 89% yield (16.7 mg, 0.022 mmol). H NMR (300 MHz, CDCl3): δ = -0.04 (s, 3H) 0.08 (s,

3H), 0.89 (s, 9H), 1.16-1.19 (t, J = 7.2, 6H), 1.44 (s, 9H), 1.46-1.90 (m, 9H), 2.41 (s, 3H),

2.80-3.06 (m, 4H), 3.37-3.42 (q, J = 7.2 Hz, 3H), 3.58-3.63 (m, 2H), 4.90-4.92 (dd, Ja = 6.4

Hz, Jb = 4.0 Hz, 1H), 6.12 (s, 1H), 6.44-6.45 (d, J = 2.8 Hz, 1H), 6.52-6.55 (dd, Ja = 9.2 Hz,

Jb = 2.8 Hz, 1H), 7.26-7.29 (d, J = 7.8 Hz, 2H), 7.47-7.49 (d, J = 9.2 Hz, 1H), 7.66-7.69 (d, J

= 7.8 Hz, 2H).

tert-Butyl (4-((N-(4-((tert-butyldimethylsilyl)oxy)-4-(7-(diethylamino)-2-thioxo-2H- chromen-4-yl)butyl)-4-methylphenyl)sulfonamido)butyl)carbamate (190). Lawesson’s reagent (1.5 eq., 11 mg, 0.027 mmol) was added to a solution of the coumarin 189 (1 eq., 13 mg, 0.018 mmol) in dry toluene (0.3 ml). The reaction mixture was heated to 90 ˚C for 24 hours and concentrated under reduced pressure. The crude product was purified by silica gel

155

chromatography with hexane/EtOAc (2:1) as the eluent, affording 190 as an orange solid in

1 37% yield (5 mg, 0.007 mmol). H NMR (300 MHz, CDCl3): δ = -0.04 (s, 3H) 0.09 (s, 3H),

0.90 (s, 9H), 1.21-1.26 (t, J = 7.2, 6H), 1.44 (s, 9H), 1.49-1.95 (m, 9H), 2.47 (s, 3H), 2.82-

3.09 (m, 4H), 3.40-3.45 (q, J = 7.2 Hz, 3H), 3.65-3.73 (m, 2H), 4.88-4.91 (m, 1H), 6.84-6.89

(m, 2H), 7.17 (s, 1H), 7.23-7.26 (d, J = 7.8 Hz, 2H), 7.63-7.69 (m, 3H).

N-(4-Aminobutyl)-4-methylbenzenesulfonamide TFA salt (192). TFA (10 eq., 0.16 ml,

2.04 mmol) and Et3SiH (2 eq., 64 µl, 0.4 mmol) were added to a solution of the protected amine 188 (1 eq, 70 mg, 0.2 mmol) in DCM (3.2 ml). The reaction mixture was stirred for 2 hours at room temperature and was concentrated under reduced pressure. The crude product

192 was confirmed by NMR and used without further purification (68 mg, 0.2 mmol). 1H

NMR (300 MHz, DMSO): δ = 1.44-1.60 (m, 4H), 2.36 (s, 3H), 2.75-3.05 (m, 4H), 7.30-7.33

(d, J = 7.8 Hz, 2H), 7.64-7.67 (d, J = 7.8 Hz, 2H). The analytical data matched reported results.241

2-Chloro-N-(4-((4-methylphenyl)sulfonamido)butyl)acetamide (193). DIPEA (1.5 eq., 52

µl, 0.3 mmol) was added to a solution of the amine 192 (1 eq., 68 mg, 0.2 mmol) in DCM (4 ml). The mixture was stirred at room temperature for 10 minutes and cooled to 0˚C.

Chloroacetyl chloride (3 eq., 48 µl, 0.6 mmol) was added and the resulting mixture was stirred at 0 ˚C for 10 minutes. The reaction was quenched with sat. NaHCO3 (1 ml) and the mixture was extracted with DCM (3 × 2 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, and concentrated, affording 193 as a light yellow oil in 96%

156

yield (45 mg, 0.14 mmol). 1H NMR (300 MHz, DMSO): δ = 1.38-1.54 (m, 4H), 2.42 (s, 3H),

2.73-3.02 (m, 4H), 4.01 (s, 2H), 7.31-7.34 (d, J = 7.8 Hz, 2H), 7.68-7.69 (d, J = 7.8 Hz, 2H).

The analytical data matched reported results.242

4-(1-((tert-Butyldimethylsilyl)oxy)-2-nitroethyl)-7-(diethylamino)-2H-chromen-2-one

(196). TBDMSCl (1.5 eq., 110 mg, 0.73 mmol) and imidazole (1.9 eq., 60 mg, 0.9 mmol) were added to a solution of the alcohol 167 (1 eq., 140 mg, 0.46 mmol) in DMF (4.5 ml). The reaction mixture was stirred overnight at room temperature and quenched with sat. NaHCO3

(10 ml). The aqueous layer was extracted with EtOAc (3 × 5 ml). The combined organic layer was washed with water (5 ml) and brine (5 ml), dried over Na2SO4 and concentrated.

The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 196 as an off-white solid in 84% yield (162 g, 0.38 mmol). 1H NMR (300

MHz, CDCl3): δ = -0.01 (s, 3H), 0.07 (s, 3H), 0.87 (s, 9H), 1.18-1.22 (t, J = 5.4 Hz, 6H),

3.38-3.44 (q, J = 5.4 Hz, 4H), 4.51-4.53 (d, J = 4.5 Hz, 2H), 5.67-5.70 (t, J = 4.5 Hz, 1H),

6.27 (s, 1H), 6.51-6.52 (d, J = 2.1 Hz, 1H), 6.61-6.63 (dd, Ja = 6.9 Hz, Jb = 1.8 Hz, 1H), 7.49-

13 7.51 (d, J = 6.6 Hz, 1H). C NMR (400 MHz, CDCl3): δ = -5.3, -4.4, 12.8, 18.4, 25.9, 45.2,

69.7, 81.5, 98.5, 105.2, 107.4, 109.4, 124.4, 151.2, 152.8, 157.0, 162.0. HRMS-LC: m/z

+ calcd for C21H32N2O5Si [M+H] : 421.2159; found: 421.2173.

4-(1-((tert-Butyldimethylsilyl)oxy)-2-nitroethyl)-7-(diethylamino)-2H-chromene-2- thione (197). Lawesson’s reagent (1.5 eq., 61 mg, 0.15 mmol) was added to a solution of the coumarin 196 (1 eq., 42 mg, 0.1 mmol) in dry toluene (1 ml). The reaction mixture was

157

heated to 90 ˚C for 24 hours and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 197 as an orange solid in

1 79% yield (35 g, 0.08 mmol). H NMR (300 MHz, CDCl3): δ = -0.09 (s, 3H), 0.07 (s, 3H),

0.87 (s, 9H), 1.21-1.24 (t, J = 5.4 Hz, 6H), 3.41-3.46 (q, J = 5.4 Hz, 4H), 4.46-4.59 (m, 2H),

5.63-5.66 (t, J = 7.2 Hz, 1H), 6.68-6.71 (m, 2H), 7.13 (s, 1H), 7.57-7.58 (d, J = 7.2 Hz, 1H).

13 C NMR (400 MHz, CDCl3): δ = -5.8, -4.7, 12.5, 18.2, 25.6, 45.1, 69.2, 80.9, 97.9, 107.2,

110.6, 121.1, 124.4, 144.1, 151.2, 159.5, 197.0. HRMS-LC: m/z calcd for C21H32N2O4SSi

[M+H]+: 437.1930; found: 437.1933.

2-(4-(1-((tert-Butyldimethylsilyl)oxy)-2-nitroethyl)-7-(diethylamino)-2H-chromen-2- ylidene)malononitrile (198). The thiocoumarin 197 (1 eq., 74 mg, 0.17 mmol), malononitrile (1.2 eq., 13.5 mg, 1.2 mmol) and TEA (3.6 eq., 88 µl, 0.6 mmol) were dissolved in dry CH3CN (0.8 ml) and silver nitrate (2.5 eq., 72 mg, 0.43 mmol) was added.

The reaction mixture was stirred for 3 hours at room temperature and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 198 as an orange solid in 89% yield (72 g, 0.15 mmol). 1H NMR (300 MHz,

CDCl3): δ = 0.02 (s, 3H), 0.08 (s, 3H), 0.91 (s, 9H), 1.23-1.27 (t, J = 5.4, 6H), 3.44-3.49 (q, J

= 5.4 Hz, 4H), 4.51-4.53 (m, 2H), 5.71-5.74 (t, J = 4.8 Hz, 1H), 6.62-6.63 (d, J = 1.5 Hz,

1H), 6.69-6.72 (dd, Ja = 6.9 Hz, Jb = 1.5 Hz, 1H), 6.94 (s, 1H), 7.51-7.53 (d, J = 6.9 Hz, 1H).

13 C NMR (400 MHz, CDCl3): δ = -5.3, -4.6, 12.6, 18.1, 25.6, 45.2, 56.6, 68.9, 81.0, 97.9,

105.9, 106.6, 111.0, 113.6, 114.2, 124.5, 148.9, 151.9, 155.3, 171.7. HRMS-LC: m/z calcd

+ for C24H32N4O4Si [M+H] : 469.2271; found: 469.2274.

158

2-(4-(2-Amino-1-((tert-butyldimethylsilyl)oxy)ethyl)-7-(diethylamino)-2H-chromen-2- ylidene)malononitrile (199). Zinc powder (40 eq., 167 mg, 2.5 mmol) was added to a solution of the coumarin 198 (1 eq., 30 mg, 0.06 mmol) in acetic acid (2 ml). The reaction mixture was stirred at room temperature for 1 hour and filtered through celite. The solution was neutralized to pH 7.0 by sat. NaHCO3, and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with chloroform/MeOH (9:1) as the eluent, affording 199 as a red solid in 70% yield (20 mg, 0.04

1 mmol). H NMR (400 MHz, CDCl3): δ = 0.02 (s, 3H), 0.13 (s, 3H), 0.96 (s, 9H), 1.21-1.25 (t,

J = 5.4, 6H), 2.24 (s, 2H), 2.89-2.93 (m, 1H), 3.05-3.08 (m, 1H), 3.42-3.47 (q, J = 5.4 Hz,

4H), 5.06 (br, 1H), 6.59-6.60 (d, J = 1.5 Hz, 1H), 6.64-6.66 (dd, Ja = 6.9 Hz, Jb = 1.5 Hz,

13 1H), 6.88 (s, 1H), 7.49-7.51 (d, J = 6.9 Hz, 1H). C NMR (400 MHz, CDCl3): δ = -4.9, -4.5,

12.6, 18.2, 25.8, 45.0, 48.2, 55.6, 71.3, 97.6, 106.1, 106.9, 111.7, 114.2, 114.7, 125.4, 152.9,

+ 155.3, 171.9. HRMS-LC: m/z calcd for C24H34N4O2Si [M+H] : 439.2524; found: 439.2525.

Methyl-6-aminohexanoate hydrochloride (201) was prepared according to previously reported procedure.243 Thionyl chloride (1.5 eq., 0.11 ml, 1.5 mmol) was added to a solution of ɛ-amino-n-caproic acid 200 (1 eq., 131 mg, 1 mmol) in MeOH (5 ml). The reaction was heated to reflux overnight and concentrated under reduced pressure. The crude product 201 was obtained as a white solid in 96% yield (1.5 eq., 174 mg, 0.96 mmol) and used in next step without further purification. 1H NMR (400 MHz, DMSO): δ = 1.29-1.33 (m, 2 H), 1.49-

159

1.57 (m, 4H), 2.28-2.33 (t, J = 7.2 Hz, 2H), 2.68-2.77 (q, J = 6.9 Hz, 2H), 3.58 (s, 3H). The analytical data matched reported results.243

Methyl 6-(2-chloroacetamido)hexanoate (202). DIPEA (3 eq., 104 µl, 0.6 mmol) was added to a solution of the amine 201 (1 eq., 36 mg, 0.2 mmol) in DCM (2 ml), and the mixture was stirred at room temperature for 10 minutes. Chloroacetyl chloride (2 eq., 45 µl,

0.4 mmol) was added at 0 ˚C and the mixture was stirred at 0 ˚C for 10 minutes. The reaction was quenched with sat. NaHCO3 (1 ml) and the aqueous phase was extracted with DCM (3 ×

2 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4 and concentrated, affording 202 as a light yellow oil in 96% yield (42 mg, 0.19 mmol). 1H NMR

(300 MHz, CDCl3): δ = 1.29-1.67 (m, 6 H), 2.28-2.33 (t, J = 7.2 Hz, 2H), 3.25-3.33 (q, J =

13 6.9 Hz, 2H), 3.64 (s, 3H), 4.03 (s, 2H). C NMR (400 MHz, CDCl3): δ = 24.5, 26.4, 29.1,

- 33.1, 39.7, 42.8, 51.6, 165.9, 174.1. HRMS-LC: m/z calcd for C9H16ClNO3 [M-H] :

220.0735; found: 220.0743.

6-(2-Chloroacetamido)hexanoic acid (203). A 2 M solution of LiOH in water (5 eq., 0.5 ml, 1 mmol) was added to a solution of the methyl ester 202 (1 eq., 44 mg, 0.2 mmol) in

MeOH (2 ml). The mixture was stirred at room temperature for 1hour and concentrated under reduced pressure. The residue was redissolved in water (1 ml), acidified to pH 3-4 with 1 M citric acid, and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4 and concentrated, affording 203 as an off-white solid in

1 84% yield (32 mg, 0.17 mmol). H NMR (400 MHz, CD3CN): δ = 1.31-1.38 (m, 2 H), 1.47-

160

1.63 (m, 4H), 2.25-2.31 (t, J = 7.2 Hz, 2H), 3.18-3.22 (q, J = 6.8 Hz, 2H), 4.02 (s, 2H). 13C

NMR (400 MHz, CD3CN): δ = 25.1, 26.8, 29.6, 34.0, 40.0, 43.6, 167.2, 175.4. HRMS-LC:

- m/z calcd for C8H14ClNO3 [M-H] : 206.0581; found: 206.0578.

6-(2-Chloroacetamido)hexanoyl chloride (204). Oxalyl chloride (1.1 eq., 1 µl, 0.011 mmol) was added to a solution of the acid 203 (1 eq., 2 mg, 0.01 mmol) in DCM (0.1 ml), followed by 2 drops of DMF. The mixture was stirred for 30 minutes at room temperature and concentrated under reduced pressure. The crude product was confirmed by NMR and

1 used without further purification. H NMR (300 MHz, CDCl3): δ = 1.31-1.38 (m, 2 H), 1.47-

1.63 (m, 4H), 2.85-2.91 (t, J = 7.2 Hz, 2H), 3.18-3.22 (q, J = 6.8 Hz, 2H), 4.01 (s, 2H). The analytical data matched reported results.244

2,5-Dioxopyrrolidin-1-yl 6-(2-chloroacetamido)hexanoate (205). N-Hydroxysuccinimide

(1.2 eq., 2.6 mg, 0.023 mmol), EDCI (1.2 eq., 4.4 mg, 0.023 mmol), HOBt (1.5 eq., 3.9 mg,

0.028 mmol), DIPEA (1.2 eq., 4.2 µl, 0.023 mmol), and catalytic amount of DMAP were added to a solution of the acid 203 (1 eq., 4 mg, 0.02 mmol) in DCM (0.5 ml). The mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with chloroform/acetone (4:1) as the eluent, affording 205 as a white solid in 58% yield (3.4 mg, 0.011 mmol). 1H NMR (300

MHz, CDCl3): δ = 1.31-1.38 (m, 2 H), 1.47-1.63 (m, 4H), 2.65-2.61 (t, J = 7.2 Hz, 2H), 2.68

(s, 4H), 3.28-3.32 (q, J = 6.8 Hz, 2H), 4.02 (s, 2H). The analytical data matched reported results.245

161

N-(2-((tert-Butyldimethylsilyl)oxy)-2-(2-(dicyanomethylene)-7-(diethylamino)-2H- chromen-4-yl)ethyl)-6-(2-chloroacetamido)hexanamide (206). DIPEA (1.5 eq., 9.9 µl,

0.054 mmol) and HATU (1.2 eq., 13.7 mg, 0.043 mmol) were added to a solution of the amine 199 (1 eq., 15 mg, 0.036 mmol) and the acid 203 (1.2 eq., 8.9 mg, 0.043 mmol) in

THF (0.4 ml). The reaction was stirred for 1 hour at room temperature and quenched with sat. NaHCO3 (1 ml). The aqueous layer was extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with

DCM/acetone (9:1) as the eluent, affording 206 as an orange film in 44% yield (10.3 mg,

1 0.016 mmol). H NMR (300 MHz, CDCl3): δ = 0.01 (s, 3H), 0.06 (s, 3H), 0.96 (s, 9H), 1.21-

1.25 (t, J = 7.2 Hz, 6H), 1.40-1.60 (m, 6H), 2.23-2.29 (t, J = 7.8 Hz, 2H), 2.90-2.98 (m, 1H),

3.30-3.36 (m, 2H), 3.41-3.48 (q, J = 7.2 Hz, 4H), 3.81-3.91 (m, 1H), 4.05 (s, 2H), 5.18-5.20

(m, 1H), 5.96 (m, 1H), 6.59-6.60 (d, J = 3.0 Hz, 1H), 6.73-6.77 (dd, Ja = 9.3 Hz, Jb = 2.7 Hz,

13 1H), 6.91 (s, 1H), 7.87-7.91 (d, J = 9.3 Hz, 1H). C NMR (400 MHz, CDCl3): δ = -4.6, -4.7,

12.5, 18.1, 24.1, 25.8, 26.0, 28.9, 31.0, 39.5, 42.8, 44.9, 54.3, 69.3, 97.2, 105.4, 107.1, 111.1,

114.3, 114.9, 125.9, 151.8, 155.1, 166.1, 172.1, 173.8. HRMS-LC: m/z calcd for

+ C21H32N2O5Si [M+H] : 628.3086; found: 628.3058.

2-(6-(2-Chloroacetamido)hexanamido)-1-(2-(dicyanomethylene)-7-(diethylamino)-2H- chromen-4-yl)ethyl (2,5-dioxopyrrolidin-1-yl) carbonate (208). TBAF in THF (1.5 eq., 1

M, 24 µl, 0.024 mmol) was added to a solution of the silyl ether 206 (1 eq., 10.3 mg, 0.016 mmol) in THF (0.5 ml) at 0 ˚C. The reaction mixture was stirred at 0 ˚C for 1 hour and N,Nʹ-

162

disuccinimidyl carbonate (2 eq., 5.8 mg, 0.02 mmol) was added followed by DIPEA (3 eq., 4

µl, 0.03 mmol). The reaction was stirred for 2 hours at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with

Et2O/acetone (2:1) as the eluent, affording 208 as an orange foam in 32% yield (3.1 mg,

1 0.005 mmol). H NMR (400 MHz, CDCl3): δ = 1.21-1.25 (t, J = 7.2 Hz, 6H), 1.40-1.60 (m,

6H), 2.25-2.31 (m, 2H), 2.76 (s, 4H), 3.20-3.36 (m, 4H), 3.41-3.48 (m, 4H), 4.03 (s, 2H),

6.31-6.38 (m, 2H), 6.58-6.59 (d, J = 3.0 Hz, 1H), 6.72-6.78 (m, 2H), 7.91-7.93 (d, J = 9.3 Hz,

13 1H). C NMR (400 MHz, CDCl3): δ = 12.5, 24.1, 25.8, 26.3, 28.9, 29.9, 39.5, 42.4, 45.2,

54.3, 69.3, 97.2, 105.4, 107.1, 111.1, 114.3, 114.9, 125.9, 151.8, 152.2, 155.1, 166.1, 167.9,

172.1, 173.8.

4-Azidobenzaldehyde (209). Pyridinium chlorochromate (2 eq., 431 mg, 2 mmol) was added to a solution of the 4-azidobenzyl alcohol 104 (1 eq., 150 mg, 1 mmol) in DCM (5 ml).

The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 209 as an off-white solid in 85% yield (125 mg,

1 0.85 mmol). H NMR (400 MHz, CDCl3): δ = 7.14-7.16 (d, J = 8.4, 2H), 7.87-7.89 (d, J =

13 8.4, 2H), 9.94 (s, 1H). C NMR (400 MHz, CDCl3): δ = 108.7, 119.6, 131.7, 164.4, 190.8.

The analytical data matched reported results.246

1-(4-Azidophenyl)but-3-en-1-ol (210). Allyltri-n-butyl tin (1.5 eq., 0.99 ml, 3 mmol) and zinc chloride (1.5 eq., 409 mg, 3 mmol) were added to a solution of the aldehyde 209 (1 eq.,

163

294 mg, 2 mmol) in CH3CN/water (4:1, 10 ml). The reaction mixture was stirred overnight at room temperature and was concentrated under reduced pressure. The residue was taken up in water (10 ml) and extracted with DCM (3 × 10ml). The combined organic layer was washed with brine (10 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatograph with hexane/EtOAc (4:1) as the eluent, affording 210 as a white

1 solid in 97% yield (366 mg, 1.94 mmol). H NMR (400 MHz, CDCl3): δ = 2.37-2.44 (m,

2H), 4.62-4.66 (m, 1H), 5.07-5.11 (m, 2H), 5.66-5.75 (m, 1H), 6.93-6.95 (d, J = 8.4, 2H),

13 7.32-7.34 (d, J = 8.4, 2H). C NMR (400 MHz, CDCl3): δ = 43.9, 72.7, 118.8, 119.1, 127.4,

+ 134.2, 139.2, 140.7. LRMS-LC: m/z calcd for C10H11N3O [M+H] : 190.1; found: 190.7.

((1-(4-Azidophenyl)but-3-en-1-yl)oxy)(tert-butyl)dimethylsilane (211). TBDMSCl (2 eq.,

602 mg, 4 mmol) and imidazole (3 eq., 408 mg, 6 mmol) were added to a solution of the alcohol 210 (1 eq., 378 mg, 2 mmol) in DMF (2 ml). The reaction mixture was stirred overnight at room temperature and quenched with sat. NaHCO3 (5 ml). The aqueous layer was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (5 ml) and brine (5 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 211 as a white

1 solid in 98% yield (588 g, 1.9 mmol). H NMR (400 MHz, CDCl3): δ = -0.13 (s, 3H), 0.03 (s,

3H), 0.87 (s, 9H), 2.33-2.45 (m, 2H), 4.64-4.68 (m, 1H), 4.97-5.02 (m, 2H), 5.69-5.78 (m,

13 1H), 6.96-6.98 (d, J = 8.4, 2H), 7.27-7.29 (d, J = 8.4, 2H). C NMR (400 MHz, CDCl3): δ =

-4.8, 18.3, 25.9, 45.6, 74.7, 117.3, 118.8, 127.5, 134.3, 139.5, 142.1. LRMS-LC: m/z calcd

+ for C16H25N3OSi [M+H] : 304.2; found: 304.1.

164

4-(4-Azidophenyl)-4-((tert-butyldimethylsilyl)oxy)butan-1-ol (212). A 2M solution of

BH3•Me2S complex in THF (10 eq., 3.3 ml, 6.6 mmol) was added to a solution of the alkene

211 (1 eq., 200 mg, 0.66 mmol) in THF (1.5 ml) at 0 ˚C, and the mixture was stirred for 3 hours at 0 ˚C. A 3 M aq. NaOH (6.7 eq., 1.5 ml, 4.4 mmol) and 30% hydrogen peroxide in water (1.8 ml/mmol, 1.5 ml) were added. The mixture was warm to room temperature over 2 hours and the aqueous phase was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over Na2SO4 and concentrated.

The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 212 as a colorless foam in 46% yield (98 g, 0.30 mmol).1H NMR (400

MHz, CDCl3): δ = -0.14 (s, 3H), 0.03 (s, 3H), 0.88 (s, 9H), 1.52-1.77 (m, 5H), 3.59-3.63 (m,

2H), 4.68-4.71 (t, J = 5.6, 1H), 6.96-6.98 (d, J = 8.4, 2H), 7.28-7.29 (d, J = 8.4, 2H). 13C

NMR (400 MHz, CDCl3): δ = -4.6, -4.2, 18.6, 26.2, 28.9, 37.6, 63.4, 74.7, 119.2, 127.7,

+ 138.9, 142.6. LRMS-LC: m/z calcd for C16H27N3O2Si [M+H] : 322.2; found: 322.2.

4-(4-Azidophenyl)-4-((tert-butyldimethylsilyl)oxy)butyl (2-(2-chloroacetamido)ethyl)- carbamate (213). 1,1ʹ-Carbonyldiimidazole (2.5 eq., 230 mg, 1.42 mmol) was added to a solution of the alcohol 212 (1 eq., 182 mg, 0.6 mmol) in DCM (6 ml). The reaction mixture was stirred at room temperature for 3 hours and diluted with DCM (4 ml). The solution was washed with water (5 ml) and brine (5 ml), dried over Na2SO4 and concentrated. The crude product was confirmed by 1H NMR and used in next step without further purification. The activated alcohol was dissolved in DCM (26 ml) and ethylene diamine (5.3 eq., 0.22 ml, 3.22 mmol) was added. The resulting mixture was stirred for 2 hours at room temperature and

165

concentrated under reduced pressure. The crude product was confirmed by 1H NMR and subjected to next step without further purification. The crude amine was dissolved in DCM

(12 ml) and DIPEA (0.5 eq., 55 µl, 0.32 mmol) was added. The mixture was stirred at room temperature for 10 minutes and cooled to 0˚C. Chloroacetyl chloride (3.2 eq., 150 µl, 1.9 mmol) was added and the mixture was stirred at 0˚C for 10 minutes. The reaction was quenched with sat. NaHCO3 (10 ml) and the aqueous layer was extracted with DCM (3 × 10 ml). The combined organic layer was washed with brine (10 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 213 as an off-white solid in 77% yield (224 mg,

1 0.47 mmol). H NMR (400 MHz, CDCl3): δ = -0.06 (s, 3H), 0.06 (s, 3H), 0.89 (s, 9H), 1.70-

1.84 (m, 4H), 3.34-3.37 (m, 2H), 3.42-3.47 (m, 2H), 4.01 (s, 2H), 4.05-4.07 (t, J = 5.6, 2H),

4.65-4.68 (t, J = 6.8, 1H), 5.15 (br, 1H), 6.93-6.95 (d, J = 8.4, 2H), 7.17 (br, 1H), 7.34-7.36

+ (d, J = 8.4, 2H). LRMS-LC: m/z calcd for C21H34ClN5O4Si [M+H] : 484.2; found: 484.8.

4-(4-Azidophenyl)-4-hydroxybutyl (2-(2-chloroacetamido)ethyl)carbamate (214). TBAF in THF (1.5 eq., 1 M, 0.72 ml, 0.72 mmol) was added to a solution of the silyl ether 213 (1 eq., 224 mg, 0.48 mmol) in THF (11 ml). The reaction was stirred at room temperature for 1 hour and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (1:1) as the eluent, affording 214 as a white solid in 92%

1 yield (156 mg, 0.44 mmol). H NMR (400 MHz, CDCl3): δ = 1.72-1.84 (m, 4H),3.32-3.35

(m, 2H), 3.40-3.33 (m, 2H), 4.02 (s, 2H), 4.08-4.10 (t, J = 5.6, 2H), 4.67-4.70 (t, J = 6.8, 1H),

5.17 (br, 1H), 6.98-7.00 (d, J = 8.4, 2H), 7.12 (br, 1H), 7.31-7.33 (d, J = 8.4, 2H). 13C NMR

166

(400 MHz, CDCl3): δ = 25.8, 35.7, 40.9, 41.0, 42.9, 65.4, 73.8, 119.5, 127.7, 139.6, 141.7,

+ 157.8, 167.4. LRMS-LC: m/z calcd for C15H20ClN5O4 [M+H] : 370.1; found: 370.7.

4-(4-Azidophenyl)-4-((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)butyl (2-(2-chloroacetamido)ethyl)carbamate (215). N,Nʹ-Disuccinimidyl carbonate (5 eq., 223 mg, 0.87 mmol) and a catalytic amount of DMAP were added to a solution of the alcohol 214 (1 eq.,

62 mg, 0.17 mmol) in DCM (3 ml). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (1:1) as the eluent, affording 215 as a white

1 solid in 71% yield (63 mg, 0.12 mmol). H NMR (400 MHz, CDCl3): δ = 1.64-1.74 (m, 4H),

2.80 (s, 4H), 3.28-3.36 (m, 2H), 3.37-3.42 (m, 2H), 4.04 (s, 2H), 4.05-4.07 (t, J = 5.6, 2H),

4.54-4.57 (t, J = 6.8, 1H), 5.62 (br, 1H), 6.99-7.01 (d, J = 8.4, 2H), 7.09 (br, 1H), 7.33-7.35

13 (d, J = 8.4, 2H). C NMR (400 MHz, CDCl3): δ = 25.4, 26.7, 36.7, 42.9, 43.0, 44.9, 62.3,

71.8, 119.4, 127.1, 139.4, 142.0, 152.8, 157.8, 167.4, 173.4.

167

CHAPTER 5: Engineering Protein Function through Unnatural Amino Acid

Mutagenesis

5.1 Post transcriptional modification of protein

The genetic code of eukaryotes consists of the same 20 common amino acids.247 The functional groups in these 20 amino acids, however, limited the flexibility in the design and improvement of protein functions. To overcome this drawback, chemical modifications have been made on amino acid residues. Early modifications were based on the special reactivity of certain amino acids, in which no genetic manipulation is needed.248 For instance, the thiol group of cysteine can react with maleimides,249 iodoacetamide,250 allyl halides,251 and dithiols252 under mild conditions. Taking advantage of this nucleophilic side chain, many active molecules and probes have been introduced onto cysteine.253 Lysine, as another good nucleophile, has been modified through reactions with N-hydroxysuccinimide (NHS) esters, isothiocyanates, aldehydes,254 and other electrophiles.248, 255 Modifications on other residues include the oxidation of the hydroxyl groups on serine and threonine,256 substitution on tyrosine,257, 258 and cyclization of arginine259 et al. However, these post translational modifications rely on the high reactivity of reagents, thus, poor selectivity and limited efficiency are potential problems. Additionally, these non-specific modifications may cause dramatic changes in protein folding, leading to unexpected problems.

The development of the stepwise solid-phase peptide synthesis (SPPS) and semisynthesis through peptide ligation allow a large variety of chemical synthesized amino acids to be incorporated into peptides.260 Amino acids that are not among the 20 canonical amino acids found in nature are named unnatural amino acids (UAAs). These UAAs bring

168

new functions to peptides and have shown great potential in the development of new drugs.

Although SPPS and peptide ligation are successful in vitro, these chemical methods cannot be easily applied in vivo due to the inherent extracellular nature of these synthetic techniques.261 Moreover, the high cost and low yields in peptide synthesis, the restriction on ligation sites, and the constrains on protein folding limit its application in the synthesis of large proteins.262 Efforts have been made to develop novel ligation techniques and total chemical synthesis of proteins.263

5.2 Unnatural amino acid mutagenesis

Methods using the existing biosynthetic machinery in cells have been developed and tested both in vitro and in vivo to site-specifically incorporate unnatural amino acids.262 The most popular method is via an exogenous transfer RNA (tRNA) / aminoacyl-tRNA synthetase (aaRSs) pair. The 20 canonical amino acids are esterified to their cognate tRNA through specific aaRSs and are incorporated according to the anticodon-codon recognition between tRNA and mRNA (Figure 5.1B).264 UAA takes advantage of unique codons and exogenous tRNAs targeting at these stop codons for incorporation. The unique codons can be one of the three stop codons on mRNA (ochre UAA, amber UAG, and opal UGA) which serve to terminate amino acid incorporation in nature,261 or a frameshift suppression such as quadruplet codons.265 The exogenous tRNAs, on the other hand, need to be functional in the host organism and orthogonal to other endogenous tRNA / aaRSs counter pairs to selectively recognize UAAs without incorporating canonical amino acids. These orthogonal tRNA / aaRS pairs can be imported from a different domain of life,266 but most of the tRNA / aaRS

169

pairs are evolved through mutations followed by selections.262, 267 The evolved orthogonal tRNA / aaRS pair and target gene with the corresponding unique codon are introduced to the host organism fed with UAA. Once the orthogonal tRNA is charged with the UAA by the aaRS, the UAA is transferred to the ribosome and incorporated into the growing peptide chain just like canonical amino acids (Figure 5.1A). This method provides a general approach to incorporate UAAs into protein in relatively high yield. Since the first report site- specific incorporation of UAAs in 1989,268 over seventy UAAs including phenylalanine, tyrosine and lysine analogs have been incorporated.262 These UAAs contain a variety of functional groups and introduced many useful physical, chemical, and biological properties into proteins, making UAAs an important tool in the study of protein function and enzymatic mechanisms.

