Synthesis and Antitumor Activities of Aquayamycin and Analogues Of

A Dissertation

entitled

Synthesis and Antitumor Activities of Aquayamycin and Analogues of

Derhodinosylurdamycin A and Synthesis of S-Linked Trisaccharide

Glycal of Derhodinosylurdamycin A

Padam Prasad Acharya

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Chemistry

______Dr. Jianglong Zhu, Committee Chair

______Dr. Steven J. Sucheck, Committee Member

______Dr. Peter R. Andreana, Committee Member

______Dr. L. M. Viranga Tillekeratne, Committee Member

______Dr. Cyndee Gruden, Dean College of Graduate Studies

The University of Toledo

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Synthesis and Antitumor Activities of Aquayamycin and Analogues of Derhodinosylurdamycin A and Synthesis of S-Linked Trisaccharide Glycal of Derhodinosylurdamycin A

Padam Prasad Acharya

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry

The University of Toledo August 2019

Angucycline natural antibiotics are one class of bioactive natural products which contain a tetracyclic ring frame assembled in an angular manner and are diversely glycosylated. These rapidly growing natural products exhibit diverse biological activities including enzymatic inhibitions, antitumor activities, antimicrobial activities, and platelet aggregation inhibition. The urdamycins are a subclass of angucyclines natural products and were first detected during the chemical screening of secondary metabolites produced by Streptomyces fradiae by Drautz and Co-workers in 1986. They are structurally related with an aquayamycin type of angucycline natural products which contains a C- glycosylated benz[a]anthraquinone core and a cis-diol at the ring junction. Urdamycins are bioactive natural products but the mode of action is unknown, therefore it would be appealing to prepare aquayamycin and analogs of derhodinosylurdamycin A for extensive

SAR studies. Recently, we have completed the total syntheses of aquayamycin and analogues of derhodinosylurdamycin A with and without 2-deoxy sugar subunits. The synthesis was carried out on the basis of the convergent synthesis of derhodinosylurdamycin A as previously reported from our group. The mild cationic gold-

iii catalyzed glycosylation with S-but-3-ynyl thioglycoside donors was employed for the synthesis of analogues bearing disaccharide subunits containing α-L-olivoside and α-L- olioside moiety, respectively. After the syntheses of these natural and unnatural compounds, we submitted aquayamycin, four different analogues, and our previously synthesized derhodinosylurdamycin A to the Development Therapeutics Program of the

National Cancer Institute of National Institutes of Health for the NCI-60 Human Tumor

Cell Lines Screening using standard protocols. It was found that the analogue bearing no sugar subunit demonstrates good potential for growth inhibition and cytotoxic activity against a variety of human cancer cell lines, however, aquayamycin, derhodinosylurdamycin A, and three other analogues bearing sugar subunits did not show significant antiproliferative activity against those human cancer cell lines. Thus we concluded that the sugar is not helpful to improve the anticancer activity of aquayamycin type of angucycline natural products.

Derhodinosylurdamycin A, a bioactive natural product was discovered in 1989 by

Zeeck et al., which was later found as the main urdamycin metabolite from S. fradiae

Tü2717 mutant lacking UrdGT1a. Dehodinosylurdamycin A exhibits cytotoxic activity against L1210 leukemia cell lines (IC50 = 0.75 μg/mL). Structurally, it consists of tetracyclic aglycon core and a trisaccharide subunit connected via β-C-aryl glycosidic linkage. This trisaccharide subunit is composed of deoxy sugar subunits including two D- olivose and one L-rhodinose. Deoxy sugars are the important constituents of bioactive natural products, however, stereoselective synthesis of 2-deoxyglycosides remains a challenge due to lack of directing group at C2 position and are susceptible towards hydrolysis. Carbohydrate mimics such as S-linked glycosides are known to be resistant

iv towards enzyme or acid-mediated hydrolysis. Therefore it is appealing to synthesize S- linked 2-deoxyglycosides. We successfully achieved the stereoselective synthesis of S- linked trisaccharide glycal of antitumor antibiotic derhodinosylurdamycin A. This synthesis was accomplished based on our group previously developed umpolung S- glycosylation strategy which utilizes the glycosyl lithium species for streoselective sulfenylation. We also found out that an alkyl thiocyanate is better electrophile than corresponding asymmetric disulfide for stereoselective sulfenylation of 2-deoxy glcosyl lithium.

This work is dedicated to my parents Mr. Tanka Prasad Acharya and Mrs. Nyaya Kumari

Acharya.

Acknowledgements

My sincere gratitude goes to my advisor, Prof. Dr. Jianglong Zhu, for his sanguine support and constructive guidance since my first day in a laboratory. I sincerely appreciate his worthy of imitation training and encouragement that helped me to develop the proper skill required to conduct research. This attainment wouldn’t have been possible without his direction. I am grateful to the members of my research committee

Prof. Dr. Steven J. Sucheck, Prof. Dr. Peter R. Andreana and Asso. Prof. Dr. L.M.

Viranga Tillekeratne for their intuitive comments and valuable suggestions during my study. My thanks go to an undergraduate Sandip Janda whose hard work helped me to accomplish my project on time. I would also like to thank Dr. Kim for NMR and MS assistance. My previous and current lab members are the characters of admiration, the discussion with them was always fruitful. I thank the Department of Chemistry and

Biochemistry, the University of Toledo for this opportunity. I would also like to appreciate NSF (CHE-1213352 and CHE-1464787) for partially funding my projects. My thanks also go to my parents and family members for their moral and emotional support so far. At last but not least, I am thankful to my wife Jiwan for her relentless support, care, and love throughout.

vii

Table of Contents

Abstract ...... iii

Acknowledgements ...... vii

Table of Contents ...... viii

List of Schemes ...... ix

List of Tables ...... xi

List of Figures ...... xii

List of Abbreviations ...... xiii

Chapter 1 Synthesis and Antitumor Activities of Aquayamycin and Analogues of

Derhodinosylurdamycin A

1.1 Background and Significance ...... 1

1.1.1 Introduction of angucycline natural products ...... 1

1.1.2 Urdamycin natural products ...... 3

1.1.3 Biological activities and mode of action of urdamycin natural products ....5

1.1.4 Earlier syntheses of aquayamycin type natural products ...... 6

1.1.5 Analogues designed ...... 10

1.2 Results and Discussion ...... 10

1.2.1 Synthetic strategy ...... 10

1.2.2 Synthesis of tetracyclic aryliodide ...... 12

1.2.2.1 Synthesis of enantiopure epoxide and vinyl bromide ...... 12

viii 1.2.2.2 Synthesis of enone ...... 13

1.2.2.3 Synthesis of cyanophthalide ...... 14

1.2.2.4 Synthesis of tetracyclic aryliodide ...... 15

1.2.3 Synthesis of aquayamycin...... 16

1.2.3.1 Synthesis of glycal stannane ...... 16

1.2.3.2 Synthesis of aquayamycin...... 17

1.2.4 Synthesis of analogue A...... 19

1.2.5 Synthesis of analogue B ...... 19

1.2.5.1 Synthesis of D-fucal derived glycal stannane ...... 19

1.2.5.2 Synthesis of analogue B ...... 20

1.2.6 Synthesis of analogue C ...... 22

1.2.6.1 Synthesis of L-olivose derived thioglycoside donor ...... 22

1.2.6.2 Synthesis of analogue C ...... 22

1.2.7 Synthesis of analogue D...... 23

1.2.7.1 Synthesis of L-oliose derived thioglycoside donor ...... 23

1.2.7.2 Synthesis of analogue D...... 24

1.2.8 Biological studies ...... 25

1.3 Experimental ...... 30

1.3.1 General considerations ...... 30

1.3.2 Synthesis of epoxide 38 ...... 31

1.3.3 Synthesis of vinyl bromide 36 ...... 32

1.3.4 Synthesis of enone 22 ...... 34

1.3.5 Synthesis of cyanophthalide 39 ...... 40

ix 1.3.6 Synthesis of aryliodide ...... 45

1.3.7 Synthesis of D-rhamnal stannane...... 49

1.3.8 Synthesis of aquayamycin...... 51

1.3.9 Synthesis of analogue A...... 55

1.3.10 Synthesis of D-fucal derived glycal stannane ...... 58

1.3.11 Synthesis of analogue B ...... 60

1.3.12 Synthesis of L-olivose derived thioglycoside donor ...... 65

1.3.13 Synthesis of analogue C ...... 67

1.3.14 Synthesis of L-oliose derived thioglycoside donor ...... 70

1.3.15 Synthesis of analogue D...... 72

1.4 NMR Spectra for selected compounds ...... 76

Chapter 2 Synthesis of S-Linked Trisaccharide Glycal of

Derhodinosylurdamycin A

2.1 Introduction ...... 112

2.1.1 Angucycline antitumor antibiotic derhodinosylurdamycin A...... 112

2.1.2 2-Deoxy glycosides and carbohydrate mimics ...... 113

2.2 Results and Discussion ...... 115

2.2.1 Retrosynthesis ...... 115

2.2.2 Synthesis of disulfide acceptor 124 ...... 116

2.2.3 Synthesis of S-linked trisaccharide 125 ...... 118

2.2.4 Improved Synthesis of S-linked trisaccharide 125 and Synthesis of S-

linked glycal 120 ...... 119

x 2.3 Experimental ...... 121

2.3.1 General considerations ...... 122

2.3.2 Synthesis of allopyranoside 14 ...... 122

2.3.3 Synthesis of asymmetric disulfide acceptor……………………………124

2.3.4 Synthesis of phenyl 3,4-di-O-tert-butyldimethylsilyl-1-thio-D-olivoside

121…...... 128

2.3.5 Synthesis of S-linked disaccharide 123...... 129

2.3.6 Synthesis of S-linked trisaccharide 125 ...... 131

2.3.7 Synthesis of thiocyanate 138 ...... 132

2.3.8 Synthesis of S-linked trisaccharide 125 from S-linked disaccharide 123

and thiocyanate 138 ...... 134

2.3.9 Synthesis of S-linked trisaccharide glycal 139 ...... 134

2.3.10 Synthesis of S-linked trisaccharide glycal of derhodinosylurdamycin A

(120) ...... 136

2.4 NMR Spectra for Selected Compounds ...... 137

References ...... 155

List of Schemes

1.1 Total synthesis of aquayamycin by Suzuki et al ...... 7

1.2 Total synthesis of aquayamycin by Toshima et al ...... 8

1.3 Total synthesis of derhodinosylurdamycin A by Zhu et al ...... 9

1.4 Syntheses of epoxide and vinyl bromide ...... 12

1.5 Synthesis of enone ...... 13

1.6 Synthesis of cyanopthalide ...... 15

1.7 Synthesis of aryliodide ...... 16

1.8 Synthesis of glycal stannane ...... 17

1.9 Stille coupling and synthesis of aquayamycin ...... 18

1.10 Synthesis of analogue A...... 19

1.11 Synthesis of D-fucal derived glycal stannane ...... 20

1.12 Synthesis of analogue B ...... 21

1.13 Synthesis of L-olivose derived S-but-3-ynyl thioglycoside donor ...... 22

1.14 Synthesis of analogue C ...... 22

1.15 Synthesis of L-oliose derived S-but-3-ynyl thioglycoside donor ...... 23

1.16 Synthesis of analogue D...... 24

2.1 Strategies employed for the construction of S-linked 2-deoxyglycosides by Zhu

et al...... 114

2.2 Retrosynthetic analysis ...... 116

xii 2.3 Synthesis of asymmetric disulfide acceptor ...... 117

2.4 Synthesis of S-linked trisaccharide ...... 118

2.5 Improved synthesis of S-linked trisaccharide 125 and synthesis of S-linked

glycal 120 ...... 119

xiii

List of Tables

1.1 Selected Results showing the antiproliferative activity of compounds

aquayamycin (13), derhodinosylurdamycin A (10), analogue A (47), analogue B

(48), analogue C (49) and analogue D (50) in the NCI-60 human tumor cell lines

screening at 10μM………………………………………………………………..27

1.2 GI50 values of MTS assay on survival of NCI-60 human tumor cell lines treated

with analogue A (47), at 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM…………..29

xiv

List of Figures

1-1 First reported angucyclines ...... 1

1-2 Five major types of angucyclines and glycosylation positions ...... 2

1-3 Examples representing each type of angucycline ...... 3

1-4 Representative angucyclines natural products ...... 4

1-5 Monosaccharides present in urdamycins ...... 5

1-6 Analogues designed for synthesis and biological evaluation ...... 10

1-7 Synthetic strategy for aquayamycin and analogues of derhodinosylurdamycin A.11

2-1 Urdamycin A and derhodinosylurdamycin A ...... 112

2-2 Representative naturally occurring deoxy-glycosides ...... 113

List of Abbreviations

[훼] specific rotation Ac2O acetic anhydride AcOH acetic acid

Bn benzyl br broad c concentration CAN ceric ammonium nitrate cat catalytic COSY correlation spectroscopy

δ chemical shift d doublet dd doublet of doublet

(DHQ)2PHAL phthalazine adduct with dihydoquinine DIAD diisopropylazidodicarbozylate DIEA diisopropylethylamine DMF dimethylformamide DMAP 4-(dimethylamino)pyridine DMSO dimethylsulfoxide d.r. diastereomeric ratio

EtOAc ethylacetate FTIR fourier transform infrared spectroscopy g gram

GI50 growth inhibitory dose 50% h hour HRMS high resolution mass spectrometry HSQC heteronuclear single quantum coherence spectroscopy

xvi IC50 inhibitory concentration 50%

KHMDS potassium bis(trimethylsilyl)amide

LiDBB lithium 4,4’-di-tert-butylbiphenyl LiHMDS lithium bis(trimethylsilyl)amide LRMS low resolution mass spectrometry

µL microliter m multiplet M molar Mg milligram MHz megahertz Min minutes mL milliliter mmol millimole MOM methoxy methyl Ms mesyl MS molecular sieves

NIS N-iodosuccinimide NMR nuclear magnetic resonance ovrlp overlapping

Ph phenyl PMP para-methoxyphenyl ppm parts per million psi pounds per square inch p-TsOH para-toluene sulfonic acid q quartet

RT room temperature s singlet

SN2 bimolecular nucleophilic substitution t triplet

xvii TBAF tetra-n-butylammonium fluoride TBS tertiary-butyldimethylsilyl TBDPS tertiary-butyldiphenylsilyl tBu tertiary-butyl THF tetrahydrofuran TMEDA tetramethylethylenediamine TLC thin layer chromatography Ts tosyl

Urd urdamycin

xviii Chapter 1

Synthesis and Antitumor Activities of Aquayamycin and Analogues of

Derhodinosylurdamycin A

1.1 Background and Significance

1.1.1 Introduction of Angucycline Natural Products

Angucycline type of natural products include hundreds of secondary metabolites of microbial origin.1 Structurally, they contain a tetracyclic frame1 assembled in angular manner with benzanthrene nucleus beautified with glycosyl subunits.2 These rapidly growing natural products were discovered by the random process during antibacterial, antitumor and anticancer screening1 and they escalated in 1980. Tetrangomycin3 and tetragulol4 (Figure 1-1) were the first reported members of angucycline natural products.

These initially reported angucycline type natural products (angucyclinones) lack sugar moieties; however, later discovered angucycline natural products were found to be beautified with glycosyl subunits.2

Figure 1-1. First reported angucyclines. 1

Tetracycline-analogues, peptides, aminoglycosides and angucyclines are some of the examples of chemical class of glycosylated bacteria-derived secondary metabolites where angucyclines cover 4.6% of the entire chemical class.2

Figure 1-2. Five major types of angucyclines and glycosylation positions.

Large numbers of synthetic chemist have been attracted to this field because of their complex structures and interesting biological activities1,2,5,6 including enzymatic inhibitions, antitumor activities, antimicrobial activities, and platelet aggregation inhibition. Regardless of interesting biological activities, none of the angucycline has been advanced to clinical phase of development6 due to poor pharmacological properties.

On the other hand, chemical preparation of angucycline remains a challenge due to difficulty in stereoselective installation of sugar side chain and handling of tetracyclic core.5 In spite of this difficulty, it is always alluring to develop an effective protocol to synthesize anguycline natural products and analogues thereof. This effort would help to

advance these angucyline to clinical phase development by improving solubility and toxicity issues. The major types of angucycline natural products are depicted in above

Figure 1-2.2 Landomycin A7, saquayamycin8, gilvocarcin V9, jadomycin B10 and benzanthrin A11 are representative examples for respective tetrangomycin, aquayamycin, gilvocarcin, jadomycin and benzanthrene type angucycline natural products (Figure 1-3).

Figure 1-3. Examples representing each type of angucycline.

1.1.2 Urdamycin Natural Products

The urdamycins12-16 are a subclass of angucyclines natural products and were first detected during the chemical screening12 of secondary metabolites produced by

Streptomyces fradiae. Under different culture conditions, these colored urdamycins (A-F)

were first isolated by Drautz and Co-workers in 1986.12 They are structurally related to aquayamycin17 type of angucycline natural products which contains a C-glycosylated benz[a]anthraquinone core and a cis-diol at the ring junction (Figure 1-4).

Figure 1-4. Representative angucyclines natural products

β-D-olivose, α-L-rhodinose, α-L-aculose, α-L-cinerulose, β-D-rhodinose, and β-

D-kerriose are the six monosaccharides adorning the urdamycins.2 Among these monosaccharides β-D-olivose, α-L-rhodinose and β-D-rhodinose are present as C-9 4

glycosides2 in urdamycins (Figure 1-5). Other known aquayamycin-type angucycline

8 18 19 antibiotics include saquayamycins, vineomycin A1, and PI-080. Urdamycins and derhodinosylurdamycin A shares the same C-glycosidic moiety consisting of two D- olivoses and one L-rhodinose sugar subunit attached to tetracyclic core (Figure 1-4).