170

A B orthogonal endogenous tRNA tRNA

canonical evolved UAA amino acid aaRS

ATP ATP

endogenous orthogonal tRNA/aaRS tRNA/aaRS

orthogonal tRNA

AMP + PPi AMP + PPi

ribosome target mRNA gene unique codon

Figure 5.1 Incorporation of UAA through an orthogonal tRNA-aaRSs pair 262, 269

Pyrrolysyl-tRNA (tRNAPyl) and pyrrolysyl-tRNA synthetases (PylRS) found in

Methanosarcinaceae can selectively incorporate pyrrolysine (216, Figure 5.2) in respond to the amber stop codon UAG.270 This Methanosarcina barkeri tRNApyl-PylRS pair as well as some of its mutants were reported to be fully functional in E. coli yet orthogonal to the endogenous tRNA-aaRSs pairs.271 Through the tRNApyl-PylRS pair and its mutations, lysine analogues with novel functional groups such as azido group, alkyl group, thiol group, keto group, diazirine group and nobornene group (217-225) were introduced into proteins serving

171

as reactive sites for Staudinger Ligation, click reaction, native chemical ligation, bio conjugation and photo crosslinking.264, 272-277

Figure 5.2 Chemical structures of selected encoded unnatural lysine amino acids by tRNApyl-PylRS pairs.

Furthermore, some tRNApyl-PylRS pairs have been applied to incorporate unnatural lysine derivatives including photo-caged lysines (226-227) in yeast and mammalian cells.278-

280 Unnatural tyrosine derivatives such as o-nitrobenzyl caged tyrosine (228) have also been incorporated in vivo through tRNApyl-PylRS pairs.281 These photo-caged unnatural amino acids allow for directly control of protein function by light (Figure 5.3). With caged UAA

172

incorporated at the active site of a protein, the caging group (red) either interferes with protein folding or blocks the binding of substrate, thus inactivates the protein. After light irradiation which cleaves the caging group, the protein regains its activity and binds to its substrate. Compared with gene silencing approaches targeting at DNA and RNA, this level of regulation provides the best spatial and temporal resolution for controlling protein function.

substrate

caging group

caged protein active protein caging group

Figure 5.3 Photo-regulation over protein function.

To further explore protein engineering with UAAs, CHAPTER 6 presents the synthesis of novel photo-caged and small molecule-triggered amino acids, as well as lysines bearing physical probes such as fluorescent and isotopic labels.

173

CHAPTER 6: Synthesis of Unnatural Amino Acids

6.1 Synthsis of two-photon caged lysine

As mentioned in CHAPTER 5, photocaged unnatural amino acids (UAAs) have been incorporated into proteins as phototriggers of protein functions.278, 279 However, the caging groups used are o-nitrobenzyl derivatives that require UV irradiation of 365 nm for their cleavage which may cause potential photodamage over time and has limited spatial focus. To overcome these drawbacks, a 3-nitro-2-ethyldibenzofuran (NDBF) caged lysine (231) was synthesized in an effort to control protein activity with IR irradiation.

The synthesis followed a reported procedure developed in the Deiters lab.276 The activation of 1-(3-nitrodibenzofuran-2-yl)-ethanol (149) via succinimidyl carbonate 229 was achieved in 31% yield by treatment with DSC and TEA in acetonitrile (Scheme 6.1). The resulting carbonate 229 was reacted with the α-Boc-protected lysine (Boc-Lys) in DMF, affording the protected NDBF-caged lysine 230 in 97% yield. The TFA salt of NDBF lysine

231 was obtained in 83% yield through the removal of Boc group by TFA.

Scheme 6.1 Synthesis of the NDBF lysine 231.

174

The NDBF lysine 231 has been successfully incorporated into proteins in both E. coli

(experiment was performed in Jason Chin’s lab at MRC) and mammalian cells (experiments were performed in Deiters lab by Dr. Chou and Ji Luo). The mammalian cells were transfected with a reporter plasmid in which the EGFP gene was fused to the mCherry gene through a TAG stop codon (Figure 6.1A). Without any UAA, the TAG codon terminated the protein expression and only red fluorescent of mCherry was seen (Figure 6.1B). In the cells incubated with 231, the NDBF lysine 231 was incorporated in response to the TAG codon and the EGFP protein was expressed together with mCherry. Thus, both red and green fluorescent were observed (Figure 6.1C). With the successful incorporation, this NDBF lysine 231 is now under functional investigation in the Deiters lab.

A mCherry TAG EGFP

mCherry EGFP B

- 231

+ 231

Figure 6.1 Incorporation of the NDBF lysine 231 in mammalian cells with the mCherry- TAG-EGFP reporter system (A). In the absence of NDBF lysine 231, only mCherry was expressed resulting in only red fluorescence (B). In the presence of NDBF lysine 231, both mCherry and EGFP were expressed, thus both red and green fluorescence was observed (C).

175

While the NDBF lysine 231 and other photocaged UAAs allow for the activation of protein function with light, a photocleavable hydroxyl acid 232 was synthesized which we hope can inactivate protein function through light induced protein cleavage upon incorporation. The initial attempts to make this hydroxyl acid 232 followed a reported procedure (Scheme 6.2).282 The commercially available 2-nitrobenzaldehyde (156) was treated with potassium cyanide followed by hydrochloric acid affording the hydroxyl acid

232 in 14% yield. The low yield was due to the problematic recrystallization purification. To simplify the purification, the hydroxyl acid methyl ester 233 was obtained in 68% yield from

2-nitrobenzaldehyde (156) where the hydroxyl group was installed followed by the formation of a methyl ester.283 The methyl ester was then hydrolyzed by 6 M HCl in water delivering the hydroxyl acid 232 in 90% yield of which the incorporation in E.Coli is under investigation in the Deiters lab by Jihe Liu.284

Scheme 6.2 Synthesis of photo-cleavable hydroxyl acid 232.

176

6.2 Synthesis of azido lysine and tyrosine analogs

Apart from light, small molecules are also important triggers of protein function.1 The existing small molecule regulatory approaches to biological processes rely on molecular switches, which recognize the changes of small molecule effector binding and transduce it into downstream outputs. The major challenge in this approach is that a specific molecule needs to be synthesized and identified as a small molecule effector for each protein of interest. Although some artificial molecular switches have been developed that provide better general applicability,285, 286 these artificial switches bear slower kinetics compared to natural switches.1 Inspired by the photocaging approach, UAAs were designed to bear organic azides which are labile to nontoxic phosphines and have been used to control the biological active molecules (95-98) in Figure 2.5.175, 177, 178, 287-289 The small molecule-triggered azide UAAs can be readily incorporated into any protein of interest through PyltRNA-PylRS pairs, and regulate protein activity in response to phosphine reduction.

6.2.1 Development of the azidomethylene and the azidoethyl lysine and tyrosine analogs

The azidomethylene (AzM) group has been used to control the fluorescence of rhodamine and coumarin in oligonucleotide detection,175, 176 and considering its small size the

AzM modified UAAs are likely to be recognized by the PylRS as to get incorporated into proteins. An AzM lysine (234, Figure 6.2) was designed, as well as a more stable azidoethyl

(AzE) lysine (235).290

177

Figure 6.2 Proposed structures of the AzM- and the AzE-modified lysines 234 and 235.

To test the cleavage of AzM carbamate upon PPh3 reduction, a model study was conducted with the AzM naphthylmethylamine 238 (Scheme 6.3). The synthesis commenced with the reaction of 1-naphthylmethylamine (236) and chloromethyl chloroformate in the presence of triethylamine to give the carbamate 237 in 57% yield.174 The resulting carbamate

237 was then treated by sodium azide affording the AzM naphthylmethylamine 238 in 94% yield. The cleavage of the AzM group was carried out with 0.1 mM of 238 and 0.11 mM of

PPh3 in a tris-HCl buffer at pH 7.2 The mixture was stirred in the dark at room temperature and the reaction was followed by TLC. The AzM naphthylmethylamine 238 disappeared 5 hours after the addition of PPh3. After 10 hours, only free naphthylmethylamine 236 and phosphorus byproduct were observed on TLC. The cleavage of the AzM group could not be followed by HPLC due to the acid sensitivity.

178

Scheme 6.3 Synthesis and cleavage of the AzM naphthylmethylamine 238.

The initial attempt to make AzM lysine 234 started with the commercially available

Boc Lys OMe 239, following a similar approach as the AzM naphthylmethylamine (Scheme

6.4). The first step was to treat Boc Lys OMe 239 with chloromethyl chloroformate and TEA affording the carbamate 240 in 46% yield. This was followed by a substitution of the chloride with azide using NaN3 in DMF to give the AzM lysine 241 (90% yield). The subsequent deprotection of either the Boc group on the amine or the methyl group on the carboxylic acid of 241 was unsuccessful due to the instability of the AzM carbamate.

Scheme 6.4 Attempts to make the AzM lysine 234 from Boc Lys 239.

179

The staring material was changed to Fmoc Lys 242 since the cleavage of the Fmoc group is milder compared to the cleavage of the Boc group (Scheme 6.5).291 The carboxyl group in the commercial available Fmoc Lys 242 was masked through the formation of a

292 methyl ester 243 (SOCl2, methanol, 90% yield). The Fmoc-protected AzM lysine 245 was obtained by treatment of the methylester 243 with chloromethyl chloroformate in the presence of potassium carbonate, followed by an azide displacement of the chloride.

Cleavage of the Fmoc group in 245 delivered the methyl ester of AzM lysine 246 in 79% yield (20% piperidine, DCM).291 As shown in Figure 6.3, this AzM 246 was successfully incorporated into sfGFP by the wild-type PylRS in E. coli, where the methyl group can be cleaved in situ (experiment was performed by Jihe Liu in the Deiters lab).293 The alloc lysine

247 was used as positive control. The incorporation of AzM lysine 246 in mammalian cells, however, lead to unexpected toxicity and resulted in cell death (experiment was performed by Ji Luo in the Deiters lab).

Scheme 6.5 Synthesis of the AzM lysine 246 from Fmoc Lys 242.

180

UAA - UAA 247 246 30 KDa

25 KDa

Figure 6.3 The SDS-PAGE of sfGFP bearing the AzM lysine 246 incorporated by the wild- type tRNApyl-PylRS pair in E. coli. The lysine 247 was used as positive control.

The reduction of the AzE carbamate in 235 was also subjected to a model study with naphthylmethylamine 236. The installation of chloromethyl carbamate was achieved in 94% yield to furnish 248, which underwent a substitution generating the AzE naphthylamine 249 in 93% yield (Scheme 6.6). To a 0.1 mM solution of this AzE naphthylamine 249 in the tris-

HCl buffer (pH 7.2) was added PPh3 (0.11 mM). The mixture was stirred in darkness at room temperature and the reaction was followed by TLC. The cleavage of the AzE group was faster than the AzM group with the disappearance of 249 in 3 hours after the addition of PPh3 and the complete recovery of naphthylmethylamine 236 in 7 hours. The AzE group is also sensitive to acid, thus the reduction could not be analyzed by HPLC.

Scheme 6.6 Synthesis and cleavage of the AzE naphthylmethylamine 249.

181

The AzE group was first applied to Boc Lys OMe 239 which was reacted with 1- chloroethyl chloroformate affording the carbamate 250 in a high yield of 84% (Scheme 6.7).

This was followed by the substitution of the chloride with the azide to produce the AzE lysine 251 in 90% yield. The cleavage of either the Boc group or the methyl ester was attempted using a variety of conditions. Owing to the same stability issue of the azido carbamate in 251, none of these attempts formed any desired product, and only lysine bearing a free ɛ-amine was observed.

Scheme 6.7 Attempts to synthesize the AzEK 235 from Boc Lys OMe 239.

With the knowledge from the synthesis of AzM lysine 246, the azido carbamate can survive Fmoc deprotection with piperidine. Thus the synthesis of AzE lysine 235 was attempted proceeding from the α-N-Fmoc lysine derivatives bearing different protecting groups on the carboxylic acid including 9-Fluorenemethyl (Fm), allyl and an acetate methyl group (Scheme 6.8). The initial step was the protection of the ɛ-amine group on Fmoc Lys

182

242 with either a Boc (252) or a Cbz (253) group in high yields (79-87%).294, 295 This was followed by the installation of above protecting groups on the carboxylic acid affording the esters 254-256.296 The subsequent deprotection was carried out either by TFA in case of Boc group (57-77% yields) or by Pd-catalyzed hydrogenation for Cbz group (96% yield) to deliver Fmoc Lys esters 257-259.297 The formation of carbamate 260-262 suffered from low yields (23-43%), furthermore, the subsequent treatment with NaN3 resulted in no azide product in any case. As the last attempt, the methyl ester 243 was tested and the carbamate

263 was obtained in 94% yield. The chloride in 263 was subtituted by NaN3 to afford the azide 264, which was treated with piperidine cleaving the Fmoc group and forming the AzE lysine 265 in 79% yield. The incorporation of this AzE lysine 265 was accomplished in E. coli and the SDS-PAGE is shown in Figure 6.4 (the experiment was performed by Jihe Liu in the Deiters lab).

183

Scheme 6.8 Attempts to synthesize AzE lysine 235 with via different esters. Fm = 9- fluorenemethyl.

UAA 247 - UAA 265 30 KDa

25 KDa

Figure 6.4 The SDS-PAGE of sfGFP bearing the AzE lysine 265 incorporated by the wild- type PylRS in E. coli. The lysine 247 served as positive control.

Compared to the AzM lysine 246, the AzE lysine 247 provides the following two advantages. First, the cleavage of the AzE group by PPh3 proceeded faster based on the model study with naphthylmethylamine 238 and 249. Second, the AzE lysine 265 provided a higher expression yield of sfGFP when been tested in E. coli. The incorporation of AzE

184

lysine 265 in mammalian cells as well as its biological applications are currently being pursued in the Deiters lab.

Meanwhile, a AzM protected tyrosine was designed to expand our panel of small- molecule regulatory tools. Taking advantage of a reported route with minor modifications,180 the AzM tyrosine 276 was synthesized and investigated as a phosphine-trigger to protein functions. The removal of the AzM group from phenol was first tested using a coumarin fluorophore substrate. It has been shown that the 7-hydroxy-4-methylcoumarin is non- fluorescent when the phenol group is protected, and a fluorescent signal can be produced upon deprotection.176, 298 The synthesis of AzM coumarin 271 began with the condensation of commercially available resorcinol (266) with ethyl-4-chloroacetoacetate (267) to form the coumarin ring structure of 268 in 83% yield (Scheme 6.9).299 The acetylation of chloride gave 269 (KOAc, DMF, 90% yield),300 which was deprotonated by KOtBu and reacted with chloromethyl methylsulfide in the presence of NaI to afford the thioether 270 in 24% yield.180 The thioether 270 was then subjected to an in situ chlorination by NCS followed by a subsequent substitution with NaN3, delivering the AzM coumarin 271 in 23% yield over two steps.180 The AzM coumarin 271 was dissolved in a 9:1 mixture of MeOH and PBS buffer (pH 7.4) at a concentration of 10 mM, and treated with triphenylphosphine (250 mM).

The fluorescence of the reaction mixture was recorded on a plate reader with an excitation at

360 nm and an emission at 480 nm (Figure 6.5). The fluorescence of the AzM coumarin 271 was only 20% of the coumarin 269 fluorescence in the absence of PPh3, which proved the quench of fluorescence with the AzM group. A full recovery of fluorescence was observed in

2 hours and the release of the free coumarin 269 was confirmed by NMR.

185

Scheme 6.9 Synthesis of AzM coumarin 271.

100 90 80 70 60 50 40 30 20

fluorescence recovery/% fluorescence 10 0 0 0.5 1 1.5 2 time/hr

Figure 6.5 PPh3 reduction of the AzM coumarin 271 (10 mM) in a 9:1 mixture of MeOH/PBS buffer (pH = 7.4). The reaction was followed by fluorescence recovery (360 nm/480 nm).

With the successful cleavage of the AzM group on coumarin 271, the AzM tyrosine

276 was synthesized from the commercially available Boc Tyr OMe 272 following a published procedure.180 The thioether was installed on the phenol group of 272 in 71% yield,

186

which was transformed to an azidomethylene group delivering the protected AzM tyrosine

274 in 51% yield over three-steps. The methyl ester was hydrolyzed with 1 M LiOH in

MeOH/water affording the acid 275 in 87% yield (Scheme 6.10).236 The removal of the Boc group by 5% TFA in DCM followed by a salt exchange delivered the final product 276 as an

HCl salt which provides better solubility in water.277 The incorporation of this AzMY 276 was achieved in E. coli with the double-mutated PylRS EV-16 (N311A/C313A) in the

Deiters lab and the SDS-PAGE is shown in Figure 6.6, in which the propargyl tyrosine 277 serves as a positive control. The subsequent tests in mammalian cells are ongoing in the

Deiters lab.

Scheme 6.10 Synthesis of AzMY 276.

UAA - UAA 277 276 30 KDa

25 KDa

Figure 6.6 The SDS-PAGE of sfGFP bearing the AzM tyrosine 276 incorporated by the EV- 16 PylRS in E. coli. The tyrosine 277 was used as positive control.

187

6.2.2 Synthesis of ortho-azidobenzyl (oAzBn) and para-azidobenzyl (pAzBn) lysine analogs

The application of para-azidobenzyl (pAzBn) group as a regulator of biological function has been reported before,177, 178 and ortho-azidobenzyl (oAzBn) lysine has been incorporated by Yokoyama et al. using the mutated PylRS (Y384F/Y306A).264 Thus the oAzBnK and pAzBnK were synthesized and selected to be potential regulatory elements of protein functions.

The reduction of the oAzBn group with phosphines was first tested on 1- naphthylmethylamine 236 and analyzed by HPLC. The synthesis of the oAzBn naphthylmethyl amine 280 proceeded from the commercial available ortho-aminobenzyl alcohol 278 (Scheme 6.11). The amino group in 278 was converted into an azide through treatment with sodium nitrite followed by sodium azide in 76% yield.182 The resulting alcohol 279 was activated by diphosgene yielding a chloroformate intermediate,279 which was confirmed by 1H NMR, and coupled to naphthylmethylamine 236 to give the final product 280. The oAzBn naphthylmethylamine 280 (0.1 mM) and a water soluble phosphine, triphenylphosphine-3,3′,3′′-trisulfonic acid trisodium salt (TPPTS, 0.2 mM), were dissolved in PBS buffer at pH 7.4. The HPLC data confirmed the recovery of the naphthylmethyl amine 236 10 hours after the addition of TPPTS (Figure 6.7). A higher equivlent of phosphine than the one stated can accelerate the Staudinger reduction which shall be analyzed in the future.301

188

Scheme 6.11 Synthesis and decaging of oAzBn naphthylmethyl amine 280.

A. oAzBn naphthylemethylamine

B. 1-naphthylemethylamine

C. oAzBn naphthylemethylamine + TPPTS

Figure 6.7 HPLC analysis of oAzBn naphthylmethylamine 280 cleavage upon TPPTS reduction.

189

Although TPPTS was efficient in reducing 280, the high polarity makes TPPTS hard

302 to cross cell membrances. The PPh3 used to reduce AzM and AzE groups above, however, has limited solubility in aqueous solutions. Thus a screen of different phosphines was conducted to determine the most suitable one for the Staudinger reaction. To this end, a more direct way to analyze the reaction kinetics is favored. In precious reports, the AzM protected rhodamine 96 (Figure 2.5) have been developed as reduction-triggered fluorescent reporter.175 Taking advantage of the commercially available rhodamine 110 (281) whose fluorescence is known to be quenched through acylation on both amines,303 the oAzBn rhodamine 287 was synthesized.

Treatment of rhodamine 110 (281) with trifluoroacetic anhydride (TFAA) generated the protected rhodamine 282 in 64% yield (Scheme 6.12). The lactone ring in 282 was reduced by hydrogen gas in the presence of Pd/C to give the acid 283 (97% yield) which was protected by the PMB group under DCC coupling conditions to form the ester 284 in 53% yield. The amine 285 was generated upon cleavage of the trifluoroacetyl group in 284 with hydroxylamine in 67% yield. The amine 285 was reacted with chloroformate 294 delivering the carbamate 286 in 36% yield, followed by the deprotection of PMB group with DDQ furnishing the oAzBn rhodamine 287 in 74% yield.

190

Scheme 6.12 Synthesis of the oAzBn rhodamine 287.

The oAzBn rhodamine 287 was subjected to the reduction by a variety of phosphines,

302 including PPh3, TPPTS, 2-(diphenylphosphino)benzoic acid (2-DPBA), 4-

(diphenylphosphino)benzoic acid (4-DPBA),302 and tris(hydroxypropyl)phosphine (THP).304

To a 10 µM solution of oAzBn rhodamine 287 in PBS buffer (pH 7) was added one of above phosphines in 25 equivalent (0.25 mM), and the reduction was monitored by the fluorescence of rhodamine 110 (281) (Figure 6.8). The PPh3, 2-DPBA and 4-DBPA all give promising results with a 15-30 fold fluorescence increase in 30 hours after the addition of phosphines.

The AzM and AzE modified rhodamine can provide a faster fluorescent recovery after the addition of phosphines compared to the AzBn rhodamine, which shall be tested in the future.

Although significant fluorescence increases were observed, the recovery of fluorescence was

191

less than 30% compared to rhodamine 110 (281). This is consistent with the previously reported low fluorescent recovery in the photolysis of caged rhodamine derivatives owing to the generation of non-fluorescent byproduct.305, 306 While the fluorescence recovery of AzBn rhodamine is not ideal and the synthesis is relatively long, the AzBn coumarines 290-291 were synthesized and used in the kinetic studies.

20 18 16 14 12 PPh3 10 TPPTS 8 2-DPBA 6 4-DPBA 4

relative fluorescence/%relative 2 THP 0 0 10 20 30 time/hr

Figure 6.8 Fluorescence (ex 495 nm / em 520 nm) of oAzBn rhodamine 287 reduction with different phosphines. The reductions were carried out in PBS buffer (pH 7) with 287 at a 10 µM concentration and 25 equivalents (0.25mM) of corresponding phosphine.

To install the AzBn groups on the phenol of coumarin 269 via a carbamate functionality, an aminomethoxyl linker was introduced to the AzBn alcohols 279 and 104

(Scheme 6.13). Starting with the activation of alcohols through diphosgene treatment, the chloroformate intermediates were obtained. The intermediates were reacted with NH4OH followed by paraformaldehyde, delivering the aminomethanols 288-289 in low to medium yields over three steps. These aminomethanols were tosylated and substituted with the deprotonated coumarin phenol 269 to give the AzBn coumarins 290-291.

192

Scheme 6.13 Synthesis of AzBn coumarins 290-291.

The oAzBn coumarin 290 was then used to conduct a phosphine screen for the

Staudinger reduction. The oAzBn was dissolved in a 9:1 mixture of MeOH and PBS buffer

(pH 7.4) at a 10 mM concentration. Then 25 equivalents of different phosphines, including

TPP, 2-DPBA and 4-DPBA, which were promising in the reduction of oAzBn rhodamine

287, were added to a final concentration of 250 mM. The reaction was followed by the recovery of fluorescence, and PPh3 released 74% coumarin 269 in 30 hours which is the highest among the three phosphines (Figure 6.9).

193

90 80 70 60

50 PPh3 40 2DPBA 30 4DPBA

relative fluorescence/%relative 20 10 0 0 10 20 30 time/hr Figure 6.9 Fluorescence (360 nm / 480 nm) of oAzBn coumarin 290 reduction through different phosphines. The reductions were carried out in a 9:1 mixture of MeOH and PBS buffer (pH 7.4) with 290 at a 10 µM concentration and 25 equivalents (250 mM) of the corresponding phosphine.

In the reduction of pAzBn coumarin 291 (10 mM) with PPh3 in MeOH/PBS buffer, however, only 20% fluorescence recovery was obtained (Figure 6.10). Thus a solvent screen was carried out and 90% of coumarin 269 was recovered after 50 hours in a 9:1 mixture of

EtOH/PBS buffer (pH 7.4). This is possibly because the pAzBn coumarin is more soluble in

EtOH/PBS solvent compared to the other solvents tried in which suspensions were observed.

100.0 90.0 80.0 70.0 60.0 50.0 MeOH+PBS 40.0 iPrOH + PBS 30.0 EtOH + PBS

20.0 fluorescence recovery/% fluorescence 10.0 0.0 0 10 20 30 40 50 time/hr

Figure 6.10 Fluorescence (360 nm/ 480 nm) of pAzBn coumarin 291 reduction in different solvents. The reductions of 291 (10 mM) and PPh3 (25 eq., 250 mM) were carried out in a 9:1 mixture of alcohol (including MeOH, EtOH and i-PrOH) and PBS buffer (pH 7.4).

194

It is noteworthy that the reduction of the AzBn coumarin (290-291) is not a good mimic of the Staudinger reduction in biological system. Firstly, organic solvents were used in above tests due to the poor solubility of 290-291 in aqueous solution. More importantly, the cell permeability of the phosphines cannot be predicted in these model studies. On the positive side, however, the cleavage of AzBn coumarin (290-291) proved that the AzBn groups can be cleaved by phosphines in good yields.

Thus, the corresponding lysine analogs (299 and 302) were synthesized (Scheme

6.14). The initial attempt was the activation of the alcohol 279 and 104 with 4-nitrophenyl chloroformate, which was used in the synthesis of the reported pAzBn prodrugs,177, 178 affording the carbonate 292-293 in 74-79% yield. This was followed by the coupling of the carbonate 292-293 to Boc Lys resulting in the Boc protected AzBn lysine 296-297 in medium to high yields, but the purification was difficult due to the similar solubility of the product 296-297 and the 4-nitrophenol byproduct. As an alternative, diphosgene was applied to activate the alcohol 279 and 104. The resulting chloroformate intermediates (294-295) were reacted with Boc Lys in DMF to give the protected AzBn lysine 296-297 (82-88% yield).

195

Scheme 6.14 Synthesis of the Boc protected AzBn lysine 296-297.

The Boc group in the protected oAzBn lysine 296 was cleaved with 5% TFA in the presence of Et3SiH in DCM affording the TFA salt of oAzBnK 298 in 81% yield (Scheme

6.15). This TFA salt 298, however, was toxic to E. coli, thus the HCl salt of oAzBnK 299 was obtained after a salt exchange in 93% yield. The HCl salt 299 demonstrated better solubility and less toxicity compared to the TFA salt, however, some toxicity was still observed. Since no toxicity was reported in the previous incorporation of this oAzBn lysine,264 the toxicity is likely to come from unknown, NMR-silent impurities.

Scheme 6.15 Synthesis of the TFA salt 298 and the HCl salt 299 of oAzBn lysine.

196

Thus, the route was changed to DSC activation which was accomplished through treatment with DSC in presence of TEA in CH3CN and the resulting succinimidyl carbonate

300 was purified by column chromatograph in 78% yield (Scheme 6.16). The subsequent coupling of the carbonate 300 with Boc Lys was tested in a variety of solvents and bases, among which NaHCO3 in a 1:1 mixture of H2O and DMF gave the highest yields (76%). The subsequent Boc deprotection was performed in aqueous HCl (1 M) instead of organic solvents, affording the HCl salt 299 in 72% yield.

Scheme 6.16 Synthesis of the HCl salt of oAzBn lysine 299 via succinimidyl carbonate.

The HCl salt 299 (synthesized via a succinimidyl carbonate) showed no toxicity and its incorporation was tested in mammalian cells transfected with the mCherry-TAG-EGFP reporter plasmid by Dr. Lockney (Figure 6.11A). Without the oAzBn lysine 299, only red fluorescence was observed (Figure 6.11B) while both mCherry and EGFP were expressed in the presence of oAzBnK 299 (Figure 6.11C), which indicated the successful incorporation of

197

299 in mammalian cells.

A mCherry TAG EGFP

mCherry EGFP B

- 299

+ 299

Figure 6.11 Incorporation of the oAzBn lysine 299 in mammalian cells with the mCherry- TAG-EGFP reporter system (A). In absence of 299, only mCherry was expressed resulting in only red fluorescence (B). In the presence of 299, both mCherry and EGFP were expression, thus both red and green fluorescence was observed (C). Experiment performed by Dr. Lockney.

The oAzBn lysine 299 was then incorporated into firefly luciferase at the K206 position in HEK293T cells and these cells were incubated with no phosphine, PPh3 or 2-

DPBA. In absence of phosphines, the inactive firefly luciferase produced no luminescence signal, which suggested the successful blocking of firefly luciferase activity with the oAzBn group. Meanwhile, with PPh3 and 2-DPBA treatments, chemiluminescence was observed, indicating that the oAzBn groups were cleaved through Staudinger reduction and the firefly luciferase regained its activity (Figure 6.12). This demonstrated the ability of the oAzBn

198

lysine 299 as small molecule-trigger to protein functions and more applications are been pursued in the Deiters Lab.

1400

1200

1000

800

600 uciferasesignal/A.U. l 400

200

no + TPP + 2-DPBA phosphine

Figure 6.12 Small molecule activation of firefly luciferase. HEK293T cells expressing luciferase with oAzBn lysine 299 in the K206 position, inhibiting enzymatic activity, were grown in a 96-well plate. Staudinger reduction of the oAzBn-luciferase variant to the wild type luciferase variant was performed by incubating the cells with 250 uM of PPh3 or 2- DPPB for 8 h at 37 °C, 5% CO2. The cells were then washed with 1×PBS and lysed with 1× passive lysis buffer, prior to the Bright-Glo chemiluminescence assay. Experiment performed by Dr. Lockney.

The pAzBn lysine 302 was synthesized in a similar way as the oAzBn lysine 299 starting from the pAzBn alcohol 104 which was activated by DSC to form the carbonate 301 in 56% yield (Scheme 6.17). The Boc protected pAzBn lysine 297 was obtained in 76% yield by coupling the carbonate 301 to Boc Lys with NaHCO3 as base in DMF and water (1:1).

However, the removal of the Boc group with either TFA or HCl cleaved the pAzBn group at

199

the same time. Therefore, the deprotection was carried out with formic acid in chloroform and the following addition of HCl allowed the pAzBn lysine to be purified as a HCl salt 302 in 66% yield. This pAzBnK 302 is under investigation for incorporation in the Deiters lab.

Scheme 6.17 Synthesis of the HCl salt of pAzBn lysine 302.

6.3 Synthesis of nitrobenzofuran lysine

Since the introduction of fluorescent proteins as protein tags in 1994,307 fluorescent labeling of proteins provides us a useful tool to analyze the structure, function, location and conformational changes of proteins.308-310 The application of the fluorescent protein labels however, suffers from several limitations. Due to the size of the fluorescent proteins, the fusion of fluorescent protein might disrupt the assembly and function of the target protein.311

Furthermore, the fluorescent proteins can only be added to the two ends of the target protein, thus the labeling can be unfeasible depending on the positions of the N- and C- termini in the target protein.312 The resolution of fluorescent proteins is limited especially in multicolor

200

imaging because of their broad absorption and emission spectra.313 Recently, small peptide tags with an organic fluorophore are introduced to label protein through reaction with certain amino acid side chain in proteins.314, 315 These chemical tags are relatively small and the fluorophores usually provide better photo-properties compared to fluorescent proteins.315

Another novel approach to label a protein is through biological conjugation via UAA.277, 316

This approach allows the site-specific labeling of protein with high selectivity. Meanwhile, fluorescent UAAs have also been used to introduce fluorophores to protein site-specifically, which saved the conjugation step.312, 317, 318

Apart from the dansyl, coumarin and 6-propionyl-2-(dimethylamino)naphthalene fluorophores incorporated by Schultz group,312, 317, 319-321 nitrobenzofuran (NBD) group is another widely used fluorescent group that have been applied to label biologically active molecules.322, 323 The synthesis of NBD lysine analogs with different carbon linkers is discussed here.