Figure 1-5. Monosaccharides present in urdamycins.

1.1.3 Biological Activities and Mode of Action of Urdamycin Natural Products

The urdamycin-type of natural products were found to be active against Gram- positive bacteria and murine L1210 leukemia stem cells. For example, urdamycin A and derhodinosylurdamycin A show significant anticancer activity against L1210 Leukemia

12,20 stem cells with an IC50 value of 0.55 and 0.75 μg/ml respectively. In addition, aquayamycin was found to be a tyrosine hydroxylase enzyme inhibitor and is active against Gram-positive bacteria Ehrlich ascites carcinoma, and Yoshida rat sarcoma cells.21 However, the actual mode of action is not known. There have been limited structure and activity relationship (SAR) studies on urdamycins and their derivatives. It was reported that diversification in sugar molecules have no significant changes in activity, however, acylation increased the activity.20 Studies also showed that 5, 6 double

bond is important and changes resulting in loss of this double bond lead to the disruption of cytostatic activity.1 In addition to the unknown mode of action, there is no specific protocol for biological evaluation. Lack of this information requires an effective synthetic method to build up libraries of these types of natural products for extensive structure and activity relationship (SAR) studies.

1.1.4 Earlier Syntheses of Aquayamycin Type Natural Products.

Due to the complex structures and interesting biological properties of urdamycins, a large number of synthetic chemists are attracted towards this field. Since their isolation, numerous synthetic studies have been reported toward the urdamycin antibiotics family including aquayamycin (urdamycinone A) tetracyclic core structure22 as well as urdamycinone B23-27 and related structures.28, 29 However, due to the highly labile nature of the aquayamycin core system,30 thus far only two groups have independently accomplished the total synthesis of aquayamycin.

Suzuki and co-workers in 2000 reported the first total synthesis of aquayamycin.31-33 One coupling partner enone 22 was synthesized from commercially available 2-cyclohexenone in 23 steps and another coupling partner phthalide 23 was furnished from another precursor 21 in 18 steps. They constructed tricyclic core 24 via

Hauser annulation between β-C-glycosylated 3-(benzenesulfonyl)phthalide 23 and enone

22. After employing vanadium mediated intramolecular Pinacol coupling to keto- aldehyde 25, tetracyclic angular aglycon 26 was achieved. Swern oxidation followed by certain functional group modification led to the first synthesis of aquayamycin.

Scheme 1.1 Total synthesis of aquayamycin by Suzuki et al. 31-33

Recently in 2016, Toshima and co-workers30 reported the second total synthesis of aquayamycin in 44 steps from commercially available precursors. It was 16 steps shorter than the former synthesis by Suzuki et al31-33. The preparation of highly functionalized ketone 30 was carried out from the commercially available precursor D-(-)-quinic acid in

10 steps. Another building block bromonaphthyl C-glycoside 29 was synthesized from compound 27 in 12 steps. One of the key step during synthesis was a diastereoselective

1,2-addition of C-glycosyl naphthyllithium derived from 29 to prepare compound 31.

Another key step involved an indium mediated site-selective allylation- rearrangement of napthoquinone 32 to afford 33. Finally vanadium mediated diastereoselective intramolecular pinacol coupling of compound 34 followed by certain functional group

manipulation afforded aquayamycin. This synthetic route (Scheme 1.2) was relatively shorter and may be more practical to access aquayamycin-type angucycline antibiotics.

Scheme 1.2 Total synthesis of aquayamycin Toshima et al. 30

Our group in 2015 accomplished the first total synthesis of derhodinosylurdamycin A34 as shown in Scheme 1.3. The highly functionalized enone 22 was synthesized from compound 36 in 14 steps and next building block cyanophthalide

39 was prepared from meta-hydroxy benzoic acid in 10 steps. Hauser annulation of cyanophthalide 39 and complex enone 22 afforded quinol which was quickly methylated to prepare compound 40. TBDPS deprotection of compound 40 generated primary alcohol which was nicely converted to compound 41 under Swern oxidation condition. 8

Scheme 1.3 Total synthesis of derhodinosylurdamycin A by Zhu et al. 34

Pinnacol coupling of keto-aldehyde 41 in highly stereoselective manner, subsequent oxidation and acetonide deprotection under acidic condition furnished tetracyclic aryliodide 43. Stille coupling between tetracyclic aryliodide 43 and glycal stannane 44, subsequent mesylation, stereoselective reduction of the resulting enol ether and desilylation provided β-C-arylglycoside 45. Late stage stereoselective α- glycosylation between disaccharide derived acetate donor 46 and acceptor 45 and necessary functional group manipulations provided derhodinosylurdamycin A 10. With the successful development of a convergent strategy amenable to the preparation of analogues of derhodinosylurdamycin A, we decided to carry out the synthesis and

explore the structure and activity relationship by varying the structures of the 2-deoxy sugar subunits.

1.1.5 Analogues designed.

These analogues as shown in Figure 1-6 were proposed for the synthesis as well as antitumor studies. The analogues are composed of angucycline core and a 2-deoxy disaccharide subunits or 2-deoxy monosaccharides subunits or without 2-deoxy sugar subunit. Several modifications were made to design natural and unnatural compounds in the hope of discovering new molecules with better biological activity.

Aquayamycin (13) Analogue A (47) Analogue B (48)

Analogue D (50) Analogue C (49)

Figure 1-6. Analogues designed for synthesis and biological evaluation.

1.2 Results and Discussion

1.2.1 Synthetic Strategy

Based on our previously reported strategy for the total synthesis of derhodinosylurdamycin A34 and our expertise towards 2-deoxy sugar subunits and 10

glycosylation strategies, we propose the following synthetic strategy so as to accomplish the syntheses of the targets.

Figure 1-7. Synthetic strategy for aquayamycin and analogues of derhodinosylurdamycin

The highly functionalized building blocks, including tetracyclic aryliodide 43 and partially protected β-C-arylglycoside 45, can be prepared in good quantities from commercially available starting substrates following previously disclosed strategies and procedures.34 The synthesis of aquayamycin 13 could be achieved from β-C- 11

arylglycoside 45 by certain functional group modifications and analogue A can be accessed from tetracyclic aryliodide 43. Analogue B can be obtained from partially protected β-C-arylglycoside 56 which in turn could be constructed by the Stille coupling of tetracyclic aryliodide 43 and D-fucal derived glycal stannane 55. Gold-catalyzed glycosylation35 between β-C-olivosyl C-3 acceptor 45 and respective thioglycosides donors 51 or 53 followed by global debenzylation, selective benzylation of phenol will afford respective benzyl ethers. Subsequent CAN oxidative deprotection, debenzylation, and mesylate elimination will lead to the preparation of respective analogues C and D.

1.2.2 Synthesis of Tetracyclic Aryliodide

1.2.2.1 Synthesis of Enantiopure Epoxide and Vinyl Bromide

The enantiopure epoxide 38 was synthesized from commercially available 3- methyl-3-buten-1-ol 57 as shown in Scheme 1.4. 34,36

Scheme 1.4 Syntheses of Epoxide and Vinyl Bromide.34

Under Mitsunobu condition compound 57 was converted to the p-methoxyphenyl ether 58 in 68% yields which underwent Sharpless asymmetric dihydroxylation reaction 12

so as to produce diol 59 quantitatively. The mesylation of primary alcohol of compound

59 followed by ring closure via SN2 attack furnished chiral epoxide 38. The preparation of

MOM protected vinyl bromide 36 began from 2-cyclohexenone 20. Bromination of compound 20 followed by elimination provided vinyl bromide 60 (68% yield).37 2- bromoenone under CBS reduction38 furnished chiral alcohol 61 in 85% yields which was subjected to MOM protection to afford MOM protected vinyl bromide compound 36.

1.2.2.2 Synthesis of Enone

The preparation Enone 22 was carried out as shown in the Scheme 1.5.34

Scheme 1.5 Synthesis of Enone.34

As soon as two coupling partners epoxide 38 and vinyl bromide 36 were synthesized, preparation of structurally complex enone 22 began with a lithium-halogen 13

exchange of vinyl bromide 36 followed by regioselective ring opening of epoxide 38 to produce chiral tertiary alcohol 62. Subsequent benzylation of tertiary alcohol 62 followed by cerium (IV) ammonium nitrate (CAN)-oxidative deprotection of PMP ether and

TBDPS protection of resulting primary alcohol afforded 64. Next, compound 64 was subjected to diastereoselective dihydroxylation39 to give diol 65 (71% yield) as a major diastereomer (dr = 4.34:1).34 Then diol 65 was subjected to MOM deprotection and selective protection of cis-diol to generate acetonide 66. Swern oxidation of secondary alcohol 66 furnished ketone 67 which upon reaction with potassium hexamethyldisilazide afforded correspomdimg enolate. This enolate was trapped with ally chloroformate to produce allyl vinyl carbonate 68. Compound 68 upon subjection to palladium mediated

Saegusa-Ito type reaction32,40 furnished desired enone 22 in 87% yield.

1.2.2.3 Synthesis of Cyanophthalide

The transformation of commercially available 3-hydroxybenzoic acid 37 to the corresponding amide 69 was accomplished in 94% yield by successive reaction with thionyl chloride and diethylamine. The phenol derivative 69 was subjected to MOM protection generating 70 which under chelation controlled lithiation followed by formylation41 produced benzaldehyde 71. The MOM protecting group in compound 71

42 was subsequently removed by MgBr2 in ether to provide phenol 72. After the iodination of phenol43 72, an inseparable mixture containing two regioisomeric mono-iodo benzenes, a bis-iodo benzene as well as unreacted starting material was obtained.

Therefore, MOM protection of this mixture was the strategy employed so as to isolate desired compound 73 (50% yield over 2 steps). Subsequent MOM deprotection of 73 14

followed by benzylation of phenol 74 provided benzyl ether 75. Finally, cyanophthalide

39 was obtained via intramolecular condensation44 of 75 in presence of trimethylsilyl cyanide and a catalytic amount of potassium cyanide in 18-crown-6 and after being stirred in acetic acid at room temperature as shown in the Scheme 1.6.

Scheme 1.6 Synthesis of Cyanopthalide34

1.2.2.4 Synthesis of Tetracyclic Aryliodide 30

The Hauser annulation33,45 reaction between complex enone 22 and cyanopthalide

39 was effected using lithium tert-butoxide as a base as shown in Scheme 1.7.34 Thus resulting crude quinol 76 is air-sensitive, thus it was quickly methylated to obtain methylated quinol 40. The varying range of yield for this reaction relied on the length of exposure of quinol 76 in air. Deprotection of TBDPS ether of compound 40 gave primary alcohol 77 which under Swern oxidation condition generated keto-aldehyde 41. A 15

diastereomeric mixture of diol 42 was produced from compound 41 by employing

Pinnacol coupling condition33,46 which cleanly furnished single diastereomeric ketone 78 upon Swern oxidation. Acetonide deprotection under acidic condition was carried out to generate tetracyclic aryl iodide core 43 in 96% yield.

Scheme 1.7 Synthesis of a Tetracyclic Aryliodide.34

1.2.3 Synthesis of Aquayamycin

1.2.3.1 Synthesis of Glycal Stannane

The chemical preparation of glycal stannane 44 commenced with bis-TBS protected D-rhamnal 79 as shown in the Scheme 1.834 below. Bis-TBS protected D- rhamnal47 was stannylated thereby affording stannane 80 (80% yields). Compound 80 underwent global TBS-deprotection followed by selective TBS-protection of C3-OH and

benzylation of C4-OH so as to furnish desired glycal stannane 44 for the following Stille coupling reaction

Scheme 1.8 Synthesis of Glycal Stannane

1.2.3.2 Synthesis of Aquayamycin

The Stille coupling between the highly functionalized tetracyclic aryliodide 43 and glycal stannane 44 afforded 85% coupling product 82. Mesylation of secondary alcohol of thus obtained coupling product 82 followed by one-pot reduction of a double bond and TBS deprotection led to the formation of previously reported β-C- arylglycoside34 45 in 78% yield over the two steps. The global debenzylation of known β-

C-arylglycoside 45 via palladium on carbon-catalyzed global hydrogenolysis afforded desired product 84 which upon selective protection of the phenol provided key intermediate 85 (79% yield over 2 steps). Next, compound 85 was converted to aquayamycin48 13 in 68% yield following our previously reported34 three-step sequence:

(1) cerium(IV) ammonium nitrate (CAN)-mediated oxidation of 85 to the corresponding quinone; (2) quick Pd/C-catalyzed hydrogenolysis to remove the phenolic benzyl ether and concomitant reduction of the quinone to hydroquinone followed by oxidation of the hydroquinone back to quinone via exposure to the air; (3) N,N-diisopropylethylamine- mediated elimination of the mesylate.

Scheme 1.9 Stille Coupling and Synthesis of Aquayamycin

1.2.4 Synthesis of Analogue A

Scheme 1.10 Synthesis of Analogue A

The preparation of analogue A (47) was accomplished starting from with highly functionalized tetracyclic aryliodide 43 as shown in the Scheme 1.10 in five steps: (1) selective mesylation of the secondary alcohol of 43 to give 88; (2) Pd/C-catalyzed global hydrogenolysis of 88 for removal of aryl iodide and benzyl ethers to produce 89; (3) selective protection of the phenol as its benzyl ether 90 (51% yield for 3 steps); (4) CAN oxidation of 90 to the corresponding quinone; (5) Pd/C-catalyzed hydrogenolysis to remove the phenolic benzyl ether and concomitant reduction of the quinone to hydroquinone followed by air mediated oxidation of the hydroquinone back to quinone and subsequent spontaneous elimination (66% over 2 steps).

1.2.5 Synthesis of Analogue B

1.2.5.1 Synthesis of D-Fucal derived Glycal Stannane

Scheme 1.11 Synthesis of D-Fucal derived Glycal Stannane

As shown in Scheme 1.11 the D-fucal stannane 55 was obtained from 3,4-di-O- tert-butyldimethylsilyl-D-fucal49 92 in four steps: (1) lithiation of the C1 of glycal followed by stannylation; (2) global silyl ether deprotection to the diol; (3) regioselective silylation of the hydroxyl group at C3; and (4) benzylation of the remaining C4-hydroxyl group.

1.2.5.2 Synthesis of Analogue B

Next, Stille coupling of D-fucal derived stannane 55 with tetracyclic aryliodide 43 provided C-arylated D-fucal 84 in 70% yield (Scheme 1.11). Mesylation of the secondary alcohol of 96 followed by stereoselective reduction of the enol ether moiety and concomitant acid-mediated desilylation afforded β-C-arylglycoside 56 (70% yield over 2 steps). Likewise, analogue B was prepared from 56 in five steps: (1) removal of all three benzyl ethers of 56 by Pd/C-catalyzed global hydrogenolysis and subsequent selective protection of the phenol as its benzyl ether 99 (65% yield over 2 steps); (2) CAN oxidation of 99 to the corresponding quinone; (3) Pd/C-catalyzed hydrogenation to remove the phenolic benzyl ether and concomitant reduction of the quinone to

hydroquinone followed by air-mediated oxidation of the hydroquinone back to quinone;

(N,N-diisopropylethylamine mediated elimination of the mesylate (69% over 3 steps).

Scheme 1.12 Synthesis of Analogue B

1.2.6 Synthesis of Analogue C

1.2.6.1 Synthesis of L-Olivose derived Thioglycoside Donor

Synthesis of S-but-3-ynyl thioglycoside donor 51 began with known compound

10235 via global deacetylation and global benzylation as presented in Scheme 1.13.

Scheme 1.13 Synthesis of L-Olivose derived S-but-3-ynyl Thioglycoside Donor

1.2.6.2 Synthesis of Analogue C

Scheme 1.14 Synthesis of Analogue C

In order to prepare analogue C bearing 2-deoxy disaccharide subunit, we chose to utilize our recently developed mild cationic gold(I)-catalyzed glycosylation methodology

35 employing S-but-3-ynyl thioglycoside donor. In the event, cationic (4-CF3Ph)3PAuOTf- catalyzed glycosylation between readily available L-olivose-derived S-but-3-ynyl thioglycoside donor 5148 and β-C-arylglycoside 45 acceptor afforded the corresponding disaccharide in 91% yield as a mixture of inseparable α/β anomers 52 (α/β, 2/1). The inseparable α/β anomers were then subjected to global debenzylation by Pd/C-catalyzed hydrogenolysis. Once all benzyl ethers were removed, α/β anomers can be separated and the α-disaccharide was obtained in 36% yield after purification. The α-anomer was then subjected to selective benzylation of the phenol to provide desired product 105 in 63% yields (21% yield over 3 steps). Next, intermediate 105 was converted to analogue C following the aforementioned strategy: (1) CAN oxidation of 105 to the corresponding quinone; (2) Pd/C-catalyzed hydrogenation to remove the phenolic benzyl ether and concomitant reduction of the quinone to hydroquinone; (3) air-mediated oxidation of the hydroquinone back to quinone followed by spontaneous elimination of the mesylate

(84% overall).

1.2.7 Synthesis of Analogue D

1.2.7.1 Synthesis of L-Oliose derived Thioglycoside Donor

Scheme 1.15 Synthesis of L-Oliose derived S-but-3-ynyl Thioglycoside Donor

L-oliose-derived S-but-3-ynyl thioglycoside donor 53 was furnished from known compound 10735 via global deacetylation and global benzylation as presented in Scheme

1.15.