The initial attempts were to install the NBD group on lysine via a two-carbon linker.

The synthesis commenced with the commercially available 4-chloro-7-nitrobenzofurazan

(303), which was reacted with ethanolamine to give the hydroxylethyl NBD 304 in 72% yield (Scheme 6.18). The activation of this alcohol 304 via either carbonate or chloroformate both failed and lead to multiple spots on TLC. To eliminate possible side reactions, a methyl group was installed on the secondary amine of 304 affording the methyl hydroxylethyl NBD

305 in 93% yield.324 Meanwhile a Boc protected hydroxylethyl NBD 306 was also obtained from 304 through the treatment with di-tert-butyl dicarbonate in EtOH (97% yield).325

Surprisingly, the activation of both alcohols 305 and 306 yielded only the starting materials.

201

Scheme 6.18 Attempts to synthesize and activate the hydroxylethyl NBDs 304-306.

The hydroxylethyl linker was then installed on Boc Lys instead. Starting with the protection of the amine in ethanolamine with a Cbz group, the protected aminoalcohol 307 was generated in 40% yield (Scheme 6.19).326 This alcohol 307 was activated by diphosgene in the presence of K2CO3, followed by Boc Lys treatment in a 1:1 mixture of 1 M NaOH and

THF affording the lysine 309 in 80% yield over two steps. The hydrogenation of the Cbz group, however, led to the cleavage of the hydroxylethyl group at the same time. The protecting group was then changed to a phthalimide group which was installed on ethanolamine through treatment with phthalic anhydride in quantitative yield.327 After the diphosgene activation and a subsequent coupling with Boc Lys, the phthalimide lysine 310 was obtained in 88% yield. The treatment of phthalimide lysine 310 with hydrazine also gave

Boc Lys as the only product rather than desired aminoethyl lysine 311.132 The failure of both

202

routes suggested that the aminoethyl lysine 311 is unstable and might spontaneously cyclize to release Boc Lys.

Scheme 6.19 Attempts to synthesize the aminoethyl lysine 311.

A NBD lysine 315 bearing a urea linker was designed which is smaller in size and thus more favorable to the PylRS. To synthesize the NBD lysine 315, 4-chloro-7- nitrobenzofurazan (303) was treated with NH4OH in MeOH affording NPE-NH2 (312) which was reacted with ethyl chloroformate to give the carbamate 313 in 82% yield (Scheme

6.20).328 The subsequent coupling step was optimized through a base and solvent screen and was accomplished in 91% yield affording the protected NDB lysine 314 (Boc lysine, K2CO3,

THF/water). The HCl salt of NBD lysine 315 was delivered through treatment of the Boc protected NBD lysine 314 with 1 M HCl in 3:1 THF/dioxane in 96% yield. The incorporation of 315 is being pursued in the Deiters lab.

203

Scheme 6.20 Synthesis of the urea NBD lysine 315.

Meanwhile, since the carbamate linker was reported to be essential for the incorporation of unnatural lysine analogs through wild-type PylRS,273 a carbamate NBD lysine 316 was designed. The synthesis of 316 commenced with the reaction of the chloromethyl carbamate 240 with the NBD-NH2 312 (Scheme 21). A variety of bases and solvents were screened for this reaction and the lysine 316 was generated in 26% yield in the presence of DIPEA and TABI in DCM, which is the best condition so far. This carbamate

NBD lysine 316 can be readily tested after the removal of the methyl ester and the Boc group.

Scheme 6.21 Synthesis of the carbamate NBD lysine 316.

204

6.4 Synthesis of p-nitrobenzyl ε-15N lysine

Coupled with mass spectrometry, Fourier-Transform Infrared (FTIR) spectroscopy and NMR spectroscopy, isotope labeling is one of the crucial tools to identify proteins, study protein structure, and to reveal enzyme reaction mechanisms.329-334 However, the commonly used group-specific approach to label proteins is problematic when applied to large proteins due to excessive labeling.332 Unnatural amino acid mutagenesis allows us to site-specifically incorporate isotope labeled UAAs into proteins without changing protein sequence.335, 336

Previous studies have shown that the nitro group of p-nitrobenzyl (pNBn) derivatives can reduced to an amino group in cellular systems,178, 337, 338 and the resulting p-aminobenzyl group will spontaneously degrade to afford free ε-15N lysine.177, 182, 339 Therefore, a pNBn ε-

15N lysine was designed which can site-specifically label a protein in a traceless fashion upon incorporation.

Efforts were made to synthesize the ε-15N lysine from regular Boc Lys 317 following a route developed by Dr. Lusic in the Deiters lab. The Boc Lys 317 was treated with sodium

166 nitroprusside in a Na2CO3 and NaOH basified water (pH 9.5). Instead of the desired alcohol 319, however, the alkene 318 was obtained which was transformed into alcohol 319 via hydroboration in 37% yield (Scheme 6.22). It was found that the alcohol 319 could be generated in 65% yield from Boc Lys 317 simply by using NaOH as the only base.340 This was followed by the formation of two different esters (320-321) in medium to high yield.341

Surprisingly, all attempts to oxidize the ε-OH group of the acid 319 and the esters 320-321 failed to give any desired product.

205

Scheme 6.22 Synthesis of alcohol 319-321.

An alternate route was designed using phthalimide-15N potassium salt as an isotopic source and a model study was conducted with phthalimide potassium salt. The benzyl ester

320 underwent an Appel reaction affording the bromine 322 in 75% yield,342 which was treated with phthalimide potassium salt to give the phthalimide lysine 323 in 94% yield

(Scheme 6.23).343 The cleavage of phthalimide with hydrazine delivered lysine 324 in 60% yield. Owing to the low yield of this route (15% through five steps), it is more efficient to purchase 15N lysine directly.

206

Scheme 6.23 Synthesis of Lys 324 as a model study for 15N Lys.

Two synthetic routes were firstly subjected to a model study with the HCl salt of lysine (327). In the shorter route,294 the α-amine and the acid were masked through the formation of a copper complex followed by coupling with chloroformate 326 which was generated by the diphosgene activation of p-nitrobenzyl alcohol 325 (Scheme 6.24). Upon addition of EDTA to the reaction mixture breaking up the copper complex, however, no desired product was obtained.

Scheme 6.24 Attempts to synthesize pNBn lysine 328 via a copper complex.

207

In the other route, 9-borabicyclo[3.3.1]nonane (9-BBN) was installed onto the α-NH2 and the carboxylic acid of lysine as a protecting group (1. aqueous NH3 solution; 2. 9-BBN, methanol) (Scheme 6.25).344 The 9-BBN protected lysine 329 was coupled to the chloroformate 326 (H2O/dioxane, 67% yield over two steps). Several conditions were tested for the cleavage of 9-BBN group,345 and the highest yield was obtained with HCl in dioxane affording the HCl salt of pNBn lysine 328 in 79% yield.

Scheme 6.25 Synthesis of pNBn lysine 328 via 9-BBN protected Lys.

The synthesis of pNBn15N lysine 332 proceeded following the same route as regular lysine (Scheme 6.26). The three-step transformation including neutralization, protection and coupling converted the commercially available ε-15N lysine 330 to the 9-BBN protected pNBn15N lysine 331 in 44% yield. After the cleavage of 9-BBN, pNBn15N lysine 332 was purified as a HCl salt in 87% yield. The incorporation and application of this lysine is currently under investigation.

208

Scheme 6.26 Synthesis of the pNBn15N lysine 332.

6.5 Conclusion

A small collection of UAAs was synthesized which upon incorporation in cells with an expanded genetic code can either control protein function or serve as protein labels

(Figure 6.13). The NDBF-caged lysine 231 can provide the regulation of protein functions with IR irradiation. Additionally, the four azide modified lysine derivatives (246, 265, 299 and 302) and the AzM tyrosine 276 were synthesized to allow small molecules such as PPh3 to triggers protein function. Biophysical probes were also installed on lysine, such as the fluorescent NBD lysine 315 and the isotope labeled lysine 332 which allow for parallel analysis of labeled proteins in addition to methods more commonly used in biological sciences. These unnatural amino acids are being actively investigated in the Deiters lab.

209

Figure 6.13 Unnatural amino acid analogs.

6.6 Experimental

University of Pittsburgh. The 1H NMR and 13C NMR spectra were recorded on a 300 MHz or

210

a 400 MHz Varian NMR spectrometer. Chemical shifts are given in δ units (ppm) for 1H

NMR spectra and 13C NMR spectra.

2,5-Dioxopyrrolidin-1-yl (1-(3-nitrodibenzo[b,d]furan-2-yl)ethyl) carbonate (229). N,Nʹ-

Disuccinimidyl carbonate (2 eq., 2 g, 7.8 mmol) was added to a solution of the alcohol 149 (1 eq., 1 g, 3.9 mmol) in CH3CN (21.6 ml), followed by TEA (3 eq., 1.6 ml, 11.7 mmol). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc

(2:1) as the eluent, affording 229 as a yellow solid in 31% yield (472 mg, 1.2 mmol). 1H

NMR (300 MHz, CDCl3): δ = 1.85-1.87 (d, J = 6.3, 3H), 2.78 (s, 4H), 6.51-6.57 (q, J = 6.3

Hz, 1H), 7.44-7.47 (t, J = 6.8, 1H), 7.59-7.62 (m, 2H), 8.08-8.11 (d, J = 7.8, 1H), 8.26-8.27

(d, J = 2.1 2H). The analytical data matched reported results.346

N2-(tert-butoxycarbonyl)-N6-((1-(3-nitrodibenzo[b,d]furan-2-yl)ethoxy)carbonyl)-L- lysine (230). Boc Lys (1.5 eq., 436 mg, 1.77 mmol) was added to a solution of the carbonate

229 (1 eq., 469 mg, 1.2 mmol) in DMF (3.7 ml). The mixture was stirred overnight at room temperature, quenched with sat. NaHCO3 (5 ml), and extracted with Et2O (3 × 5 ml). The combined organic layer was washed with water (3 ml) and brine (3 ml), and dried over

Na2SO4. After filtration, the solvent was removed affording 230 as a yellow foam in 97%

1 yield (604 mg, 1.14 mmol). H NMR (300 MHz, CDCl3): δ = 1.40 (s, 9H), 1.41-1.90 (m,

6H), 1.66-1.68 (d, J = 6.3 Hz, 3H), 3.00-3.20 (m, 2H), 4.19-4.28 (m, 1H), 6.28-6.40 (q, J =

211

6.3 Hz, 1H), 7.39-7.42 (m, 2H), 7.51-7.62 (m, 2H), 8.01-8.04 (d, J = 7.8, 1H), 8.14 (s, 2H).

- LRMS-LC: m/z calcd for C26H31N3O9 [M-H] : 528.2; found: 528.4.

N6-((1-(3-Nitrodibenzo[b,d]furan-2-yl)ethoxy)carbonyl)-L-lysine TFA salt (231). TFA

(10 eq., 0.95 ml, 12.3 mmol) and Et3SiH (2 eq., 0.39 ml, 2.47 mmol) were added to a solution of the Boc Lys 230 (1 eq, 652.8 mg, 1.23 mmol) in DCM (19 ml). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The residue was redissolved in MeOH (0.5 ml) and the solution was added drop-wise to Et2O (200 ml) with vigorous stirring. The precipitate was colltected through filtration and dried, affording the product 231 as a white solid in 83% yield (439 mg, 1.02 mmol). 1H NMR (300 MHz,

CD3OD): δ = 1.40 (s, 9H), 1.37-1.58 (m, 4H), 1.70-1.72 (d, J = 6.3 Hz, 3H), 1.73-1.94 (m,

2H), 3.00-3.11 (m, 2H), 3.58-3.80 (m, 1H), 6.21-6.30 (q, J = 6.3 Hz, 1H), 7.41-7.47 (m, 1H),

7.59-7.67 (m, 2H), 7.71-7.78 (m, 1H), 7.87-7.90 (d, J = 7.8, 1H), 7.97 (s, 1H), 8.13 (s, 1H).

+ LRMS-LC: m/z calcd for C44H40N4O8 [M+H] : 430.1614; found: 430.18.

2-Hydroxy-2-(2-nitrophenyl)acetic acid (232). The methyl ester 233 (1 eq., 100 mg, 0.47 mmol) was suspended in 6 M aq. HCl (2 ml) and heated to reflux for 2 hours. After been cooled to room temperature, the mixture was washed with EtOAc (3 × 5 ml). The aqueous layer was concentrated under reduced pressure, affording the product 232 as a brown solid in

1 90% yield (83 mg, 0.42 mmol). H NMR (300 MHz, CD3OD): δ = 5.84 (s, 1H), 7.51-7.57

(m, 1H), 7.68-7.73 (m, 1H), 7.81-7.84 (dd, Ja = 7.8 Hz, Jb = 1.2 Hz, 1H), 7.97-8.00 (dd, Ja =

347 7.8 Hz, Jb = 1.2 Hz, 1H). The analytical data matched reported results.

212

Methyl 2-hydroxy-2-(2-nitrophenyl)acetate (233). A catalytic amount of I2 (0.1 eq., 127 mg, 0.5 mmol) and trimethylsilylcyanid (1.2 eq., 0.75 ml, 6 mmol) were added to a solution of the 2-nitrobenzaldehyde 156 (1 eq., 756 mg, 5 mmol) in DCM (10 ml). The reaction mixture was stirred for 2 hours at room temperature, quenched with water (5 ml), and extracted with DCM (3 × 10 ml). The combined organic layer was washed with brine (30 ml), dried over Na2SO4, filtered, and concentrated. The crude product was dissolved in

MeOH (10 ml) and acetyl chloride (0.5 ml) was added. The reaction mixture was stirred overnight at room temperature and concentrated. The residue was taken up in aq. NaHCO3

(10 ml), and the aqueous layer was extracted with DCM (3 × 10 ml). The combined organic layer was washed with 10% Na2S2O3 (10 ml), water (30 ml) and brine (30 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 233 as a yellow solid in

1 68% yield (723 mg, 3.4 mmol). H NMR (300 MHz, CDCl3): δ = 3.76 (s, 3H), 5.83 (s, 1H),

7.45-7.58 (m, 1H), 7.80-7.81 (m, 1H), 7.96-7.98 (m, 1H), 8.19-8.22 (d, J = 8.4 Hz, 1H). The analytical data matched reported results.68

Chloromethyl (naphthalen-1-ylmethyl)carbamate (237). TEA (1.5 eq, 0.76 ml, 5.4 mmol) was added to a solution of the 1-naphthalene methyl amine 236 (1 eq, 0.4 ml, 2.7 mmol) in

DCM (10 ml) at –20 ˚C, and the mixture was stirred for 10 minutes. Chloromethyl chloroformate (1.1 eq, 0.35 ml, 4 mmol) was added drop-wise and the reaction mixture was stirred for 30 minutes at –20 ˚C. The mixture was acidified to pH 3-4 with 1 M aq. HCl, and the aqueous layer was extracted with chloroform (3 × 5 ml). The combined organic layer was

213

washed with sat. NaHCO3 (5 ml), water (5 ml) and brine (5 ml), and dried over Na2SO4.

After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 237 as a clear foam in

1 27% yield (185 mg, 0.74 mmol). H NMR (300 MHz, CDCl3): δ = 4.85-4.87 (d, J = 5.7 Hz,

2H), 5.77 (s, 2H), 7.41-7.43 (m, 2H), 7.50-7.56 (m, 2H), 7.75-7.82 (m, 1H), 7.86-7.89 (m,

+ 1H), 7.96-8.01 (m, 1H). LRMS-LC: m/z calcd for C13H12ClNO2 [M+H] : 250.1; found:

250.1.

Azidomethyl (naphthalen-1-ylmethyl)carbamate (238). NaN3 (1.5 eq, 49 mg, 0.75 mmol) was added to a solution of the naphthalene 237 (1 eq, 125 mg, 0.5 mmol) in CH3CN/H2O

(1:1, 2 ml). The mixture was stirred at room temperature overnight and diluted with diethyl ether (5 ml). The organic layer was washed with sat. NaHCO3 (5 ml), water (5 ml) and brine

(5 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 238 as a white solid in 94% yield (120 mg, 0.47 mmol). 1H NMR (300 MHz,

CDCl3): δ = 4.85-4.87 (d, J = 6.0 Hz, 2H), 5.16 (s, 2H), 7.42-7.46 (m, 2H), 7.48-7.55 (m,

- 2H), 7.80-7.89 (m, 3H), 7.99-8.02 (m, 1H). HRMS-LC: m/z calcd for C13H12N4O2 [M-H]

255.0882; found: 255.0888.

Methyl N2-(tert-Butoxycarbonyl)-N6-((chloromethoxy)carbonyl)-L-lysinate (240). TEA

(4 eq, 1.1 ml, 7.8 mmol) was added to a solution of the Boc Lys OMe 239 (1 eq, 580 mg,

1.95 mmol) in DCM (5 ml) at –20 ˚C and the mixture was stirred for 10 minutes.

214

Chloromethyl chloroformate (1.1 eq, 0.35 ml, 2.15 mmol) was added drop-wise and the reaction mixture was stirred at –20 ˚C for 30 minutes. The mixture was acidified to pH 3-4 with 1 M aq. HCl, and the aqueous layer was extracted with chloroform (3 × 5 ml). The combined organic layer was washed with sat. NaHCO3 (10 ml), water (10 ml) and brine (10 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording

1 240 as a clear foam in 46% yield (316 mg, 0.90 mmol). H NMR (300 MHz, CDCl3): δ =

1.41 (s, 9H), 1.42-1.93 (m, 6H), 3.16-3.22 (m, 2H), 3.71 (s, 3H), 4.11-4.19 (br, 1H), 5.71 (s,

+ 2H). LRMS-LC: m/z calcd for C14H25ClN2O6 [M+H] : 353.1; found: 353.3.

Methyl N6-((azidomethoxy)carbonyl)-N2-(tert-butoxycarbonyl)-L-lysinate (241). The Boc

Lys methyl ester 240 (1 eq, 268 mg, 0.76 mmol) was dissolved in DMF (4 ml), and NaN3

(1.5 eq, 74 mg, 1.14 mmol) was added. The mixture was stirred at room temperature overnight and diluted with diethyl ether (5 ml). The organic layer was washed with sat.

NaHCO3 (5 ml), water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 241 as a clear foam in 90% yield (277 mg, 0.69

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.36 (s, 9H), 1.40-1.93 (m, 6H), 3.09-3.20 (m, 2H),

1 3.66 (s, 3H), 4.17-4.23 (br, 1H), 5.05 (s, 2H). HRMS-LC: m/z calcd for C15H27ClN2O6

[M+Na]+: 382.1703; found: 382.2000.

215

Methyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysinate hydrochloride (243). Thionyl chloride (1.5 eq., 0.29 ml, 4.1 mmol) was added to a solution of the ɛ-Fmoc Lys 242 (1 eq., 1 g, 2.71 mmol) in MeOH (13.5 ml). The reaction mixture was heated to reflux overnight and concentrated under reduced pressure. The residue was redissolved in MeOH (1 ml) and the solution was added drop-wise to Et2O (300 ml) with vigorous stirring. The precipitate was colltected and dried, affording the Fmoc Lys methyl ester 243 as a colorless foam in 91% yield (1.03 g, 2.5 mmol) and used in next step without further purification. 1H NMR (300

MHz, DMSO): δ = 1.28-1.79 (m, 6H), 2.68-2.80 (m, 2H), 3.63 (s, 3H), 3.94-4.07 (m, 1H),

4.19-4.40 (m, 3H), 7.30-7.45 (m, 3H), 7.67-7.92 (m, 5H). The analytical data matched reported results.348

Methyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((chloromethoxy)carbonyl)-L- lysinate (244). The Fmoc lysine methyl ester 243 (1 eq, 1.24 g, 3.25 mmol) was dissolved in

DCM (40 ml) and K2CO3 (2.5 eq, 1.12 g, 8.1 mmol) was added at 0 ˚C. The mixture was stirred at 0 ˚C for 10 minutes, and chloromethyl chloroformate (1.1 eq, 0.32 ml, 3.6 mmol) was added drop-wise. The reaction mixture was warmed from 0 ˚C to room temperature overnight and was acidified to pH 3-4 with 1 M aq. HCl, and the aqueous layer was extracted with chloroform (3 × 30 ml). The combined organic layer was washed with sat. NaHCO3 (50 ml), water (50 ml) and brine (50 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 244 as a clear foam in 75% yield (1.1 g, 2.42

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.31-1.40 (m, 2H), 1.51-1.59 (m, 2H), 1.62-1.78

216

(m, 2H), 3.18-3.22 (m, 2H), 3.74 (s, 3H), 4.20-4.22 (m, 1H), 4.38-4.40 (m, 3H), 5.67 (s, 2H),

7.27-7.38 (m, 4H), 7.57-7.60 (d, J = 6.9 Hz, 2H), 7.74-7.76 (d, J = 7.5 Hz, 2H). LRMS-LC:

+ m/z calcd for C24H27ClN2O6 [M+H] : 475.2; found: 475.1.

Methyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((azidomethoxy)carbonyl)-L- lysinate (245). The Fmoc lysine methyl ester 244 (1 eq, 1.11 g, 2.4 mmol) was dissolved in

DMF (30 ml), and NaN3 (1.5 eq, 236 mg, 3.6 mmol) was added. The mixture was stirred at room temperature overnight and diluted with diethyl ether (30 ml). The organic layer was washed with sat. NaHCO3 (15 ml), water (15 ml) and brine (15 ml), and dried over Na2SO4.

After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 245 as a white solid in

1 62% yield (725 mg, 1.51 mmol). H NMR (300 MHz, CDCl3): δ = 1.30-1.98 (m, 6H), 3.16-

3.21 (m, 2H), 3.74 (s, 3H), 4.18-4.22 (m, 1H), 4.38-4.45 (m, 3H), 5.04-5.06 (m, 2H), 7.30-

7.32 (m, 2H), 7.36-7.41 (m, 2H), 7.57-7.60 (d, J = 7.5 Hz, 2H), 7.74-7.76 (d, J = 7.5 Hz, 2H).

+ HRMS-LC: m/z calcd for C24H27N5O6 [M+Na] : 504.1859; found: 504.24.

Methyl N6-((azidomethoxy)carbonyl)-L-lysinate (246). Piperidine (10 eq., 1.1 ml, 11.2 mmol) was added to a solution of the Fmoc lysine methyl ester 245 (1 eq., 527 mg, 1.13 mmol) in DCM (4.4 ml). The reaction mixture was stirred for 5 minutes at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 246 as a white solid in 79%

1 yield (229 mg, 0.08 mmol). H NMR (300 MHz, CDCl3): δ = 1.38-1.60 (m, 6H), 3.14-3.20

217

(q, J = 6.6 Hz, 2H), 3.39-3.44 (t, J = 6.6 Hz, 1H), 3.69 (s, 3H), 5.08 (s, 2H). 13C NMR (400

MHz, CDCl3): δ = 20.1, 22.8, 29.5, 34.4, 40.8, 52.1, 54.3, 82.8, 155.1, 176.5. HRMS-LC:

+ m/z calcd for C9H17N5O4 [M+H] : 260.1359; found: 260.10.

1-Chloroethyl (naphthalen-1-ylmethyl)carbamate (248). Potassium carbonate (2 eq, 387 mg, 2.8 mmol) was added to a solution of the 1-naphthalene methyl amine 236 (1 eq, 0.18 ml, 1.4 mmol) in DCM (5 ml) at –20 ˚C. The mixture was stirred for 10 minutes and 1- chloroethyl chloroformate (1.1 eq, 0.17 ml, 1.54 mmol) was added drop wise. The reaction mixture was stirred at –20 ˚C for 30 minutes, acidified to pH 3-4 with 1 M aq. HCl, and extracted with chloroform (3 × 5 ml). The combined organic layer was washed with sat.

NaHCO3 (5 ml), water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 248 as a clear foam in 94% yield (347 mg, 1.32

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.74-1.76 (d, J = 5.7 Hz, 3H), 4.84-4.87 (m, 2H),

6.58-6.62 (q, J = 5.7 Hz, 1H), 7.41-7.44 (m, 2H), 7.50-7.59 (m, 2H), 7.78-7.82 (m, 1H), 7.82-

+ 7.85 (m, 1H), 7.97-8.01 (m, 1H). LRMS-LC: m/z calcd for C14H14ClNO2 [M+H] : 264.1; found: 264.2.

1-Azidoethyl (naphthalen-1-ylmethyl)carbamate (249). NaN3 (1.5 eq, 15 mg, 0.23 mmol) was added to a solution of the naphthalene 248 (1 eq, 40 mg, 0.15 mmol) in DMF (1 ml). The mixture was stirred at room temperature overnight and diluted with diethyl ether (2 ml). The organic layer was washed with sat. NaHCO3 (1 ml), water (1 ml) and brine (1 ml), and dried

218

over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 249 as a white

1 solid in 93% yield (37 mg, 0.14 mmol). H NMR (300 MHz, CDCl3): δ = 1.39-1.40 (d, J =

5.7 Hz, 3H), 4.84-4.86 (d, J = 5.7 Hz, 2H), 5.95-6.03 (q, J = 5.7 Hz, 1H), 7.42-7.44 (m, 2H),

7.49-7.58 (m, 2H), 7.79-7.84 (m, 1H), 7.86-7.89 (m, 1H), 7.99-8.03 (m, 1H). HRMS-LC: m/z

+ calcd for C14H14N4O2 [M+H] : 271.1195; found: 271.1.

Methyl N2-(tert-butoxycarbonyl)-N6-((1-chloroethoxy)carbonyl)-L-lysinate (250).

Potassium carbonate (2 eq, 464 mg, 3.4 mmol) was added to a solution of the Boc Lys OMe

239 (1 eq, 500 mg, 1.68 mmol) in DCM (6 ml) at –20 ˚C and the mixture was stirred for 10 minutes. 1-Chloroethyl chloroformate (1.1 eq, 0.2 ml, 1.85 mmol) was added drop-wise and the reaction was warmed overnight from –20 ˚C to room temperature. The solution was acidified to pH 3-4 with 1 M aq. HCl, and extracted with chloroform (3 × 5 ml). The combined organic layer was washed with sat. NaHCO3 (10 ml), water (10 ml) and brine (10 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording

1 250 as a clear foam in 84% yield (517 mg, 1.41 mmol). H NMR (300 MHz, CDCl3): δ =

1.42 (s, 9H), 1.43-1.73 (m, 4H), 1.74-1.80 (m, 5H), 3.17-3.22 (q, J = 6.8 Hz, 2H), 3.72 (s,

3H), 4.20-4.27 (br, 1H), 6.50-6.58 (q, J = 5.7 Hz, 1H). LRMS-LC: m/z calcd for

+ C15H27ClN2O6 [M+H] : 367.2; found: 367.5.

219

Methyl N6-((1-azidoethoxy)carbonyl)-N2-(tert-butoxycarbonyl)-L-lysinate (251). The

Boc Lys methyl ester 250 (1 eq, 517 mg, 1.41 mmol) was dissolved in DMF (11 ml), and to

NaN3 (1.5 eq, 178 mg, 2.74 mmol) was added. The mixture was stirred at room temperature overnight and diluted with diethyl ether (10 ml). The organic layer was washed with sat.

NaHCO3 (5 ml), water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography eluting with hexane/EtOAc (2:1) affording 251 as a clear foam in 90% yield (615.3 mg, 1.65

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.40-1.42 (d, J = 5.7 Hz, 3H), 1.44 (s, 9H), 1.45-

1.95 (m, 6H), 3.17-3.22 (q, J = 6.8 Hz, 2H), 3.74 (s, 3H), 4.25-4.35 (br, 1H), 5.86-5.95 (q, J

+ = 5.7 Hz, 1H). LRMS-LC: m/z calcd for C15H27ClN2O6 [M+H] : 374.2; found: 374.2.

N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (252). The

Fmoc lysine 242 (1 eq, 500 mg, 1.4 mmol) was dissolved in aq. NaOH/THF (1:1, 4 ml), and di-tert-butyl dicarbonate (1 eq, 296 mg, 1.4 mmol) was added. The mixture was stirred at room temperature overnight, concentrated, acidified to pH 3-4, and extracted with EtOAc (3

× 2 ml). The organic layer was combined, washed with water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed, affording 252 as a white solid

1 in 87% yield (546 mg, 1.16 mmol). H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.46-1.89

(m, 6H), 3.07-3.14 (m, 2H), 4.17-4.21 (t, J = 6.4 Hz, 1H), 4.32-4.46 (m, 3H), 7.24-7.28 (m,

2H), 7.34-7.38 (m, 2H), 7.51-7.59 (m, 2H), 7.71-7.74 (d, J = 7.5 Hz, 2H). The analytical data matched reported results.349

220

N2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-N6-((benzyloxy)carbonyl)-L-lysine (253). The

Fmoc lysine 242 (1 eq, 500 mg, 1.36 mmol) was dissolved in aq. NaOH/THF (1:1, 4 ml), and benzyl chloroformate (1 eq, 0.73 ml, 1.36 mmol) was added. The mixture was stirred at room temperature overnight, concentrated, acidified to pH 3-4, and extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed affording 253 as a white solid in 79%

1 yield (542 mg, 1.1 mmol). H NMR (300 MHz, CDCl3): δ = 1.26-1.98 (m, 6H), 3.07-3.22

(m, 2H), 4.22-4.30 (m, 1H), 4.49-4.60 (m, 3H), 5.03 (s, 2H), 7.29-7.36 (m, 9H), 7.49-7.60

(m, 2H), 7.71-7.73 (d, J = 7.5 Hz, 2H). The analytical data matched reported results.350

(9H-Fluoren-9-yl)methyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysinate (254). 9-Fluoroenemethanol (2 eq., 42 mg, 0.2 mmol) was added to a solution of the Fmoc Lys 252 (1 eq., 50 mg, 0.11 mmol) in DCM (1 ml), followed by N,Nʹ- dicyclohexylcarbodiimide (1 eq., 22 g, 0.11 mmol) and a catalytic amount of 4-

(dimethylamino)pyridine. The reaction mixture was stirred overnight at room temperature and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 254 as a clear foam in 87% yield (62 mg, 0.09

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.43-1.82 (m, 6H), 3.01-3.09 (m, 2H),

4.16-4.22 (t, J = 6.4 Hz, 1H), 4.30-4.40 (m, 3H), 4.48-4.56 (m, 3H), 7.31-7.41 (m, 8H), 7.59-

7.64 (m, 4H), 7.64-7.77 (m, 4H). The analytical data matched reported results.351

221

Allyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysinate

(255). K2CO3 (3 eq, 178 mg, 1.3 mmol) and allylbromide (2 eq, 74µl, 0.86 mmol) were added to a solution of the Fmoc lysine 252 (1 eq, 200 mg, 0.43 mmol) in CH3CN (0.8 ml) at

0 ˚C. The mixture was stirred overnight at room temperature and acidified to pH 3-4 with 1

M aq. HCl in water, and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with sat. NaHCO3 (2 ml), water (2 ml) and brine (2 ml), and dried over Na2SO4.

After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 255 as a clear foam in

1 83% yield (181 mg, 0.34 mmol). H NMR (300 MHz, CDCl3): δ = 1.41 (s, 9H), 1.43-1.96

(m, 6H), 3.01-3.09 (m, 2H), 4.16-4.22 (m, 1H), 4.30-4.40 (m, 2H), 4.48-4.56 (m, 3H), 5.32

(m, 2H), 5.76 (m, 1H), 7.36-7.43 (m, 4H), 7.61-7.69 (m, 2H), 7.82-7.91 (m, 2H).

2-Oxopropyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((benzyloxy)carbonyl)-L- lysinate (256). K2CO3 (4 eq, 144 mg, 1 mmol) and bromomethyl acetate (1.5 eq, 38 µl, 0.39 mmol) were added to a solution of the Fmoc lysine 253 (1 eq, 100 mg, 0.26 mmol) in

CH3CN (0.5 ml) at 0 ˚C. The mixture was stirred overnight at room temperature, acidified to pH 3-4 with 1 M HCl in water, extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with sat. NaHCO3 (2 ml), water (2 ml) and brine (2 ml), and dried over Na2SO4.

After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 256 as a clear foam in

1 16% yield (30 mg, 0.04 mmol). H NMR (300 MHz, CDCl3): δ = 1.23-1.91 (m, 6H), 2.05 (s,

3H), 3.08-3.20 (m, 2H), 4.17-4.20 (t, J = 6.4 Hz, 1H), 4.29-4.41 (m, 3H), 4.81-4.92 (m, 1H),

222

5.04 (s, 2H), 5.41-5.49 (m, 1H), 7.27-7.36 (m, 9H), 7.49-7.60 (m, 2H), 7.70-7.73 (d, J = 7.2

Hz, 2H).

(9H-Fluoren-9-yl)methyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysinate (257). TFA

(10 eq., 86 µl, 1.2 mmol) was added to a solution of the Fmoc Lys 254 (1 eq, 79 mg, 0.11 mmol) in DCM (1.6 ml), followed by Et3SiH (2 eq., 39 µl, 0.22 mmol). The reaction was stirred overnight at room temperature and concentrated. The residue was redissolved in

MeOH (0.1 ml) and the solution was added drop-wise to Et2O (5 ml) with vigorous stirring.

The precipitate was colltected and dried, affording the product 257 as a white solid in 57%

1 yield (51 mg, 0.07 mmol). H NMR (400 MHz, CDCl3): δ = 1.20-1.65 (m, 6H), 2.80-2.97

(m, 2H), 3.96-4.02 (m, 1H), 4.05-4.15 (m, 2H), 4.16-4.21 (m, 4H), 7.10-7.18 (m, 4H), 7.27-

7.43 (m, 4H), 7.49-7.54 (m, 4H), 7.64-7.70 (m, 4H).The analytical data matched reported results.351

Allyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysinate (258). TFA (10 eq., 0.27 ml, 3.56 mmol) was added to a solution of the Fmoc Lys 255 (1 eq, 181 mg, 0.36 mmol) in DCM (4.5 ml), followed by Et3SiH (2 eq., 0.12 ml, 0.72 mmol). The reaction was stirred for 2 hours at room temperature and concentrated. The residue was redissolved in MeOH (0.2 ml) and the solution was added drop-wise to Et2O (10 ml) with vigorous stirring. The precipitate was colltected and dried, affording the product 258 as a white solid in 77% yield (168 mg, 0.28

1 mmol). H NMR (400 MHz, CDCl3): δ = 1.32-1.86 (m, 6H), 3.04-3.12 (m, 2H), 4.17-4.23

223

(m, 1H), 4.38-4.50 (m, 2H), 4.52-4.66 (m, 3H), 5.34 (m, 2H), 5.87 (m, 1H), 7.35-7.42 (m,

4H), 7.60-7.68 (m, 2H), 7.81-7.89 (m, 2H).

2-Oxopropyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysinate (259). Palladium on carbon (10% wt loading, 5 mg) was added to a solution of the Fmoc Lys 256 (1 eq., 30 mg,

0.045 mmol) in EtOAc (0.45 ml). The reaction mixture was stirred overnight under hydrogen gas, filtered through celite and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 259 as a colorless oil in

1 96% yield (19 mg, 0.044 mmol). H NMR (300 MHz, CDCl3): δ = 1.23-1.91 (m, 6H), 2.25

(s, 3H), 2.87-3.01 (m, 2H), 4.15-4.22 (m, 1H), 4.49-4.51 (m, 3H), 5.24 (s, 2H), 7.27-7.36 (m,

4H), 7.49-7.60 (m, 2H), 7.70-7.73 (m, 2H).

(9H-Fluoren-9-yl)methyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1-chloroethoxy)carbonyl)-L-lysinate (260). Potassium carbonate (2 eq, 7.7 mg, 0.06 mmol) was added to a solution of the Fmoc Lys 257 (1 eq, 20 mg, 0.028 mmol) in DCM (0.2 ml) at –20

˚C and the mixture was stirred for 10 minutes. 1-Chloroethyl chloroformate (1.1 eq, 3.4 µl,

0.03 mmol) was added drop-wise and the reaction mixture was warmed overnight from –20

˚C to room temperature. The mixture was acidified to pH 3-4 with 1 M aq. HCl, extracted with chloroform (3 × 1 ml). The combined organic layer was washed with sat. NaHCO3 (1 ml), water (1 ml) and brine (1 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 260 as a clear foam in 43% yield (8.8 mg, 0.012

224

1 mmol). H NMR (400 MHz, CDCl3): δ = 1.22-1.74 (m, 9H), 3.13-3.15 (m, 2H), 4.20-4.22

(m, 2H), 4.34-4.40 (m, 3H), 4.52-4.56 (m, 2H), 4.71-4.74 (m, 1H), 5.28-5.31 (d, J = 8.4 Hz,

1H), 6.54-6.57 (m, 1H), 7.27-7.43 (m, 8H), 7.54-7.58 (m, 4H), 7.74-7.76 (d, J = 7.2 Hz, 4H).

Allyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1-chloroethoxy)carbonyl)-L- lysinate (261). Potassium carbonate (6.2 eq, 242 mg, 1.75 mmol) was added to a solution of the Fmoc Lys 258 (1 eq, 168 mg, 0.28 mmol) in DCM (2.5 ml) at –20 ˚C and the mixture was stirred for 10 minutes. 1-Chloroethyl chloroformate (1.5 eq, 69 µl, 0.42 mmol) was added drop-wise and the reaction mixture was warmed from –20 ˚C to room temperature overnight. The mixture was acidified to pH 3-4 with 1 M aq. HCl, and extracted with chloroform (3 × 2 ml). The combined organic layer was washed with sat. NaHCO3 (1 ml), water (1 ml) and brine (1 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 261 as a clear foam in 23% yield (33 mg, 0.06

1 mmol). H NMR (400 MHz, CDCl3): δ = 1.27-1.86 (m, 9H), 3.01-3.19 (m, 2H), 4.27-4.33

(m, 1H), 4.46-4.54 (m, 2H), 4.57-4.61 (m, 3H), 5.34 (m, 2H), 5.87 (m, 1H), 6.56-6.59 (m,

1H), 7.35-7.42 (m, 4H), 7.60-7.68 (m, 2H), 7.81-7.89 (m, 2H).

2-Oxopropyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1-chloroethoxy)carbonyl)-

L-lysinate (262). Potassium carbonate (2 eq, 12 mg, 0.09 mmol) was to a solution of the

Fmoc Lys 259 (1 eq, 20 mg, 0.045 mmol) in DCM (0.5 ml) at – 20 ˚C and the mixture was stirred for 10 minutes. 1-Chloroethyl chloroformate (1.2 eq, 6 µl, 0.05 mmol) was added

225

drop-wise and the reaction was warmed from –20 ˚C to room temperature overnight. The mixture was acidified to pH 3-4 with 1 M aq. HCl and extracted with chloroform (3 ×1 ml).

The combined organic layer was washed with sat. NaHCO3 (1 ml), water (1 ml) and brine (1 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording

1 262 as a clear foam in 30% yield (7 mg, 0.014 mmol). H NMR (300 MHz, CDCl3): δ =

1.32-1.93 (m, 9H), 2.21 (s, 3H), 3.02-3.11 (m, 2H), 4.15-4.22 (m, 1H), 4.49-4.51 (m, 3H),

5.28 (s, 2H), 6.54-6.60 (m, 1H), 7.27-7.36 (m, 4H), 7.49-7.60 (m, 2H), 7.70-7.73 (m, 2H).

Methyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1-chloroethoxy)carbonyl)-L- lysinate (263). The Fmoc lysine methyl ester 243 (1 eq, 382 mg, 1 mmol) was dissolved in

DCM (4.6 ml) and K2CO3 (2.5 eq, 345 mg, 2.5 mmol) was added. The mixture was stirred at

0 ˚C for 10 minutes and 1-chloroethyl chloroformate (1.1 eq, 0.14 ml, 1.1 mmol) was added drop-wise. The reaction mixture was warmed from 0 ˚C to room temperature overnight, acidified to pH 3-4 with 1 M aq. HCl, and extracted with chloroform (3 × 5 ml). The combined organic layer was washed with sat. NaHCO3 (5 ml), water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 263

1 as a clear foam in 94% yield (459 mg, 0.94 mmol). H NMR (400 MHz, CDCl3): δ = 1.28-

1.40 (m, 2H), 1.41-1.53 (m, 2H), 1.67-1.74 (m, 5H), 3.17-3.22 (q, J = 6.8 Hz, 2H), 3.74 (s,

3H), 4.20-4.23 (t, J = 7.4 Hz, 2H), 4.34-4.43 (m, 2H), 5.46-5.48 (d, J = 8.4 Hz, 1H), 6.55-

6.58 (m, 1H), 7.29-7.32 (m, 2H), 7.37-7.41 (m, 2H), 7.58-7.61 (m, 2H), 7.75-7.76 (d, J = 7.2

226

13 Hz, 2H). C NMR (400 MHz, CDCl3): δ = 14.4, 22.4, 25.6, 29.2, 32.4, 40.8, 47.4, 52.7,

53.8, 67.3, 83.0, 120.2, 125.3, 127.3, 127.9, 141.5, 144.1, 153.8, 156.3, 173.1. HRMS-LC:

- m/z calcd for C25H29ClN2O6 [M-H] 487.1714; found: 487.1643.

Methyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-((1-azidoethoxy)carbonyl)-L- lysinate (264). The Fmoc lysine methyl ester 263 (1 eq, 250 mg, 0.5 mmol) was dissolved in

DMF (2.8 ml), and NaN3 (1.5 eq, 51.3 mg, 0.79 mmol) was added. The mixture was stirred at room temperature overnight and diluted with diethyl ether (3 ml). The solution was washed with sat. NaHCO3 (5 ml), water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 264 as a white solid in

1 43% yield (104 mg, 0.22 mmol). H NMR (400 MHz, CDCl3): δ = 1.35-1.44 (m, 5H), 1.52-

1.56 (m, 2H), 1.66-1.72 (m, 1H), 1.85-1.89 (m, 1H), 3.17-3.22 (q, J = 6.8 Hz, 2H), 3.75 (s,

3H), 4.20-4.24 (t, J = 7.2 Hz, 2H), 4.37-4.43 (m, 2H), 5.42-5.44 (d, J = 8.4 Hz, 1H), 5.90-

5.92 (m, 1H), 7.31-7.33 (m, 2H), 7.38-7.42 (m, 2H), 7.58-7.61 (m, 2H), 7.75-7.77 (d, J = 7.2

13 Hz, 2H). C NMR (400 MHz, CDCl3): δ = 14.4, 20.3, 22.5, 29.4, 32.4, 40.8, 47.4, 52.7,

53.8, 67.2, 83.1, 120.2, 125.3, 127.3, 127.9, 141.5, 144.1, 155.4, 156.3, 173.1. HRMS-LC:

+ m/z calcd for C25H29N5O6 [M+Na] : 518.2016; found: 518.1981.

Methyl N6-((1-azidoethoxy)carbonyl)-L-lysinate (265). Piperidine (10 eq., 0.29 ml, 2.9 mmol) was added to a solution of the Fmoc lysine methyl ester 264 (1 eq., 141 mg, 0.29 mmol) in DCM (1.2 ml). The reaction was stirred for 5 minutes at room temperature and

227

concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/MeOH (19:1) as the eluent, affording 265 as a white solid in

1 54% yield (43 mg, 0.16 mmol). H NMR (400 MHz, CDCl3): δ = 1.36-1.44 (m, 5H), 1.50-

1.56 (m, 2H), 1.58-1.62 (m, 1H), 1.67-1.72 (m, 1H), 3.16-3.19 (q, J = 6.8 Hz, 2H), 3.40-3.42

13 (t, J = 6.4 Hz, 1H), 3.68 (s, 3H), 5.86-5.90 (m, 1H). C NMR (400 MHz, CDCl3): δ = 20.1,

22.8, 29.5, 34.4, 40.8, 52.1, 54.3, 82.8, 155.1, 176.5. HRMS-LC: m/z calcd for C10H19N5O4

[M+H]+: 274.1515; found: 274.3530.

4-(Chloromethyl)-7-hydroxy-2H-chromen-2-one (268). Ethyl 4-chloroacetoacetate (267)

(1 eq., 1.36 g, 10 mmol) was added to a solution of the resorcinol (266) (1 eq., 1.1 g, 10 mmol) in con. H2SO4 (10 ml) at 0 ˚C. The reaction mixture was warmed from 0 ˚C to room temperature in 24 hours, and the solution was poured into ice-cold water (100 ml) and filtered. The solid was collected, washed with ice cold water (3 × 50 ml ) and dried, affording

1 268 as an off-white solid in 94% yield (1.98 g, 9.4 mmol). H NMR (300 MHz, CDCl3): δ =

4.63 (s, 2H), 6.41 (s, 1H), 6.82-6.86 (dd, Ja = 8.4 Hz, Jb = 1.8 Hz, 1H), 6.92-6.93 (d, J = 1.8

Hz, 1H), 7.54-7.56 (d, J = 8.4 Hz, 1H). The analytical data matched reported results.299

(7-Hydroxy-2-oxo-2H-chromen-4-yl)methyl acetate (269). Ethyl potassium acetate (1.2 eq., 235 mg, 2.4 mmol) was added to a solution of the coumarin 268 (1 eq., 420 mg, 2 mmol) in DMF (2 ml), followed by a catalytic amount of TBAI. The reaction mixture was warmed to 50 ˚C until the solution is clear. The resulting solution was stirred for 24 hours at room temperature, poured into ice cold water (10 ml), and filtered. The solid was collected, washed

228

with ice cold water (3 × 2 ml) and dried, affording 269 as an off white solid in 90% yield

1 (420 mg, 1.8 mmol). H NMR (300 MHz, CDCl3): δ = 2.19 (s, 3H), 5.21 (s, 2H), 6.43 (s,

1H), 6.68-6.80 (m, 2H), 7.49-7.52 (d, J = 8.7 Hz, 1H).The analytical data matched reported results.92

(7-((Methylthio)methoxy)-2-oxo-2H-chromen-4-yl)methyl acetate (270). t-BuOK (1.1 eq.,

26.3 mg, 0.23 mmol) was added to a solution of the coumarin 269 (1 eq., 50 mg, 0.21 mmol) in DMF/THF (2:1, 0.6 ml) at 0 ˚C, followed by chloromethyl methylsulfide (1.1 eq., 20 µl,

0.23 mmol) and NaI (0.1 eq., 3.2 mg, 0.02 mmol). The mixture was warmed to room temperature and stirred overnight. The solution was diluted with EtOAc (2 ml) and washed with washed with water (2 ml), 10% w/v citric acid (2 ml), and brine (2 ml), dried over

Na2SO4, filtered and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 270 as an off-white solid

1 in 24% yield (15 mg, 0,05 mmol). H NMR (300 MHz, CDCl3): δ = 2.18 (s, 3H), 2.24 (s,

3H), 5.18 (s, 2H), 5.24 (s, 2H), 6.33 (s, 1H), 6.88-6.90 (m, 2H), 7.39-7.42 (d, J = 8.7 Hz,

+ 1H). LRMS-LC: m/z calcd for C14H14O5S [M+H] : 295.1; found: 295.3.

(7-(Azidomethoxy)-2-oxo-2H-chromen-4-yl)methyl acetate (271). NCS (1.1 eq., 7.3 mg,

0.055 mmol) was added to a solution of the thioether 270 (1 eq., 15 mg, 0.05 mmol) in DCM

(0.5 ml) and the reaction was stirred at room temperature for 2 hours. TMSCl (1.1 eq., 7 µl,

0.055 mmol) was added and the mixture was stirred for 3 hours. The reaction was quenched with sat. NaHCO3 (2 ml) and extracted with DCM (3 × 1 ml). The combined organic layer

229

was washed with water (1 ml), and brine (1 ml), and concentrated under reduced pressure.

The crude product was confirmed by 1H NMR and used in next step without further purification. The crude coumarin was dissolved in DMF (0.4 ml) and sodium azide (1.5 eq.,

4.8 mg, 0.075 mmol) was added. The resulting mixture was stirred overnight at room temperature, and the reaction was quenched with sat. NaHCO3 (2 ml) and extracted with

EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 271 as an off-white solid

1 in 23% yield (3.3 mg, 3.4 mmol). H NMR (300 MHz, CDCl3): δ = 2.19 (s, 3H), 5.21 (s, 2H),

5.25 (s, 2H), 6.37 (s, 1H), 6.93-6.96 (m, 2H), 7.43-7.46 (d, J = 8.7 Hz, 1H). LRMS-LC: m/z

+ calcd for C13H11N3O5 [M+H] : 290.1; found: 290.2.

Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-((methylthio)methoxy)phenyl)- propanoate (273). t-BuOK (1.2 eq., 1.07 g, 9.6 mmol) was added to a solution of the Boc

Tyr methyl ester 272 (1 eq., 2.36 g, 8 mmol) in DMF/THF (2:1, 24 ml) at 0 ˚C, followed by chloromethyl methylsulfide (1.2 eq., 0.8 ml, 9.6 mmol) and NaI (0.1 eq., 112 mg, 0.4 mmol).

The mixture was warmed to room temperature and stirred overnight. The solution was diluted with EtOAc (40 ml) and washed with water (20 ml), 10% w/v citric acid (20 ml), and brine (20 ml), dried over Na2SO4, filtered and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 273 as a

1 colorless foam in 71% yield (2 g, 5.7 mmol). H NMR (400 MHz, CDCl3): δ = 1.40 (s, 9H),

230

2.33 (s, 3H), 2.97-3.05 (m, 2H), 3.69 (s, 3H), 4.49-4.58 (m, 1H), 5.10 (s, 2H), 6.84-6.87 (d, J

= 8.4 Hz, 2H), 7.02-7.04 (d, J = 8.4 Hz, 2H). The analytical data matched reported results.180

Methyl (S)-3-(4-(azidomethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate

(274). NCS (1.1 eq., 827 mg, 6.2 mmol) was added to a solution of the thioether 273 (1 eq., 2 g, 5.7 mmol) in DCM (18 ml) and the reaction was stirred at room temperature for 2 hours.

TMSCl (1.1 eq., 0.79 ml, 6.2 mmol) was added and the mixture was stirred for 3 hours. The reaction was quenched with sat. NaHCO3 (20 ml) and aqueous layer was extracted with

DCM (10 ml × 3). The combined organic layer was washed with water (20 ml), and brine (20 ml), and concentrated under reduced pressure. The crude product was confirmed by 1H NMR and used in next step without further purification. The crude product was dissolved in

DMF/water (1:1, 27 ml), and sodium azide (1.5 eq., 549 mg, 8.45 mmol) was added. The resulting mixture was stirred for overnight at room temperature. The reaction was quenched with sat. NaHCO3 (20 ml) and the aqueous layer was extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (20 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 274 as a colorless foam in 51% yield (1 g, 2.9

1 mmol). H NMR (300 MHz, CDCl3): δ = 1.40 (s, 9H), 2.93-3.11 (m, 2H), 3.70 (s, 3H), 4.49-

4.60 (m, 1H), 5.12 (s, 2H), 6.89-6.92 (d, J = 8.4 Hz, 2H), 7.04-7.07 (d, J = 8.4 Hz, 2H).

+ HRMS-LC: m/z calcd for C16H22N4O5 [M+H] : 373.1488; found: 373.2000.

231

(S)-3-(4-(Azidomethoxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (275). A

2 M solution of LiOH in water (5 eq., 7.6 ml, 15 mmol) was added to the solution of methyl ester 274 (1 eq., 1 g, 2.9 mmol) in MeOH (7.6 ml). The mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was dissolved in water (5 ml), acidified to pH 3-4 with 1 M citric acid, and extracted with EtOAc (3 × 10 ml). The combined organic layer was washed with brine (5 ml), dried over Na2SO4, filtered, and concentrated, affording 275 as a light yellow foam in 84% yield (848 mg, 2.5 mmol). 1H

NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 3.01-3.20 (m, 2H), 4.47-4.63 (m, 1H), 5.14 (s,

2H), 6.93-6.95 (d, J = 8.4 Hz, 2H), 7.12-7.15 (d, J = 8.4 Hz, 2H). HRMS-LC: m/z calcd for

- C15H20N4O5 [M-H] : 335.1350; found: 335.1367.

(S)-2-Amino-3-(4-(azidomethoxy)phenyl)propanoic acid hydrochloride salt (276). TFA

(10 eq., 0.46 ml, 6 mmol) and Et3SiH (2 eq., 0.2 ml, 1.2 mmol) were added to a solution of the Boc Tyr 275 (1 eq, 200 mg, 0.6 mmol) in DCM (8 ml). The reaction mixture was stirred overnight at room temperature and concentrated. The residue was redissolved in MeOH (0.5 ml) and the solution was added drop-wise to Et2O (100 ml) with vigorous stirring. The precipitate was colltected and dried, affording the TFA salt of Tyr as a white solid. The TFA salt was dissolved in DCM (0.7 ml) and 4 M HCl in dioxane (1.5 eq., 0.225 ml, 0.9 mmol) was added. The reaction mixture was stirred for 30 minutes and concentrated. The residue was dissolved in water (4 ml) and washed with Et2O (3 × 10 ml). The aqueous layer was concentrated under reduced pressure affording 276 as a white solid in 96% yield (129 mg,

1 0.47 mmol). H NMR (300 MHz, CD3OD): δ = 3.18-3.28 (m, 2H), 4.24-4.27 (m, 1H), 5.28

232

(s, 2H), 7.06-7.09 (d, J = 8.4 Hz, 2H), 7.27-7.29 (d, J = 8.4 Hz, 2H). HRMS-LC: m/z calcd

˗ for C10H12N4O3 [M˗H] : 235.0826; found: 235.0836.

(2-Azidophenyl)methanol (279). A solution of sodium nitrite (1.125 eq., 1.26 g, 18.3 mmol) in water (10 ml) was added to a solution of the 2-aminobenzyl alcohol (278) (1 eq., 2 g, 16.2 mmol) in 5 M HCl (32 ml) at 0 ˚C. The mixture was stirred for 30 minutes and sodium azide

(4 eq., 4 × 1.05 g, 65 nnol) was added in four portion over 1 hour with vigorous stirring. The resulting mixture was stirred at 0 ˚C for 2 hours and poured into ice-cold water (100 ml). The solution was basified to pH 8 with solid NaHCO3 and the aqueous layer was extracted with

EtOAc (3 × 50 ml). The combined organic layer was washed with brine (100 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by recrystallization in

EtOAc/hexane (1:9) affording the product 279 as a white solid in 76% yield (1.8 g, 12.3

1 mmol). H NMR (300 MHz, CDCl3): δ = 4.63 (s, 2H), 7.13-7.19 (m, 2H), 7.33-7.36 (m, 2H).

The analytical data matched reported results.352

2-Azidobenzyl (naphthalen-1-ylmethyl)carbamate (280). Diphosgene (1.2 eq., 0.12 ml,

1.1 mmol) was added to a solution of the alcohol 279 (1.1 eq., 149 mg, 1 mmol) in THF (3.7 ml) at 0 ˚C, followed by potassium carbonate (3.3 eq., 414 mg, 3 mmol). The mixture was stirred overnight at room temperature, filtered and concentrated delivering the crude product as a brown oil which was confirmed by NMR and used without purification. TEA (1.5 eq, 0.2 ml, 1.36 mmol) was added to a solution of the 1-naphthalene methyl amine 236 (1 eq, 0.13 ml, 0.9 mmol) in DCM (2 m l) at 0 ˚C and the mixture was stirred for 10 minutes. The

233

chloroformate generated in previous step was dissolved in DCM (1ml) and the solution was added drop-wise at 0 ˚C. The reaction was warmed from 0 ˚C to room temperature over night. The solution was acidified to pH 3-4 with 1 M aq. HCl, extracted with chloroform (3 ×

2 ml). The combined organic layer was washed with sat. NaHCO3 (5 ml), water (5 ml) and brine (5 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 280 as an off-white solid in 25% yield (76 mg, 0.23 mmol). 1H NMR (300 MHz,

CDCl3): δ = 3.86 (s, 2H), 4.20 (s, 2H), 7.10-7.17 (m, 2H), 7.33-7.50 (m, 6H), 7.74-7.76 (m,

+ 1H), 7.81-7.83 (m, 1H), 8.04-8.05 (m, 1H). LRMS-LC: m/z calcd for C19H16N4O2 [M+H] :

333.1; found: 333.2.

N,N'-(3-Oxo-3H-spiro(isobenzofuran-1,9'-xanthene)-3',6'-diyl)bis(2,2,2-trifluoroacet- amide) (282). Pyridine (4 eq., 44 µl, 0.55 mmol) was added to a solution of Rhodamine 110

(1 eq., 50 mg, 0.14 mmol) in DCM (1 ml), followed by trifluoroacetic anhydride (4 eq., 76

µl, 0.55 mmol). The reaction mixture was stirred for 3 hours at room temperature, quenched with 10% w/v aq. citric acid (1 ml), and extracted with DCM (3 × 1 ml). The combined organic layer was washed with water (2 ml), and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (3:1) as the eluent, affording 282 as a red solid in 64% yield (45 mg, 0.09

1 mmol). H NMR (300 MHz, CDCl3): δ = 6.80-6.83 (m, 2H), 7.08-7.17 (m, 2H), 7.63-7.71

(m, 1H), 7.76-7.78 (m, 2H), 7.93-8.04 (m, 2H). The analytical data matched reported results.305

234

2-(3,6-Bis(2,2,2-trifluoroacetamido)-9H-xanthen-9-yl)benzoic acid (283). Palladium on carbon (10% wt loading, 23 mg) was added to a solution of the rhodamine 282 (1 eq., 45 mg,

0.086 mmol) in THF (2 ml). The reaction was stirred overnight under hydrogen, filtered through celite and concentrated. The crude product 283 was used in the next step without further purification (44 mg, 0.08 mmol).

2-(3,6-Bis(2,2,2-trifluoroacetamido)-9H-xanthen-9-yl)benzoic acid (284). 4-

Methoxylbenzyl alcohol (4.4 eq., 47 µl, 0.38 mmol) was added to a solution of the acid 283

(1 eq., 44 mg, 0.08 mmol) in DCM (2 ml), followed by N,Nʹ-dicyclohexylcarbodiimide (4.4 eq., 78.1 g, 0.38 mmol) and a catalytic amount of 4-(dimethylamino)pyridine. The reaction was stirred overnight at room temperature and concentrated. The crude product was purified by silica gel chromatography with DCM/acetone (9:1) as the eluent, affording 284 as a red

1 oil in 53% yield (27 mg, 0.042 mmol). H NMR (300 MHz, CDCl3): δ = 3.82 (s, 3H), 5.30 (s,

2H), 6.33 (s, 1H), 6.83-6.92 (m, 2H), 7.01-7.10 (m, 4H), 7.19-7.23 (m, 2H), 7.27-7.34 (m,

1H), 7.36-7.40 (m, 2H), 7.47 (s, 1H), 7.79-7.84 (m, 2H).305

4-Methoxybenzyl 2-(3,6-diamino-9H-xanthen-9-yl)benzoate (285). Hydroxylamine (0.13 ml) was added to a solution of the rhodamine 284 (1 eq., 27 mg, 0.042 mmol) in MeOH (3 ml). The reaction mixture was stirred for 24 hours at room temperature, quenched with sat.

NaHCO3 (2 ml), concentrated, and extracted with EtOAc (3 × 2 ml). The combined organic layer washed with water (2 ml), and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with

235

DCM/acetone (19:1) as the eluent, affording 285 as a red oil in 67% yield (13 mg, 0.028

1 mmol). H NMR (300 MHz, CDCl3): δ = 3.83 (s, 3H), 5.33 (s, 2H), 6.29 (s, 1H), 6.63-6.67

(m, 2H), 6.83-6.84 (m, 1H), 6.91-6.93 (m, 3H), 7.00-7.06 (m, 2H), 7.24-7.25 (m, 2H), 7.30-

7.41 (m, 3H), 7.80-7.84 (m, 1H). The analytical data matched reported results.305

4-Methoxybenzyl 2-(3,6-bis((((2-azidobenzyl)oxy)carbonyl)amino)-9H-xanthen-9-yl) benzoate (286). DIPEA (2.5 eq., 20 µl, 0.114 mmol) was added to a solution of the diamine

285 (1 eq., 13 mg, 0.028 mmol) and the chloroformate 294 (2.2 eq., 0.1 mmol) in DCM (1.8 ml). The mixture was stirred for 18 hours in dark. The reaction quenched with 10% w/v aq. citric acid (1 ml) and the aqueous layer was extracted with DCM (3 × 1 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (9:1) as the eluent, affording 286 as a red oil in 36% yield (8 mg, 0.01 mmol).

1 H NMR (300 MHz, CDCl3): δ = 3.79 (s, 3H), 5.16 (s, 4H), 5.32 (s, 2H), 6.21 (s, 1H), 6.62

(s, 2H), 6.82-6.90 (m, 6H), 6.70-6.72 (m, 1H), 7.13-7.23 (m, 6H), 7.36-7.42 (m, 6H), 7.76-

7.80 (m, 1H).

Bis(2-azidobenzyl) (3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-3',6'-diyl)dicarba- mate (287). DDQ (10 eq., 18 mg, 0.081 mmol) was added to a solution of the rhodamine 286

(1 eq., 6.5 mg, 0.008 mmol) in DCM/PBS buffer (1X, pH = 7.0) (10:1, 1.3 ml). The reaction mixture was stirred overnight at 40 ˚C, cooled to room temperature and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the

236

eluent, affording 287 as a red oil in 74% yield (4.1 mg, 0.006 mmol). 1H NMR (300 MHz,

CDCl3): δ = 5.86 (s, 4H), 6.67-6.82 (m, 3H), 6.85-6.93 (m, 2H), 6.70-6.72 (m, 1H), 7.13-7.23

(m, 6H), 7.36-7.42 (m, 6H), 7.76-7.80 (m, 1H).

2-Azidobenzyl (hydroxymethyl)carbamate (288). Diphosgene (1.2 eq., 133 µl, 1.1 mmol) was added to a solution of the 2-azidobenzyl alcohol 279 (1 eq., 137 mg, 0.9 mmol) in THF

(0.5 ml) at 0 ˚C, followed by potassium carbonate (4 eq., 507 mg, 4 mmol). The reaction was stirred at room temperature overnight, filtered and concentrated. The crude product was confirmed by 1H NMR and used in next step without further purification. The acid chloride was dissolved in THF (1 ml), and 30% NH4OH (1 ml) was added drop-wise at 0 ˚C. The reaction was stirred for 2 hours, concentrated, and filtered. The solid was collected, washed with water (3 × 1 ml), and dried affording the crude product as an off white solid which was used in the next step without further purification. The carbamate was dissolved in DMSO

(2.5 ml) and para formaldehyde (1.1 eq., 30.3 mg, 1.01 mmol) was added, followed by potassium carbonate (0.12, 15.2 mg, 0.11 mmol). The resulting mixture was stirred for 2 hours at room temperature and diluted with EtOAc (5 ml). The solution was washed with brine (5 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 288 as an off-

1 white solid in 36% yield (85 mg, 0.32 mmol). H NMR (300 MHz, CDCl3): δ = 4.76 (s, 2H),

5.12 (s, 2H), 7.14-7.20 (m, 2H), 7.36-7.41 (m, 2H). HRMS-LC: m/z calcd for C9H10N4O3

[M˗H]˗: 221.0669; found: 235.0679.

237

4-Azidobenzyl (hydroxymethyl)carbamate (289). Diphosgene (1.1 eq., 131 µl, 1.1 mmol) was added to a solution of the 4-azidobenzyl alcohol 104 (1 eq., 149 mg, 1 mmol) in THF (1 ml) at 0 ˚C, followed by potassium carbonate (4 eq., 507 mg, 4 mmol). The reaction was stirred at room temperature overnight, filtered and concentrated. The crude product was confirmed by 1H NMR and used in the next step without further purification. The acid chloride generated in previous step was dissolved in THF (1 ml), and 30% NH4OH (1 ml) was added drop-wise at 0 ˚C. The reaction mixture was stirred for 2 hours, concentrated, and filtered. The solid was collected, washed with water (3 × 1 ml) and dried, affording the crude product as an off-white solid which was used in next step without further purification. The carbamate generated in previous step was dissolved in DMSO (2.5 ml) and para formaldehyde (1.1 eq., 30.3 mg, 1.01 mmol) was added, followed by potassium carbonate

(0.12, 15.2 mg, 0.11 mmol). The resulting mixture was stirred for 2 hours at room temperature, diluted with EtOAc (5 ml), washed with brine (5 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 289 as an off-white solid in 28% yield (62 mg,

1 0.32 mmol). H NMR (300 MHz, CDCl3): δ = 4.93 (s, 2H), 5.16 (s, 2H), 7.01-7.03 (d, J = 8.1

˗ Hz, 1H), 7.35-7.37 (d, J = 7.8 Hz, 1H). HRMS-LC: m/z calcd for C9H10N4O3 [M˗H] : 221.1; found: 221.1.