1.2.7.2 Synthesis of Analogue D

Scheme 1.16 Synthesis of Analogue D

The cationic (4-CF3Ph)3PAuOTf-catalyzed glycosylation between readily available L-oliose-derived S-but-3-ynyl thioglycoside donor48 53 and β-C-arylglycoside

45 acceptor afforded corresponding disaccharide in 84% yield as a mixture of inseparable

α/β anomers (α/β, 2/1). Upon global debenzylation of this mixture of inseparable α/β anomers by Pd/C-catalyzed hydrogenolysis, the α-disaccharide was separated from its β- 24

anomer and obtained in 56% yield after purification. The α-anomer was then subjected to selective benzylation of the phenol to provide desired product 110 in 57% yields (27% yield over three steps). Likewise, intermediate 110 was converted to analogue D following the aforementioned two-step sequence (75% overall) (Scheme 1.16).

1.2.8 Biological Studies

With newly synthesized aquayamycin, analogues A–D and previously prepared derhodinosylurdamycin A in hand, we decided to evaluate their antiproliferative activities against various cancer cell lines. After careful consideration, we submitted these molecules to the Development Therapeutics Program of the National Cancer Institute of

National Institutes of Health for the NCI-60 Human Tumor Cell Lines Screening using standard protocols. This known 60-cell panel contains a variety of human cell lines of leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer, would be ideal for in vitro assay of our synthesized molecules. Initially, six compounds including aquayamycin, analogues A–D and previously prepared derhodinosylurdamycin A were evaluated for their antiproliferative activities against 60 human cancer cell lines at 10 μM concentration. The growth percent for selected cancer cells in the presence of each of these six molecules at 10 μM concentration are shown in Table 1 and also illustrated using by dynamic heat map presentation. Despite that it was known that derhodinosylurdamycin A is active against L1210 Leukemia stem cells and aquayamycin is active against Ehrlich ascites carcinoma and Yoshida rat sarcoma cells,21 to our

surprise, in this NCI-60 Human Tumor Cell Lines Screening none of, aquayamycin 13, derhodinosylurdamycin A 10 and analogues B-D bearing various 2-deoxysugar subunits showed significant antiproliferative activities against these 60 human cancer cell lines.

Gratifyingly, analogue A demonstrated potent growth inhibition against a broad range of human cancer cell lines, especially for some colon, melanoma, ovarian, renal, prostate, and breast cancer cell lines. Despite numerous previous reports indicating that the sugar play critical roles in the bioactivity of their carrier molecules,50, 51 these biological studies herein indicated that sugars do not help to improve the anticancer activity of these aquayamycin-type of angucycline antibiotics. Next, due to its promise analogue A was advanced to the next level and subjected to five dose testing at 10 nM, 100 nM, 1 μM, 10

μM, and 100 μM. The GI50 values are provided in Table 2 and also illustrated using by dynamic heat map presentation. As shown in Table 2, the GI50 values of analogue A range from approximately 0.23 μM to 18 μM against all the 60 human cancer cell lines. It was found that analogue A was most active against colon cancer cell COLO 205 (GI50

0.229 μM), Leukemia cell CCRF-CEM (GI50 0.342 μM), Melanoma MALME-3M (GI50

0.492 μM), Renal cancer cell ACHN (GI50 0.5 μM), and breast cancer cell MCF7 (GI50

0.257 μM). In general, analogue A was found not very active against CNS cancer cell lines, some of the non-small cell lung cancer cell lines and ovarian cancer cell lines.

Overall, analogue A showed good potential for growth inhibition and cytotoxic activity against a variety of human cancer cell lines. Despite that the cytotoxic mechanism for these aquayamycin-type molecules is not clear, it may be due to the electrophilic property of the 2-vinylnaphthoquinone moiety. In principle, protein or cellular nucleophiles may add to the 2-vinylnaphthoquinone moiety via Michael-type addition and, therefore, get 26

deactivated. This relatively reactive 2-vinylnaphthoquinone moiety may also be responsible to the labile nature of these molecules. In addition, analogue A lacking the sugar subunit contains less polar hydroxyl groups and may be more lipophilic than aquayamycin 13, derhodinosylurdamycin A 10 and analogues B-D. The more potent cytotoxicity of analogue A discovered according to our experiments is also in agreement with the previous report that increase of the lipophilicity of organic molecules improved their cytotoxicity.52

Table 1 Selected Results showing the antiproliferative activity of compounds aquayamycin (13), derhodinosylurdamycin A (10), analogue A (47), analogue B (48), analogue C (49) and analogue D (50) in the NCI-60 human tumor cell lines screening at

10μM.

10 13 4747 48 49 50

Table 2 GI50 values of MTS assay on survival of NCI-60 human tumor cell lines treated with analogue A (47), at 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM.

Reproduced from Acharya, P. P.; Khatri, H. R.; Janda, S.; Zhu, J. “Synthesis and antitumor activities of aquayamycin and analogues of derhodinosylurdamycin A,” Organic & biomolecular chemistry 2019, 17, 2691-2704 with the permission from Royal Society of Chemistry.

1.3 Experimental

1.3.1 General Considerations

Proton and carbon nuclear magnetic resonance spectra (1H NMR and 13C NMR) were recorded on either Bruker 600 (1H NMR-600 MHz; 13C NMR 150 MHz) at ambient temperature with CDCl3 as the solvent unless otherwise stated. Chemical shifts are reported in parts per million relative to residual protic solvent internal standard CDCl3

1 13 1 13 1 ( H, δ 7.26; C, δ 77.36), CD3OD ( H, δ 3.31; C δ 49.00). Data for H NMR are reported as follows: chemical shift, integration, multiplicity (app = apparent, par obsc = partially obscure, ovrlp = overlapping, s = singlet, d = doublet, dd = doublet of doublet, t

= triplet, q = quartet, m = multiplet) and coupling constants in Hertz. All 13C NMR spectra were recorded with complete proton decoupling. High resolution mass spectrometry (HRMS) was performed on a TOF mass spectrometer. Optical rotations were measured with Autopol-IV digital polarimeter; concentrations are expressed as g/100 mL. All reagents and chemicals were purchased from Acros Organics, Sigma

Aldrich, Fisher Scientific, Alfa Aesar, and Strem Chemicals and used without further purification. THF, methylene chloride, toluene, and diethyl ether were purified by passing through two packed columns of neutral alumina (Innovative Technology).

Anhydrous DMF and benzene were purchased from Acros Organics and Sigma-Aldrich and used without further drying. All reactions were carried out in oven-dried glassware under an argon atmosphere unless otherwise noted. Analytical thin layer chromatography was performed using 0.25 mm silica gel 60-F plates. Flash column chromatography was performed using 200-400 mesh silica gels (Scientific Absorbents, Inc.). Yields refer to chromatographically and spectroscopically pure materials, unless otherwise stated. 30

1.3.2 Synthesis of Epoxide 38

1-(4-methoxyphenoxy)-3-methyl-3-butene (58)

To a solution of commercially available 3-methyl-3-buten-1-ol 57 (10 g, 116 mmol) in 387 mL THF, p-methoxyphenol (21.6 g, 174 mmol) and triphenyl phosphine

(39.5 g, 150.8 mmol) were added and cooled at 0 ºC. After diisopropylazidocarboxylate

(29.7 mL, 150.8 mmol) being added slowly, the reaction mixture was refluxed for 6 h.

The solvent was concentrated under reduce pressure and dissolved in 200 mL of ether and filtered. The filtrate after being concentrated in vacuo, purification of crude was carried out via flash column chromatography (hexanes:dichloromethane = 10:1 to 4:1) to afford 15.2 g (65% yield) of product 58. The spectroscopic data were in good agreement with the literature reported data for this compound.

(S)-2-methyl-4-phenoxybutane-1, 2-diol (59)

A solution of compound 58 (9 g, 46.81 mmol) in 231 mL tert-BuOH was added to a solution of DHQ2PHAL (0.37 g, 0.48 mmol), K2OsO4.2H2O (0.03 g, 0.09 mmol),

K3[Fe(CN)6] (46.2 g, 140.4 mmol) and K2CO3 (19.4 g, 140.4 mmol) in 230 mL H2O at 0

º º C. The reaction mixture was stirred at 0 C for 5 h before 12 g of Na2S2O3 was added, stirred for 20 min and brine was added. After extraction was carried out with hexanes:ethyl acetate (1:2), the combined organic fractions were washed with water,

brine, dried over sodium sulfate, concentrated under reduce pressure and azeotroped with toluene so as to furnish crude diol 59 quantitatively. The spectroscopic data were in good agreement with the literature reported data for this compound.

S-2-methyl-2-(2-phenoxyethyl) oxirane (38)

Crude compound 59 (10.58g, 46.81 mmol) was dissolved in 100 mL dichloromethane before being cooled at 0 ºC, trimethylamine (7.8 mL, 56.17 mmol) and methane sulfonyl chloride (3.8 mL, 49.15 mmol) were added. The reaction mixture at

0 ºC was stirred for 45 min then the solvent was removed under vacuo. Thus obtained residue was dissolved in ether, washed with water, brine, dried over sodium sulfate and concentrated under reduce pressure. The crude residue was dissolved in 90 mL methanol,

º cooled at 0 C, and K2CO3 (19.41 g, 140.43 mmol) was added. The reaction was stirred at room temperature for 30 min before methanol was removed under vacuo, water was added to the residue and extracted with ether. The combined organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo. Purification on flash column chromatography (hexanes:ethyl acetate = 15:1) provided 5.5 g (56% yield) of corresponding epoxide 38. The spectroscopic data were in good agreement with the literature reported data for this compound.

1.3.3 Synthesis of Vinyl Bromide 36

2-bromocyclohex-2-en-1-one (60)

A solution of compound 20 (10 mL, 103.3 mmol) in 258 mL of CH2Cl2 was

º cooled at 0 C and Br2 solution (5.56 mL, 108.47 mmol in 100 mL CH2Cl2) was cannulated over 1.5 h. The resulting reaction mixture was stirred at 0 ºC for 2 h before trimethylamine (23.8 mL, 171.49 mmol) was added. The reaction mixture after being stirred at room temperature for 3 h was quenched with 100 mL of 1 M HCl. The organic layer was washed with 50 mL 1 M NaOH, brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude compound was re-dissolved in ether and filtered. The filtrate was concentrated under vacuo to get solid residue which was later recrystallized to afford 11.93 g (yield 65%) of compound 60. The spectroscopic data were in good agreement with the literature reported data for this compound.

R-2-bromocyclohex-2-en-1-ol (61)

To a solution of (S)-α,α-diphenylprolinol (1.45 g, 5.71 mmol) in 95 mL THF was added B(OMe)3 (765 µL, 6.74 mmol) and stirred for 1 h at room temperature before

º being cooled at -10 C and addition of BH3. N,N-diethylaniline (9.32 mL, 57.14 mmol). A solution of compound 60 (10 g, 57.14 mmol) in 95 mL THF was added to the reaction mixture at -10 ºC and stirred for 12 h before being quenched with 1M HCl. The aqueous mixture was extracted with ether and combined organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. Purification on flash column chromatography (hexanes:ethyl acetate = 20:1 to 10:1) provided 8.6 g of corresponding compound 61 (85% yield). The spectroscopic data were in good agreement with the literature reported data for this compound. 33

R-1-bromo-6-(methoxymethoxy) cyclohex-1-ene (36)

To a solution of compound 61 (4.46 g, 25.2 mmol) in 84 mL CH2Cl2, N,N- diisopropylethylamine (16.6 mL, 100.8 mmol) was added and cooled at 0 ºC. After

MOMCl (4 mL, 50.4 mmol) was added, the reaction mixture was stirred at room temperature for 12 h before being quenched with NaHCO3. The aqueous solution was extracted with CH2Cl2, the combined organic fractions were washed with water, brine, dried over sodium sulfate and concentrated under reduce pressure. The crude compound was purified by flash column chromatography (hexanes:ethyl acetate = 30:1) to furnish

5.07 g of desired compound 36 (yield 91%). The spectroscopic data were in good agreement with the literature reported data for this compound.

1.3.4 Synthesis of Enone 22

Compound 62

Vinyl bromide 36 (9.83 g, 44.5 mmol) and epoxide 38 (3.1 g, 14.83 mmol) were taken separately in flame dried flasks and kept in vacuum overnight for drying. THF was dried by adding a pinch of 1, 10-phenanthroline indicator, cooled to 0 ºC and n-BuLi (2.5

M) was added dropwise until dark color persisted. 127 mL of dry THF was added to vinyl bromide and cooled at -78 ºC and n-BuLi (44.5 mmol, 17.8 mL) was added dropwise. The resulting mixture was stirred at -70 ºC for 1.5 h then after it was transferred back to -78 ºC and epoxide solution (3.1 g, 14.83 mmol dissolved in 33 mL dry THF) was added followed by BF3.OEt2 (2.8 mL, 22.24 mmol). The reaction mixture was stirred at -

º 78 C for 2 h before being quenched with NaHCO3 and THF was removed under reduce pressure. The remaining aqueous portion was extracted with ethyl acetate, combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 10:1 to 4:1) to provide

4.05 g of compound 62 (78% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 63

KH (2.5 g, 18.72 mmol, 30% in mineral oil) was taken in flame dried flask and 47 mL of N,N-dimethylformamide was added and cooled at 0 ºC. The solution of compound

62 (3.28 g, 9.36 mmol) in 27 mL N,N-dimethylformamide was added slowly to the emulsion of KH in N,N-dimethylformamide. The resulting mixture was stirred at 0 ºC for 35

10 min, BnBr (1.7 mL, 14.04 mmol) and tetra-butylammonium iodide (0.35 g, 0.93 mmol) was added. The reaction mixture was stirred at 0 ºC for 30 min before being quenched with slow addition of water. The aqueous mixture was extracted with ether and combined organic extracts were washed with water, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification on flash column chromatography (hexanes:ethyl acetate = 5:1) provided 3.41 g (83% yield) of the corresponding benzyl ether 63. The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 64

Compound 63 (3.41 g, 7.74 mmol) was dissolved in 77 mL of acetonitrile and cooled at -15 ºC. The solution of ceric ammonium nitrate (12.73 g, 22.23 mmol in 12 mL

H2O) was added in the reaction mixture and stirred for 15 min. The reaction mixture was diluted with ether and the organic layer was washed sequentially with water, saturated

NaHCO3 and brine, dried over anhydrous sodium sulfate, filtered, concentrated to afford crude compound. The crude residue was purified by flash column chromatography

(hexanes:ethyl acetate = 4:1 to 2:1) to provide 2.57 g of primary alcohol. This alcohol

(2.57 g, 7.69 mmol) after being dissolved in 15 mL DMF was cooled at 0 ºC, imidazole

(0.78 g, 11.54 mmol) and tert-butyl (chloro) diphenylsilane (2.1 mL, 8.07 mmol) were added. The reaction mixture before being quenched with water was stirred at room temperature for 2 h. The remaining aqueous portion was extracted with ethyl acetate, combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude 36

residue was purified by flash column chromatography (hexanes:acetone = 100:1 to 50:1) to provide 4.25 g of compound 64 (96% yield over 2 steps). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 65

Potassium hexacyanoferrate (7.9 g, 24.06 mmol), potassium carbonate (3.33 g,

24.06 mmol), quiniclidine (0.066 g, 0.6 mmol), and methanesulfonamide (0.57 g, 6.01 mmol) were taken in flask containing 32 mL water and solution of compound 64 (3.44 g,

6.01 mmol) in 53 mL tert-butanol was added. After potassium osmate (VI) dihydrate

(0.044 g, 0.012 mmol) was added, the resulting mixture was stirred for 24 h at room temperature. The reaction was quenched with saturated sodium thiosulfate solution, aqueous portion was extracted with ethyl acetate, combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 10:1 to 5:1) and provided 2.57 g (71% yield) of the corresponding diol 65. The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 66

Anhydrous zinc chloride (1.16 g, 8.5 mmol) was taken in flame dried flask and the solution of diol 65 (2.57 g, 4.25 mmol) in 57 mL CH2Cl2 was transferred to the flask.

The reaction mixture was stirred for three hours at room temperature after octane thiol

(1.5 mL, 8.5 mmol) was added and quenched with saturated NaHCO3 solution. The 37

aqueous solution was extracted with CH2Cl2, the combined organic fractions were washed with water, brine, dried over sodium sulfate and concentrated under reduce pressure. The crude compound was passed through a small silica gel column

(CH2Cl2:MeOH = 20:1) to collect triol. This crude triol was dissolved in 14.2 mL of dimethoxypropane, camphorsulfonic acid (0.049 g, 0.21 mmol) and stirred at room temperature for 12 h. The reaction mixture was quenched with NaHCO3, an aqueous portion was extracted with ethyl acetate, combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (toluene:ethyl acetate = 50:1 to 30:1) to provide 1.51 g of compound 66

(59% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 67

A solution of DMSO (1.1 mL, 14.49 mmol) in 18 mL CH2Cl2 was cooled at -78

ºC, oxalyl chloride (632 μL, 7.24 mmol) was added and left stirring for 15 min then after the solution of compound 66 (2.18 g, 3.62 mmol) in 18 mL CH2Cl2 was added. The reaction mixture was stirred at -78 ºC for 2 h, triethylamine (3 mL, 21.72 mmol) was added, and stirred at 0 ºC for 45 min before being quenched with brine solution. After the reaction mixture was extracted with CH2Cl2, the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude compound was purified by flash column chromatography

(hexanes:ethyl acetate = 15:1) to provide 1.74 g of ketone 67 (80% yield). The 38

spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 22

Compound 67 (2.19 g, 3.65 mmol) was taken in a flame dried flask, evacuated overnight and filled with argon. THF was dried by adding a pinch of 1, 10-phenanthroline indicator, cooled to 0 ºC and n-BuLi (2.5 M) was added dropwise until dark color persisted. 37 mL of dry THF was added to ketone 67, cooled at -78 ºC and KHMDS solution (6.1 mL, 5.47 mmol) was added. The resulting reaction mixture was transferred to -40 ºC, stirred for 45 min and cooled back to -78 ºC. The reaction mixture was stirred for 30 min at -78 ºC after allyl chloroformate (584 μL, 5.47 mmol) was added. The reaction mixture was quenched with saturated NaHCO3, THF was removed, aqueous portion was extracted with ethyl acetate, combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 30:1, 2% triethylamine) after column being buffered with solvent system to obtain 1.82 g allyl vinyl carbonate 68 (73% yield). Thus furnished carbonate (1.08 g, 1.6 mmol) was dissolved in 5.3 mL degassed acetonitrile,

Pd(OAc)2 (0.53 g, 2.4 mmol) was added and stirred at room temperature for 24 h. The reaction mixture was filtered through a small pad of celite, filtrate was concentrated and crude residue was purified through flash column chromatography (hexanes:acetone =

40:1) so as to afford 0.84 g of enone 22 (87% yield).