(7-(((((2-Azidobenzyl)oxy)carbonyl)amino)methoxy)-2-oxo-2H-chromen-4-yl)methyl acetate (290). Solution A: NaH (1.5 eq., 3.6 mg, 0.15 mmol) was added to a solution of the coumarin 269 (1 eq., 23 mg, 0.1 mmol) in THF (0.5 ml). The resulting solution was heated to

238

60 ˚C for 10 minutes. Solution B: p-Toluene sulfonyl chloride (1 eq., 19 mg, 0.1 mmol) was added to a solution of the alcohol 288 (1 eq., 22 mg, 0.1 mmol) in THF (0.5 ml), followed by

Cs2CO3 (1.5 eq., 49 mg, 0.15 mmol). The resulting solution was heated to 60 ˚C for 10 minutes. Solution B was added to solution A and the reaction mixture was stirred at 60 ˚C overnight. The reaction was quenched with water (2 ml), and the aqueous layer was extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording the 290 as an off-white

1 solid in 52% yield (23 mg, 0.052 mmol). H NMR (300 MHz, CDCl3): δ = 2.46 (s, 3H), 4.70-

4.73 (m, 2H), 5.08-5.10 (m, 2H), 5.24 (s, 2H), 6.46 (s, 1H), 6.90 (s, 1H), 7.05-7.19 (m, 2H),

7.33-7.37 (m, 2H), 7.46-7.48 (d, J = 8.7 Hz, 1H), 7.72-7.75 (d, J = 8.4 Hz, 1H). LRMS-LC:

+ m/z calcd for C21H18N4O7 [M+H] : 439.1; found: 439.1.

(7-(((((4-Azidobenzyl)oxy)carbonyl)amino)methoxy)-2-oxo-2H-chromen-4-yl)methyl acetate (291). Solution A: NaH (1.5 eq., 3.5 mg, 0.15 mmol) was added to a solution of the coumarin 269 (1 eq., 23 mg, 0.1 mmol) in THF (0.5 ml). The resulting solution was heated to

60 ˚C for 10 minutes. Solution B: p-Toluene sulfonyl chloride (1 eq., 19 mg, 0.1 mmol) was added to a solution of the alcohol 289 (1 eq., 22 mg, 0.1 mmol) in THF (0.5 ml), followed by

239

over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 291 as an off-white solid

1 in 23% yield (10 mg, 0.023 mmol). H NMR (300 MHz, CDCl3): δ = 2.43 (s, 3H), 4.83 (s,

2H), 5.10-5.13 (m, 2H), 5.21-5.26 (m, 2H), 6.57 (s, 1H), 6.89 (s, 1H), 7.05-7.07 (d, J = 8.1

Hz, 1H), 7.34-7.36 (d, J = 7.8 Hz, 1H), 7.42-7.46 (m, 2H), 7.75-7.78 (m, 2H). LRMS-LC:

+ m/z calcd for C21H18N4O7 [M+H] : 439.1; found: 439.1.

2-Azidobenzyl (4-nitrophenyl) carbonate (292). A solution of the alcohol 279 (1 eq., 149 mg, 1 mmol) in THF (5 ml) was added drop-wise to a solution of 4-nitrophenyl chloroformate (1 eq., 203 mg, 1 mmol) and pyridine (2.1 eq., 0.17 ml, 2.1 mmol) in THF (3.5 ml) at 0 ˚C. The mixture was stirred for 72 hours at room temperature and concentrated. The residue was dissolved in EtOAc (5 ml), and the solution was washed with water (3 ml) and brine (3 ml), dried over Na2SO4, filtered and concentrated. The crude product was purified by recrystallization from 10 ml of EtOAc/hexane (1:4), and the solid was collected through filtration and dried, affording the product 292 as a white solid in 74% yield (233 mg, 0.74

1 mmol). H NMR (300 MHz, CDCl3): δ = 5.27 (s, 2H), 7.15-7.25 (m, 2H), 7.37-7.49 (m, 4H),

8.25-8.28 (d, J = 9.3 Hz, 2H). The analytical data matched reported results.353

4-Azidobenzyl (4-nitrophenyl) carbonate (293). A solution of the alcohol 104 (1 eq., 149 mg, 1 mmol) in THF (5 ml) was added drop-wise to a solution of 4-nitrophenyl chloroformate (1 eq., 203 mg, 1 mmol) and pyridine (2.1 eq., 0.17 ml, 2.1 mmol) in THF (3.5 ml) at 0 ˚C. The mixture was stirred for 72 hours at room temperature and concentrated. The

240

residue was dissolved in EtOAc (5 ml), and the solution was washed with water (3 ml) and brine (3 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by recrystallization from 10 ml of EtOAc/hexane (1:4). The solid was collected through filtration and dried, affording the product 293 as a white solid in 79% yield

1 (250 mg, 0.79 mmol). H NMR (300 MHz, CDCl3): δ = 5.26 (s, 2H), 7.05-7.08 (d, J = 8.4

Hz, 2H), 7.36-7.39 (d, J = 9.3 Hz, 2H), 7.43-7.46 (d, J = 8.4 Hz, 2H), 8.26-8.29 (d, J = 9.3

Hz, 2H). The analytical data matched reported results.182

2-Azidobenzyl carbonochloridate (294). Diphosgene (1.1 eq., 0.7 ml, 5.9 mmol) was added to a solution of the alcohol 279 (1 eq., 800 mg, 5.4 mmol) in THF (20 ml) at 0 ˚C, followed by potassium carbonate (3 eq., 2.23 g, 16 mmol). The mixture was stirred overnight at room temperature and diluted with EtOAc (20 ml). The solution was washed with water (20 ml) and brine (20 ml), dried over Na2SO4, filtered and concentrated, delivering 294 as a brown oil which was confirmed by NMR and used without purification. 1H NMR (300 MHz,

CDCl3): δ = 5.29 (s, 2H), 7.15-7.23 (m, 2H), 7.38-7.48 (m, 2H).

4-Azidobenzyl carbonochloridate (295). Diphosgene (1.1 eq., 0.5 ml, 4.35 mmol) was added to a solution of the alcohol 104 (1 eq., 590 mg, 3.95 mmol) in THF (15 ml) at 0 ˚C, followed by potassium carbonate (3 eq., 1.64 g, 11.8 mmol). The mixture was stirred overnight at room temperature and diluted with EtOAc (20 ml). The solution was washed with water (20 ml) and brine (20 ml), dried over Na2SO4, filtered and concentrated delivering

295 as a brown oil which was confirmed by NMR and used without purification. 1H NMR

241

(300 MHz, CDCl3): δ = 5.26 (s, 2H), 7.04-7.07 (d, J = 8.4 Hz, 2H), 7.38-7.41 (d, J = 8.4 Hz,

2H).

N6-(((2-Azidobenzyl)oxy)carbonyl)-N2-(tert-butoxycarbonyl)-L-lysine (296). Method A:

Boc Lys (1.5 eq., 118 mg, 0.48 mmol) was added to a solution of the carbonate 292 (1 eq.,

100 mg, 0.32 mmol) in DMF (1 ml). The mixture was stirred overnight at room temperature and poured into water (3 ml). The aqueous layer was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (3 ml) and brine (3 ml), and dried over

Na2SO4. After filtration, the solvent was removed affording the product 296 as a yellow foam in 69% yield (92 mg, 0.22 mmol). Method B: Boc Lys (1 eq., 1.3 g, 5.37 mmol) was added to a solution of the chloroformate 294 (1 eq., 1.1 g, 5.37 mmol) in THF/1M NaOH (1:1, 20 ml).

The mixture was stirred overnight at room temperature, concentrated, acidified to pH 3-4 with 1M aq. HCl, and extracted with EtOAc (3 × 20 ml). The combined organic layer was washed with water (30 ml) and brine (30 ml), dried over Na2SO4 and filtered. The solvent was removed under reduced pressure, affording the product 296 as a yellow foam in 82% yield (1.9 g, 4.4 mmol). Method C: Boc Lys (1.2 eq., 2.3 g, 9.4 mmol) was added to a solution of the carbonate 300 (1 eq., 2.28 g, 7.8 mmol) in 80 ml of DMF/water (1:1), followed by potassium carbonate (3 eq., 3.2 g, 23.4 mmol). The mixture was stirred overnight at room temperature, concentrated, acidified to pH 3-4 with 1 M aq. HCl, and extracted with Et2O (3 × 20 ml). The combined organic layer was washed with water (30 ml) and brine (30 ml), and dried over Na2SO4. After filtration, the solvent was removed under reduced pressure, affording the product 296 as a yellow foam in 76% yield (2.6 g, 5.9 mmol).

242

1 H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.42-1.90 (m, 6H), 3.15-3.23 (m, 2H), 4.23-

4.50 (m, 1H), 5.06 (s, 2H), 6.95-7.02 (m, 2H), 7.35-7.37 (m, 2H). 13C NMR (400 MHz,

CDCl3): δ = 13.7, 22.1, 27.8, 28.3, 28.4, 38.7, 52.9, 59.9, 72.4, 78.9, 117.6, 124.3, 126.5,

129.4, 130.9, 155.3, 161.9, 174.2. The analytical data matched reported results. 353

N6-(((4-Azidobenzyl)oxy)carbonyl)-N2-(tert-butoxycarbonyl)-L-lysine (297). Method A:

Boc Lys (1.5 eq., 118 mg, 0.48 mmol) was added to a solution of the carbonate 293 (1 eq.,

100 mg, 0.32 mmol) in DMF (1 ml). The mixture was stirred overnight at room temperature and was poured into water (3 ml). The aqueous solution was extracted with EtOAc (3 × 2 ml), and the combined organic layer was washed with water (3 ml) and brine (3 ml), and dried over Na2SO4. After filtration, the solvent was removed affording the product 297 as a yellow foam in 87% yield (117 mg, 0.27 mmol). Method B: Boc Lys (1 eq., 973 mg, 3.95 mmol) was added to a solution of the chloroformate 295 (1 eq., 1.6 g, 3.95 mmol) in

THF/1M NaOH (1:1, 15 ml). The mixture was stirred overnight at room temperature, concentrated, acidified to pH 3-4 with 1 M HCl, and extracted with EtOAc (3 × 10 ml). The combined organic layer was washed with water (20 ml) and brine (20 ml), and dried over

Na2SO4. After filtration, the solvent was removed affording the product 297 as a yellow foam in 88% yield (1.46 g, 3.47 mmol). Method C: Boc Lys (1.2 eq., 148 mg, 0.6 mmol) was added to a solution of the carbonate 301 (1 eq., 145 mg, 0.5 mmol) in DMF/water (1:1, 7.2 ml) was added Boc Lys (1.2 eq., 148 mg, 0.6 mmol) followed by potassium carbonate (3 eq.,

204 mg, 1.5 mmol). The mixture was stirred overnight at room temperature, concentrated, acidified to pH 3-4 with 1 M HCl, and extracted with Et2O (3 × 5 ml). The combined organic

243

layer was washed with water (3 ml) and brine (3 ml), and dried over Na2SO4. After filtration, the solvent was removed affording the product 297 as a yellow foam in 69% yield (145 mg,

1 0.345 mmol). H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.42-1.90 (m, 6H), 3.15-3.23

(m, 2H), 4.21-4.32 (m, 1H), 5.06 (s, 2H), 7.13-7.16 (d, J = 8.4 Hz, 2H), 7.31-7.34 (d, J = 8.4

- Hz, 2H). HRMS-LC: m/z calcd for C19H27N5O6 [M-H] : 420.1883; found: 420.1845.

N6-(((4-Azidobenzyl)oxy)carbonyl)-L-lysine trifluoroacetic acid salt (298). TFA (10 eq.,

2.7 ml, 35.6 mmol) and Et3SiH (2 eq., 1.14 ml, 7.12 mmol) were added to a solution of the

Boc Lys 296 (1 eq, 1 g, 3.56 mmol) in DCM (50 ml). The reaction was stirred overnight at room temperature and concentrated. The residue was redissolved in MeOH (1 ml) and the solution was added drop-wise to Et2O (500 ml) with vigorous stirring. The precipitate was colltected and dried, affording the product 298 as a white solid in 41% yield (612 mg, 1.46 mmol). 1H NMR (400 MHz, DMSO): δ = 1.23-1.49 (m, 4H), 1.69-1.71 (m, 2H), 2.97-2.99

(q, J = 5.7 Hz, 2H), 3.80-3.85 (t, J = 5.7 Hz, 1H), 4.96 (s, 2H), 7.18-7.46 (m, 4H). The analytical data matched reported results.264

N6-(((4-Azidobenzyl)oxy)carbonyl)-L-lysine hydrochloride salt (299). Method A: A 4 M

HCl in dioxane (1.5 eq., 0.55 ml, 2.19 mmol) was added to a solution of the Lys 298 (1 eq,

612 mg, 1.46 mmol) in dioxane (1.64 ml). The reaction mixture was stirred for 30 minutes and concentrated. The residue was dissolved in water (2 ml) and washed with Et2O (3 × 2 ml). The aqueous layer was concentrated under reduced pressure, affording the product 299 as a white solid in 43% yield (225 mg, 0.63 mmol). Method B: A solution of 1 M aq. HCl

244

(2.4 ml) was added to the Boc Lys 296 (1 eq, 100 mg, 0.24 mmol) in Et2O (2.4 ml). The reaction mixture was stirred for 2 days and washed with Et2O (3 × 5 ml). The aquious layer was concentrated under reduced pressure, affording the product 299 as a white solid in 72% yield (61.7 mg, 0.17 mmol). 1H NMR (400 MHz, DMSO): δ = 1.23-1.49 (m, 4H), 1.69-1.71

(m, 2H), 2.97-2.99 (q, J = 5.7 Hz, 2H), 3.80-3.85 (t, J = 5.7 Hz, 1H), 4.96 (s, 2H), 7.18-7.46

+ (m, 4H). HRMS-LC: m/z calcd for C14H19N5O4 [M+H] : 322.1515; found: 322.1544.

2-Azidobenzyl (2,5-dioxopyrrolidin-1-yl) carbonate (300). N,Nʹ-Disuccinimidyl carbonate

(2 eq., 5.2 g, 20 mmol) was added to a solution of the alcohol 279 (1 eq., 1.5 g, 10 mmol) in

CH3CN (44 ml), followed by TEA (3 eq., 4.2 ml, 30 mmol). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (19:1) as the eluent, affording

1 300 as a white solid in 78% yield (2.28 g, 7.8 mmol). H NMR (300 MHz, CDCl3): δ = 2.66

13 (s, 4H), 5.11 (s, 2H), 7.13-7.19 (m, 2H), 7.39-7.50 (m, 2H). C NMR (400 MHz, CDCl3): δ

= 25.6, 73.6, 118.5, 125.0, 125.1, 131.2, 132.4, 139.8, 171.2.

4-Azidobenzyl (2,5-dioxopyrrolidin-1-yl) carbonate (301). N,Nʹ-Disuccinimidyl carbonate

(2 eq., 51.2 g, 2 mmol) was added to a solution of the alcohol 104 (1 eq., 149 mg, 1 mmol) in

CH3CN (4.4 ml), followed by TEA (3 eq., 0.42 ml, 3 mmol). The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with DCM/acetone (19:1) as the eluent, affording 301 as a white solid in 56% yield (164 mg, 0.56 mmol). 1H NMR (300 MHz,

245

CDCl3): δ = 2.64 (s, 4H), 5.05 (s, 2H), 6.99-7.02 (d, J = 8.4 Hz, 2H), 7.44-7.47 (d, J = 8.4

13 Hz, 2H). C NMR (400 MHz, CDCl3): δ = 25.6, 78.1, 119.3, 130.2, 131.8, 141.4, 150.1,

171.4.

N6-(((4-Azidobenzyl)oxy)carbonyl)-L-lysine hydrochloride salt (302). Formic acid (2 eq.,

0.05 ml, 1.3 mmol) was added to a solution of the Boc Lys 297 (1 eq, 281 mg, 0.67 mmol) in chloroform (0.3 ml). The reaction mixture was stirred for 72 hours at room temperature and concentrated. The residue was taken up in 0.1 M of HCl in dioxane (1.7 ml) and concentrated. The crude product was dissolved in MeOH (0.3 ml) and the solution was added drop-wise to Et2O (100 ml) with vigorous stirring. The solid was collected through filtration and dried, affording the product 302 as an off-white solid in 66% yield (156 mg, 0.44 mmol).

1H NMR (400 MHz, DMSO): δ = 1.38-1.42 (m, 4H), 1.79-1.81 (m, 2H), 2.96-3.00 (m, 2H),

3.80-3.85 (m, 1H), 4.99 (s, 2H), 7.11-7.14 (d, J = 8.4 Hz, 2H), 7.34-7.36 (d, J = 8.4 Hz, 2H),

8.76 (s(br), 2H). 13C NMR (400 MHz, DMSO): δ = 21.6, 28.9, 29.6, 34.0, 51.8, 64.6, 119.1,

- 129.7, 134.2, 138.9, 156.1, 171.0. HRMS-LC: m/z calcd for C14H19N5O4 [M-H] 320.1359; found: 320.1364.

2-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethan-1-ol (304). A solution of ethanolamine (2 eq., 60 µl, 1 mmol) and NaHCO3 (3.3 eq., 139 mg, 1.65 mmol) in water (2 ml) was added to 4-chloro-7-nitrobenzofuran 030 (1 eq., 100 mg, 0.5 mmol) in MeOH (8 ml). The reaction mixture was heated to 50 ˚C for 1 hour and concentrated. The residue was dissolved in EtOAc (5 ml), and the solution was washed with water (2 ml) and brine (2 ml),

246

dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with DCM/MeOH (9:1) as the eluent, affording 304 as a red solid in 72%

1 yield (81 mg, 0.48 mmol). H NMR (300 MHz, CDCl3): δ = 3.64-3.67 (m, 2H), 4.02-4.04

(m, 2H), 6.20-6.23 (d, J = 8.4 Hz, 1H), 8.47-8.50 (d, J = 8.4 Hz, 1H). HRMS-LC: m/z calcd

+ for C8H8N4O4 [M+Na] : 247.0443; found: 247.0421. The analytical data matched reported results.354

2-(Methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethan-1-ol (305). A mixture of the amine 304 (1 eq., 20 mg, 0.09 mmol), formaldehyde (37% w/w in water, 50 µl) and formic acid (25 µl) was heated to 90 ˚C and stirred overnight. The reaction was quenched with sat.

NaHCO3 (2 ml) and the aqueous solution was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (2 ml) and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 305 as a red solid in 93% yield (20 mg, 0.084

1 mmol). H NMR (300 MHz, CDCl3): δ = 2.79 (s, 3H), 3.69-3.71 (m, 2H), 4.05-4.08 (m, 2H),

6.21-6.24 (d, J = 8.4 Hz, 1H), 8.48-8.51 (d, J = 8.4 Hz, 1H). The analytical data matched reported results.355

tert-Butyl (2-hydroxyethyl)(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)carbamate (306). Di- tert-butyl dicarbonate (1.5 eq, 73 mg, 0.33 mmol) was added to a solution of the amine 304

(1 eq., 50 mg, 0.22 mmol) in EtOH (0.5 ml). The mixture was heated to 30 ˚C, stirred for 72 hours, and concentrated. The residue was dissolved in EtOAc (5 ml), and the solution was

247

washed with water (2 ml) and brine (2 ml), dried over Na2SO4, filtered, and concentrated, affording the product 306 as a red solid in 97% yield (70 mg, 0.22 mmol). 1H NMR (300

MHz, CDCl3): δ = 1.42 (s, 9H), 3.65-3.67 (m, 2H), 4.01-4.05 (t, J = 5.4 Hz, 2H), 6.20-6.23

(d, J = 8.7 Hz, 1H), 8.47-8.50 (d, J = 8.7 Hz, 1H). HRMS-LC: m/z calcd for C13H16N4O6

[M+Na]+: 347.0968; found: 347.0943.

Benzyl (2-hydroxyethyl)carbamate (307). Benzyl chloroformate (1 eq, 0.26 ml, 1.85 mmol) was added to ethanolamine (1 eq, 0.11 ml, 1.85 mmol) in DCM (3 ml). The mixture was stirred at room temperature overnight and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 307 as a colorless oil in 40% yield (144 mg, 0.74 mmol). 1H NMR (300

MHz, CDCl3): δ = 3.19-3.27 (m, 2H), 3.56-3.63 (m, 2H), 4.99 (s, 2H), 7.23-7.25 (m, 5H).

The analytical data matched reported results.356

2-(2-Hydroxyethyl)isoindoline-1,3-dione (308). A mixture of ethanolamine (1 eq., 200 mg,

1.35 mmol) and phthalic anhydride (1 eq., 81 µl, 1.35 mmol) was heated for 30 mins at 200

˚C and the resulting thick melt was allowed to cool to room temperature over 30 minutes affording the crude product in quantitative yield (258 mg, 1.35 mmol). The crude product

308 was confirmed by NMR and used without further purification. 1H NMR (300 MHz,

CDCl3): δ = 2.81-2.84(t, J = 5.1 Hz, 2H), 3.56-3.60 (t, J = 5.4 Hz, 2H), 7.72-7.75 (m, 2H),

7.85-7.88 (m, 2H). The analytical data matched reported results.357

248

(S)-1,4-((tert-butoxycarbonyl)amino)-3,8-dioxo-1-phenyl-2,7-dioxa-4,9-diazapenta- decan-15-oic acid (309). Diphosgene (1.1 eq., 34 µl, 0.28 mmol) was added to the alcohol

307 (1 eq., 50 mg, 0.26 mmol) in THF (1 ml) at 0 ˚C, followed by potassium carbonate (3 eq., 106.1 mg, 0.77 mmol). The mixture was stirred overnight at room temperature and diluted with EtOAc (2 ml). The solution was washed with water (2 ml) and brine (2 ml), dried over Na2SO4, filtered and concentrated, delivering the crude product as a colorless oil which was confirmed by NMR and used without purification. Boc Lys (1.5 eq., 95 mg, 0.38 mmol) was added to a solution of the chloroformate generated above in THF/1M NaOH (1:1,

0.8 ml). The mixture was stirred overnight at room temperature, concentrated, acidified to pH

3-4 with 1 M aq. HCl, and extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (3 ml) and brine (3 ml), and dried over Na2SO4. After filtration, the solvent was removed under reduced pressure, affording the product 309 as a colorless foam

1 in 80% yield (96 mg, 0.21 mmol). H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.42-1.90

(m, 6H), 3.02-3.21 (m, 2H), 3.33-3.46 (m, 2H), 4.12-4.18 (m, 2H), 4.20-4.32 (m, 1H), 5.09

(s, 2H), 7.33 (s, 5H).

N2-(tert-Butoxycarbonyl)-N6-((2-(1,3-dioxoisoindolin-2-yl)ethoxy)carbonyl)-L-lysine

(310). Diphosgene (1.1 eq., 35 µl, 0.29 mmol) was added to a solution of the alcohol 308 (1 eq., 50 mg, 0.26 mmol) in THF (1 ml) at 0 ˚C, followed by potassium carbonate (3 eq., 109 mg, 0.79 mmol). The mixture was stirred overnight at room temperature and diluted with

EtOAc (2 ml). The solution was washed with water (2 ml) and brine (2 ml), dried over

Na2SO4, filtered and concentrated, delivering the crude product as a colorless oil which was

249

confirmed by NMR and used without purification. Boc Lys (1 eq., 65 mg, 0.26 mmol) was added to a solution of the chloroformate generated above in THF/1M NaOH (1:1, 0.8 ml).

The mixture was stirred overnight at room temperature, concentrated, acidified to pH 3-4 with 1 M HCl, and extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (3 ml) and brine (3 ml), and dried over Na2SO4. After filtration, the solvent was removed under reduced pressure, affording the product 310 as a colorless foam

1 in 88% yield (103 mg, 0.23 mmol). H NMR (300 MHz, CDCl3): δ = 1.39 (s, 9H), 1.42-1.90

(m, 6H), 3.02-3.17 (m, 2H), 3.59-3.69 (m, 2H), 4.19-4.27 (m, 2H), 4.39-4.48 (m, 1H), 7.65-

7.73 (m, 2H), 7.79-7.84 (m, 2H).

7-Nitrobenzo[c][1,2,5]oxadiazol-4-amine (312). A solution of 30% aq. NH4OH (16.4 ml) was added to 4-chloro-7-nitrobenzo-furan 303 (1 eq, 820 mg, 4.11 mmol) in MeOH (80 ml).

The mixture was stirred at room temperature for 24 hours and concentrated under reduced pressure. The crude product was purified by silica gel chromatography with hexane/EtOAc

(1:1) as the eluent, affording 312 as a red solid in 77% yield (569 mg, 0.74 mmol). 1H NMR

(300 MHz, CDCl3): δ = 6.38-6.41 (d, J = 9.0 Hz, 1H), 8.49-8.52 (d, J = 8.7 Hz, 1H). The analytical data matched reported results.328

Ethyl (7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)carbamate (313). NaH (1.5 eq, 87 mg, 3.63 mmol) was added to a solution of the amine 312 (1 eq., 435 mg, 2.42 mmol) in THF (37 ml) at 0 ˚C. The mixture was stirred for 10 minutes and ethyl chloroformate (1.2 eq, 0.28 ml,

2.90 mmol) was added drop-wise. The reaction mixture was allowed to warm to room

250

temperature and stirred overnight. The reaction was quenched with sat. NaHCO3 (50 ml), and the aqueous solution was extracted with EtOAc (3 × 30 ml). The combined organic layer was washed with water (30 ml) and brine (30 ml), dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent,

1 affording 313 as a red solid in 82% yield (694 mg, 2 mmol). H NMR (300 MHz, CDCl3): δ

= 1.36-1.41 (t, J = 7.2 Hz, 3H), 4.32-4.40 (q, J = 7.2 Hz, 2H), 8.12-8.14 (d, J = 8.4 Hz, 1H),

+ 8.54-8.57 (d, J = 8.4 Hz, 1H). HRMS-LC: m/z calcd for C10H10N4O4 [M+Na] : 275.0392; found: 275.0387.

N2-(tert-Butoxycarbonyl)-N6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)carbamoyl)-L-lysine

(314). Boc Lys (1.5 eq., 157 mg, 0.64 mmol) was added to the carbonate 313 (1 eq., 150 mg,

0.43 mmol) in THF/water (1:1, 2.6 ml), followed by potassium carbonate (4 eq., 235 mg, 1.7 mmol). The mixture was stirred overnight at room temperature, concentrated, acidified to pH

3-4 with 1 M aq. HCl, and extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with water (3 ml) and brine (3 ml), dried over Na2SO4, filtered, and concentrated, affording the product 314 as a red solid in 91% yield (163 mg, 0.39 mmol). 1H NMR (300

MHz, CDCl3): δ = 1.44 (s, 9H), 1.45-2.00 (m, 6H), 3.43-3.59 (m, 2H), 4.31-4.42 (m, 1H),

6.15-6.18 (d, J = 8.4 Hz, 1H), 8.43-8.46 (d, J = 8.4 Hz, 1H). LRMS-LC: m/z calcd for

+ C18H24N6O8 [M+Na] : 475.1; found: 475.2.

N6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)carbamoyl)-L-lysine HCl salt (315). A 4 M HCl solution in dioxane (10 eq, 0.57 ml, 2.3 mmol) was added to the Boc Lys 314 (1 eq, 103 mg,

251

0.23 mmol) in THF (1.7 ml). The mixture was stirred overnight at room temperature and concentrated. The crude product was dissolved in MeOH (0.2 ml) and the solution was added drop-wise to Et2O (100 ml) with vigorous stirring. The solid was collected through filtration and dried, affording 315 as a red solid in 96% yield (85 mg, 0.22 mmol). 1H NMR (300

MHz, CD3OD): δ = 1.39-2.00 (m, 6H), 3.33-3.42 (m, 2H), 3.80-3.87 (m, 1H), 4.31-4.42 (m,

1H), 6.18-6.21 (d, J = 9.3 Hz, 1H), 8.33-8.36 (d, J = 8.4 Hz, 1H). LRMS-LC: m/z calcd for

+ C13H16N6O6 [M+H] : 353.1; found: 353.1.

Methyl N2-(tert-butoxycarbonyl)-N6-((((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)- methoxy)carbonyl)-L-lysinate (316). DIPEA (1.1 eq, 25 µl, 0.14 mmol) was added to the

Boc Lys 240 (1.1 eq, 50 mg, 0.14 mmol) in DCM (1 ml), followed by TBAI (1.1 eq, 52 mg,

0.14 mmol) and the amine 312 (1 eq, 23 mg, 0.13 mmol). The mixture was stirred overnight at room temperature, and the reaction was quenched with sat. NaHCO3 (1 ml). The aqueous layer was extracted with DCM (3 × 1 ml). The combined organic layer was washed with water (1 ml) and brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent,

1 affording 316 as a red solid in 26% yield (17 mg, 0.033 mmol). H NMR (300 MHz, CDCl3):

δ = 1.43 (s, 9H), 1.45-1.90 (m, 6H), 3.21-3.40 (m, 2H), 3.73 (s, 3H), 4.21-4.32 (m, 1H), 5.27-

5.32 (m, 2H), 8.22-8.25 (d, J = 8.4 Hz, 1H), 8.49-8.52 (d, J = 8.7 Hz, 1H).

(S)-2-((tert-Butoxycarbonyl)amino)hex-5-enoic acid (318). Sodium carbonate (1 eq,. 862 mg, 8.13 mmol) was added to the Boc Lys 317 (1 eq., 2 g, 8.1 mmol) in water (40 ml) and

252

the mixture was heated to 60 ˚C. Sodium nitroprusside (2 eq., 5 × 960 mg, 16.2 mmol) was added in five portion over 40 minutes with vigrous effervescence. The pH of the resulting mixture was maintained at pH 9 by addition of 4 M NaOH and the reaction mixture was stirred at 60 ˚C overnight. The solution was cooled to 0 ˚C, acidified to pH 1 with the addition of 1 M aq. HCl, and extracted with EtOAc (3 × 50 ml). The combined organic layer was washed with brine (100 ml), dried over Na2SO4, filtered, and concentrated, affording the product 318 as an off-white solid in 47% yield (873 mg, 3.8 mmol). 1H NMR (300 MHz,

CDCl3): δ = 1.41 (s, 9H), 1.42-1.90 (m, 6H), 4.22-4.38 (m, 1H), 4.20-4.32 (m, 1H), 4.93-5.09

(m, 2H), 5.70-5.83 (m, 1H). The analytical data matched reported results.358

(S)-2-((tert-Butoxycarbonyl)amino)-6-hydroxyhexanoic acid (319). Method A: NaOH (4

M, 4.3 ml) was added to the Boc Lys 317 (1 eq., 2 g, 8.1 mmol) in water (30 ml) at 60 ˚C, followed by sodium nitroprusside (1.53 eq., 4 × 925 mg, 12.4 mmol) in four portion over 1 hour with vigrous stirring. The pH of the resulting mixture was maintained at pH 9 by adding

4 M NaOH and the reaction mixture was stirred at 60 ˚C overnight. The solution was cooled to 0 ˚C, acidified to pH 1 with 1 M aq. HCl, and extracted with EtOAc (3 × 50 ml). The combined organic layer was washed with brine (100 ml), dried over Na2SO4, filtered, and concentrated, affording the product 319 as an off-white solid in 65% yield (1.3 g, 5.3 mmol).