1.3.5 Synthesis of Cyanophthalide 39

Amide 69

N,N-dimethylformamide (210 μL, 2.7 mmol) was added to a solution of 3- hydroxybenzoic acid 37 (11.3 g, 81.8 mmol) in thionyl chloride (41.75 mL, 572 mmol) and refluxed for 1 h by maintaining oil bath temperature 70 ºC. The reaction mixture was cooled, thionyl chloride was removed under reduce pressure and crude compound was azeotroped with toluene. This crude oil was cooled to 0 ºC after it was dissolved in 80 mL

CH2Cl2 and diethylamine (31.3 mL, 302 mmol) was added dropwise. The reaction

º mixture was stirred at 0 C for 1 h, concentrated, diluted with CH2Cl2, sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column

chromatography (hexanes:ethyl acetate = 2:1 to 1:1) to provide 14.86 g of compound 69

(94% yield for 2 steps). The spectroscopic data were in good agreement with the literature reported data for this compound.

Methoxymethyl ether 70

To a solution of compound 69 (10.29 g, 53.25 mmol) dissolved in 152 mL

CH2Cl2, N,N-diisopropylethylamine (15.84 mL, 95.85 mmol) was added and cooled at 0

ºC. After MOMCl (4.85 mL, 63.9 mmol) was added, the reaction mixture was stirred at room temperature for 12 h before being quenched with NaHCO3. The aqueous solution was extracted with CH2Cl2, the combined organic fractions were washed with water, brine, dried over sodium sulfate and concentrated under reduce pressure. The crude compound was purified by flash column chromatography (hexanes:ethyl acetate = 3:1) to furnish 11.61 g of desired compound 70 (yield 94%). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 71

THF was dried by adding a pinch of 1, 10-phenanthroline indicator, cooled to 0 ºC and n-BuLi (2.5 M) was added dropwise until dark color persisted. In a flame dried flask

(Flask X), 79 mL of dry THF was taken cooled at -78 ºC, tetramethylethylenediamine

(9.51 mL, 63.4 mmol) was added. After Sec-butyllithium (51.2 mL, 66.5 mmol, 1.3 M in cyclohexanes) being added to the reaction mixture it was left stirring at -78 ºC for 25 min.

In another flame dried flask (Flask Y) compound 70 (10 g, 42.2 mmol) was dissolved in

64 mL dry THF, after being cooled at -78 ºC it was cannulated to Flask X and left stirring 41

at -78 ºC for 1.5 h. After N,N-dimethylformamide (4.1 mL, 52.74 mmol) being added, the reaction mixture was warmed to room temperature and stirred for 1 h before it was quenched with slow addition of water. THF was removed under vacuo, the aqueous layer was extracted with ether, the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude compound was purified by flash column chromatography (hexanes:ethyl acetate =

3:1 to 1:3) to provide 8.05 g of aldehyde 71 (72% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 72

Magnesium turnings (5 g, 136.6 mmol) was taken in 1 litre flask and 234 mL of ether was added followed by addition of 1,2 dibromoethane (7.1 mL, 82 mmol). The reaction mixture was stirred at room temperature for an hour with open flask until the effervescence ceases to form. This etherial solution of magnesium bromide was transferred into another flask containing the solution of compound 71 (7.24 g, 27.3 mmol) in 55 mL THF. The reaction mixture was quenched with saturated NaHCO3 after stirring at room temperature for 24 h. The aqueous layer was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 3:1 to 2:1) to provide 5.31 g of phenol 72 (88% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 73

To a solution of compound 72 (4.96 g, 22.44 mmol) in 135 mL of CH2Cl2, AlCl3

(6 g, 44.88 mmol) in three portions was added and stirred at room temperature for 15 min. The reaction mixture was stirred at room temperature for 12 h after N- iodosuccinimide (6.06 g, 26.93 mmol) was added in three in potions. The reaction was quenched with 1 M HCl, extracted with CH2Cl2 and concentrated under vacuo so as to afford crude residue. This crude material was passed through a short bed of silica and the combined fractions were concentrated and subjected for the next step. To a solution of crude compound in 75 mL CH2Cl2, N,N-diisopropylethylamine (15 mL, 89.76 mmol) was added and cooled at 0 ºC. After MOMCl (3.6 mL, 44.88 mmol) was added, the reaction mixture was stirred at room temperature for 12 h before being quenched with NaHCO3.

The aqueous layer was extracted with CH2Cl2, the combined organic fractions were washed with water, brine, dried over sodium sulfate and concentrated under reduce pressure. The crude compound was purified by flash column chromatography

(hexanes:ethyl acetate = 5:1 to 2:1, 1% Et3N) to furnish 4.38 g of desired compound 73

(50% yield for 2 steps). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 74

Magnesium turnings (2.1 g, 86 mmol) was taken in 500 mL flask and 147 mL of ether was added followed by the addition of 1,2 dibromoethane (4.5 mL, 51.6 mmol).

The reaction mixture was stirred at room temperature for an hour with open flask until the effervescence ceases to form. This etherial solution of magnesium bromide was 43

transferred into another flask containing the solution of compound 73 (6.71 g, 17.2 mmol) in 34 mL THF. The reaction mixture was quenched with saturated NaHCO3 after stirring at room temperature for 5 h. The aqueous layer was extracted with ethyl acetate, the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was then purified by flash column chromatography (hexanes:ethyl acetate = 3:1 to 2:1) to provide

4.1 g of phenol 74 (69% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 75

To a solution of compound 74 (1.9 g, 5.5 mmol) in 11 mL acetone, potassium carbonate (1.52 g, 11 mmol) and benzyl bromide (1 mL, 8.25 mmol) were added respectively. The reaction was stirred at room temperature for 12 h before being quenched with water. The solvent was concentrated under vacuo and extracted with ethyl acetate. The combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure.

The crude residue was purified by flash column chromatography (hexanes:ethyl acetate =

5:1 to 3:1) to provide 1.8 g of compound 75 (75% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 39

KCN/18-crown-6 (6.6 mg/26.5 mg in 5 mL CH2Cl2,) solution and trimethylsilyl cyanide (635 µL, 5 mmol) were added to a solution of compound 75 (1.46 g, 3.34 mmol) 44

º º in 6.7 mL CH2Cl2 at 0 C. The reaction mixture was stirred at 0 C for 20 minutes and the solvent was removed under reduce pressure. Thus obtained crude compound was dissolved in 6.7 mL acetic acid and left stirring at room temperature for 24 h. The reaction mixture was quenched with water, the white precipitate formed was filtered, washed with enough water and recrystallized with hexanes:CHCl3 (3:1) to generate 1.1 g of cyanophthalide 39 (85% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

1.3.6 Synthesis of Aryliodide

Compound 40

To a flame dry flask tBuOLi (1.41 g, 17.61 mmol) was taken, 41 mL THF was added before it was cooled at -78 ºC and stirred for 15 min. The cyanophthalide solution

(2.3 g, 5.87 mmol) in 45 mL THF was added dropwise and the reaction mixture was stirred at -78 ºC for 30 min. After the addition of enone solution (3.51 g, 5.87 mmol) the reaction mixture was left stirring at -78 ºC for 1 h. The reaction mixture was let warm to room temperature over 30 min before stirring it at 45 ºC for 1 h. The reaction mixture was quenched with saturated ammonium chloride and the solvent was removed under vacuo.

The extraction of an organic compound from the aqueous layer was carried out with ethyl acetate, the combined organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. Thus furnished crude compound

76 was dissolved in 38 mL acetone, K2CO3 (41 g, 293.5 mmol) and dimethylsulfate (22.3 mL, 234.8 mmol) were added. The reaction mixture was refluxed at 62 ºC for 24 h before

it was quenched with saturated ammonium chloride solution. The solvent was concentrated under vacuo and extracted with ethyl acetate. The combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 40:1 to 10:1) to provide 5.82 g of compound 27 (80% yield). The varying yield (62-80%) for this reaction depends on the exposure of quinol 76 to air. The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 77

To a solution of compound 40 (4.24 g, 4.28 mmol) in 43 mL tetrahydrofuran at 0

ºC was added 1 M tetra-n-butylammonium fluoride in THF (4.71 mL, 4.71 mmol). The resulting mixture was stirred at 0 ºC for 12 h before it was quenched with saturated 46

sodium bicarbonate and extracted with ethyl acetate. The combined organic extracts were sequentially washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure and purified via flash column chromatography

(hexanes:ethyl acetate = 1:1) to produce 3.22 g of desired compound 77 quantitatively.

The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 42

A solution of DMSO (1.9 mL, 26.68 mmol) in 33 mL CH2Cl2 was cooled at -78

ºC, oxalyl chloride (1.2 mL, 13.34 mmol) was added and left stirring for 15 min then after the solution of compound 77 (5.02 g, 6.67 mmol) in 33 mL CH2Cl2 was added. The reaction mixture was stirred at -78 ºC for 2 h, triethylamine (5.6 mL, 40 mmol) was added, and stirred at 0 ºC for 45 min before being quenched with brine solution. After the reaction mixture was extracted with CH2Cl2, the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure so as to afford crude compound 41 which was directly used for next step.

Zn powder (1.05 g, 16 mmol), VCl3(thf)3 (10.96 g, 29.35 mmol) were weighed in a flame dried flask under inert atmosphere and 100 mL degassed CH2Cl2 was added.

After the reaction mixture was stirred at room temperature for 30 min, degassed N,N- dimethylformamide (5.5 mL, 73.37 mmol) was added dropwise and stirred for 15 min.

The solution of crude compound 41 in 17 mL CH2Cl2 was added to the reaction mixture and stirred for 15 min before being quenched with saturated sodium potassium tartrate solution followed by extraction with extracted with CH2Cl2. The combined organic 47

extracts were sequentially washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 10:1 to 3:1) to provide 3.77 g of compound 42 (75% yield over 2 steps). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 78

A solution of DMSO (1.52 mL, 21.4 mmol) in 27 mL CH2Cl2 was cooled at -78

ºC, oxalyl chloride (933 μL, 7.24 mmol) was added and left stirring for 15 min then after the solution of compound 42 (4.03 g, 5.35 mmol) in 27 mL CH2Cl2 was added. The reaction mixture was stirred at -78 ºC for 2 h, triethylamine (4.5 mL, 32.1 mmol) was added, and stirred at 0 ºC for 45 min before being quenched with brine solution. After the reaction mixture was extracted with CH2Cl2, the combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude compound was purified by flash column chromatography

(hexanes:ethyl acetate = 15:1 to 10:1) to provide 3.37 g of ketone 78 (84% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 43

To a solution of compound 78 (3.08 g, 4.1 mmol) in 157 mL 1,4 dioxane was

º added 8.4 mL of 5% H2SO4. The reaction mixture was stirred at 80 C for 24 h before being quenched with saturated sodium bicarbonate and extracted with ethyl acetate. The 48

combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude compound was the purified by flash column chromatography (hexanes:ethyl acetate = 2:1) to provide 2.1 g of aryliodide 43 (96% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

1.3.7 Synthesis of D-rhamnal Stannane

Compound 80

To a solution of compound 79 (4.5 g, 12.55 mmol) in dry 94 mL THF (dried with n-BuLi using 1,10-phenanthroline as an indicator) cooled at -78 ºC was added tert- butyllithium (23 mL, 43.9 mmol) dropwise cautiously. The reaction mixture was warmed up to 0 ºC and stirred for 25 min before it was transferred back to -78 ºC. After tributyltin chloride (11.8 mL, 43.9 mmol) was added, the reaction mixture was stirred at -78 ºC for

20 min, quenched with water and extracted with ethyl acetate. The combined organic fractions were washed with water and brine, dried over sodium sulfate, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography

(hexanes:CH2Cl2 = 40:1, 1% Et3N) using preflushed silica gel column with the solvent system to provide 6.51 g of compound 80 (80% yield). The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 44

To a solution of glycal stannane 80 (2.29 g, 3.53 mmol) in 12 mL THF cooled at 0

ºC was added 1 M tetra-n-butyl ammonium fluoride (10.6 mL, 10.6 mmol) and the resulting mixture was stirred at room temperature for 12 h. Saturated aqueous NaHCO3 solution was added and THF was removed under reduce pressure. The aqueous layer was extracted with ethyl acetate and combined organic extracts were washed sequentially with water and brine, dried over sodium sulfate, and concentrated. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 5:1, with 2% Et3N) using preflushed silica gel column to provide 1.48 g of diol 81 quantitatively. To a solution of diol 81 (0.5 g, 1.19 mmol) in 1.2 mL DMF were added Et3N (827 µL, 5.95 mmol) and tert-butyldimethylsilyl chloride (0.196 g, 1.31 mmol). The resulting mixture was stirred at room temperature for 10 h before being quenched with water. The mixture was extracted with ethyl acetate, and combined organic extracts were washed with water and brine, dried over sodium sulfate, and concentrated under reduce pressure. To a solution of this crude compound in 4 mL DMF cooled at 0 ºC was added sodium hydride

(0.095 g, 2.38 mmol) and the mixture was stirred at 0 ºC for 45 minutes. Benzyl bromide

(245 µL, 1.43 mmol) was then added and the resulting mixture was warmed up to room temperature and stirred overnight before being quenched with water. The aqueous mixture was extracted with ethyl acetate and combined organic extracts were washed with water, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification on flash column chromatography (hexanes:dichloromethane = 30:1, 2% Et3N) using preflushed silica gel column to provide provided 0.52 g (70% yield over 2 steps) of 50

corresponding glycal stannane 44. The spectroscopic data were in good agreement with the literature reported data for this compound.

1.3.8 Synthesis of Aquayamycin

Compound 82

Glycal stannane 44 (0.88 g, 1.41 mmol), aryl iodide 43 (0.50 g, 0.70 mmol) and

Pd(PPh3)2Cl2 (0.1 g, 0.141 mmol) were taken into a flame dried flask, evacuated, and filled with argon. 7 mL of degassed dry toluene was added to the flask and the resulting mixture was stirred at 100 ºC for 6 h. The reaction mixture was cooled to room temperature and directly subjected to purification via silica gel flash column chromatography (hexanes:ethyl acetate = 5:1, 2% Et3N) to affording 548 mg (85% yield) of the C1-arylated glycal 82. The spectroscopic data were in good agreement with the literature reported data for this compound.

Compound 45

To a solution of C1-arylated glycal 82 (443 mg, 0.483 mmol) in 2.4 mL pyridine were added methanesulfonyl chloride (56 µL, 0.72 mmol) and 4-dimethylaminopyridine

(6 mg, 0.048 mmol). After the resulting mixture was stirred at room temperature for 24 h, pyridine was removed by air flow. The residue was diluted with CH2Cl2, washed sequentially with aqueous saturated CuSO4 solution, water, and brine. The organic solution was dried over sodium sulfate, filtered, and concentrated under reduce pressure to produce the crude mesylate 83 which was used directly in the next step.

The mesylate 83 was dissolved in 9.6 mL ethanol and a pinch of bromocresol green was added as an indicator. To the mixture was added sodium cyanoborohydride

(0.061 g, 0.966 mmol) followed by addition of 0.5 M HCl in methanol (3.9 mL, 1.93 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes.

The second batch of sodium cyanoborohydride (0.061 g, 0.966 mmol) and 0.5 M HCl in 52

methanol (3.9 mL, 1.93 mmol) was added, and the reaction mixture was stirred at room temperature for 24 h before being quenched with saturated NaHCO3 solution. The aqueous mixture was extracted with ethyl acetate and combined organic extracts were washed with water, dried over sodium sulfate, filtered, and concentrated under reduce pressure. Purification on silica gel flash column chromatography (toluene:ethyl acetate =

20:1 to 5:1) furnished 332 mg (78% yield for 2 steps) of 2-deoxy β-C-glycoside 45. The spectroscopic data were in good agreement with the literature reported data for this compound.

Aquayamycin (13)

To a solution of partially protected β-C-arylglycoside 45 (30 mg, 0.034 mmol) in

1.4 mL of mixed solvents (EtOAc:MeOH = 1:1, v:v), 10% palladium on carbon (36.2 mg, 0.034 mmol) was added. After being evacuated and filled with hydrogen five times, the reaction mixture was stirred at room temperature under positive hydrogen pressure

(40 psi) for 32 h. The reaction mixture was then diluted with CH2Cl2:MeOH (10:1, v:v), filtered through celite, concentrated. The crude compound 84 (20.8 mg, quantitative) was used directly in the next step without purification.

A solution of crude compound 84 (20.8 mg, 0.034 mmol) in 0.74 mL DMF was

º cooled at 0 C. To this solution was added Cs2CO3 (13.3 mg, 0.041 mmol) followed by addition of 37 µL stock solution of benzyl bromide in DMF (0.051 mmol, 1.5 eq.) (Note: the stock solution was prepared by adding 40 µL of benzyl bromide in 200 µL DMF).