Method B: BH3•THF complex in THF (1.5 eq., 1 M, 0.45 ml, 0.45 mmol) was added to a solution of the alkene 318 (1 eq., 72 mg, 0.3 mmol) in THF (0.5 ml) at 0 ˚C, and the mixture was stirred for 1 hour at 0 ˚C. A 3 M aq. NaOH (6.7 eq., 0.67 ml, 2 mmol) and 30% hydrogen peroxide in water (1.8 ml/mmol, 0.54 ml) were added. The solution was allowed to

253

warm to room temperature over one hour and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with water (1 ml) and brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 319 as an off-white solid in 37% yield (29 mg,

1 0.11 mmol). H NMR (300 MHz, CDCl3): δ = 1.41 (s, 9H), 1.42-1.90 (m, 6H), 3.61-3.65 (m,

+ 2H), 4.21-4.39 (m, 1H), 4.20-4.32 (m, 1H). LRMS-LC: m/z calcd for C11H21NO5 [M+Na] :

270.13; found: 270.08. The analytical data matched reported results.340

Benzyl (S)-2-((tert-butoxycarbonyl)amino)-6-hydroxyhexanoate (320). Benzyl bromide

(1.5 eq., 260 mg, 1.5 mmol) was added to a solution of the acid 319 (1 eq., 250 mg, 1 mmol) and KHCO3 (1.5 eq., 152 mg, 1.5 mmol) in DMSO (2 ml), followed by TBAI (0.1 eq., 37 mg, 0.1 mmol). The mixture was stirred overnight and the reaction was quenched by the addition of water (5 ml). The aqueous solution was extracted with EtOAc (3 × 2 ml). The combined organic layer was washed with sat. NaHCO3 (2 ml), water (2 ml) and brine (2 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 320 as a colorless foam in

1 57% yield (194 mg, 0.57 mmol). H NMR (300 MHz, CDCl3): δ = 1.42 (s, 9H), 1.42-1.90

(m, 6H), 3.42-3.52 (m, 2H), 4.29-4.42 (m, 1H), 5.12-5.19 (m, 2H), 5.67-5.79 (m, 1H), 7.34

+ (s, 5H). LRMS-LC: m/z calcd for C18H27NO5 [M+Na] : 260.18; found: 260.2. The analytical data matched reported results.359

254

Methyl (S)-2-((tert-butoxycarbonyl)amino)-6-hydroxyhexanoate (321). Iodomethane (1.1 eq., 56 µl, 0.89 mmol) was added to a solution of the acid 319 (1 eq., 200 mg, 0.81 mmol) and DIPEA (2.2 eq., 0.31 ml, 1.78 mmol) in DMF (2.4 ml) at 0 ˚C, and the reaction mixture was stirred overnight at room temperature. The mixture was diluted with EtOAc (5 ml) and the solution was washed with sat. NaHCO3 (2 ml), water (2 ml) and brine (2 ml), dried over

Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 321 as a colorless foam in

1 59% yield (125 mg, 0.48 mmol). H NMR (300 MHz, CDCl3): δ = 1.40 (s, 9H), 1.42-1.90

(m, 6H), 3.56-3.60 (m, 2H), 3.69 (s, 3H), 4.21-4.32 (m, 1H), 5.03-5.11 (m, 1H). The analytical data matched reported results.360

Benzyl (S)-6-bromo-2-((tert-butoxycarbonyl)amino)hexanoate (322). The alcohol 320 (1 eq., 80 mg, 0.24 mmol) and CBr4 (2 eq., 158 mg, 0.48 mmol) was dissolved in THF (1 ml) and triphenylphosphine (2 eq., 125 mg, 0.48 mmol) was added. The resulting mixture was stirred for 1 hour at room temperature and concentrated under reduced pressure. The solution was diluted with water (1 ml) and extracted with EtOAc (3 × 1 ml). The crude product was purified by silica gel chromatography with hexane/EtOAc (1:1) as the eluent, affording 322

1 as a colorless foam in 75% yield (72 mg, 0.18 mmol). H NMR (300 MHz, CDCl3): δ = 1.42

(s, 9H), 1.42-1.90 (m, 6H), 3.30-3.34 (t, J = 5.4 Hz, 2H), 4.31-4.40 (m, 1H), 4.99-5.21 (m,

3H), 7.34 (s, 5H). The analytical data matched reported results.361

255

Benzyl (S)-2-((tert-butoxycarbonyl)amino)-6-(1,3-dioxoisoindolin-2-yl)hexanoate (323).

Potassium phthalimide (1 eq., 31 mg, 0.17 mmol) was added to a solution of the hexanoate

322 (1 eq., 67 mg, 0.17 mmol) in DMF (0.3 ml). The reaction mixture was stirred for 1 hour at 70 ˚C and filtered through silica gel. The filtrate was taken up in water (2 ml) and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (4:1) as the eluent, affording 323 as a colorless foam in

1 94% yield (73 mg, 0.16 mmol). H NMR (300 MHz, CDCl3): δ = 1.40 (s, 9H), 1.60-1.90 (m,

6H), 3.58-3.62 (t, J = 5.4 Hz, 2H), 4.24-4.36 (m, 1H), 4.99-5.20 (m, 3H), 7.33 (s, 5H), 7.65-

7.70 (m, 2H), 7.79-7.83 (m, 2H).

Benzyl (tert-butoxycarbonyl)-L-lysinate (324). Hydrazine (2.5 eq., 8.5 µl, 0.27 mmol) was added to a solution of the phthalimide 323 (1 eq., 50 mg, 0.11 mmol) in EtOH (0.5 ml). The mixture was heated to reflux for 1 hour and concentrated under reduced pressure. The residue was taken up in water (1 ml), and the solution was basified to pH 8 with 1M aq. NaOH and extracted with EtOAc (3 × 1 ml). The combined organic layer was washed with brine (1 ml), dried over Na2SO4, filtered, and concentrated, affording the product 324 as a yellow foam in

1 60% yield (22 mg, 0.065 mmol). H NMR (300 MHz, CDCl3): δ = 1.41 (s, 9H), 1.42-1.97

(m, 6H), 3.41-3.50 (m, 2H), 4.29-4.30 (m, 1H), 4.97-5.20 (m, 2H), 7.33 (s, 5H). The analytical data matched reported results.362

256

4-Nitrobenzyl carbonochloridate (326). Diphosgene (1.1 eq., 0.26 ml, 2.16 mmol) was added to a solution of the p-nitrobenzyl alcohol (1 eq., 300 mg, 1.96 mmol) in THF (7.5 ml) at 0 ˚C, followed by potassium carbonate (3 eq., 812 mg, 5.88 mmol). The mixture was stirred overnight at room temperature, filtered and concentrated delivering the crude product

326 as a yellow oil which was confirmed by NMR and used without purification. 1H NMR

(300 MHz, CDCl3): δ = 5.37 (s, 2H), 7.53-7.59 (m, 2H), 8.20-8.27 (m, 2H). The analytical data matched reported results.363

4-Nitrobenzyl (S)-(4-(5'-oxo-9λ4-boraspiro[bicyclo[3.3.1]nonane-9,2'-[1,3,2]oxaza- borolidin]-4'-yl)butyl)carbamate (328). The HCl salt of lysine (327) (1 eq., 298 mg, 1.63 mmol) was dissolved in 7 M NH3 in MeOH (4.1 ml) at 0 ˚C. The solution was stirred for 30 minutes and concentrated, affording a white solid. The solid was added to a stirring solution of 9-BBN (1.5 eq., 3.9 ml of 0.5 M solution in THF, 1.96 mmol) in MeOH (12 ml). The reaction mixture was heated to reflux overnight and concentrated. The residue was dissolved in hot THF and filtered. The filtrate was concentrated under reduced pressure and dried, affording the 9-BBN protected Lys which was confirmed by NMR and used in next step without further purification. The crude 9-BBN Lys was dissolved in a mixture of dioxane

(1.4 ml) and water (2ml), and cooled to 0 ˚C. The 4-nitrobenzyl chloroformate 326 (1.2 eq.,

1.96 mmol) in dioxane (0.4 ml) was added, and the mixture was stirred overnight at room temperature and concentrated. The residue was dissolved in EtOAc (10 ml) and washed with water (5 ml) and brine (5 ml), dried over Na2SO4, filtered and concentrated. The crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent,

257

affording 328 as a colorless foam in 67% yield (486 mg, 1.09 mmol). 1H NMR (300 MHz,

CDCl3): δ = 0.53 (s, 2H), 1.37-1.98 (m, 18H), 3.15-3.23 (m, 2H), 4.52-4.63 (m, 1H), 5.14-

5.27 (m, 3H), 7.43-7.46 (d, J = 8.7 Hz, 2H), 8.16-8.19 (d, J = 8.7 Hz, 2H).

N6-(((4-Nitrobenzyl)oxy)carbonyl)-L-lysine HCl salt (329). The 9-BBN Lys 328 (1 eq, 200 mg, 0.45 mmol) was dissolved in 4 M HCl in dioxane (6 ml). The mixture was stirred overnight at room temperature and concentrated. The crude product was dissolved in MeOH

(0.2 ml) and the solution was added drop-wise to Et2O (200 ml) with vigorous stirring. The solid was collected through filtration and dried, affording the product 329 as a white solid in

1 79% yield (128 mg, 0.35 mmol). H NMR (300 MHz, CDCl3): δ = 1.23-1.50 (m, 4H), 1.69-

1.89 (m, 2H), 2.99-3.08 (m, 2H), 3.80-3.84 (m, 1H), 5.04 (s, 2H), 7.41-7.44 (d, J = 8.4 Hz,

2H), 8.04-8.07 (d, J = 9.0 Hz, 2H). The analytical data matched reported results.339

4-Nitrobenzyl (S)-(4-(5'-oxo-9λ4-boraspiro[bicyclo[3.3.1]nonane-9,2'-[1,3,2]oxazaboro- lidin]-4'-yl)butyl)carbamate-15N (331). The HCl salt of 15N lysine 330 (1 eq., 500 mg, 2.27 mmol) was dissolved in 7 M NH3 (5.8 ml) in MeOH at 0 ˚C. The solution was stirred for 30 minutes and concentrated affording a white solid. The solid was added to a stirring solution of 9-BBN (1.5 eq., 5.5 ml of 0.5 M solution in THF, 2.72 mmol) in MeOH (17 ml). The reaction mixture was heated to reflux overnight and concentrated. The residue generated was dissolved in hot THF and filtered. The filtrate was concentrated under reduced pressure and dried, affording 9-BBN protected Lys which was confirmed by NMR and used in next step without further purification. The crude 9-BBN Lys was dissolved in a mixture of dioxane (2

258

ml) and water (2.7 ml) and cooled to 0 ˚C. A solution of 4-nitrobenzyl chloroformate 326

(1.2 eq., 2.72 mmol) in 0.7 ml of dioxane was added. The mixture was stirred overnight at room temperature and concentrated. The residue was dissolved in EtOAc (20 ml) and washed with water (15 ml) and brine (15 ml), and dried over Na2SO4. After filtration, the solvent was removed and the crude product was purified by silica gel chromatography with hexane/EtOAc (2:1) as the eluent, affording 331 as a colorless foam in 37% yield (385 mg,

1 0.85 mmol). H NMR (300 MHz, CDCl3): δ = 0.55-0.58 (m, 2H), 1.28-2.00 (m, 18H), 3.24-

3.26 (m, 2H), 4.57-4.65 (m, 1H), 5.16-5.26 (m, 3H), 7.46-7.49 (d, J = 8.4 Hz, 2H), 8.22-8.24

15 + (d, J = 8.4 Hz, 2H). LRMS-LC: m/z calcd for C22H32BN2 NO6 [M+H] : 447.2; found: 447.2.

N6-(((4-Nitrobenzyl)oxy)carbonyl)-L-lysine-N-15N HCl salt (332). A solution of the 9-

BBN Lys 331 (1 eq, 385 mg, 0.86 mmol) in 4 M HCl in dioxane (12 ml) was stirred overnight at room temperature and concentrated. The crude product was dissolved in MeOH

(0.3 ml) and the solution was added drop-wise to Et2O (200 ml) with vigorous stirring. The solid was collected through filtration and dried, affording the product 332 as a white solid in

1 87% yield (273 mg, 0.75 mmol). H NMR (300 MHz, CDCl3): δ = 1.20-1.43 (m, 4H), 1.71-

1.80 (m, 2H), 2.97-3.03 (m, 2H), 3.80-3.90 (m, 1H), 5.14 (s, 2H), 7.56-7.59 (d, J = 8.4 Hz,

15 + 2H), 8.22-8.25 (d, J = 9.0 Hz, 2H). HRMS-LC: m/z calcd for C14H19N2 NO6 [M+H] :

327.1322; found: 327.1308.

259

REFERENCES

1. Buskirk, A. R.; Liu, D. R., Creating small-molecule-dependent switches to modulate biological functions. Chem Biol 2005, 12, 151-61.

2. Seidman, M. M.; Glazer, P. M., The potential for gene repair via triple helix formation. J Clin Invest 2003, 112, 487-94.

3. Sargent, R. G.; Kim, S.; Gruenert, D. C., Oligo/polynucleotide-based gene modification: strategies and therapeutic potential. Oligonucleotides 2011, 21, 55-75.

4. Diviacco, S.; Rapozzi, V.; Xodo, L.; Helene, C.; Quadrifoglio, F.; Giovannangeli, C., Site-directed inhibition of DNA replication by triple helix formation. FASEB J 2001, 15, 2660-8.

5. Patterson, A.; Caprio, F.; Vallée-Bélisle, A.; Moscone, D.; Plaxco, K. W.; Palleschi, G.; Ricci, F., Using triplex-forming oligonucleotide probes for the reagentless, electrochemical detection of double-stranded DNA. Anal Chem 2010, 82, 9109-15.

6. Brennan, P.; Donev, R.; Hewamana, S., Targeting transcription factors for therapeutic benefit. Mol. Biosyst. 2008, 4, 909-919.

7. Morishita, R.; Higaki, J.; Tomita, N.; Ogihara, T., Application of transcription factor "decoy" strategy as means of gene therapy and study of gene expression in cardiovascular disease. Circ Res 1998, 82, 1023-8.

8. Kurreck, J., Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 2003, 270, 1628-44.

9. Shestopalov, I. A.; Chen, J. K., Oligonucleotide-Based Tools for Studying Zebrafish Development. Zebrafish 2010, 7, 31-40.

10. Breaker, R. R.; Joyce, G. F., A DNA enzyme that cleaves RNA. Chem Biol 1994, 1, 223-9.

11. Joyce, G. F., RNA cleavage by the 10-23 DNA enzyme. Ribonucleases, Pt A 2001, 341, 503-517.

12. Willner, I.; Shlyahovsky, B.; Zayats, M.; Willner, B., DNAzymes for sensing, nanobiotechnology and logic gate applications. Chem. Soc. Rev. 2008, 37.

13. Silverman, S. K., DNA as a Versatile Chemical Component for Catalysis, Encoding, and Stereocontrol. Angew. Chem. Int. Ed. 2010, 49, 7180-7201.

260

14. Samanta, B.; Hobartner, C., Combinatorial Nucleoside-Deletion-Scanning Mutagenesis of Functional DNA. Angew. Chem. Int. Ed. 2013, 52, 2995-2999.

15. Koizumi, M.; Soukup, G. A.; Kerr, J. N. Q.; Breaker, R. R., Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nature Struct Biol 1999, 6, 1062-1071.

16. Xiang, S.; Fruehauf, J.; Li, C. J., Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nat Biotechnol 2006, 24, 697-702.

17. Elbashir, S.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001, 411, 494-8.

18. Tiemann, K.; Rossi, J. J., RNAi-based therapeutics-current status, challenges and prospects. EMBO Mol Med 2009, 1, 142-51.

19. Shrivastava, N.; Srivastava, A., RNA interference: an emerging generation of biologicals. Biotechnol J 2008, 3, 339-53.

20. Bartel, D. P., MicroRNAs: Target Recognition and Regulatory Functions. Cell 2009, 136.

21. Calin, G. A.; Croce, C. M., MicroRNA signatures in human cancers. Nat Rev Cancer 2006, 6.

22. Williams, A. E., Functional aspects of animal microRNAs. Cell Mol Life Sci 2008, 65, 545-62.

23. Krutzfeldt, J.; Rajewsky, N.; Braich, R.; Rajeev, K. G.; Tuschl, T.; Manoharan, M.; Stoffel, M., Silencing of microRNAs in vivo with 'antagomirs'. Nature 2005, 438.

24. Thomas, M.; Deiters, A., MicroRNA miR-122 as a therapeutic target for oligonucleotides and small molecules. Curr Med Chem 2013, 20, 3629-40.

25. Mayer, G., The Chemical Biology of Aptamers. Angew. Chem. Int. Ed. 2009, 48, 2672-2689.

26. Famulok, M.; Mayer, G., Aptamer Modules as Sensors and Detectors. Acc. Chem. Res. 2011, 44, 1349-1358.

27. Zhou, J. H.; Rossi, J. J., Cell-Specific Aptamer-Mediated Targeted Drug Delivery. Oligonucleotides 2011, 21, 1-10.

261

28. Zhang, J. N.; Liu, B.; Liu, H. X.; Zhang, X. B.; Tan, W. H., Aptamer-conjugated gold nanoparticles for bioanalysis. Nanomedicine 2013, 8, 983-993.

29. Pednekar, P. P.; Jadhav, K. R.; Kadam, V. J., Aptamer-dendrimer bioconjugate: a nanotool for therapeutics, diagnosis, and imaging. Exp. Opin. Drug Deliv. 2012, 9, 1273- 1288.

30. Wang, T. J.; Ray, J., Aptamer-based molecular imaging. Protein & Cell 2012, 3, 739- 754.

31. Chen, X.; Ellington, A., Shaping up nucleic acid computation. Curr. Opin. Biotechnol. 2010, 21, 392-400.

32. Zamecnik, P. C.; Stephenson, M. L., Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci U S A 1978, 75, 280-4.

33. Prakash, T. P., An Overview of Sugar-Modified Oligonucleotides for Antisense Therapeutics. Chem. Biodiversity 2011, 8, 1616-1641.

34. Eckstein, F., Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev 2000, 10, 117-21.

35. Guga, P.; Koziolkiewicz, M., Phosphorothioate Nucleotides and Oligonucleotides - Recent Progress in Synthesis and Application. Chem. Biodiversity 2011, 8, 1642-1681.

36. Deleavey, G. F.; Damha, M. J., Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing. Chem. Biol. 2012, 19, 937-954.

37. Rayburn, E. R.; Zhang, R., Antisense, RNAi, and gene silencing strategies for therapy: mission possible or impossible? Drug Discov Today 2008, 13, 513-21.

38. Karkare, S.; Bhatnagar, D., Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino. Appl Microbiol Biotechnol 2006, 71, 575-86.

39. Karkare, S.; Bhatnagar, D., Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino. Appl. Microbiol. Biotechnol. 2006, 71, 575-586.

40. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E., PNA Hybridizes to complementary

262

oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 1993, 365, 566- 568.

41. Nielsen, P. E., Applications of peptide nucleic acids. Curr. Opin. Biotechnol. 1999, 10, 71-75.

42. Briones, C.; Moreno, M., Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development. Anal. Bioanal. Chem.y 2012, 402, 3071- 3089.

43. Lennox, K. A.; Behlke, M. A., Chemical modification and design of anti-miRNA oligonucleotides. Gene Ther. 2011, 18, 1111-1120.

44. Arzumanov, A.; Walsh, A. P.; Rajwanshi, V. K.; Kumar, R.; Wengel, J.; Gait, M. J., Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2'-O- methyl/LNA oligoribonucleotides. Biochemistry 2001, 40, 14645-54.

45. Stenvang, J.; Silahtaroglu, A. N.; Lindow, M.; Elmen, J.; Kauppinen, S., The utility of LNA in microRNA-based cancer diagnostics and therapeutics. Semin Cancer Biol 2008, 18, 89-102.

46. Veedu, R. N.; Wengel, J., Locked nucleic acid nucleoside triphosphates and polymerases: on the way towards evolution of LNA aptamers. Mol. Biosyst. 2009, 5, 787-792.

47. Campbell, M. A.; Wengel, J., Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals. Chem. Soc. Rev. 2011, 40, 5680-5689.

48. Bill, B. R.; Petzold, A. M.; Clark, K. J.; Schimmenti, L. A.; Ekker, S. C., A Primer for Morpholino Use in Zebrafish. Zebrafish 2009, 6, 69-77.

49. Bruno, I. G.; Jin, W.; Cote, G. J., Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements. Hum Mol Genet 2004, 13, 2409-20.

50. Pase, L.; Layton, J. E.; Kloosterman, W. P.; Carradice, D.; Waterhouse, P. M.; Lieschke, G. J., miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2. Blood 2009, 113, 1794-804.

51. Du, L. T.; Gatti, R. A., Potential therapeutic applications of antisense morpholino oligonucleotides in modulation of splicing in primary immunodeficiency diseases. J. Immunol. Methods 2011, 365, 1-7.

263

52. Stein, D. A., Inhibition of RNA Virus Infections with Peptide-Conjugated Morpholino Oligomers. Curr. Pharm. Des. 2008, 14, 2619-2634.

53. Jain, M. L.; Bruice, P. Y.; Szabo, I. E.; Bruice, T. C., Incorporation of Positively Charged Linkages into DNA and RNA Backbones: A Novel Strategy for Antigene and Antisense Agents. Chem. Rev. 2012, 112, 1284-1309.

54. Carell, T.; Brandmayr, C.; Hienzsch, A.; Muller, M.; Pearson, D.; Reiter, V.; Thoma, I.; Thumbs, P.; Wagner, M., Structure and Function of Noncanonical Nucleobases. Angew. Chem. Int. Ed. 2012, 51, 7110-7131.

55. Mayer, G.; Heckel, A., Biologically active molecules with a "light switch". Angew. Chem. Int. Ed. 2006, 45, 4900-4921.

56. Tomasini, A. J.; Schuler, A. D.; Zebala, J. A.; Mayer, A. N., PhotoMorphs (TM): A Novel Light-Activated Reagent for Controlling Gene Expression in Zebrafish. Genesis 2009, 47, 736-743.

57. Kaplan, J. H.; Forbush, B.; Hoffman, J. F., Rapid photolytic release of adenosine 5'- triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 1978, 17, 1929-35.

58. Klán, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J., Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy. Chem Rev 2013, 113, 119-91.

59. Young, D.; Deiters, A., Photochemical control of biological processes. Org. Biomol. Chem. 2007, 999-1005.

60. Gardner, L.; Deiters, A., Light-controlled synthetic gene circuits. Curr Opin Chem Biol 2012, 16, 292-9.

61. Riggsbee, C.; Deiters, A., Recent advances in the photochemical control of protein function. Trends in Biotechnol. 2010, 468-475.

62. Deiters, A., Principles and Applications of the Photochemical Control of Cellular Processes. Chembiochem 2010, 47-53.

63. Lawrence, D. S., The preparation and in vivo applications of caged peptides and proteins. Curr Opin Chem Biol 2005, 9, 570-5.

64. Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A., Light-controlled tools. Angew Chem Int Ed Engl 2012, 51, 8446-76.

264

65. Lee, H. M.; Larson, D. R.; Lawrence, D. S., Illuminating the chemistry of life: design, synthesis, and applications of "caged" and related photoresponsive compounds. ACS Chem Biol 2009, 4, 409-27.

66. Adams, S. R.; Tsien, R. Y., Controlling cell chemistry with caged compounds. Annu Rev Physiol 1993, 55, 755-84.

67. Cummings, R. T.; Krafft, G. A., Photoactivable fluorophores. 1. Synthesis and photoactivation of ortho-nitrobenzyl-quenched fluorescent carbamates. Tetrahedron Lett. 1988, 29, 65-68.

68. Rossi, F. M.; Kao, J. P. Y., Nmoc-DBHQ, a new caged molecule for modulating sarcoplasmic/endoplasmic reticulum Ca2+ ATPase activity with light flashes. J. Biol. Chem. 1997, 272, 3266-3271.

69. Specht, A.; Goeldner, M., 1-(o-nitrophenyl)-2,2,2-trifluoroethyl ether derivatives as stable and efficient photoremovable alcohol-protecting groups. Angew. Chem. Int. Ed. 2004, 43, 2008-2012.

70. Zehavi, U.; Amit, B.; Patchorn.A, Light-sensitive glycosides. 1. 6-Nitroveratryl beta- D-glucopyranoside and 2-nitrobenzyl beta-D-glucopyranoside. J. Org. Chem. 1972, 37, 2281-&. 71. Martina, S.; Macdonald, S. A.; Enkelmann, V., Photosensitive tetramethylpiperidine urethanes-synthesis and characterization. J. Org. Chem. 1994, 59, 3281-3283.

72. Lusic, H.; Deiters, A., A new photocaging group for aromatic N-heterocycles. Synthesis 2006, 2147-2150.

73. Wu, L.; Wang, Y.; Wu, J. Z.; Lv, C.; Wang, J.; Tang, X. J., Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells. Nucleic Acids Ress 2013, 41, 677-686.

74. Govan, J. M.; Uprety, R.; Thomas, M.; Lusic, H.; Lively, M. O.; Deiters, A., Cellular Delivery and Photochemical Activation of Antisense Agents through a Nucleobase Caging Strategy. Acs Chem. Biol. 2013, 8, 2272-2282.

75. Aujard, I.; Benbrahim, C.; Gouget, M.; Ruel, O.; Baudin, J. B.; Neveu, P.; Jullien, L., o-nitrobenzyl photolabile protecting groups with red-shifted absorption: syntheses and uncaging cross-sections for one- and two-photon excitation. Chemistry 2006, 12, 6865-79.

76. Pirrung, M. C.; Lee, Y. R.; Park, K.; Springer, J. B., Pentadienylnitrobenzyl and pentadienylnitropiperonyl photochemically removable protecting groups. J. Org. Chem. 1999, 64, 5042-5047.

265

77. Pirrung, M. C.; Wang, L. X.; Montague-Smith, M. P., 3 '- nitrophenylpropyloxycarbonyl (NPPOC) protecting groups for high-fidelity automated 5 ' -> 3 ' photochemical DNA synthesis. Org. Lett. 2001, 3, 1105-1108.

78. Specht, A.; Thomann, J. S.; Alarcon, K.; Wittayanan, W.; Ogden, D.; Furuta, T.; Kurakawa, Y.; Goeldner, M., New photoremovable protecting groups for carboxylic acids with high photolytic efficiencies at near-UV irradiation. Application to the photocontrolled release of L-glutamate. Chembiochem 2006, 7, 1690-1695.

79. Berroy, P.; Viriot, M. L.; Carre, M. C., Photolabile group for 5 '-OH protection of nucleosides: synthesis and photodeprotection rate. Sens. Actuators, B 2001, 74, 186-189.

80. San Miguel, V.; Bochet, C. G.; del Campo, A., Wavelength-Selective Caged Surfaces: How Many Functional Levels Are Possible? J. Am. Chem. Soc. 2011, 133, 5380-5388.

81. Fonseca, A. S. C.; Goncalves, M. S. T.; Costa, S. P. G., Photocleavage studies of fluorescent amino acid conjugates bearing different types of linkages. Tetrahedron 2007, 63, 1353-1359.

82. Eckardt, T.; Hagen, V.; Schade, B.; Schmidt, R.; Schweitzer, C.; Bendig, J., Deactivation Behavior and excited-state properties of (coumarin-4-yl)methyl derivatives. 2. Photocleavage of selected (coumarin-4-yl)methyl-Caged adenosine cyclic 3 ',5 '- monophosphates with fluorescence enhancement. J. Org. Chem. 2002, 67, 703-710.

83. Fournier, L.; Aujard, I.; Le Saux, T.; Maurin, S.; Beaupierre, S.; Baudin, J. B.; Jullien, L., Coumarinylmethyl Caging Groups with Redshifted Absorption. Chem. Eur. J. 2013, 19, 17494-17507.

84. Fonseca, A. S. C.; Soares, A. M. S.; Goncalves, M. S. T.; Costa, S. P. G., Thionated coumarins and quinolones in the light triggered release of a model amino acid: synthesis and photolysis studies. Tetrahedron 2012, 68, 7892-7900.

85. Hasan, A.; Stengele, K. P.; Giegrich, H.; Cornwell, P.; Isham, K. R.; Sachleben, R. A.; Pfleiderer, W.; Foote, R. S., Photolabile protecting groups for nucleosides: Synthesis and photodeprotection rates. Tetrahedron 1997, 53, 4247-4264.

86. Givens, R. S.; Rubina, M.; Wirz, J., Applications of p-hydroxyphenacyl (pHP) and coumarin-4-ylmethyl photoremovable protecting groups. Photochem. Photobiol. Sci. 2012, 11, 472-488.

87. Fournier, L.; Gauron, C.; Xu, L.; Aujard, I.; Le Saux, T.; Gagey-Eilstein, N.; Maurin, S.; Dubruille, S.; Baudin, J. B.; Bensimon, D.; Volovitch, M.; Vriz, S.; Jullien, L., A Blue-

266

Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation. ACS Chem Biol 2013.

88. Schmidt, R.; Geissler, D.; Hagen, V.; Bendig, J., Mechanism of photocleavage of (coumarin-4-yl)methyl esters. J Phys Chem A 2007, 111, 5768-74.

89. Piston, D., Imaging living cells and tissues by two-photon excitation microscopy. Trends in Cell Biol. 1999, 66-69.

90. Augustine, G., Illuminating the location of brain glutamate receptors. Nat. Neurosci. 2001, 1051-1052.

91. Helmchen, F.; Denk, W., Deep tissue two-photon microscopy. Nat Methods 2005, 2, 932-40.

92. Furuta, T.; Wang, S.; Dantzker, J.; Dore, T.; Bybee, W.; Callaway, E.; Denk, W.; Tsien, R., Brominated 7-hydroxycoumarin-4-ylmethyls: Photolabile protecting groups with biologically useful cross-sections for two photon photolysis. Proc. Natl. Acad Sci. U. S. A. 1999, 1193-1200.

93. LaFratta, C. N.; Fourkas, J. T.; Baldacchini, T.; Farrer, R. A., Multiphoton fabrication. Angew Chem Int Ed Engl 2007, 46, 6238-58.

94. Gug, S.; Bolze, F.; Specht, A.; Bourgogne, C.; Goeldner, M.; Nicoud, J., Molecular Engineering of Photoremovable Protecting Groups for Two-Photon Uncaging. Angew. Chem. Int. Ed. 2008, 9525-9529.

95. Warther, D.; Gug, S.; Specht, A.; Bolze, F.; Nicoud, J. F.; Mourot, A.; Goeldner, M., Two-photon uncaging: New prospects in neuroscience and cellular biology. Bioorg Med Chem 2010, 18, 7753-8.

96. Kotzur, N.; Briand, B.; Beyermann, M.; Hagen, V., Wavelength-selective photoactivatable protecting groups for thiols. J Am Chem Soc 2009, 131, 16927-31.

97. Davis, M. J.; Kragor, C. H.; Reddie, K. G.; Wilson, H. C.; Zhu, Y.; Dore, T. M., Substituent effects on the sensitivity of a quinoline photoremovable protecting group to one- and two-photon excitation. J Org Chem 2009, 74, 1721-9.

98. Momotake, A.; Lindegger, N.; Niggli, E.; Barsotti, R.; Ellis-Davies, G., The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells. Nat. Methods2006, 35-40.

267

99. Gug, S.; Charon, S.; Specht, A.; Alarcon, K.; Ogden, D.; Zietz, B.; Leonard, J.; Haacke, S.; Bolze, F.; Nicoud, J.; Goeldner, M., Photolabile glutamate protecting group with high one- and two-photon uncaging efficiencies. Chembiochem 2008, 1303-1307.

100. Donato, L.; Mourot, A.; Davenport, C. M.; Herbivo, C.; Warther, D.; Leonard, J.; Bolze, F.; Nicoud, J. F.; Kramer, R. H.; Goeldner, M.; Specht, A., Water-Soluble, Donor- Acceptor Biphenyl Derivatives in the 2-(o-Nitrophenyl)propyl Series: Highly Efficient Two- Photon Uncaging of the Neurotransmitter ?-Aminobutyric Acid at ?=800 nm. Angew. Chem. Int. Ed. 2012, 51, 1840-1843.

101. Melandri, B. A.; Pupillo, P.; Baccarin.A, D-Glyceraldehyde-3-phosphate dehydrogenase in photosynthetic cells. 2. Reversible light-induced activation in-vivo of NADP-depencent enzyme and its relationship to NAD-dependent activities. Biochim. Biophys. Acta 1970, 220, 178-&.

102. Zatsepin, T. S.; Abrosimova, L. A.; Monakhova, M. V.; Hien, L. T.; Pingoud, A.; Kubareva, E. A.; Oretskaya, T. S., Design of photocontrolled biomolecules based on azobenzene derivatives. Russian Chem. Rev. 2013, 82, 942-963.