The reaction mixture was stirred at room temperature for 5 h before being quenched with a pinch of solid ammonium chloride. DMF was removed by air flow and the residue was 53

purified by using preparative TLC in CH2Cl2:MeOH (10:1, v:v) to afford 18.9 mg (79% yield for 2 steps) of compound 85.

Next, A solution of compound 85 (14.6 mg, 0.021 mmol) in 1.3 mL of acetonitrile was cooled at 0 ºC and 83 µL of stock solution of cerium ammonium nitrate in water

(0.0624 mmol, 3 eq.) was added (Note: the stock solution was prepared by adding 174 mg of cerium ammonium nitrate in 400 µL water). The reaction mixture was stirred at 0

ºC for 35 min before being diluted with 4 mL ethyl acetate. 0.5 mL of ice cooled saturated NaHCO3 was added and the resulting mixture was stirred for 2 minutes. The organic layer was separated and passed through a small pad of Na2SO4, concentrated under reduce pressure and kept in vacuum for 10 minutes to give the crude compound 86.

This crude material was dissolved in 0.26 mL of mixed solvents (EtOAc:MeOH, 1:1, v:v) and 10% palladium on carbon (4.4 mg, 0.0042 mmol) was added. The mixture was evacuated and filled with hydrogen for three times. After being stirring at room temperature under positive hydrogen pressure for 30 min, the reaction mixture was diluted with methanol, filtered through celite, and concentrated under reduced pressure.

The resulting crude compound 87 was dissolved in 0.8 mL of 1,4-dioxane and N,N- diisopropylethylamine (7.3 µL, 0.042 mmol) was added. After being stirred at 40 ºC for 1 h, the reaction mixture was cooled down. Dioxane was removed by air flow and the residue was purified by preparative TLC in CH2Cl2:MeOH (10:1, v:v) to furnish 6.9 mg

ퟐퟑ º of aquayamycin 13 (68% yield for 3 steps). [휶]퐃 = 119.8 (c = 0.1, CH3OH); FT-IR

(thin film): 3367, 2963, 2921, 2852, 1723, 1637, 1563, 1260, 1055, 650 cm-1; 1H NMR

(600 MHz,CD3OD)  7.86 (d, J=7.9 Hz, 1 H), 7.59 (d, J=7.7 Hz, 1 H), 6.87 (d, J=9.7 Hz,

1 H), 6.40 (d, J=9.7 Hz, 1 H), 4.90 (br. s., 1 H), 3.69 (ddd, J=11.2, 8.8, 5.0 Hz, 1 H), 3.42 54

- 3.46 (m, 1 H), 3.03 (t, J=9.0 Hz, 1 H), 2.82 (d, J=12.8 Hz, 1 H), 2.66 (dd, J=12.9, 2.1

Hz, 1 H), 2.37 - 2.46 (m, 1 H), 2.01 - 2.06 (m, 2 H), 1.37 (d, J=6.2 Hz, 4 H), 1.24 (s, 3 H)

13 ppm; C NMR (150 MHz, CD3OD)  206.94, 190.43, 183.60, 158.92, 145.91, 140.48,

139.89, 139.31, 134.29, 132.24, 120.03, 118.12, 115.44, 82.09, 78.80, 78.64, 77.76,

+ 77.67, 73.57, 72.49, 53.25, 44.73, 41.11, 30.17, 18.61 ppm; ESI-HRMS [M+Na] calculated for C25H26NaO10 509.1424, found 509.1438.

1.3.9 Synthesis of Analogue A

Compound 90

To a solution of tetracyclic aryliodide 43 (115 mg, 0.162 mmol) in 0.8 mL anhydrous pyridine, methanesulfonyl chloride (19 µL, 0.243 mmol) and 4- dimethylaminopyridine (2 mg, 0.0162 mmol) were added. The resulting mixture was stirred at room temperature for 24 h before pyridine was removed by air flow. The reaction mixture was diluted with CH2Cl2, washed sequentially with saturated CuSO4 solution, water, and brine. The organic layer was separated, dried over sodium sulfate, 55

filtered, and concentrated under reduce pressure to produce crude compound 88 which was directly used in the next step.

To a solution of crude compound 88 in 2 mL of EtOAc:MeOH (4:1, v:v), 10% palladium on carbon (172 mg, 0.162 mmol) was added. After the reaction mixture was evacuated and filled with hydrogen three times, it was stirred at room temperature under positive hydrogen pressure for 13 days. The reaction mixture was diluted with

CH2Cl2:MeOH (10:1, v:v), filtered through celite and concentrated under reduce pressure. The residue was purified by using preparative TLC in CH2Cl2:MeOH (15:1, v:v) to afford 48.8 mg (51% yield for 3 steps) of desired compound 89.

To a solution of compound 89 (48.8 mg, 0.101 mmol) in 2.2 mL DMF cooled at 0

º C was added Cs2CO3 (40 mg, 0.122 mmol). After the addition of benzyl bromide (18 µL,

0.152 mmol), the resulting mixture was stirred at room temperature for 6 h. The reaction mixture was quenched with a pinch of solid NaHCO3 and DMF was removed by air flow.

The residue was purified via preparative TLC (hexanes:ethyl acetate, 1:1, v:v) to give

23 º 40.5 mg (70% yield) of the desired compound 90. [훼]D = -75.0 (c = 0.1, CHCl3); FT-IR

(thin film): 3445, 2972, 2861, 1720, 1572, 1326, 1167, 1053, 926, 697 cm-1; 1H NMR

(600 MHz, CD3OD)  7.63 - 7.65 (m, 1 H), 7.57 - 7.60 (m, 2 H), 7.39 - 7.44 (m, 3 H),

7.32 - 7.36 (m, 1 H), 7.11 (d, J=7.3 Hz, 1 H), 5.21 - 5.26 (m, 2 H), 5.03 (dd, J=5.8, 1.4

Hz, 1 H), 3.77 (s, 3 H), 3.69 - 3.74 (m, 1 H), 3.68 (s, 3 H), 3.51 - 3.58 (m, 1 H), 3.18 (s, 3

H), 2.76 (d, J=12.7 Hz, 1 H), 2.56 (dd, J=12.7, 2.9 Hz, 1 H), 1.98 (dd, J=14.9, 2.9 Hz, 1

13 H), 1.88 (d, J=14.7 Hz, 1 H), 1.16 (s, 3 H) ppm; C NMR (150 MHz, CD3OD) 

207.23, 156.29, 152.24, 151.19, 138.41, 131.73, 129.52, 128.99, 128.90, 127.72, 123.00,

122.56, 116.46, 110.27, 82.08, 79.14, 79.05, 76.07, 72.46, 63.32, 62.05, 51.46, 42.98, 56

+ 38.31, 31.04, 30.47 ppm; ESI-HRMS [M+Na] calculated for C29H32NaO10S 595.1614, found 595.1637.

Analogue A

A solution of compound 90 (15 mg, 0.026 mmol) in 2.0 mL of CH3CN:CH2Cl2

(5:1, v:v) was cooled to 0 ºC. 104 µL of stock solution of cerium ammonium nitrate in water (0.079 mmol, 3 eq.) was added (Note: the stock solution was prepared by adding

174 mg of cerium ammonium nitrate in 400 µL water). The reaction mixture was stirred at 0 ºC for 1 h before being diluted with 2 mL ethyl acetate. 0.5 mL of ice cooled saturated NaHCO3 was added and the resulting mixture was stirred for 5 minutes. The organic layer was separated and passed through a small pad of Na2SO4, concentrated under reduce pressure, and kept in vacuum for 10 minutes to give the crude compound

91. This crude material was dissolved in 0.34 mL of mixed solvents (EtOAc:MeOH, 1:1, v:v) and 10% palladium on carbon (28 mg, 0.0262 mmol) was added. The mixture was evacuated and filled with hydrogen three times. After being stirring at room temperature under positive hydrogen pressure for 1.5 h, the reaction mixture was diluted with methanol, filtered through celite, and concentrated under reduced pressure. The residue was purified by preparative TLC in CH2Cl2:MeOH (20:1, v:v) to furnish 6.2 mg of

ퟐퟑ º compound 34 (66 % yield for 2 steps). [휶]퐃 = -48.0 (c = 0.1, CH3OH); FT-IR (thin film): 3369, 2976, 2930, 1725, 1635, 1456, 1086, 1045, 696 cm-1; 1H NMR (600 MHz,

CD3OD)  7.71 (dd, J=8.3, 7.5 Hz, 1 H), 7.58 (dd, J=7.5, 1.1 Hz, 1 H), 7.31 (dd, J=8.4,

0.9 Hz, 1 H), 6.87 (d, J=9.9 Hz, 1 H), 6.41 (d, J=9.7 Hz, 1 H), 2.84 (d, J=12.8 Hz, 1 H),

2.67 (dd, J=12.9, 2.3 Hz, 1 H), 2.03 - 2.05 (m, 2 H), 1.24 (s, 3 H) ppm; 13C NMR (150 57

MHz, CD3OD)  206.96, 189.47, 183.73, 162.69, 146.35, 140.37, 139.90, 138.00,

133.59, 125.29, 120.07, 118.22, 115.95, 82.10, 78.67, 77.72, 53.24, 44.72, 30.16 ppm;

+ ESI-HRMS [M+Na] calculated for C19H16NaO7 379.0794, found 379.0796.

1.3.10 Synthesis of D-fucal derived Glycal Stannane

Compound 93

To a flame-dried round-bottomed flask containing potassium tert-butoxide (2.4 g,

21.2 mmol) in 15 mL dry THF (dried with n-BuLi using 1,10-phenanthroline as an indicator) cooled at -78 ºC, was added 22 mL of 1.6 M n-BuLi (35.4 mmol). To this mixture was added a solution of 3,4-di-O-tert-butyldimethylsilyl-D-fucal (4.23 g, 11.8 mmol) in 8.5 mL dry THF and the resulting mixture was stirred at -78 ºC for 1 h. Tri-n- butyltin chloride (9.5 mL, 35.4 mmol) was then added, and the resulting mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was quenched with saturated NaHCO3 and extracted with ethyl acetate. The combined organic fractions were washed with water and brine, dried over sodium sulfate, and concentrated under reduce pressure. The residue was then passed through a small pad of silica using hexanes as the

eluent, and the organic fractions were concentrated to afford crude glycal stannane 93 which was used directly in the next step.

To a solution of crude glycal stannane 93 in 39 mL THF cooled at 0 ºC was added

1.0 M tetra-n-butyl ammonium fluoride (35.4 mL, 35.4 mmol) and the resulting mixture was stirred at room temperature for 10 h. Saturated aqueous NaHCO3 solution was added and THF was removed under reduce pressure. The aqueous layer was extracted with ethyl acetate and combined organic extracts were washed sequentially with water and brine, dried over sodium sulfate, and concentrated. The crude residue was purified by silica gel flash column chromatography (hexanes:ethyl acetate = 10:1 to 4:1, with 1% Et3N) to provide 2.13 g (43% yield for 2 steps) of diol 94.

To a solution of diol 94 (1.13 g, 2.69 mmol) in 2.7 mL DMF were added Et3N

(1.87 mL, 13.5 mmol) and tert-butyldimethylsilyl chloride (0.44 g, 2.96 mmol). The resulting mixture was stirred at room temperature for 10 h before being quenched with water. The mixture was extracted with ethyl acetate, and combined organic extracts were washed with water and brine, dried over sodium sulfate, and concentrated under reduce pressure. The crude residue was purified by silica gel flash chromatography

(hexanes:ethyl acetate, 10:1, with 1% Et3N) to afford 1.33 g (93% yield) of glycal stannane 95.

To a solution of 95 (1.33 g, 2.49 mmol) in 8.3 mL DMF cooled at 0 ºC was added sodium hydride (0.2 g, 4.98 mmol) and the mixture was stirred at 0 ºC for 45 minutes.

Benzyl bromide (0.36 mL, 2.99 mmol) was then added and the resulting mixture was warmed up to room temperature and stirred for 30 h before being quenched with water.

The aqueous mixture was extracted with ethyl acetate and combined organic extracts 59

were washed with water, dried over sodium sulfate, filtered, and concentrated in vacuo.

Purification on silica gel flash column chromatography (hexanes:dichloromethane = 40:1,

ퟐퟑ with 1% Et3N) provided 1.1 g (71% yield) of corresponding glycal stannane 55. [휶]퐃 = -

º 60.7 (c = 0.1, CHCl3); FT-IR (thin film): 3071, 2953, 2925, 2856, 2674, 2559, 1677,

-1 1 1288, 1072, 702 cm ; H NMR (600 MHz, CDCl3)  7.36 - 7.40 (m, 2 H), 7.29 - 7.33

(m, 2 H), 7.23 - 7.26 (m, 1 H), 4.97 (d, J=12.1 Hz, 1 H), 4.66 (dd, J=2.8, 1.3 Hz, 1 H),

4.61 (d, J=11.9 Hz, 1 H), 4.46 - 4.51 (m, 1 H), 3.99 - 4.05 (m, 1 H), 3.51 (dt, J=3.7, 2.0

Hz, 1 H), 1.48 - 1.55 (m, 6 H), 1.31 (dq, J=14.8, 7.4 Hz, 6 H), 1.23 (d, J=6.8 Hz, 3 H),

13 0.86 - 0.94 (m, 24 H), 0.11 (d, J=4.2 Hz, 6 H) ppm; C NMR (150 MHz, CDCl3) 

163.09, 139.66, 128.39, 128.02, 127.50, 113.71, 76.05, 73.34, 73.00, 29.27, 27.55, 26.27,

18.60, 16.79, 14.07, 10.02, -4.02, -4.32 ppm; ESI-HRMS [M+H]+ Calculated for

C31H57O3SiSn 625.3099, found 625.3113.

1.3.11 Synthesis of Analogue B

Compound 56

Glycal stannane 55 (1.32 g, 2.11 mmol), aryl iodide 43 (0.50 g, 0.70 mmol) and

Pd(PPh3)2Cl2 (0.1 g, 0.141 mmol) were taken into a flame dried flask, evacuated, and filled with argon. 7 mL of degassed dry toluene was added to the flask and the resulting mixture was stirred at 100 ºC for 8 h. The reaction mixture was cooled to room temperature and directly subjected to purification via silica gel flash column chromatography (hexanes:ethyl acetate = 10:1, 2% Et3N) to affording 452 mg (70% yield) of the C1-arylated glycal 96.

To a solution of C1-arylated glycal 96 (424 mg, 0.463 mmol) in 2.3 mL pyridine were added methanesulfonyl chloride (54 µL, 0.70 mmol) and 4-dimethylaminopyridine

(5.6 mg, 0.046 mmol). After the resulting mixture was stirred at room temperature for 36 h, pyridine was removed by air flow. The residue was diluted with CH2Cl2, washed sequentially with aqueous saturated CuSO4 solution, water, and brine. The organic solution was dried over sodium sulfate, filtered, and concentrated under reduce pressure to produce the crude mesylate 97 which was used directly in the next step.

The mesylate 97 was dissolved in 8.8 mL ethanol and a pinch of bromocresol green was added as an indicator. To the mixture was added sodium cyanoborohydride

(0.056 g, 0.89 mmol) followed by addition of 0.5 M HCl in methanol (3.6 mL, 1.8 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes. A second batch of sodium cyanoborohydride (0.056 g, 0.886 mmol) and 0.5 M HCl in methanol (3.6 mL, 1.772 mmol) was added, and the reaction mixture was stirred at room temperature for 30 h before being quenched with saturated NaHCO3 solution. The aqueous mixture was extracted with ethyl acetate and combined organic extracts were washed with water, dried over sodium sulfate, filtered, and concentrated under reduce pressure. Purification on silica gel flash column chromatography (toluene:ethyl acetate =

20:1 to 5:1) furnished 310 mg (70% yield for 2 steps) of 2-deoxy β-C-glycoside 56.

ퟐퟑ º [휶]퐃 = -26.0 (c = 0.1, CHCl3); FT-IR (thin film): 3407, 2934, 2883, 1682, 1453, 1326,

-1 1 1027, 698 cm ; H NMR (600 MHz, MHz, CDCl3)  7.88 (d, J=8.8 Hz, 1 H), 7.74 (d,

J=8.8 Hz, 1 H), 7.31 - 7.50 (m, 15 H), 5.04 - 5.09 (m, 2 H), 5.02 (s, 1 H), 4.81 - 4.92 (m,

3 H), 4.68 - 4.72 (m, 2 H), 4.41 (d, J=9.5 Hz, 1 H), 4.34 (s, 1 H), 3.96 (d, J=19.6 Hz, 1

H), 3.84 (s, 3 H), 3.80 (d, J=3.5 Hz, 1 H), 3.72 (s, 3 H), 3.57 (d, J=6.6 Hz, 1 H), 3.54 (d,

J=3.1 Hz, 1 H), 3.38 (dd, J=19.8, 5.9 Hz, 1 H), 3.18 (dd, J=13.4, 2.9 Hz, 1 H), 3.13 (s, 3

H), 2.75 (d, J=13.4 Hz, 1 H), 2.15 (dd, J=14.9, 2.9 Hz, 1 H), 1.90 - 2.00 (m, 2 H), 1.81 - 62

13 1.88 (m, 2 H), 1.38 (d, J=6.4 Hz, 3 H), 1.32 (s, 3 H) ppm; C NMR (150 MHz, CDCl3)

 205.48, 150.72, 150.59, 150.52, 138.77, 137.86, 137.06, 134.04, 130.29, 129.03,

128.96, 128.88, 128.76, 128.73, 128.57, 128.41, 128.33, 128.18, 127.27, 125.56, 123.86,

121.72, 119.87, 81.52, 80.98, 79.55, 78.72, 78.48, 76.30, 75.40, 72.57, 70.78, 65.02,

63.55, 61.76, 44.69, 43.38, 38.87, 37.29, 30.39, 25.74, 18.34 ppm; ESI-HRMS [M+Na]+ calculated for C49H54NaO13S 905.3183, found 905.3214.

Compound 99

To a solution of 2-deoxy β-C-glycoside 56 (50 mg, 0.057 mmol) in 1.6 mL of mixed solvents (EtOAc:MeOH, 1:1, v:v) was added 10% palladium on carbon (60 mg,

0.057 mmol). The reaction mixture was evacuated and filled with hydrogen for five times and then stirred at room temperature under positive hydrogen pressure (40 psi) for 50 h.

The reaction mixture was then diluted with CH2Cl2:MeOH (10:1, v:v), filtered through celite and concentrated. The residue was purified via preparative TLC (CH2Cl2:MeOH,

10:1, v:v) to afford 31 mg (89% yield) of compound 98.

To a solution of compound 98 (29.1 mg, 0.0476 mmol) in 1 mL DMF cooled at 0

º C were added Cs2CO3 (18.6 mg, 0.0571 mmol) and benzyl bromide (8.5 µL, 0.071 mmol). The reaction mixture was stirred at room temperature for 5 h and then quenched with a pinch of solid ammonium chloride. DMF was removed by air flow and the residue was purified by using preparative TLC in CH2Cl2:MeOH (10:1, v:v) to furnish 21.7 mg

ퟐퟑ º (65% yield) of the desired product 99. [휶]퐃 = -100.0 (c = 0.1, CHCl3); FT-IR (thin film): 3407, 2934, 2978, 1716, 1331, 1166, 1041, 905, 530 cm-1; 1H NMR (600 MHz,

CDCl3)  7.85 (d, J=8.8 Hz, 1 H), 7.67 (d, J=8.8 Hz, 1 H), 7.38 - 7.48 (m, 5 H), 5.23 (s, 1 63

H), 5.08 - 5.14 (m, 2 H), 4.78 - 4.84 (m, 2 H), 4.13 - 4.18 (m, 2 H), 3.97 (d, J=19.4 Hz, 1

H), 3.77 - 3.83 (m, 1 H), 3.74 - 3.76 (m, 6 H), 3.63 - 3.67 (m, 1 H), 3.56 (d, J=6.8 Hz, 1

H), 3.39 (dd, J=19.8, 5.9 Hz, 1 H), 3.16 (s, 3 H), 2.77 - 2.83 (m, 2 H), 2.29 (d, J=7.9 Hz,

1 H), 2.09 - 2.14 (m, 1 H), 2.00 - 2.06 (m, 2 H), 1.83 (dd, J=14.7, 2.0 Hz, 1 H), 1.71 (q,

J=12.7 Hz, 1 H), 1.34 (d, J=6.4 Hz, 3 H), 1.23 (s, 3 H) ppm; 13C NMR (150 MHz,

CDCl3)  206.11, 150.70, 150.57, 150.52, 137.75, 133.89, 130.31, 128.97, 128.62,

128.53, 126.66, 125.23, 123.93, 121.42, 119.98, 80.01, 78.53, 77.72, 77.08, 75.06, 74.83,

72.91, 71.20, 70.37, 63.32, 61.84, 50.44, 41.85, 39.07, 36.09, 30.70, 29.85, 17.66 ppm;

+ ESI-HRMS [M+Na] calculated for C35H42NaO13S 725.2244, found 725.2236.

Analogue B

A solution of compound 99 (17 mg, 0.024 mmol) in 1.5 mL of acetonitrile was cooled at 0 ºC. 96 µL of stock solution of cerium ammonium nitrate in water (0.073 mmol, 3 eq.) was added (Note: the stock solution was prepared by adding 174 mg of cerium ammonium nitrate in 400 µL water). The reaction mixture was stirred at 0 ºC for

35 min before being diluted with 2 mL ethyl acetate. 0.5 mL of ice cooled saturated

NaHCO3 was added and the resulting mixture was stirred for 5 minutes. The organic layer was separated and passed through a small pad of Na2SO4, concentrated under reduce pressure, and kept in vacuum for 10 minutes to give the crude compound 100.

This crude material was dissolved in 0.3 mL of mixed solvents (EtOAc:MeOH, 1:1, v:v) and 10% palladium on carbon (5.1 mg, 0.0048 mmol) was added. The mixture was evacuated and filled with hydrogen three times. After being stirring at room temperature under positive hydrogen pressure for 1 h, the reaction mixture was diluted with methanol, 64

filtered through celite, and concentrated under reduced pressure to furnish compound

101. The crude 101 was dissolved in 0.93 mL of dioxane and N,N-diisopropylethylamine

(8.5 µL, 0.048 mmol) was added. After being stirred at 40 ºC for 1 h, the reaction mixture was cooled down. Dioxane was removed by air flow and the residue was purified by preparative TLC in CH2Cl2:MeOH (10:1, v:v) to furnish 8.1 mg of Analogue B (69%

ퟐퟑ º yield for 3 steps). [휶]퐃 = 42.3 (c = 0.1, CH3OH); FT-IR (thin film): 3384, 2961, 2923,

-1 1 2853, 1725, 1637, 1284, 1259, 1080, 652 cm ; H NMR (600 MHz, CD3OD)  7.99 (d,

J=7.7 Hz, 1 H), 7.59 (d, J=7.9 Hz, 1 H), 6.87 (d, J=9.7 Hz, 1 H), 6.40 (d, J=9.7 Hz, 1 H),

4.84 - 4.87 (m, 1 H), 3.86 - 3.90 (m, 1 H), 3.70 - 3.75 (m, 1 H), 3.61 (d, J=2.8 Hz, 1 H),

2.82 (d, J=12.8 Hz, 1 H), 2.66 (dd, J=13.0, 2.4 Hz, 1 H), 2.06 - 2.10 (m, 1 H), 2.02 - 2.05

(m, 2 H), 1.61 (q, J=11.9 Hz, 1 H), 1.33 (d, J=6.4 Hz, 3 H), 1.24 (s, 3 H) ppm; 13C NMR

(150 MHz, CD3OD)  206.94, 189.88, 183.63, 158.77, 146.24, 140.48, 139.88, 139.67,

134.93, 132.16, 120.02, 118.22, 115.36, 82.10, 78.65, 77.71, 76.11, 72.76, 71.84, 71.10,

53.26, 44.73, 35.35, 30.17, 17.71 ppm; ESI-HRMS [M+Na]+ calculated for

C25H26NaO10 509.1424, found 509.1424.

1.3.12 Synthesis of L-Olivose derived Thioglycoside Donor

Compound 51

To S-but-3-ynyl 3,4-di-O-acetyl-L-olivoside donor 10244 (150 mg, 0.50 mmol) was added 1.1 mL of 7.0 N NH3 in MeOH and the resulting mixture was stirred at room 65

temperature for 6 h. Solvent was removed under reduced pressure and the residue was azeotroped with toluene to produce crude diol 103. This crude 103 was dissolved in 1.7 mL DMF and cooled at 0 ºC. NaH (60% in mineral oil, 60 mg, 1.5 mmol) was added and the reaction mixture was stirred for 45 min at 0 ºC. Next, benzyl bromide (0.15 mL, 1.25 mmol) was added and the resulting mixture was stirred for 12 h at room temperature before being quenched with water. The aqueous mixture was extracted with ethyl acetate and combined organic extracts were washed with water, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification on flash column chromatography

(hexanes:ethyl acetate, 10:1, v:v) provided 189 mg of corresponding S-but-3-ynyl 3,4-di-

ퟐퟑ º O-benzyl-L-olivoside donor 51 (96% yield for 2 steps). [휶]퐃 = -82.3 (c = 0.3, CHCl3);

FT-IR (thin film): 3289, 3029, 2929, 2858, 1496, 1453, 1091, 734, 697 cm-1; 1H NMR

1 (600 MHz, CDCl3)  7.29 - 7.40 (ovrlp, 10 H, α and β), 5.41 (d, J=5.5 Hz, 1 H, H α),

4.98 (ovrlp, 1 H, α and β), 4.57 - 4.73 (ovrlp, 3 H, α and β and 1H, H3 β ), 4.13 (dq,

J=9.4, 6.2 Hz, 1 H, H5 α), 3.91 (ddd, J=11.6, 8.6, 4.8 Hz, 1 H, H3 α), 3.66 (ddd, J=11.2,

8.6, 5.1 Hz, 1 H, H1 β), 3.39 (dq, J=9.3, 6.1 Hz, 1 H, H5 β), 3.17 (ovrlp, 1 H, H4 α and H4

β), 2.91 - 2.97 (m, 1 H, β), 2.77 - 2.86 (m, 2 H, α), 2.67 - 2.73 (m, 1 H, β), 2.47 - 2.64

(ovrlp, 2 H, α and β), 2.41 (ddd, J=12.7, 5.1, 1.7 Hz, 1 H, H2 β), 2.31 - 2.36 (m, 1 H, H2

α), 2.02 - 2.09 (ovrlp, 1 H, α and β and 1 H, H2 α), 1.71 - 1.79 (q, 1 H, H2 β), 1.37 (d,

6 6 13 J=6.1 Hz, 3 H, C – CH3 β), 1.33 (d, J=6.2 Hz, 3 H, C – CH3 α) ppm; C NMR (150

MHz, CDCl3)  138.72, 138.64, 138.60, 138.47, 128.69, 128.66, 128.64, 128.61, 128.33,

128.21, 128.00, 127.95, 127.94, 127.90, 84.61, 83.65, 82.86, 80.73, 80.56, 79.88, 75.90,

75.61, 75.41, 72.04, 71.72, 69.75, 69.72, 67.93, 37.38, 36.30, 30.13, 29.96, 20.69, 20.16,

+ 18.66, 18.31 ppm; ESI-HRMS [M+Na] calculated for C24H28NaO3S 419.1657, found

419.1667.

1.3.13 Synthesis of Analogue C

Compound 93

A mixture of S-but-3-ynyl 3,4-di-O-benzyl-L-olivoside donor 51 (33.6 mg, 0.085 mmol) and partially protected β-C-arylglycoside acceptor 45 (50 mg, 0.057 mmol) was azeotroped with 2 mL benzene and kept in high vacuum for 30 minutes. To this mixture were sequentially added 23 mg of freshly activated 4 Ǻ molecular sieves, silver triflate

(1.5 mg, 0.0057 mmol) and a freshly prepared solution of gold catalyst in dry CH2Cl2 67

(prepared by dissolving 2.0 mg (0.0028 mmol) of chloro[tris(para- trifluoromethylphenyl)phosphine]gold(I) in 0.56 mL CH2Cl2 ). The resulting mixture was stirred at room temperature for 1 h before being quenched with a pinch of solid NaHCO3.

The reaction mixture was diluted with CH2Cl2, filtered through a small pad of Na2SO4, concentrated under vacuo, and purified using preparative TLC (hexanes:ethyl acetate,

2:1, v:v, with 1% MeOH) to afford 61.5 mg (91% yield) of mixture of inseparable anomers 52.

To the compound 52 (50 mg, 0.042 mmol) dissolved in 1.6 mL of EtOAc:MeOH

(1:1, v:v) was added 10% palladium on carbon (45 mg, 0.042 mmol). The resulting mixture was evacuated and filled with hydrogen for five times and stirred at room temperature under positive hydrogen pressure (40 psi) for 40 h. The reaction mixture was diluted with CH2Cl2:MeOH (10:1, v:v), filtered through celite, concentrated, and purified via preparative TLC (CH2Cl2:MeOH, 10:1, v:v) to give 11.2 mg (36% yield) of desired α- disaccharide 104 (α:β, 2:1).

To a solution of 104 (15.5 mg, 0.021 mmol) in 0.45 mL DMF cooled at 0 ºC was added Cs2CO3 (8.2 mg, 0.025 mmol) followed by addition of 22 µL stock solution of benzyl bromide in DMF (0.031 mmol, 1.5 eq.) (Note: the stock solution was prepared by adding 40 µL of benzyl bromide in 200 µL DMF). The reaction mixture was stirred at room temperature for 7 h and quenched with a pinch of solid ammonium chloride. DMF was removed by air flow and the residue was purified by using preparative TLC in

CH2Cl2:MeOH (10:1, v:v) to furnish 11.1 mg (63% yield) of the desired product 105.

ퟐퟑ º [휶]퐃 = -69.3 (c = 0.1, CHCl3); FT-IR (thin film): 3391, 2923, 2856, 1721, 1330, 1041,

-1 1 973, 908, 529 cm ; H NMR (600 MHz, CD3OD)  7.88 (d, J=8.8 Hz, 1 H), 7.62 (d, 68

J=8.8 Hz, 1 H), 7.47 - 7.50 (m, 2 H), 7.41 - 7.45 (m, 2 H), 7.35 - 7.39 (m, 1 H), 5.04 -

5.08 (m, 2 H), 4.99 (d, J=3.1 Hz, 1 H), 4.89 - 4.92 (m, 2 H), 3.90 - 3.95 (m, 1 H), 3.82 -

3.87 (m, 2 H), 3.79 (s, 3 H), 3.77 (s, 3 H), 3.56 - 3.66 (m, 2 H), 3.27 - 3.30 (m, 1 H), 3.18

- 3.21 (m, 3 H), 3.13 (t, J=9.0 Hz, 1 H), 2.97 (t, J=9.2 Hz, 1 H), 2.80 (d, J=12.7 Hz, 1 H),

2.59 (dd, J=12.7, 2.8 Hz, 1 H), 2.22 (dd, J=12.6, 3.6 Hz, 1 H), 1.93 - 2.04 (m, 3 H), 1.55 -

1.65 (m, 2 H), 1.31 (d, J=6.1 Hz, 3 H), 1.28 (d, J=6.2 Hz, 3 H), 1.20 (s, 3 H) ppm; 13C

NMR (150 MHz, CD3OD)  207.17, 151.68, 151.52, 151.37, 138.92, 134.78, 131.26,

129.72, 129.67, 129.66, 129.25, 128.92, 126.15, 124.59, 123.52, 120.45, 95.16, 82.07,

79.25, 79.17, 79.08, 79.03, 78.08, 77.89, 76.66, 76.10, 73.10, 69.44, 69.34, 63.60, 62.05,

51.52, 43.04, 39.39, 38.31, 37.98, 30.97, 30.49, 18.81, 18.24 ppm; ESI-HRMS [M+Na]+ calculated for C41H52NaO16S 855.2874, found 855.2869

Analogue C

To a solution of 105 (10 mg, 0.012 mmol) in 0.76 mL acetonitrile cooled at 0 ºC was added 48 µL of stock solution of cerium ammonium nitrate in water (0.036 mmol, 3 eq.) was added (Note: the stock solution was prepared by adding 174 mg of cerium ammonium nitrate in 400 µL water). The reaction mixture was stirred at 0 ºC for 30 min before being diluted with 2 mL ethyl acetate. 0.5 mL of ice cooled saturated NaHCO3 was added and the resulting mixture was stirred for 5 minutes. The organic layer was separated and passed through a small pad of Na2SO4, concentrated under reduce pressure, and kept in vacuum for 10 minutes to give the crude compound 106. This crude material was dissolved in 0.15 mL of mixed solvents (EtOAc:MeOH, 1:1, v:v) and 10% palladium on carbon (2.6 mg, 0.0024 mmol) was added. The mixture was evacuated and filled with 69

hydrogen for three times. After being stirring at room temperature under positive hydrogen pressure for 1 h, the reaction mixture was diluted with methanol, filtered through celite, and concentrated under reduced pressure. The residue was purified through preparative TLC in CH2Cl2:MeOH (10:1, v:v) to afford 6.2 mg of analogue C

ퟐퟑ º (84% yield for 2 steps). [휶]퐃 = 99.3 (c = 0.1, CH3OH); FT-IR (thin film): 3380, 2958,

-1 1 2921, 2854, 1725, 1636, 1260, 1055, 476 cm ; H NMR (600 MHz, CD3OD)  7.86 (d,

J=7.9 Hz, 1 H), 7.58 (d, J=7.9 Hz, 1 H), 6.87 (d, J=9.7 Hz, 1 H), 6.40 (d, J=9.7 Hz, 1 H),

5.04 (d, J=3.1 Hz, 1 H), 4.85 - 4.87 (m, 1 H), 3.91 - 3.97 (m, 1 H), 3.85 (ddd, J=11.6, 9.0,

5.0 Hz, 1 H), 3.73 - 3.79 (m, 1 H), 3.45 - 3.51 (m, 1 H), 3.11 - 3.18 (m, 1 H), 2.96 (t,

J=9.3 Hz, 1 H), 2.82 (d, J=12.8 Hz, 1 H), 2.67 (dd, J=13.0, 2.6 Hz, 1 H), 2.56 (ddd,

J=12.7, 4.8, 1.7 Hz, 1 H), 1.95 - 2.08 (m, 3 H), 1.61 - 1.67 (m, 1 H), 1.38 (d, J=6.1 Hz, 3

13 H), 1.27 (d, J=6.1 Hz, 3 H), 1.24 (s, 3 H) ppm; C NMR (150 MHz, CD3OD)  206.91,

189.82, 183.58, 158.75, 146.29, 140.46, 139.89, 139.13, 134.37, 132.26, 120.02, 118.19,

115.43, 95.33, 82.09, 79.00, 78.64, 77.83, 77.79, 77.68, 76.65, 72.32, 69.42, 69.39, 53.26,

44.72, 39.40, 37.55, 30.76, 30.17, 18.79, 18.16 ppm; ESI-HRMS [M+Na]+ calculated for C31H36NaO13 639.2054, found 639.2084.