103. Minkin, V. I., Light-controlled molecular switches based on bistable spirocyclic organic and coordination compounds. Russian Chem. Rev. 2013, 82, 1-26.

104. Wenger, O. S., Photoswitchable mixed valence. Chem. Soc. Rev. 2012, 41, 3772-3779. 105. Renth, F.; Siewertsen, R.; Temps, F., Enhanced photoswitching and ultrafast dynamics in structurally modified photochromic fulgides. Int. Rev. Phys. Chem. 2013, 32, 1- 38.

106. Perez-Hernandez, G.; Gonzalez, L., Mechanistic insight into light-driven molecular rotors: a conformational search in chiral overcrowded alkenes by a pseudo-random approach. Phys. Chem. Chem. Phys. 2010, 12, 12279-12289.

107. Bandara, H. M. D.; Burdette, S. C., Photoisomerization in different classes of azobenzene. Chem. Soc. Rev. 2012, 41, 1809-1825.

108. Ordoukhanian, P.; Taylor, J. S., Design and synthesis of a versatile photocleavable DNA building-block - application to phototriggered hybridization. J. Am. Chem. Soc. 1995, 117, 9570-9571.

109. Shestopalov, I. A.; Sinha, S.; Chen, J. K., Light-controlled gene silencing in zebrafish embryos. Nat Chem Biol 2007, 3, 650-1.

110. Richards, J. L.; Tang, X. J.; Turetsky, A.; Dmochowski, I. J., RNA bandages for photoregulating in vitro protein synthesis. Bioorg. Med. Chem. Lett. 2008, 18, 6255-6258.

268

111. Tallafuss, A.; Gibson, D.; Morcos, P.; Li, Y.; Seredick, S.; Eisen, J.; Washbourne, P., Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish. Development 2012, 139.

112. Ouyang, X. H.; Shestopalov, I. A.; Sinha, S.; Zheng, G. H.; Pitt, C. L. W.; Li, W. H.; Olson, A. J.; Chen, J. K., Versatile Synthesis and Rational Design of Caged Morpholinos. J. Am. Chem. Soc. 2009, 131, 13255-13269.

113. Zheng, G.; Cochella, L.; Liu, J.; Hobert, O.; Li, W. H., Temporal and spatial regulation of microRNA activity with photoactivatable cantimirs. ACS Chem Biol 2011, 6, 1332-8.

114. Tang, X.; Maegawa, S.; Weinberg, E. S.; Dmochowski, I. J., Regulating gene expression in zebrafish embryos using light-activated, negatively charged peptide nucleic acids. J Am Chem Soc 2007, 129, 11000-1.

115. Tang, X.; Swaminathan, J.; Gewirtz, A. M.; Dmochowski, I. J., Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides. Nucleic Acids Ress 2008, 36, 559-569.

116. Yamazoe, S.; Shestopalov, I. A.; Provost, E.; Leach, S. D.; Chen, J. K., Cyclic Caged Morpholinos: Conformationally Gated Probes of Embryonic Gene Function. Angew. Chem. Int. Ed. 2012, 51, 6908-6911.

117. Tang, X. J.; Su, M.; Yu, L. L.; Lv, C.; Wang, J.; Li, Z. J., Photomodulating RNA cleavage using photolabile circular antisense oligodeoxynucleotides. Nucleic Acids Ress 2010, 38, 3848-3855.

118. Wang, Y.; Wu, L.; Wang, P.; Lv, C.; Yang, Z.; Tang, X., Manipulation of gene expression in zebrafish using caged circular morpholino oligomers. Nucleic Acids Ress 2012, 40.

119. Asanuma, H.; Ito, T.; Yoshida, T.; Liang, X.; Komiyama, M., Photoregulation of the Formation and Dissociation of a DNA Duplex by Using the cis-trans Isomerization of Azobenzene. Angew Chem Int Ed Engl 1999, 38, 2393-2395.

120. Asanuma, H.; Liang, X.; Yoshida, T.; Ito, T.; Komiyama, M., Photo-regulated duplex- and triplex-formation of the modified DNA carrying azobenzene. Nucleic Acids Symp Ser 1999, 135-6.

121. Asanuma, H.; Liang, X.; Nishioka, H.; Matsunaga, D.; Liu, M.; Komiyama, M., Synthesis of azobenzene-tethered DNA for reversible photo-regulation of DNA functions: hybridization and transcription. Nat Protoc 2007, 2, 203-12.

269

122. Tang, X. J.; Dmochowski, I. J., Regulating gene expression with light-activated oligonucleotides. Mol. Biosyst. 2007, 3, 100-110.

123. Ogasawara, S.; Maeda, M., Reversible Photoswitching of a G-Quadruplex. Angew. Chem. Int. Ed. 2009, 48, 6671-6674.

124. Cahova, H.; Jaeschke, A., Nucleoside-Based Diarylethene Photoswitches and Their Facile Incorporation into Photoswitchable DNA. Angew. Chem. Int. Ed. 2013, 52, 3186-3190.

125. Brieke, C.; Heckel, A., Spiropyran Photoswitches in the Context of DNA: Synthesis and Photochromic Properties. Chem. Eur. J. 2013, 19, 15726-15734.

126. Monroe, W. T.; McQuain, M. M.; Chang, M. S.; Alexander, J. S.; Haselton, F. R., Targeting expression with light using caged DNA. J. Biol. Chem. 1999, 274, 20895-20900.

127. Jain, P. K.; Shah, S.; Friedman, S. H., Patterning of Gene Expression Using New Photolabile Groups Applied to Light Activated RNAi. J. Am. Chem. Soc. 2011, 133, 440-446.

128. Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H., Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos. Nat. Genet. 2001, 28, 317-325.

129. Chaulk, S. G.; MacMillan, A. M., Caged RNA: photo-control of a ribozyme reaction. Nucleic Acids Ress 1998, 26, 3173-3178.

130. Chaulk, S. G.; MacMillan, A. M., Separation of spliceosome assembly from catalysis with caged pre-mRNA substrates. Angew. Chem. Int. Ed. 2001, 40, 2149-2152.

131. Ting, R.; Lermer, L.; Perrin, D. M., Triggering DNAzymes with light: A photoactive C8 thioether-linked adenosine. J. Am. Chem. Soc. 2004, 126, 12720-12721.

132. Hobartner, C.; Silverman, S. K., Modulation of RNA tertiary folding by incorporation of caged nucleotides. Angew. Chem. Int. Ed. 2005, 44, 7305-7309.

133. Kröck, L.; Heckel, A., Photoinduced transcription by using temporarily mismatched caged oligonucleotides. Angew Chem Int Ed Engl 2005, 44, 471-3.

134. Schäfer, F.; Joshi, K. B.; Fichte, M. A.; Mack, T.; Wachtveitl, J.; Heckel, A., Wavelength-selective uncaging of dA and dC residues. Org Lett 2011, 13, 1450-3.

135. Mayer, G.; Krock, L.; Mikat, V.; Engeser, M.; Heckel, A., Light-induced formation of G-quadruplex DNA secondary structures. Chembiochem 2005, 6.

270

136. Rodrigues-Correia, A.; Koeppel, M.; Schafer, F.; Joshi, K.; Mack, T.; Heckel, A., Comparison of the duplex-destabilizing effects of nucleobase-caged oligonucleotides. Anal. Bioanal. Chem.y 2011, 399, 441-447.

137. Lusic, H.; Lively, M.; Deiters, A., Light-activated deoxyguanosine: photochemical regulation of peroxidase activity. Mol. Biosyst. 2008, 508-511.

138. Lusic, H.; Young, D.; Lively, M.; Deiters, A., Photochemical DNA activation. Org. Lett. 2007, 1903-1906.

139. Govan, J. M.; Uprety, R.; Hemphill, J.; Lively, M. O.; Deiters, A., Regulation of Transcription through Light-Activation and Light-Deactivation of Triplex-Forming Oligonucleotides in Mammalian Cells. Acs Chem. Biol. 2012, 7.

140. Connelly, C. M.; Uprety, R.; Hemphill, J.; Deiters, A., Spatiotemporal control of microRNA function using light-activated antagomirs. Mol. Biosyst. 2012, 8, 2987-2993.

141. Robert, C.; Muel, B.; Benoit, A.; Dubertret, L.; Sarasin, A.; Stary, A., Cell survival and shuttle vector mutagenesis induced by ultraviolet A and ultraviolet B radiation in a human cell line. J. Invest. Dermatol. 1996, 106, 721-728.

142. Schindl, A.; Klosner, G.; Honigsmann, H.; Jori, G.; Calzavara-Pinton, P. C.; Trautinger, F., Flow cytometric quantification of UV-induced cell death in a human squamous cell carcinoma-derived cell line: dose and kinetic studies. J. Photochem. Photobiol., B 1998, 44, 97-106.

143. Priestman, M. A.; Sun, L. A.; Lawrence, D. S., Dual Wavelength Photoactivation of cAMP- and cGMP-Dependent Protein Kinase Signaling Pathways. Acs Chem. Biol. 2011, 6, 377-384.

144. Stains, C. I.; Tedford, N. C.; Walkup, T. C.; Lukovic, E.; Goguen, B. N.; Griffith, L. G.; Lauffenburger, D. A.; Imperiali, B., Interrogating Signaling Nodes Involved in Cellular Transformations Using Kinase Activity Probes. Chem. Biol. 2012, 19, 210-217.

145. Menge, C.; Heckel, A., Coumarin-Caged dG for Improved Wavelength-Selective Uncaging of DNA. Org. Lett. 2011, 13, 4620-4623.

146. Lusic, H.; Uprety, R.; Deiters, A., Improved Synthesis of the Two-Photon Caging Group 3-Nitro-2-Ethyldibenzofuran and Its Application to a Caged Thymidine Phosphoramidite. Org. Lett. 2010, 916-919.

147. Young, D.; Govan, J.; Lively, M.; Deiters, A., Photochemical Regulation of Restriction Endonuclease Activity. Chembiochem 2009, 1612-1616.

271

148. Mayer, G.; Mueller, J.; Mack, T.; Freitag, D. F.; Hoever, T.; Poetzsch, B.; Heckel, A., Differential Regulation of Protein Subdomain Activity with Caged Bivalent Ligands. Chembiochem 2009, 10, 654-657.

149. Young, D.; Lusic, H.; Lively, M.; Yoder, J.; Deiters, A., Gene Silencing in Mammalian Cells with Light-Activated Antisense Agents. Chembiochem 2008, 2937-2940.

150. Prokup, A.; Hemphill, J.; Deiters, A., DNA computation: a photochemically controlled AND gate. J Am Chem Soc 2012, 134, 3810-5.

151. Heckel, A.; Buff, M. C. R.; Raddatz, M.-S. L.; Mueller, J.; Poetzsch, B.; Mayer, G., An anticoagulant with light-triggered antidote activity. Angew. Chem. Int. Ed. 2006, 45.

152. Mikat, V.; Heckel, A., Light-dependent RNA interference with nucleobase-caged siRNAs. RNA 2007, 13, 2341-2347.

153. Liu, Q.; Deiters, A., Optochemical Control of Deoxyoligonucleotide Function via a Nucleobase-Caging Approach. Acc Chem Res 2014, 47, 45-55.

154. Hemphill, J.; Deiters, A., DNA Computation in Mammalian Cells: MicroRNA Logic Operations. J. Am. Chem. Soc. 2013.

155. Schmidt, T. L.; Koeppel, M. B.; Thevarpadam, J.; Goncalves, D. P. N.; Heckel, A., A Light Trigger for DNA Nanotechnology. Small 2011, 7, 2163-2167.

156. Joshi, K. B.; Vlachos, A.; Mikat, V.; Deller, T.; Heckel, A., Light-activatable molecular beacons with a caged loop sequence. Chem. Commun. 2012, 48, 2746-2748.

157. Höbartner, C.; Silverman, S. K., Modulation of RNA tertiary folding by incorporation of caged nucleotides. Angew Chem Int Ed Engl 2005, 44, 7305-9.

158. Fisher, W. G.; Partridge, W. P.; Dees, C.; Wachter, E. A., Simultaneous two-photon activation of type-I photodynamic therapy agents. Photochem. Photobiol. 1997, 66, 141-155.

159. Rodrigues-Correia, A.; Weyel, X. M. M.; Heckel, A., Four Levels of Wavelength- Selective Uncaging for Oligonucleotides. Org. Lett. 2013, 15, 5500-5503.

160. Furuta, T.; Takeuchi, H.; Isozaki, M.; Takahashi, Y.; Kanehara, M.; Sugimoto, M.; Watanabe, T.; Noguchi, K.; Dore, T. M.; Kurahashi, T.; Iwamura, M.; Tsien, R. Y., Bhc- cNMPs as either water-soluble or membrane-permeant photoreleasable cyclic nucleotides for both one- and two-photon excitation. Chembiochem 2004, 5, 1119-1128.

272

161. Ito, K.; Maruyama, J., Studies on Stable Diazoalkanes as Potential Fluorogenic Reagets. 1. 7-Substituted 4- Diazomethylcoumarins. Chem. Pharm. Bull. 1983, 31, 3014- 3023.

162. Su, M.; Wang, J.; Tang, X. J., Photocaging Strategy for Functionalisation of Oligonucleotides and Its Applications for Oligonucleotide Labelling and Cyclisation. Chem. Eur. J. 2012, 18, 9628-9637.

163. Thibaudeau, C.; Plavec, J.; Chattopadhyaya, J., Quantitation of the pD dependent thermodynamics of the N reversible arrow S pseudorotational equilibrium of the pentofuranose moiety in nucleosides gives a direct measurement of the strength of the tunable anomeric effect and the pK(a) of the nucleobase. J. Org. Chem. 1996, 61, 266-286.

164. Krim, J.; Taourirte, M.; Grunewald, C.; Krstic, I.; Engels, J. W., Microwave-Assisted Click Chemistry for Nucleoside Functionalization: Useful Derivatives for Analytical and Biological Applications. Synthesis 2013, 45, 396-405.

165. Bornemann, B.; Liu, S. P.; Erbe, A.; Scheer, E.; Marx, A., Thiolated nucleotides for immobilisation of DNA oligomers on gold surfaces. Chemphyschem 2008, 9, 1241-1244.

166. Lusic, H. Control of Biological Processes with Modified Nucleosides, Amino Acids, and Small-Molecule Probes. North Carolina State University, Raleigh, NC, 29-Jul-2010.

167. Donato, L.; Mourot, A.; Davenport, C. M.; Herbivo, C.; Warther, D.; Léonard, J.; Bolze, F.; Nicoud, J. F.; Kramer, R. H.; Goeldner, M.; Specht, A., Water-soluble, donor- acceptor biphenyl derivatives in the 2-(o-nitrophenyl)propyl series: highly efficient two- photon uncaging of the neurotransmitter γ-aminobutyric acid at λ = 800 nm. Angew Chem Int Ed Engl 2012, 51, 1840-3.

168. Peixoto, P. A.; Boulange, A.; Leleu, S.; Franck, X., Versatile Synthesis of Acylfuranones by Reaction of Acylketenes with alpha-Hydroxy Ketones: Application to the One-Step Multicomponent Synthesis of Cadiolide B and Its Analogues. European J. Org. Chem. 2013, 3316-3327.

169. Hale, J. J.; Doherty, G.; Toth, L.; Li, Z.; Mills, S. G.; Hajdu, R.; Keohane, C. A.; Rosenbach, M.; Milligan, J.; Shei, G. J.; Chrebet, G.; Bergstrom, J.; Card, D.; Rosen, H.; Mandala, S., The discovery of 3-(N-alkyl)aminopropylphosphonic acids as potent S1P receptor agonists. Bioorg. Med. Chem. Lett. 2004, 14, 3495-3499.

170. Blanc, A.; Bochet, C. G., Isotope effects in photochemistry: Application to chromatic orthogonality. Org. Lett. 2007, 9, 2649-2651.

273

171. Kuzuya, A.; Okada, F.; Komiyama, M., Precise site-selective termination of DNA replication by caging the 3-position of thymidine and its application to polymerase chain reaction. Bioconjug Chem 2009, 20, 1924-9.

172. Brase, S.; Gil, C.; Knepper, K.; Zimmermann, V., Organic azides: An exploding diversity of a unique class of compounds. Angew. Chem. Int. Ed. 2005, 5188-5240.

173. Fukase, K.; Hashida, M.; Kusumoto, S., APPLICATION OF 4-AZIDOBENZYL GROUP TO PROTECTION OF HYDROXYL FUNCTIONS. Tetrahedron Lett. 1991, 32, 3557-3558.

174. Pothukanuri, S.; Winssinger, N., A highly efficient azide-based protecting group for amines and alcohols. Org Lett 2007, 9, 2223-5.

175. Furukawa, K.; Abe, H.; Hibino, K.; Sako, Y.; Tsuneda, S.; Ito, Y., Reduction- triggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells. Bioconjug Chem 2009, 20, 1026-36.

176. Franzini, R. M.; Kool, E. T., 7-Azidomethoxy-coumarins as profluorophores for templated nucleic acid detection. Chembiochem 2008, 9, 2981-8.

177. van Brakel, R.; Vulders, R.; Bokdam, R.; Grull, H.; Robillard, M., A doxorubicin prodrug activated by the staudinger reaction. Bioconjugate Chem. 2008, 714-718.

178. Damen, E. W.; Nevalainen, T. J.; van den Bergh, T. J.; de Groot, F. M.; Scheeren, H. W., Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue. Bioorg Med Chem 2002, 10, 71-7.

179. Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R., Mechanistic investigation of the staudinger ligation. J Am Chem Soc 2005, 127, 2686-95. 180. Young, T.; Kiessling, L. L., A strategy for the synthesis of sulfated peptides. Angew. Chem. Int. Ed. 2002, 41, 3449-3451.

181. Khan, A. T.; Mondal, E., A highly efficient and useful synthetic protocol for the cleavage of tert-butyldimethylsilyl (TBS) ethers using a catalytic amount of acetyl chloride in dry methanol. Synlett 2003, 694-698.

182. Griffin, R.; Evers, E.; Davison, R.; Gibson, A.; Layton, D.; Irwin, W., The 4- azidoberazyloxycarbonyl function; Application as a novel protecting group and potential prodrug modification for amines. Perkin Trans. 1 1996, 1205-1211.

183. Xue, C. B.; He, X. H.; Roderick, J.; Corbett, R. L.; Duan, J. J. W.; Liu, R. Q.; Covington, M. B.; Newton, R. C.; Trzaskos, J. M.; Magolda, R. L.; Wexler, R. R.; Decicco,

274

C. P., Rational design, synthesis and structure-activity relationships of a cyclic succinate series of TNF-alpha converting enzyme inhibitors. Part 1: Lead identification. Bioorg. Med. Chem. Lett. 2003, 13, 4293-4297.

184. Aigner, M.; Hartl, M.; Fauster, K.; Steger, J.; Bister, K.; Micura, R., Chemical Synthesis of Site-Specifically 2 '-Azido-Modified RNA and Potential Applications for Bioconjugation and RNA Interference. Chembiochem 2011, 12, 47-51.

185. Schonleber, R. O.; Bendig, J.; Hagen, V.; Giese, B., Rapid photolytic release of cytidine 5-diphosphate from a coumarin derivative: A new tool for the investigation of ribonucleotide reductases. Bioorg. Med. Chem. 2002, 10, 97-101.

186. Atta, S.; Jana, A.; Ananthakirshnan, R.; Dhuleep, P. S. N., Fluorescent Caged Compounds of 2,4-Dichlorophenoxyacetic Acid (2,4-D): Photorelease Technology for Controlled Release of 2,4-D. J. Agric. Food Chem. 2010, 58, 11844-11851.

187. El Safadi, Y.; Paillart, J. C.; Laumond, G.; Aubertin, A. M.; Burger, A.; Marquet, R.; Vivet-Boudou, V., 5-Modified-2 '-dU and 2 '-dC as Mutagenic Anti HIV-1 Proliferation Agents: Synthesis and Activity. J. Med. Chem. 2010, 53, 1534-1545.

188. Friedel, M. G.; Pieck, J. C.; Klages, J.; Dauth, C.; Kessler, H.; Carell, T., Synthesis and stereochemical assignment of DNA spore photoproduct analogues. Chemistry 2006, 12, 6081-94.

189. Tsujihara, K.; Kawaguchi, T.; Harada, N.; Oohashi, M.; Akaike, Y. Preparation of 2'- deoxy-5-fluorouridine derivatives of amino acids and peptides as antitumor agents. JP 05097887, Apr 20, 1993.

190. Buhler, S.; Lagoja, I.; Giegrich, H.; Stengele, K. P.; Pfleiderer, W., New types of very efficient photolabile protecting groups based upon the 2-(2-nitrophenyi)propoxy carbonyl (NPPOC) moiety. Helv. Chim. Acta 2004, 87, 620-659.

191. Jacquart, A.; Tauc, P.; Pansu, R. B.; Ishow, E., Tunable emissive thin films through ICT photodisruption of nitro-substituted triarylamines. Chem. Commun. 2010, 46, 4360-4362.

192. Kenwright, J. L.; Galloway, W. R.; Blackwell, D. T.; Isidro-Llobet, A.; Hodgkinson, J.; Wortmann, L.; Bowden, S. D.; Welch, M.; Spring, D. R., Novel and efficient copper- catalysed synthesis of nitrogen-linked medium-ring biaryls. Chemistry 2011, 17, 2981-6.

193. Chi, S.-M.; Hah, J.-H.; Kim, K.-S.; Kim, W.-S.; Ryoo, M.-H. Photolabile compound, oligomer probe array and substrate for oligomer probe array containing the same, and manufacturing method of the same. US 20080188380, August 7, 2008.

275

194. Cummings, M. M.; Soderberg, B. C. G., Reexamination of the Bromination of 2- Nitrobenzaldehyde with NBS or NaBr-NaIO4 in Sulfuric Acid. Synth. Commun. 2014, 44, 954-958.

195. Morcos, P. A., Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos. Biochem. Biophys. Res. Commun. 2007, 358, 521-527.

196. Kloosterman, W. P.; Lagendijk, A. K.; Ketting, R. F.; Moulton, J. D.; Plasterk, R. H. A., Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. Plos Biol. 2007, 5, 1738-1749.

197. Deiters, A.; Garner, R.; Lusic, H.; Govan, J.; Dush, M.; Nascone-Yoder, N.; Yoder, J., Photocaged Morpholino Oligomers for the Light-Regulation of Gene Function in Zebrafish and Xenopus Embryos. J. Am. Chem. Soc. 2010, 15644-15650.

198. Crotwell, P. L.; Mabee, P. M., Gene expression patterns underlying proximal-distal skeletal segmentation in late-stage zebrafish, Danio rerio. Dev. Dynam. 2007, 236, 3111- 3128.

199. Summerton, J.; Weller, D., Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 1997, 7, 187-95.

200. Hadj-Bouazza, A.; Zerrouki, R.; Krausz, P.; Laumond, G.; Aubertin, A. M.; Champavier, Y., New acyclonucleosides: Synthesis and anti-HIV activity. Nucleos. Nucleot. Nucl. 2005, 24, 1249-1263.

201. Nguyen, C.; Kasinathan, G.; Leal-Cortijo, I.; Musso-Buendia, A.; Kaiser, M.; Brun, R.; Ruiz-Perez, L. M.; Johansson, N. G.; Gonzalez-Pacanowska, D.; Gilbert, I. H., Deoxyuridine triphosphate nucleotidohydrolase as a potential antiparasitic drug target. J. Med. Chem. 2005, 48, 5942-5954.

202. Weller, D. D.; Hassinger, J. N. Oligonucleotide analogs having cationic intersubunit linkages. US 2009/0088562 A1, April 2, 2009.

203. Khaled, A.; Piotrowska, O.; Dominiak, K.; Augé, C., Exploring specificity of glycosyltransferases: synthesis of new sugar nucleotide related molecules as putative donor substrates. Carbohydr Res 2008, 343, 167-78.

204. Mourani, R.; Damha, M. J., Synthesis, characterization, and biological properties of small branched RNA fragments containing chiral (Rp and Sp) 2',5'-phosphorothioate linkages. Nucleos. Nucleot. Nucl. 2006, 25, 203-29.

276

205. Ohkubo, A.; Kuwayama, Y.; Nishino, Y.; Tsunoda, H.; Seio, K.; Sekine, M., Oligonucleotide synthesis involving deprotection of amidine-type protecting groups for nucleobases under acidic conditions. Org Lett 2010, 12, 2496-9.

206. Majumdar, K. C.; Samanta, S.; Sinha, B., Recent Developments in Palladium- Catalyzed Formation of Five-and Six-Membered Fused Heterocycles. Synthesis 2012, 44, 817-847.

207. Kerti, G.; Kurtan, T.; Antus, S., Study on the reaction mechanism of Heck- oxyarylation of 2H-chromenes. Arkivoc 2009, 103-110.

208. Le Bras, J.; Muzart, J., Heck Arylations of Baylis-Hillman Adducts. Synthesis 2011, 3581-3591.

209. Kurokawa, T.; Isomura, M.; Tokuyama, H.; Fukuyama, T., Synthesis of Lysergic Acid Methyl Ester via the Double Cyclization Strategy. Synlett 2009, 775-778.

210. Debarge, S.; Balzarini, J.; Maguire, A. R., Design and synthesis of α-carboxy phosphononucleosides. J Org Chem 2011, 76, 105-26.

211. Hanson, G. J.; Rudolph, A. C.; Cai, B. Z.; Zhou, M.; Weller, D. D. Preparation of oligonucleotide analogs having morpholino modified inter-subunit linkages and/or terminal groups as antiviral agents WO 2011150408, Dec 1, 2011.

212. Summerton, J. E.; Weller, D. D. Preparation of phosphoryl-linked morpholino-based oligonucleotides. WO 9109033, Jun 27, 1991.

213. Surendra, K.; Krishnaveni, N. S.; Sridhar, R.; Srinivas, B.; Kumar, V. P.; Nageswar, Y. V. D.; Rao, K. R., ZnCl2: Simple and efficient catalyst for the allylation of aldehydes with allyltributylstannane. Synth. Commun. 2006, 36, 1-5.

214. Couty, S.; Meyer, C.; Cossy, J., Gold-catalyzed cycloisomerizations of ene-ynamides. Tetrahedron 2009, 65, 1809-1832.

215. Kiyooka, S.; Kaneko, Y.; Komura, M.; Matsuo, H.; Nakano, M., Enatioselective chiral boran-mediated aldol reactions of silyl ketene acetals with aldehydes - Novl effect of the trialkylsilyl group of the silyl ketene acetal on the reaction course. J. Org. Chem. 1991, 56, 2276-2278.

216. Rana, V. S.; Kumar, V. A.; Ganesh, K. N., Oligonucleotides with (N-thymin-1- ylacetyl)-1-arylserinol backbone: chiral acyclic analogs with restricted conformational flexibility. Tetrahedron 2001, 57, 1311-1321.

277

217. Zhou, C. Z.; Sczepanski, J. T.; Greenberg, M. M., Mechanistic Studies on Histone Catalyzed Cleavage of Apyrimidinic/Apurinic Sites in Nucleosome Core Particles. J. Am. Chem. Soc. 2012, 134, 16734-16741.

218. Lindley, H., Study of the kinetics of the reaction between thiol compounds and chloroacetamide. Biochem. J. 1960, 74, 577-584.

219. Sheikh, S.; Katiyar, S. S., Chemical modification of octopine dehydrogenase by thiol- specific reagents - Evidence for the presence of an essential cysteine at the catalytic site. Biochim. Biophys. Acta 1993, 1202, 251-257.

220. Loupy, A., Microwaves in organic synthesis. Wiley-VCH: Weinheim ; Cambridge, 2002; p xxiv, 499 p.

221. Cochi, A.; Métro, T. X.; Pardo, D. G.; Cossy, J., Enantioselective synthesis of SSR 241586 by using an organo-catalyzed Henry reaction. Org Lett 2010, 12, 3693-5.

222. Song, H. Y.; Ngai, M. H.; Song, Z. Y.; MacAry, P. A.; Hobley, J.; Lear, M. J., Practical synthesis of maleimides and coumarin-linked probes for protein and antibody labelling via reduction of native disulfides. Org Biomol Chem 2009, 7, 3400-6.

223. Brendel, J.; Schmidt, W.; Below, P. 2'-Substituted 1,1'-biphenyl-2-carboxamides, processes for their preparation, their use as medicaments, and pharmaceutical preparations comprising them. US20090192096 A1, 30 Jul 2009.

224. Borah, J. C.; Boruwa, J.; Barua, N. C., Stereoselective total synthesis of (+)-sedamine. Lett. in Org. Chem. 2006, 3, 140-142.

225. Ohtani, T.; Kawano, Y.; Kitano, K.; Matsubara, J.; Komatsu, M.; Uchida, M.; Tabusa, F.; Nagao, Y., Efficient synthesis of functionalized benzazepine derivatives utilizing intramolecular Mitsunobu reaction. Heterocycles 2005, 66, 481-502.

226. Eidamshaus, C.; Reissig, H. U., A Chiral Pool Strategy for the Synthesis of Enantiopure Hydroxymethyl-Substituted Pyridine Derivatives. European J. Org. Chem. 2011, 6056-6069.

227. Brendel, J.; Schmidt, W.; Below, P. 2'-Substituted 1,1'-biphenyl-2-carboxamides, processes for their preparation, their use as medicaments, and pharmaceutical preparations comprising them. 2003.

228. Boomer, J. A.; Qualls, M. M.; Inerowicz, H. D.; Haynes, R. H.; Patri, V. S.; Kim, J. M.; Thompson, D. H., Cytoplasmic delivery of liposomal contents mediated by an acid-labile cholesterol-vinyl ether-PEG conjugate. Bioconjug Chem 2009, 20, 47-59.

278

229. Weinberg, E. S.; Allende, M. L.; Kelly, C. S.; Abdelhamid, A.; Murakami, T.; Andermann, P.; Doerre, O. G.; Grunwald, D. J.; Riggleman, B., Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development 1996, 122, 271- 280.

230. Fournier, L.; Gauron, C.; Xu, L. J.; Aujard, I.; Le Saux, T.; Gagey-Eilstein, N.; Maurin, S.; Dubruille, S.; Baudin, J. B.; Bensimon, D.; Volovitch, M.; Vriz, S.; Jullien, L., A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation. Acs Chem. Biol. 2013, 8, 1528-1536.

231. Sergeyev, S. A.; Hesse, M., A novel synthesis of the macrocyclic spermidine alkaloid (+)-(S)-dihydroperiphylline. Helv. Chim. Acta 2002, 85, 161-167.

232. Mishra, J. K.; Samanta, K.; Jain, M.; Dikshit, M.; Panda, G., Amino acid based enantiomerically pure 3-substituted benzofused heterocycles: A new class of antithrombotic agents. Bioorg. Med. Chem. Lett. 2010, 20, 244-247.

233. Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T., Improved efficiency and selectivity in peptide synthesis - use of triethyl silane as a carbocation scavenger in deprotection of tert-butyl esters and tert-butoxycarbonyl-protected sites. Tetrahedron Lett. 1992, 33, 5441-5444.

234. Shibuya, I.; Taguchi, Y.; Tsuchiya, T.; Oishi, A.; Katoh, E., Silver(I) ion-mediated desulfurization-condensation of thiocarbonyl compounds with several nucleophiles. Bull. Chem. Soc. Jpn. 1994, 67, 3048-3052.

235. Brehm, E.; Breinbauer, R., Investigation of the origin and synthetic application of the pseudodilution effect for Pd-catalyzed macrocyclisations in concentrated solutions with immobilized catalysts. Org. Biomol. Chem. 2013, 11, 4750-4756.

236. Drager, G.; Kiss, C.; Kunz, U.; Kirschning, A., Enzyme-purification and catalytic transformations in a microstructured PASSflow reactor using a new tyrosine-based Ni-NTA linker system attached to a polyvinylpyrrolidinone-based matrix. Org. Biomol. Chem. 2007, 5, 3657-3664.

237. Habarova, O.; Bobileva, O.; Loza, E.; Romancikova, N., Synthesis of 2- quinolinecarboxamide derivatives as potential HDAC ihibitors. Chem. Heterocycl. Compd. 2011, 47, 719-727.