1.3.14 Synthesis of L-Oliose derived Thioglycoside Donor

Compound 53

To S-but-3-ynyl 3,4-di-O-acetyl-L-olioside donor 107 (120 mg, 0.40 mmol) was added 0.86 mL of 7.0 N NH3 in MeOH and the resulting mixture was stirred at room temperature for 12 h. Solvent was removed under reduced pressure and the residue was azeotroped with toluene to produce crude diol 108. This crude 108 was dissolved in 1.3 mL DMF and cooled at 0 ºC. NaH (60% in mineral oil, 48 mg, 1.2 mmol) was added and the reaction mixture was stirred for 45 min at 0 ºC. Next, benzyl bromide (0.12 mL, 1.0 mmol) was added and the resulting mixture was stirred for 12 h at room temperature before being quenched with water. The aqueous mixture was extracted with ethyl acetate and combined organic extracts were washed with water, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification on flash column chromatography

(hexanes:ethyl acetate, 10:1, v:v) provided 139 mg of corresponding S-but-3-ynyl 3,4-di-

ퟐퟐ º O-benzyl-L-olioside donor 53 (88% yield for 2 steps). [휶]퐃 = -76.5 (c = 0.3, CHCl3);

FT-IR (thin film): 3291, 3030, 2931, 1496, 1454, 1362, 1059, 733, 697 cm-1; 1H NMR

1 (600 MHz, CDCl3)  7.29 - 7.45 (ovrlp, 10 H, α and β), 5.55 (d, J=5.7 Hz, 1 H, H α),

4.97 - 5.04 (ovrlp, 1 H, α and β), 4.70 - 4.77 (ovrlp, 1 H, α and β), 4.54 - 4.69 (ovrlp, 2 H,

α and β and 1H, H1 β), 4.17 (q, J=6.5 Hz, 1 H, H5 α), 3.88 (ddd, J=12.2, 4.4, 2.5 Hz, 1 H,

H3 α), 3.64 (s, 1 H, H4 α), 3.60 (ddd, J=11.6, 4.6, 2.6 Hz, 1 H, H3 β), 3.55 - 3.58 (m, 1 H,

H4 β), 3.41 - 3.48 (m, 1 H, H5 β), 2.96 (ddd, J=13.3, 8.5, 6.6 Hz, 1 H, β), 2.77 - 2.86

(ovrlp, 1 H, α and β), 2.66 - 2.74 (m, 1 H, α), 2.50 - 2.62 (ovrlp, 2 H, α and β and 1 H, H2

α ), 2.21 (q, J=11.8 Hz, 1 H, H2 β), 2.07 - 2.14 (m, 1 H, H2 β), 2.04 - 2.06 (ovrlp, 1 H, α

2 6 and β), 2.01 - 2.04 (m, 1 H, H α), 1.25 (d, J=6.4 Hz, 3 H, C – CH3 β), 1.22 (d, J=6.4 Hz,

6 13 3 H, C – CH3 α) ppm; C NMR (150 MHz, CDCl3)  138.86, 138.77, 138.38, 128.52, 71

128.49, 128.43, 128.42, 128.39, 128.35, 128.24, 128.21, 128.16, 127.70, 127.66, 127.59,

127.47, 127.36, 127.31, 82.95, 82.77, 81.15, 80.13, 78.87, 75.91, 75.01, 74.47, 74.46,

74.25, 70.48 , 70.19, 69.37, 69.27, 67.20, 32.05, 30.93, 29.95, 29.44, 20.42, 19.93, 17.65,

+ 17.21 ppm; ESI-HRMS [M+Na] calculated for C24H28NaO3S 419.1657, found

419.1655.

1.3.15 Synthesis of Analogue D

Compound 110

A mixture of S-but-3-ynyl 3,4-di-O-benzyl-L-olioside donor 53 (33.6 mg, 0.085 mmol) and partially protected β-C-arylglycoside acceptor 45 (50 mg, 0.057 mmol) was

azeotroped with 2 mL benzene and kept in high vacuum for 30 minutes. To this mixture were sequentially added 23 mg of freshly activated 4 Ǻ molecular sieves, silver triflate

(1.5 mg, 0.0057 mmol) and a freshly prepared solution of gold catalyst in dry CH2Cl2

NaHCO3. The reaction mixture was diluted with CH2Cl2, filtered through a small pad of

Na2SO4, concentrated under vacuo, and purified using preparative TLC (hexanes:ethyl acetate, 2:1, v:v, with 1% MeOH) to afford 56.6 mg (84% yield) of the mixture of inseparable anomers 54.

To 54 (50 mg, 0.042 mmol) dissolved in 1.6 mL of EtOAc:MeOH (1:1, v:v) was added 10% palladium on carbon (45 mg, 0.042 mmol). The resulting mixture was evacuated and filled with hydrogen for five times and stirred at room temperature under positive hydrogen pressure (40 psi) for 66 h. The reaction mixture was diluted with

CH2Cl2:MeOH (10:1, v:v), filtered through celite, concentrated, and purified via preparative TLC (CH2Cl2:MeOH, 10:1, v:v) to give 17.4 mg (56% yield) of desired α- disaccharide 109 (α:β, 2:1).

To a solution of 109 (17.3 mg, 0.023 mmol) in 0.5 mL DMF cooled at 0 ºC was added Cs2CO3 (9.1 mg, 0.028 mmol) followed by addition of 25 µL stock solution of benzyl bromide in DMF (0.031 mmol, 1.5 eq.) (Note: the stock solution was prepared by adding 40 µL of benzyl bromide in 200 µL DMF). The reaction mixture was stirred at room temperature for 5 h and quenched with a pinch of solid ammonium chloride. DMF

was removed by air flow and the residue was purified by using preparative TLC in

CH2Cl2:MeOH (10:1, v:v) to furnish 11 mg (57% yield) of the desired product 110.

ퟐퟑ º [휶]퐃 = -89.7 (c = 0.1, CHCl3); FT-IR (thin film): 3393, 2976, 2938, 1718, 1329, 1167,

-1 1 1040, 699, 639, 529 cm ; H NMR (600 MHz, CD3OD)  7.87 (d, J=9.0 Hz, 1 H), 7.61

(d, J=8.8 Hz, 1 H), 7.47 (d, J=7.2 Hz, 2 H), 7.40 - 7.45 (m, 2 H), 7.37 (d, J=7.3 Hz, 1 H),

5.05 (dd, J=16.0, 4.2 Hz, 3 H), 4.90 (d, J=4.6 Hz, 2 H), 4.20 (d, J=6.8 Hz, 1 H), 4.04 (dd,

J=12.1, 1.8 Hz, 1 H), 3.84 (d, J=19.1 Hz, 1 H), 3.78 (s, 3 H), 3.76 (s, 3 H), 3.63 - 3.66

(m, 1 H), 3.55 - 3.61 (m, 2 H), 3.27 - 3.30 (m, 1 H), 3.19 (s, 3 H), 3.10 - 3.15 (m, 1 H),

2.79 (d, J=12.7 Hz, 1 H), 2.59 (dd, J=12.7, 2.8 Hz, 1 H), 2.23 (dd, J=12.38, 3.6 Hz, 1 H),

2.00 - 2.04 (m, 1 H), 1.92 - 1.96 (m, 1 H), 1.91 (d, J=3.7 Hz, 1 H), 1.67 (dd, J=12.7, 5.0

Hz, 1 H), 1.57 (q, J=12.5 Hz, 1 H), 1.30 (d, J=6.1 Hz, 3 H), 1.25 (d, J=6.6 Hz, 3 H), 1.18

13 (s, 3 H) ppm; C NMR (150 MHz, CD3OD)  207.15, 151.65, 151.50, 151.35, 138.87,

134.77, 131.22, 129.66, 129.64, 129.25, 128.90 , 126.16 , 124.58 , 123.51 , 120.44 , 95.34

, 82.04 , 79.25, 79.13 , 79.06 , 77.84 , 77.77, 76.69, 76.08 , 73.06, 72.40 , 71.52 , 67.67,

66.70 , 63.60 , 62.07 , 51.50 , 43.01, 38.32 , 37.94, 33.47, 30.96 , 30.49, 18.82, 17.26

+ ppm; ESI-HRMS [M+Na] calculated for C41H52NaO16S 855.2874, found 855.2867.

Analogue D

To a solution of 110 (15.2 mg, 0.0183 mmol) in 1.2 mL acetonitrile cooled at 0 ºC was added 73 µL of stock solution of cerium ammonium nitrate in water (0.055 mmol, 3 eq.) was added (Note: the stock solution was prepared by adding 174 mg of cerium ammonium nitrate in 400 µL water). The reaction mixture was stirred at 0 ºC for 30 min before being diluted with 2 mL ethyl acetate. 0.5 mL of ice cooled saturated NaHCO3 74

was added and the resulting mixture was stirred for 5 minutes. The organic layer was separated and passed through a small pad of Na2SO4, concentrated under reduce pressure, and kept in vacuum for 10 minutes to give the crude compound 111. This crude material was dissolved in 0.23 mL of mixed solvents (EtOAc:MeOH, 1:1, v:v) and 10% palladium on carbon (3.9 mg, 0.0037 mmol) was added. The mixture was evacuated and filled with hydrogen for three times. After being stirring at room temperature under positive hydrogen pressure for 1 h, the reaction mixture was diluted with methanol, filtered through celite, and concentrated under reduced pressure. The residue was purified through preparative TLC in CH2Cl2:MeOH (10:1, v:v) to afford 8.4 mg of compound 7

ퟐퟑ º (75% yield for 2 steps). [휶]퐃 = 41.3 (c = 0.1, CH3OH); FT-IR (thin film): 3389, 2974,

-1 1 2927, 1723, 1637, 1436, 1284, 1082, 1056, 598 cm ; H NMR (600 MHz, CD3OD) 

7.85 (d, J=7.7 Hz, 1 H), 7.57 (d, J=7.9 Hz, 1 H), 6.87 (d, J=9.9 Hz, 1 H), 6.40 (d, J=9.7

Hz, 1 H), 5.07 (d, J=3.3 Hz, 1 H), 4.85 (s, 1 H), 4.21 (q, J=6.5 Hz, 1 H), 4.01 - 4.05 (m, 1

H), 3.72 - 3.79 (m, 1 H), 3.55 - 3.57 (m, 1 H), 3.45 - 3.51 (m, 1 H), 3.10 - 3.16 (m, 1 H),

2.80 - 2.88 (m, 1 H), 2.67 (dd, J=12.9, 2.7 Hz, 1 H), 2.55 (ddd, J=12.7, 4.8, 1.7 Hz, 1 H),

2.00 - 2.08 (m, 2 H), 1.93 (td, J=12.5, 3.9 Hz, 1 H), 1.68 (dd, J=12.7, 5 Hz, 1 H), 1.38 (d,

13 J=6.1 Hz, 3 H), 1.23 - 1.25 (m, 7 H) ppm; C NMR (150 MHz, CD3OD)  206.91,

189.82, 183.57, 158.74, 146.29, 140.45, 139.88, 139.14, 134.35, 132.23, 120.03, 118.19,

115.41, 95.50, 82.08, 78.62, 77.76, 77.69, 77.50, 76.70, 72.43, 72.32, 67.75, 66.70, 53.26,

44.71, 37.49, 33.48, 30.18, 18.81, 17.18 ppm; ESI-HRMS [M+Na]+ calculated for

C31H36NaO13 639.2054, found 639.2068.

1.4 NMR Spectra for Selected Compounds

1H NMR for Aquayamycin

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)

13C NMR for Aquayamycin

200 180 160 140 120 100 80 60 40 20 Chemical Shift (ppm)

COSY for Aquayamycin

6 F1 Chemical Shift (ppm) Shift Chemical F1 7

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

HSQC for Aquayamycin

100

120 (ppm) Shift Chemical F1

140

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

1H NMR for Compound 88

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13C NMR for Compound 88

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

1H NMR for Analogue A

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)

13C NMR for Analogue A

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 Chemical Shift (ppm)

COSY for Analogue A

6 F1 Chemical Shift (ppm) Shift Chemical F1

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shift (ppm)

HSQC for Analogue A

100

150 F1 Chemical Shift (ppm) Shift Chemical F1

200

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

1H NMR for Compound 55

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13C NMR for Compound 55

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Chemical Shift (ppm)

1H NMR for Compound 56

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13C NMR for Compound 56

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

1H NMR for Compound 99

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13C NMR for Compound 99

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

1H NMR for Analogue B

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Chemical Shift (ppm)

13C NMR for Analogue B

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

COSY for Analogue B

6 F1 Chemical Shift (ppm) Shift Chemical F1 7

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

HSQC for Analogue B

100

150 F1 Chemical Shift (ppm) Shift Chemical F1

200

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

1H NMR for Compound 51

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13C NMR for Compound 51

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1H NMR for Compound 105

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13C NMR for Compound 105

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

1H NMR for Analogue C

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)

100

13C NMR for Analogue C

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

101

COSY for Analogue C

6 F1 Chemical Shift (ppm) Shift Chemical F1 7

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

102

HSQC for Analogue C

100

150 F1 Chemical Shift (ppm) Shift Chemical F1

200

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

103

1H NMR for Compound 53

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

104

13C NMR for Compound 53

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

105

1H NMR for Compound 110

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

106

13C NMR for Compound 110

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

107

1H NMR for Analogue D

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Chemical Shift (ppm)

108

13C NMR for Analogue D

200 180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)

109

COSY for Analogue D

6 F1 Chemical Shift (ppm) Shift Chemical F1 7

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

110

HSQC for Analogue D

100

150 F1 Chemical Shift (ppm) Shift Chemical F1

200

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

111

Chapter 2

Synthesis of S-Linked Trisaccharide Glycal of Derhodinosylurdamycin

2.1 Introduction

2.1.1 Angucycline Antitumor Antibiotic Derhodinosylurdamycin A

Figure 2-1. Urdamycin A and derhodinosylurdamycin A

The urdamycins12-16 are a subclass of angucycline natural products and were first isolated by Zeeck12 and co-workers in 1986 from Streptomyces fradiae. Methanolysis20 of urdamycin A in presence of dichloroacetic acid lead to the first identification of bioactive natural product derhodinosylurdamycin A which was later found as the main urdamycin metabolite from S. fradiae Tü2717 mutant lacking UrdGT1a.53

Derhodinosylurdamycin A is composed of teracyclic aglycon core, trisaccharide subunit containing two D-olivose and one L-rhodinose sugar subunits linked together via a β-C-

112

aryl glycosidic linkage.54 Derhodinosylurdamycin A exhibits significant cytotoxic

20 activity against L1210 leukemia cell lines (IC50 value of 0.75 μg/mL) comparable to

12 urdamycin A (IC50 value of 0.55 μg/mL). In 2015, our group reported the first asymmetric synthesis of derhodinosylurdamycin A.34

2.1.2 2-Deoxy Glycosides and Carbohydrate Mimics

The monosaccharides in which the hydroxyl group is being replaced by H-atom are referred as deoxysugars.55 Deoxy sugars are important constituents of bioactive natural products. In particular 2,6-dideoxy sugar and 2,3,6-trideoxy sugar subunits are present in numerous bioactive natural products like anthracyclines56, angucycline1, 5, 6 and cardiac glycosides.57

Figure 2-2 Representative naturally occurring deoxy-glycosides.

113

These deoxy sugar subunits are known to play an important role in biological activities of their parent molecules.51, 58 Both the composition and length of sugar chain are known to influence bioactivity, for instance, the anticancer activity of Landomycins is dependent on the length of oligosaccharide side chain.7 Despite their biological importance, the stereoselective preparation of 2-deoxyglycosidic linkages is of great challenge due to lack of C-2 directing groups. On the other hand 2-deoxy glycosides are known to be susceptible to hydrolysis, however, carbohydrate mimics, such as thioglycosides (S-linked glycosides),59-61 are resistant towards acid-mediated or enzymatic hydrolysis. Recently, our group has developed an umpolung reactivity-based

S-glycosylation62, 63 for stereoselective preparation of S-linked 2-deoxyglycosides (2- deoxythioglycosides).64-66 The scheme shown below (Scheme 2.1) is the strategy employed by our research group for the stereoselective construction of S-linked 2- deoxyglycosides.

Scheme 2.1 Strategies Employed for the Stereoselective Construction of S-linked 2-

deoxyglycosides by Zhu et al.62

114

According to this strategy, reductive lithiation of a mixture of 2-deoxy α- and β- glycosyl phenylsulfide 114 affords predominantly axially lithiated species 115 at -78 ºC which upon warming epimerizes to equatorially lithiated species 116. This 2- deoxyglycosyl lithium species (115 or 116) can now be used to react with asymmetric disulfide acceptor 117 to provide corresponding S-linked 2-deoxyoligosaccharide.

Temperature control strategy was the beauty of this approach where desired glycosidic linkage can be formed by altering the temperature of reaction system. Use of the bulky group like tert-butyl at one end of disulfide 117 to control regioselectivity was another successful approach utilized. In addition, this umpolung S-glycosylation was successfully applied to the synthesis of S-linked hexasaccharide of Landomycin A.67 Based on those accomplishments, we decided to prepare S-linked derhodinosylurdamycin A for biological studies. Towards this goal, the synthesis of S-linked trisaccharide glycal68 of derhodinosylurdamycin A was carried out by employing the aforementioned umpolung S- glycosylation.

2.2 Results and Discussion

2.2.1 Retrosynthesis

The synthetic strategy for S-linked trisaccharide glycal of derhodinosylurdamycin

A was devised based on our experiences in the synthesis of S-linked hexasaccharide of

Landomycin A.67 As shown in Scheme 2.2, S-linked trisaccharide glycal 120 may be obtained from thioglycoside 125 via reductive lithiation-elimination followed by deprotection.69, 70 Trisaccharide thioglycoside 125 should be obtained from disaccharide phenylsulfide donor 123 and disulfide acceptor 124 via our previously developed 115

umpolung S-glycosylation. In turn, disaccharide phenylsulfide donor 123 should be obtained from phenylsulfide donor 121 and known disulfide acceptor 12267 via umpolung

S-glycosylation.

Scheme 2.2 Retrosynthetic Analysis.