238. Mukherjee, S.; Robinson, C. A.; Howe, A. G.; Mazor, T.; Wood, P. A.; Urgaonkar, S.; Hebert, A. M.; RayChaudhuri, D.; Shaw, J. T., N-Benzyl-3-sulfonamidopyrrolidines as novel inhibitors of cell division in E-coli. Bioorg. Med. Chem. Lett. 2007, 17, 6651-6655.

279

239. Boomer, J. A.; Qualls, M. M.; Inerowicz, H. D.; Haynes, R. H.; Patri, V. S.; Kim, J. M.; Thompson, D. H., Cytoplasmic Delivery of Liposomal Contents Mediated by an Acid- Labile Cholesterol-Vinyl Ether-PEG Conjugate. Bioconjugate Chem. 2009, 20, 47-59.

240. Chen, S.; Coward, J. K., Investigations on New Strategies for the Facile Synthesis of Polyfunctionalized Phosphinates: Phosphinopeptide Analogues of Glutathionylspermidine. J Org Chem 1998, 63, 502-509.

241. Vonkiedrowski, G.; Dorwald, F. Z., Synthesis of polyamine-carbodiimides-potential activators for chemical ligations of oligonucleotide 3ʹ-phosphates. Liebigs Annalen Der Chemie 1988, 787-794.

242. Reinhardt, G., alpha-Halogenmethyl carbonyl compounds as very potent inhibitors of factor XIIIa in vitro. Ann N Y Acad Sci 1981, 370, 836-42.

243. Lin, Y. M.; Miller, M. J., Oxidation of primary amines to oxaziridines using molecular oxygen (O2) as the ultimate oxidant. J Org Chem 2001, 66, 8282-5.

244. Ogura, N.; Ota, Y.; Sotodani; Koshiro; Aoyanagi, M. Bleach and bleach detergent compositions. JP 03287699, Dec 18, 1991.

245. Tsuboi, K.; Bachovchin, D. A.; Speers, A. E.; Spicer, T. P.; Fernandez-Vega, V.; Hodder, P.; Rosen, H.; Cravatt, B. F., Potent and selective inhibitors of glutathione S- transferase omega 1 that impair cancer drug resistance. J Am Chem Soc 2011, 133, 16605-16.

246. Pelletier, G.; Bechara, W. S.; Charette, A. B., Controlled and chemoselective reduction of secondary amides. J Am Chem Soc 2010, 132, 12817-9.

247. Schwarzer, D., Hacking the Genetic Code of Mammalian Cells. Chembiochem 2009, 10, 1602-1604.

248. Basle, E.; Joubert, N.; Pucheault, M., Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17, 213-227.

249. Kim, Y. G.; Ho, S. O.; Gassman, N. R.; Korlann, Y.; Landorf, E. V.; Collart, F. R.; Weiss, S., Efficient site-specific Labeling of proteins via cysteines. Bioconjugate Chem. 2008, 19, 786-791.

250. Nielsen, M. L.; Vermeulen, M.; Bonaldi, T.; Cox, J.; Moroder, L.; Mann, M., Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat. Methods2008, 5, 459-460.

280

251. Chalker, J. M.; Lin, Y. A.; Boutureira, O.; Davis, B. G., Enabling olefin metathesis on proteins: chemical methods for installation of S-allyl cysteine. Chem. Commun. 2009, 3714- 3716.

252. Rademann, J., Organic protein chemistry: Drug discovery through the chemical modification of proteins. Angew. Chem. Int. Ed. 2004, 43, 4554-4556.

253. Chalker, J. M.; Bernardes, G. J. L.; Lin, Y. A.; Davis, B. G., Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology. Chem. Asian J. 2009, 4, 630-640.

254. Gildersleeve, J. C.; Oyelaran, O.; Simpson, J. T.; Allred, B., Improved procedure for direct coupling of carbohydrates to proteins via reductive amination. Bioconjugate Chem. 2008, 19, 1485-1490.

255. Hooker, J. M.; Esser-Kahn, A. P.; Francis, M. B., Modification of aniline containing proteins using an oxidative coupling strategy. J. Am. Chem. Soc. 2006, 128, 15558-15559.

256. Geoghegan, K. F.; Stroh, J. G., Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine. Bioconjug Chem 1992, 3, 138-46.

257. Hooker, J. M.; Kovacs, E. W.; Francis, M. B., Interior surface modification of bacteriophage MS2. J. Am. Chem. Soc. 2004, 126, 3718-3719.

258. Guo, H. M.; Minakawa, M.; Ueno, L.; Tanaka, F., Synthesis and evaluation of a cyclic imine derivative conjugated to a fluorescent molecule for labeling of proteins. Bioorg. Med. Chem. Lett. 2009, 19, 1210-1213.

259. Oya, T.; Hattori, N.; Mizuno, Y.; Miyata, S.; Maeda, S.; Osawa, T.; Uchida, K., Methylglyoxal modification of protein - Chemical and immunochemical characterization of methylglyoxal-arginine adducts. J. Biol. Chem. 1999, 274, 18492-18502.

260. Kerr, J. M.; Banville, S. C.; Zuckermann, R. N., Encoded combinatorial peptide libraries containing nonnatural amino acids. J. Am. Chem. Soc. 1993, 115, 2529-2531.

261. Wang, L.; Schultz, P., Expanding the genetic code. Angew. Chem. Int. Ed. 2005, 34- 66.

262. Liu, C.; Schultz, P., Adding New Chemistries to the Genetic Code. Annu. Rev. Biochem., Vol 79 2010, 79, 413-444.

281

263. Hackenberger, C. P. R.; Schwarzer, D., Chemoselective Ligation and Modification Strategies for Peptides and Proteins. Angew. Chem. Int. Ed. 2008, 47, 10030-10074.

264. Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S., Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o- azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem Biol 2008, 15, 1187-97.

265. Rodriguez, E. A.; Lester, H. A.; Dougherty, D. A., In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression. Proc. Natl. Acad Sci. U. S. A. 2006, 103, 8650-8655.

266. Kiick, K. L.; van Hest, J. C. M.; Tirrell, D. A., Expanding the scope of protein biosynthesis by altering the methionyl-tRNA synthetase activity of a bacterial expression host. Angew. Chem. Int. Ed. 2000, 39, 2148.

267. Johnson, J. A.; Lu, Y. Y.; Van Deventer, J. A.; Tirrell, D. A., Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Chem. Biol. 2010, 14, 774-780.

268. Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G., A general method for site-specific incorporation of unnatural amino acids into proteins. Science 1989, 244, 182- 8.

269. Daggett, K. A.; Sakmar, T. P., Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors. Curr. Opin. Chem. Biol. 2011, 15, 392-398.

270. Srinivasan, G.; James, C. M.; Krzycki, J. A., Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA. Science 2002, 296, 1459-62.

271. Fekner, T.; Li, X.; Chan, M., Pyrrolysine Analogs for Translational Incorporation into Proteins. European J. Org. Chem. 2010, 4171-4179.

272. Fekner, T.; Li, X.; Lee, M. M.; Chan, M. K., A pyrrolysine analogue for protein click chemistry. Angew Chem Int Ed Engl 2009, 48, 1633-5.

273. Nguyen, D. P.; Lusic, H.; Neumann, H.; Kapadnis, P. B.; Deiters, A.; Chin, J. W., Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry. J Am Chem Soc 2009, 131, 8720-1.

282

274. Huang, Y.; Wan, W.; Russell, W. K.; Pai, P. J.; Wang, Z.; Russell, D. H.; Liu, W., Genetic incorporation of an aliphatic keto-containing amino acid into proteins for their site- specific modifications. Bioorg Med Chem Lett 2010, 20, 878-80.

275. Mukai, T.; Kobayashi, T.; Hino, N.; Yanagisawa, T.; Sakamoto, K.; Yokoyama, S., Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem Biophys Res Commun 2008, 371, 818-22.

276. Chou, C. J.; Uprety, R.; Davis, L.; Chin, J. W.; Deiters, A., Genetically encoding an aliphatic diazirine for protein photocrosslinking. Chem. Sci. 2011, 2, 480-483.

277. Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C. J.; Deiters, A.; Chin, J. W., Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat. Chem. 2012, 4, 298-304.

278. Chen, P. R.; Groff, D.; Guo, J.; Ou, W.; Cellitti, S.; Geierstanger, B. H.; Schultz, P. G., A facile system for encoding unnatural amino acids in mammalian cells. Angew Chem Int Ed Engl 2009, 48, 4052-5.

279. Gautier, A.; Nguyen, D.; Lusic, H.; An, W.; Deiters, A.; Chin, J., Genetically Encoded Photocontrol of Protein Localization in Mammalian Cells. J. Am. Chem. Soc. 2010, 4086.

280. Hancock, S.; Uprety, R.; Deiters, A.; Chin, J., Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair. J. Am. Chem. Soc. 2010, 14819-14824.

281. Arbely, E.; Torres-Kolbus, J.; Deiters, A.; Chin, J. W., Photocontrol of Tyrosine Phosphorylation in Mammalian Cells via Genetic Encoding of Photocaged Tyrosine. J. Am. Chem. Soc. 2012, 134, 11912-11915.

282. Alford, E. J.; Schofield, K., Cinnolines. 28. The nature of the C(3)-position. 1. The Ber-Bossel synthesis of 3-hydroxycinnoline. J. Chem. Soc. 1952, 2102-2108.

283. Chen, C. T.; Kao, J. Q.; Salunke, S. B.; Lin, Y. H., Enantioselective aerobic oxidation of α-hydroxy-ketones catalyzed by oxidovanadium(V) methoxides bearing chiral, N- salicylidene-tert-butylglycinates. Org Lett 2011, 13, 26-9.

284. Yamakawa, N.; Suemasu, S.; Matoyama, M.; Kimoto, A.; Takeda, M.; Tanaka, K.; Ishihara, T.; Katsu, T.; Okamoto, Y.; Otsuka, M.; Mizushima, T., Properties and synthesis of 2-{2-fluoro (or bromo)-4-[(2-oxocyclopentyl)methyl]phenyl}propanoic acid: nonsteroidal anti-inflammatory drugs with low membrane permeabilizing and gastric lesion-producing activities. J Med Chem 2010, 53, 7879-82.

283

285. Lin, Q.; Barbas, C. F.; Schultz, P. G., Small-molecule switches for zinc finger transcription factors. J. Am. Chem. Soc. 2003, 125, 612-613.

286. Mootz, H. D.; Muir, T. W., Protein splicing triggered by a small molecule. J Am Chem Soc 2002, 124, 9044-5.

287. Cline, D. J.; Redding, S. E.; Brohawn, S. G.; Psathas, J. N.; Schneider, J. P.; Thorpe, C., New water-soluble phosphines as reductants of peptide and protein disulfide bonds: Reactivity and membrane permeability. Biochemistry 2004, 43, 15195-15203.

288. Almasieh, M.; Lieven, C. J.; Levin, L. A.; Di Polo, A., A cell-permeable phosphine- borane complex delays retinal ganglion cell death after axonal injury through activation of the pro-survival extracellular signal-regulated kinases 1/2 pathway. J. Neurochem. 2011, 118, 1075-1086.

289. Gorska, K.; Manicardi, A.; Barluenga, S.; Winssinger, N., DNA-templated release of functional molecules with an azide-reduction-triggered immolative linker. Chem. Commun. 2011, 47, 4364-4366.

290. Amyes, T. L.; Jencks, W. P., Lifetimes of oxocarbenium ions in aqueous solution from commom ion inhibition of the solvolysis of alpha-azido ethers by added azide ion. J. Am. Chem. Soc. 1989, 111, 7888-7900.

291. Kates, S. A.; Albericio, F., Solid-phase synthesis : a practical guide. Marcel Dekker: New York, 2000; p xx, 826 p.

292. Greenstein, J. P.; Winitz, M., Chemistry of the amino acids. Wiley: New York,, 1961; p 3 v. (xxi, 2872 p.).

293. Takimoto, J. K.; Xiang, Z.; Kang, J. Y.; Wang, L., Esterification of an unnatural amino acid structurally deviating from canonical amino acids promotes its uptake and incorporation into proteins in mammalian cells. Chembiochem 2010, 11, 2268-72.

294. Casadio, Y. S.; Brown, D. H.; Chirila, T. V.; Kraatz, H. B.; Baker, M. V., Biodegradation of Poly(2-hydroxyethyl methacrylate) (PHEMA) and Poly{(2-hydroxyethyl methacrylate)-co- poly(ethylene glycol) methyl ether methacrylate } Hydrogels Containing Peptide-Based Cross-Linking Agents. Biomacromolecules 2010, 11, 2949-2959.

295. Sun, P. Y.; Lu, H. X.; Yao, X.; Tu, X. X.; Zheng, Z.; Wang, X. L., Facile and universal immobilization of L-lysine inspired by mussels. J. Mater. Chem. 2012, 22, 10035- 10041.

284

296. Shah, K.; Rana, T. M., Synthesis of N-alpha-Fmoc-N-epsilon-tetrabutyl ester-EDTA- L-Lysine: An amino acid analog to prepare affinity cleaving peptides. Synth. Commun. 1996, 26, 2695-2702.

297. Adamczyk, M.; Johnson, D. D.; Reddy, R. E., Bone collagen cross-links: an efficient one-pot synthesis of (+)-pyridinoline and (+)-deoxypyridinoline (vol 11, pg 2289, 2000). Tetrahedron-Asymmetry 2000, 11, 5017-5017.

298. Orange, C.; Specht, A.; Puliti, D.; Sakr, E.; Furuta, T.; Winsor, B.; Goeldner, M., Synthesis and photochemical properties of a light-activated fluorophore to label His-tagged proteins. Chem. Commun. 2008, 1217-1219.

299. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.; Mattevi, A., Structures of human monoamine oxidase B complexes with selective noncovalent inhibitors: Safinamide and coumarin analogs. J. Med. Chem. 2007, 50, 5848- 5852.

300. Cürten, B.; Kullmann, P. H.; Bier, M. E.; Kandler, K.; Schmidt, B. F., Synthesis, photophysical, photochemical and biological properties of caged GABA, 4-[[(2H-1- benzopyran-2-one-7-amino-4-methoxy) carbonyl] amino] butanoic acid. Photochem Photobiol 2005, 81, 641-8.

301. Gololobov, Y. G.; Zhmurova, I. N.; Kasukhin, L. F., 60 Years of Staudinger reaction. Tetrahedron 1981, 37, 437-472.

302. Schlieve, C. R.; Tam, A.; Nilsson, B. L.; Lieven, C. J.; Raines, R. T.; Levin, L. A., Synthesis and characterization of a novel class of reducing agents that are highly neuroprotective for retinal ganglion cells. Experimental Eye Research 2006, 83, 1252-1259.

303. Wysocki, L. M.; Grimm, J. B.; Tkachuk, A. N.; Brown, T. A.; Betzig, E.; Lavis, L. D., Facile and General Synthesis of Photoactivatable Xanthene Dyes. Angew. Chem. Int. Ed. 2011, 50, 11206-11209.

304. Perez-Vilar, J.; Mabolo, R.; McVaugh, C. T.; Bertozzi, C. R.; Boucher, R. C., Mucin granule intraluminal organization in living mucous/goblet cells - Roles of protein post- translational modifications and secretion. J. Biol. Chem. 2006, 281, 4844-4855.

305. McIver, A. L. Development of New Methodologies for the Synthesis of Alkaloids and Light-Cleavable Groups. North Carolina State University, Raleigh, NC, 26-Jun-2012. 306. Furukawa, K.; Abe, H.; Tsuneda, S.; Ito, Y., Photoactivatable fluorescein derivatives with azidomethyl caging groups for tracing oligonucleotides in living human cells. Org Biomol Chem 2010, 8, 2309-11.

285

307. Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C., GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE-EXPRESSION. Science 1994, 263, 802-805.

308. Tsien, R. Y., The green fluorescent protein. Annu. Rev. Biochem. 1998, 67, 509-544.

309. Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y., Review - The fluorescent toolbox for assessing protein location and function. Science 2006, 312, 217-224.

310. Shaner, N. C.; Patterson, G. H.; Davidson, M. W., Advances in fluorescent protein technology. J. Cell Sci. 2007, 120, 4247-4260.

311. Ormo, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J., Crystal structure of the Aequorea victoria green fluorescent protein. Science 1996, 273, 1392-1395.

312. Charbon, G.; Wang, J. Y.; Brustad, E.; Schultz, P. G.; Horwich, A. L.; Jacobs- Wagner, C.; Chapman, E., Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid. Bioorg. Med. Chem. Lett. 2011, 21, 6067-6070.

313. Shaner, N. C.; Steinbach, P. A.; Tsien, R. Y., A guide to choosing fluorescent proteins. Nat. Methods2005, 2, 905-909.

314. Lin, M. Z.; Wang, L., Selective labeling of proteins with chemical probes in living cells. Physiology 2008, 23, 131-141.

315. Jing, C. R.; Cornish, V. W., Chemical Tags for Labeling Proteins Inside Living Cells. Acc. Chem. Res. 2011, 44, 784-792.

316. Kim, C. H.; Axup, J. Y.; Schultz, P. G., Protein conjugation with genetically encoded unnatural amino acids. Curr. Opin. Chem. Biol. 2013, 17, 412-419.

317. Charbon, G.; Brustad, E.; Scott, K. A.; Wang, J. Y.; Lobner-Olesen, A.; Schultz, P. G.; Jacobs-Wagner, C.; Chapman, E., Subcellular Protein Localization by Using a Genetically Encoded Fluorescent Amino Acid. Chembiochem 2011, 12, 1818-1821.

318. Krueger, A. T.; Imperiali, B., Fluorescent amino acids: modular building blocks for the assembly of new tools for chemical biology. Chembiochem 2013, 14, 788-99.

319. Summerer, D.; Chen, S.; Wu, N.; Deiters, A.; Chin, J. W.; Schultz, P. G., A genetically encoded fluorescent amino acid. Proc Natl Acad Sci U S A 2006, 103, 9785-9.

286

320. Wang, J.; Xie, J.; Schultz, P. G., A genetically encoded fluorescent amino acid. J Am Chem Soc 2006, 128, 8738-9.

321. Lee, H. S.; Guo, J.; Lemke, E. A.; Dimla, R. D.; Schultz, P. G., Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. J Am Chem Soc 2009, 131, 12921-3.

322. Hope-Roberts, M.; Wainwright, M.; Horobin, R. W., A nitrobenzofuran-conjugated phosphatidycholine (C-12-NBD-PC) as a stain for, membrane lamellae for both microscopic imaging and spectrofluorimetry. Biotech. Histochem. 2008, 83, 25-28.

323. Nunnally, B. K.; He, H.; Li, L. C.; Tucker, S. A.; McGown, L. B., Characterization of visible dyes for four-decay fluorescence detection in DNA sequencing. Anal. Chem. 1997, 69, 2392-2397.

324. Dick, G. R.; Knapp, D. M.; Gillis, E. P.; Burke, M. D., General method for synthesis of 2-heterocyclic N-methyliminodiacetic acid boronates. Org Lett 2010, 12, 2314-7.

325. Vilaivan, T., A rate enhancement of tert-butoxycarbonylation of aromatic amines with Boc(2)O in alcoholic solvents. Tetrahedron Lett. 2006, 47, 6739-6742.

326. De Cola, C.; Manicardi, A.; Corradini, R.; Izzo, I.; De Riccardis, F., Carboxyalkyl peptoid PNAs: synthesis and hybridization properties. Tetrahedron 2012, 68, 499-506.

327. Billman, J. H.; Parker, E. E., Amino acids I Glycine. J. Am. Chem. Soc. 1943, 65, 761-762.

328. Jiang, W.; Fu, Q. Q.; Fan, H. Y.; Ho, J.; Wang, W., A highly selective fluorescent probe for thiophenols. Angew. Chem. Int. Ed. 2007, 46, 8445-8448.

329. Schmidt, F.; Strozynski, M.; Salus, S. S.; Nilsen, H.; Thiede, B., Rapid determination of amino acid incorporation by stable isotope labeling with amino acids in cell culture (SILAC). Rapid Commun Mass Spectrom 2007, 21, 3919-26.

330. Wang, H.; Wong, C. H.; Chin, A.; Kennedy, J.; Zhang, Q.; Hanash, S., Quantitative serum proteomics using dual stable isotope coding and nano LC-MS/MSMS. J Proteome Res 2009, 8, 5412-22.

331. Zinn, N.; Winter, D.; Lehmann, W. D., Recombinant isotope labeled and selenium quantified proteins for absolute protein quantification. Anal Chem 2010, 82, 2334-40.

287

332. Warscheid, B.; Brucker, S.; Kallenbach, A.; Meyer, H.; Gerwert, K.; Kotting, C., Systematic approach to group-specific isotopic labeling of proteins for vibrational spectroscopy. Vib. Spectrosc. 2008, 28-36.

333. Kötting, C.; Gerwert, K., Proteins in action monitored by time-resolved FTIR spectroscopy. Chemphyschem 2005, 6, 881-8.

334. Tanio, M.; Tanaka, R.; Tanaka, T.; Kohno, T., Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: proteins secreted by Brevibacillus choshinensis. Anal Biochem 2009, 386, 156-60.

335. Deiters, A.; Geierstanger, B. H.; Schultz, P. G., Site-specific in vivo labeling of proteins for NMR studies. Chembiochem 2005, 6, 55-8.

336. Cellitti, S. E.; Jones, D. H.; Lagpacan, L.; Hao, X.; Zhang, Q.; Hu, H.; Brittain, S. M.; Brinker, A.; Caldwell, J.; Bursulaya, B.; Spraggon, G.; Brock, A.; Ryu, Y.; Uno, T.; Schultz, P. G.; Geierstanger, B. H., In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy. J Am Chem Soc 2008, 130, 9268-81.

337. Schackmann, A.; Muller, R., Reduction of nitroaromatic compounds by defferent pseudomonas species under aerobic conditions. Appl. Microbiol. Biotechnol. 1991, 34, 809- 813.

338. Rafii, F.; Franklin, W.; Heflich, R. H.; Cerniglia, C. E., Reduction of nitroaromatic compounds by anaerobic-bacteria isolated from the human gastrointestinal tract. Appl. Environ. Microbiol. 1991, 57, 962-968.

339. Virdee, S.; Kapadnis, P. B.; Elliott, T.; Lang, K.; Madrzak, J.; Nguyen, D. P.; Riechmann, L.; Chin, J. W., Traceless and Site-Specific Ubiquitination of Recombinant Proteins. J. Am. Chem. Soc. 2011, 133, 10708-10711.

340. Glenn, M. P.; Pattenden, L. K.; Reid, R. C.; Tyssen, D. P.; Tyndall, J. D. A.; Birch, C. J.; Fairlie, D. P., beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity. J. Med. Chem. 2002, 45, 371-381.

341. Ripka, A. S.; Bohacek, R. S.; Rich, D. H., Synthesis of novel cyclic protease inhibitors using Grubbs olefin metathesis. Bioorg. Med. Chem. Lett. 1998, 8, 357-360.

342. Cama, E.; Shin, H.; Christianson, D. W., Design of amino acid sulfonamides as transition-state analogue inhibitors of arginase. J. Am. Chem. Soc. 2003, 125, 13052-13057.

288

343. Romuald, C.; Busseron, E.; Coutrot, F., Very Contracted to Extended co- Conformations with or without Oscillations in Two- and Three-Station c2 Daisy Chains. J. Org. Chem. 2010, 75, 6516-6531.

344. Syed, B. M.; Gustafsson, T.; Kihlberg, J., 9-BBN as a convenient protecting group in functionalisation of hydroxylysine. Tetrahedron 2004, 60, 5571-5575.

345. Schade, D.; Topker-Lehmann, K.; Kotthaus, J.; Clement, B., Synthetic approaches to N-delta-methylated L-arginine, N-omega-hydroxy-L-arginine, L-citrulline, and N-delta- cyano-L-ornithine. J. Org. Chem. 2008, 73, 1025-1030.

346. Deiters, A.; Hahn, K.; Karginov, A. Light mediated regulation of protein dimerization. WO 2012024558, Feb 23, 2012.

347. Grewer, C.; Jäger, J.; Carpenter, B. K.; Hess, G. P., A new photolabile precursor of glycine with improved properties: A tool for chemical kinetic investigations of the glycine receptor. Biochemistry 2000, 39, 2063-70.

348. Vernall, A. J.; Abell, A. D., Cross-metathesis coupling of sugars and fatty acids to lysine and cysteine. Org Biomol Chem 2004, 2, 2555-7.

349. Bonke, G.; Vedel, L.; Witt, M.; Jaroszewski, J. W.; Olsen, C. A.; Franzyk, H., Dimeric building blocks for solid-phase synthesis of alpha-peptide-beta-peptoid chimeras. Synthesis 2008, 2381-2390.

350. Rosowsky, A.; Wright, J. E., N-Epsilon-2-(trimethylsilyl)ethoxycarbonyl derivatives of tri-L-lysine and tetra-Llysine as potential intermediates in the block polymer synthesis of macromolecular drug conjugates. J. Org. Chem. 1989, 54, 5551-5558.

351. Tan, Z.; Shang, S.; Danishefsky, S. J., Rational development of a strategy for modifying the aggregatibility of proteins. Proc Natl Acad Sci U S A 2011, 108, 4297-302.

352. Alajarin, M.; Lopez-Lazaro, A.; Vidal, A.; Berna, J., Tripod-tripod coupling of triazides with triphosphanes. The synthesis, characterization, and stability in solution of new cage compounds: Chiral macrobicyclic triphosphazides. Chem. Eur. J. 1998, 4, 2558-2570.

353. Kariyuki, S.; Iida, T.; Kojima, M.; Takeyama, R.; Tanada, M.; Kojima, T.; Iikura, H.; Matsuo, A.; Shiraishi, T.; Emura, T.; Nakano, K.; Takano, K.; Asou, K.; Torizawa, T.; Takano, R.; Hisada, N.; Murao, N.; Ohta, A.; Kimura, K.; Yamagishi, Y.; Kato, T. Peptide- compound cyclization method and preparation of cyclic peptides. WO 2013100132, July 4, 2013.

289

354. Onoda, M.; Uchiyama, S.; Santa, T.; Imai, K., A photoinduced electron-transfer reagent for peroxyacetic acid, 4-ethylthioacetylamino-7-phenylsulfonyl-2,1,3-benzoxadiazole, based on the method for predicting the fluorescence quantum yields. Anal Chem 2002, 74, 4089-96.

355. Katzenstein, J. M.; Janes, D. W.; Hocker, H. E.; Chandler, J. K.; Ellison, C. J., Nanoconfined Self-Diffusion of Poly(isobutyl methacrylate) in Films with a Thickness- Independent Glass Transition. Macromolecules 2012, 45, 1544-1552.

356. Keller, M.; Jorgensen, M. R.; Perouzel, E.; Miller, A. D., Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes. Biochemistry 2003, 42, 6067-77.

357. Pierwocha, A. W.; Walczak, K., The use of tri-O-acetyl-D-glucal and -D-galactal in the synthesis of omega-aminoalkyl 2-deoxy- and 2,3-dideoxy-d-hexopyranosides. Carbohydr Res 2008, 343, 2680-6.

358. Kaul, R.; Surprenant, S.; Lubell, W. D., Systematic study of the synthesis of macrocyclic dipeptide beta-turn mimics possessing 8-, 9-, and 10- membered rings by ring- closing metathesis. J Org Chem 2005, 70, 3838-44.

359. Kelly, W. L.; Boyne, M. T.; Yeh, E.; Vosburg, D. A.; Galonić, D. P.; Kelleher, N. L.; Walsh, C. T., Characterization of the aminocarboxycyclopropane-forming enzyme CmaC. Biochemistry 2007, 46, 359-68.

360. Rodríguez, A.; Miller, D. D.; Jackson, R. F., Combined application of organozinc chemistry and one-pot hydroboration-Suzuki coupling to the synthesis of amino acids. Org Biomol Chem 2003, 1, 973-7.

361. Yoshida, M.; Nishino, N. Preparation of cyclic tetrapeptide derivatives as histone deacetylase (HDAC) inhibitors and process for producing the same. WO 2004113366, Dec 29, 2004.

362. Leplawy, M. T.; Kociolek, K.; Slomczynska, U.; Zabrocki, J.; Beusen, D. D.; Marshall, G. R., Synthetic oligomers of conformationally restricted peptides as models for membrane channels. Pol. J. Chem. 1994, 68, 969-974.

363. Tran, H. A.; Kitov, P. I.; Paszkiewicz, E.; Sadowska, J. M.; Bundle, D. R., Multifunctional multivalency: a focused library of polymeric cholera toxin antagonists. Org Biomol Chem 2011, 9, 3658-71.

290

APPENDICES

291

APPENDIX A: Additional Information on the Photolysis of DEACM Group

Decaging Time Calculation

The decaging time T can be calculated from the number of molecule to be cleaved

(Nto cleave) and the number of molecule that get cleaved per second (Ncleaved per sec) as shown in equation A1.

(A1)

The definition of quantum yield (ø) in photolysis is defined by the number of molecule underwent photolysis (Ncleaved) and the number of photon absorbed (Nabsorbed) as shown in equation A2.

(A2)

When the irradiation time is 1 second, the Ncleaved per sec is equal to the Ncleaved, thus the

Ncleaved per sec can be calculated from equation A3.

(A3)

The Nabsorbed can be calculated with the ratio (r) of the absorbed photon and total photon (Nphoton), which can be derived from the absorbance (A). And absorbance (A) is calculated from light intensity falling upon the solution (I0) and the intensity passed through the solution (I1) shown in equation A4.

(A4)

Thus the ratio of absorbed photon (r) can be derived as equation A5.

(A5)

292

Taken Beer-Lambert law (A6) into A5 gave equation A7 with ɛ as extinction coefficient, l as the length of light path, and C as the concentration of sample solution.

(A6)

(A7)

Thus, the Nabsorbed can be derived with the ratio of absorbed photon (r) and total photon (Nphoton) as equation A8 below.

( ) (A8)

The total photon number (Nphoton) can be calculated from the power of light beam (P) and the energy of each photon (E) as equation A9.

(A9)

The energy of a photon (E) can be calculated based wavelength (λ), Planck’s constant

(h), and the speed of light (c) as shown in equation A10.

(A10)

Taking A9 and A10 into A8 gives equation A11.

( ) ( ) ( ) (A11)

Taking A11 into A3 gives the equation A12 for the number of molecule that get cleaved per second (Ncleaved per sec).

( ) (A12)

If the concentration of solution is the same, all the parameters except P in A12 are constant which is defined as constant B here (A13), Thus A12 can be write as below (A14).

293

( ) (A13)

(A14)

Taking A14 into A1 gave A15.

(A15)

The number of molecule to be cleaved (Nto cleave) can be calculated from the concentration (C) and the volume (V) of solution as shown in A16.

A16

The decaging time T can be calculated from A17 derived by taking A16 into A15.

A17

Experimental Setup

The 405 nm irradiation was performed in a setup as the cartoon in Figure A.1 consists of a Hg/Xe lamp, a 405 nm filter and a cuvette. The size of the cuvette was shown in Figure

A.2.

Figure A.1 Photolysis instrument setup.

294

side view

top view L

H L = 1 cm

Figure A.2 Size of the cuvette used in photolysis experiment.

The area (a) irradiated with light is calculated from equation A18 in which L and H are shown in Figure A.2 and V refers to the volume of solution.

(A18)

Thus the power of light irradiated on the solution can be calculated with equation A19 and I refers to the light intensity.

(A19)

Taking A19 into A17, the time needed to cleave our DEACM sample can be calculated with equation A20.

(A20)

The compare of our required decaging time (Tour) and the time Heckel reported (Trep) is listed in equation A21. The concentration of samples (C) is the same in our measurement.

Thus our required decaging time (Tour) can be calculated from equation A22.

(A21)

295

(A 22)

The light intensity (I) passed the 405 nm filter is 0.5 mW/cm2 which was measured by the photometer in Dr. Ghiladi’s lab, and the length (L) of cuvette is 1 cm. The power (P) of the LED light Heckel used is 30 mW, the volume (V) they subjected to decaging is 20 uL, and the irradiation time (Trep) is 120 s. Taking these numbers into A22, the time needed to cleave our sample is 100 hours which is unrealistic.

Thus, the failure of DEACM cleavage in our lab is due to instrumental limitation, or rather the light source and the type of cuvette to be specific.

296