2.2.2 Synthesis of Disulfide Acceptor 124

3-O-allyl-1,2:5,6-di-O-isopropylidene-α-D-allofuranose 127 was used as the precursor for the synthesis of asymmetric disulfide acceptor 124 as shown in the Scheme

2.371. First, 127 was converted into 3-O-allyl-1,2,4,6-tetra-O-acetyl-α-D-allopyranose

128 following previously reported procedure72 involving acid-mediated deprotection of acetonides followed by isomerization of furanose to pyranose and subsequent peracetylation. Next, treatment of 128 with thiophenol in the presence of boron trifluoride diethyl etherate afforded corresponding glycosyl phenylsulfide (46% over three steps) which underwent global deacetylation to furnish 129 in quantitative yield as a mixture of

116

anomers. After protection of 4,6-diol of 129 as its 4,6-O-benzylidene followed by methylation of the C2-alcohol, β-thioglycoside 130 was isolated

Scheme 2.3 Synthesis of Asymmetric Disulfide Acceptor.

in 42% yield over two steps together with its α-anomer (33% yield). Although theoretically both β-thioglycoside 130 and its α-anomer can be used for the synthesis of trisaccharide glycal 120, in this work only β-thioglycoside 130 was used for the remaining synthesis in consideration of the easier characterization of the synthetic intermediates. Thus, acid-catalyzed deprotection of 4,6-O-benzylidene of β-thioglycoside

130 followed by 2-aminoethyl diphenylborinate-catalyzed selective tosylation73 of the

C6-primary alcohol gave rise to 131 in 91% yield over two steps. Lithium aluminium hydride-mediated reduction of C6-tosylate followed by benzylation of the C4-alcohol and subsequent deprotection of the allyl ether74 provided C3-axial alcohol 132 in 58% yield

117

over three steps. Standard triflation of C3-axial alcohol 132 afforded triflate 133 which then reacted with cesium thioacetate to furnish 134 in 60% yield over two steps.

Reductive removal of S-acetyl group provided desired corresponding thiol which was then converted to the asymmetric disulfide acceptor 124 in 72% yields over two steps.

2.2.3 Synthesis of S-linked Trisaccharide 125

Scheme 2.4 Synthesis of S-linked Trisaccharide 125.

D-Olivose-derived phenylsulfide donor 121 was readily prepared in 95% yield

75 from known glycal 79 and thiophenol via ReOCl3(SMe2)(OPPh3)-catalyzed thioglycosylation (Scheme 2.4).65 Next, lithium 4,4’-di-tert-butylbiphenyl (LiDBB)- mediated reductive lithiation of the glycosyl phenylsulfide of 121 followed by anomerisation (-20 ºC)63 afforded the corresponding equatorial lithium 136 which reacted

118

with known asymmetric disulfide acceptor 12267 to give rise to the desired β-S-linked disaccharide 123 in 47% yield (52% based on recovered disulfide acceptor 122). A second lithium 4,4’-di-tert-butylbiphenyl (LiDBB)-mediated reductive lithiation of the S- linked disaccharide-derived glycosyl phenylsulfide of 123 afforded the corresponding axial lithium 137 which reacted with asymmetric disulfide acceptor 124 to furnish the desired β-S-linked trisaccharide 125 in low yield (36%).

2.2.4 Improved Synthesis of S-linked Trisaccharide 125 and Synthesis of S-linked

Glycal 120.

Scheme 2.5 Improved synthesis of S-linked Trisaccharide 125 and Synthesis of S- linked Glycal 120.

We hypothesized that production of trisaccharide 125 from disulfide acceptor 122 in low yield may be due to the lower reactivity of asymmetric disulfide acceptor 122 as compared to other 2-deoxy sugar-derived asymmetric disulfide acceptors. In order to improve the S-glycosylaton efficiency, we decided to prepare the corresponding

119

thiocyanate 138 (Scheme 2.5) in the hope that thiocyanate 138 may be a better electrophile than asymmetric disulfide acceptor 124 in the sulfenylation of glycosyl lithium 137. Thus, triflate 133 was subjected to an SN2 reaction with potassium thiocyanate to afford sugar-derived thiocyanate 138 in 47% yield. Gratifyingly, lithium

4,4’-di-tert-butylbiphenyl (LiDBB)-mediated reductive lithiation of the S-linked disaccharide-derived glycosyl phenylsulfide of 123 afforded the corresponding axial lithium 137 which reacted with thiocyanate 138 to give the desired β-S-linked trisaccharide 125 in 64% yield. Next, thioglycoside 125 underwent rapid reductive lithiation-elimination and concomitant debenzylation afforded S-linked trisaccharide glycal 139 in 92% yield. Finally, tetra-n-butylammonium fluoride-mediated deprotection of two tert-butyldimethylsilyl ethers of S-linked trisaccharide glycal 139 provided the desired target, S-linked trisaccharide glycal of derhodinosylurdamycin A 120 in 87% yield.

120

2.3 Experimental

2.3.1 General Considerations

1H NMR at δ 7.26, 13C NMR at δ 77.36. Data for 1H NMR are reported as follows: chemical shift, integration, multiplicity (app = apparent, par obsc = partially obscure, ovrlp = overlapping, s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, m = multiplet) and coupling constants in Hertz. All 13C NMR spectra were recorded with complete proton decoupling. Low resolution mass spectra (LRMS) were acquired on a Waters Acuity Premiere XE TOF LC-MS by electrospray ionization.

Optical rotations were measured with Autopol-IV digital polarimeter; concentrations are expressed as g/100 mL. All reagents and chemicals were purchased from Acros Organics,

Sigma Aldrich, Fisher Scientific, Alfa Aesar, and Strem Chemicals and used without further purification. THF, methylene chloride, toluene, and diethyl ether were purified by passing through two packed columns of neutral alumina (Innovative Technology).

121

performed using 200-400 mesh silica gel (Scientific Absorbents, Inc.). Yields refer to chromatographically and spectroscopically pure materials, unless otherwise stated.

2.3.2 Synthesis of Allopyranoside 132.

To a solution of 3-O-allyl-1,2:5,6-di-O-isopropylidene-α-D-allofuranose 127

(34.6 g, 115 mmol) in 294 mL glacial acetic acid and 194 mL water was added concentrated sulfuric acid (1.5 mL). The resulting mixture was stirred at 90 ºC for 2 hours before being 2.25 g sodium hydroxide was added to neutralize the sulfuric acid. Acetic acid and water were removed under reduced pressure and the crude residue was azeotroped with toluene and dried under vacuo. The crude product was dissolved in acetic anhydride (65 mL), then 4-(dimethylamino)pyridine (704 mg, 5.8 mmol) and triethylamine (128 mL) were added. The resulting mixture was sonicated for 1.5 hours before being quenched with 10 mL of methanol via sonication. The reaction mixture was diluted with dichloromethane and the organic layer was washed sequentially with water,

122

1 N aqueous HCl, water, and brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and dried under vacuo to afford crude 3-O-allyl-1,2,4,6-tetra-O-acetyl-α-D- allopyranose 128.

To a solution of this 3-O-allyl-1,2,4,6-tetra-O-acetyl-α-D-allopyranose 128 in 288 mL of anhydrous dichloromethane was added thiophenol (23.5 mL, 230 mmol). The

º resulting reaction mixture was cooled to 0 C and BF3·OEt2 (28.5 mL, 230 mmol) was added. The mixture was stirred overnight at room temperature before being quenched with water. The aqueous layer was extracted with dichloromethane and combined organic layers were sequentially washed with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography (toluene:ethyl acetate = 50:1 to 10:1) to produce 23.2 g

(46% yield over three steps) of corresponding thioglycoside. This thioglycoside (15.5 g,

35.4 mmol) was dissolved in 71 mL methanol and 5.4 M sodium methoxide in methanol

(1.3 mL, 7.1 mmol) was added. The resulting mixture was stirred at room temperature overnight and then neutralized with Dowex H+ resin, filtered, concentrated under reduced pressure, and azeotroped with toluene to afford 11.1 g (quantitative) of 3-O-allyl-1-thio-

D-allopyranoside 129 as a mixture of α and β anomers.

To a solution of 3-O-allyl-1-thio-D-allopyranoside 129 (11.1 g, 35.4 mmol) in 70 mL N,N-dimethylformamide were added benzaldehyde dimethyl acetal (8 mL, 53 mmol) and p-TsOH (202 mg). The mixture was stirred at room temperature overnight before being quenched with saturated sodium bicarbonate. The aqueous layer was extracted with ethyl acetate, and combined organic extracts were sequentially washed with water and brine, dried with sodium sulfate, filtered, and concentrated in vacuo. The crude residue 123

was purified by flash column chromatography (hexanes:ethyl acetate = 1:1) to afford corresponding benzylidene protected compound. To a solution of this benzylidene protected compound in 118 mL N,N-dimethylformamide cooled to 0 ºC was added sodium hydride (2.83 g) and methyl iodide (3.3 mL). The reaction mixture was stirred at

0 ºC until completion. The mixture was quenched with ice cooled water and the aqueous layer was extracted with ethyl acetate. Combined organic extracts were sequentially washed with water and brine, dried with sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (hexanes: ethyl acetate = 5:1) to furnish 6.3 g of phenyl β-thioglycoside 130 (43% yield over two steps) as well as 4.86 g of its α-isomer (33% yield over two steps).

To a solution of phenyl β-thioglycoside 130 (5.25 g, 12.7 mmol) in 127 mL methanol was added p-TsOH (240 mg, 1.27 mmol). The resulting mixture was stirred at room temperature for 5 hours before being quenched with triethylamine (1 mL), concentrated in reduced pressure, and dried under vacuo to afford corresponding diol. To a mixture of this crude diol, 2-aminoethyl diphenylborinate catalyst (286 mg, 1.27 mmol), and p-toluenesulfonylchloride (3.63 g, 19.1 mmol) in 42 mL of anhydrous acetonitrile was added N,N-diisopropylethylamine (3.14 mL, 19.1 mmol). The resulting mixture was stirred at room temperature for 30 hours before being quenched with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate and combined organic extracts were washed with brine, dried with anhydrous sodium sulfate, filtered, and concentrated. The crude compound was purified via flash column chromatography (hexanes:ethylacetate = 5:1) to afford 5.55 g of compound 131 (91% yield over two steps). 124

To a solution of compound 131 (18.6 g, 38.7 mmol) in 193 mL anhydrous tetrahydrofuran cooled at 0 ºC was added 4 M lithium aluminum hydride in diethyl ether

(21.3 mL, 85 mmol). The reaction mixture was slowly warmed up and then refluxed for 1 hour before being quenched with saturated ammonium chloride. The mixture was extracted with ethyl acetate, and combined organic fractions were sequentially washed with water and brine, dried with anhydrous sodium sulfate, filtered, and concentrated.

The crude product was purified by flash column chromatography (hexanes:ethyl acetate =

5:1) to furnish 10.1 g (84% yield) of phenyl 3-O-allyl-6-deoxy-2-O-methyl-1-thio-β-D- allopyranoside. To a solution of this phenyl 3-O-allyl-6-deoxy-2-O-methyl-1-thio-β-D- allopyranoside (10.1 g, 32.6 mmol) in 65 mL N,N-dimethylformamide cooled at 0 ºC was added sodium hydride (1.83 g, 45.6 mmol). The mixture was stirred at 0 ºC for 45 minutes before benzyl bromide (4.7 mL, 39.1 mmol) was added. The resulting mixture was then stirred at room temperature for 15 hours before being quenched with water. The aqueous mixture was extracted with ethyl acetate and combined organic extracts were washed with water, dried over sodium sulfate, filtered, and concentrated in vacuo.

Purification on flash column chromatography (hexanes:ethyl acetate = 10:1) provided

10.9 g (84% yield) of the corresponding phenyl 3-O-allyl-4-O-benzyl-6-deoxy-2-O- methyl-1-thio-β-D-allopyranoside. To phenyl 3-O-allyl-4-O-benzyl-6-deoxy-2-O-methyl-

1-thio-β-D-allopyranoside (10.8 g, 27 mmol) in a mixture of MeOH (123 mL) and water

(12.3 mL) was added 1,4-diazabicyclo[2.2.2]octane (DABCO) (4.54 g, 40.5 mmol) and tris(triphenylphoshine)rhodium(I) chloride (1.25 g, 1.35 mmol). The resulting mixture was refluxed for 4 hours before 4 M HCl (13.5 mL) was added. The mixture was stirred at room temperature for 7 hours before being quenched with saturated sodium 125

bicarbonate. Methanol was evaporated and the aqueous solution was extracted with ethyl acetate. Combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated in vacuo. Purification on flash column chromatography (hexanes:ethyl acetate = 10:1 to 2:1) gave 8.04 g (83% yield) of

22 o 4-O-benzyl-6-deoxy-2-O-methyl-1-thio-β-D-allopyranoside 132. [α]D = ‒5.5 (c = 1.0,

1 CHCl3). H NMR (600 MHz, CDCl3) δ 7.55 - 7.59 (m, 2 H, aromatic of SPh), 7.25 -

7.40 (m, 8 H, aromatic), 5.03 (d, J=9.7 Hz, 1 H, H1), 4.73 (d, J=11.6 Hz, 1 H, benzylic),

4.57 (d, J=11.6 Hz, 1 H, benzylic), 4.50 (t, J=2.7 Hz, 1 H, H3), 3.91 (dd, J=9.4, 6.2 Hz, 1

5 2 4 H, H ), 3.49 (s, 3 H, C -OCH3), 3.13 (dd, J=9.4, 2.7 Hz, 1 H, H ), 3.08 (dd, J=9.7, 2.8

2 3 13 Hz, 1 H, H ), 2.58 (s, 1 H, C -OH), 1.33 (d, J=6.2 Hz, 3 H, CH3). C NMR (150 MHz,

CDCl3) δ 137.68, 133.59, 132.22, 128.91, 128.67, 128.18, 128.12, 128.07, 127.54, 83.11,

79.79, 79.14, 71.34, 71.05, 64.81, 57.78, 18.24. FT-IR (thin film): 3479, 2931, 2893,

-1 + 1733, 1242, 1075, 739 cm . ESI-LRMS [M+Na] Calculated for NaC20H24O4S 383.13, found 382.90.

2.3.3 Synthesis of Asymmetric Disulfide Acceptor 124

126

To a solution of 4-O-benzyl-6-deoxy-2-O-methyl-1-thio-β-D-allopyranoside 132

(3.8 g, 10.6 mmol) in 53 mL dichloromethane was added pyridine (8.6 mL, 106 mmol).

The mixture was cooled to 0 ºC and triflic anhydride (4.5 mL, 26.7 mmol) was added.

The reaction mixture was stirred at 0 ºC for 1.5 hours and quenched with water. The mixture was extracted with dichloromethane, and combined organic extracts were sequentially washed with saturated copper sulfate solution, water, and brine, dried over anhydrous sodium sulfate, and concentrated under vacuo to furnish triflate 133 in quantitative yield. A solution of this triflate (5.2 g, 10.6 mmol) in 35 mL anhydrous tetrahydrofuran was cooled to 0 ºC and cesium thioacetate (3.1 g, 14.8 mmol) was added.

The reaction mixture was warmed up and stirred at room temperature overnight before water was added. Tetrahydrofuran was removed under reduced pressure and the remaining aqueous mixture was extracted with ethyl acetate. Combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated in vacuo. The residue was purified by flash column chromatography (hexanes:ethyl acetate = 10:1) to furnish 2.53 g of compound 134 (60% yield over two steps). To a solution of compound 134 (2.53 g, 6.0 mmol) in 10 mL of anhydrous diethyl ether cooled at -30 ºC was added 4.0 M lithium aluminum hydride in diethyl ether (1.5 mL, 6.0 mmol). The resulting mixture was slowly warmed up and stirred at room temperature for one hour before being quenched with saturated ammonium chloride. The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated under vacuo to obtain corresponding crude mercapto-sugar which was directly used in the next step. To a mixture of this crude mercapto-sugar and triethyamine (1.7 mL, 12 mmol) in 20 mL degassed dichloromethane cooled at 0 ºC was 127

t added BuSSO2Me (1.11 g, 6.6 mmol) in 22 mL degassed dichloromethane. The reaction mixture was stirred at room temperature for 12 hours, concentrated under vacuo, and purified by flash column chromatography (hexanes:ethyl acetate = 50:1 to 15:1) to afford

22 o 2.01g of asymmetric disulfide acceptor 124 (72% yield for two steps). [α]D = ‒14.4 (c

1 = 1.0, CHCl3). H NMR (600 MHz, CDCl3) δ 7.58 (d, J=7.15 Hz, 2 H, aromatic ), 7.46

(d, J=7.2 Hz, 2 H, aromatic), 7.40 (t, J=7.3 Hz, 2 H, aromatic), 7.34 (dd, J=8.3, 6.5 Hz, 3

H, aromatic), 7.31 (d, J=7.2 Hz, 1 H, aromatic), 5.07 (d, J=10.1 Hz, 1 H, benzylic), 4.70

1 2 (d, J=10.3 Hz, 1 H, benzylic), 4.60 (d, J=9.5 Hz, 1 H, H ), 3.77 (s, 3 H, C -OCH3), 3.45

(dd, J=9.0, 6.2 Hz, 1 H, H5), 3.10 - 3.19 (m, 2 H, H2 and H4), 2.96 - 3.02 (m, 1 H, H3),

6 t 13 1.37 - 1.44 (m, 12 H, C -CH3, Bu). C NMR (150 MHz, CDCl3) δ 137.75, 134.02,

132.02, 129.11, 28.81, 128.63, 128.20, 127.68, 89.00, 82.90, 82.55, 77.86, 76.12, 65.16,

61.39, 48.30, 29.84, 18.76. FT-IR (thin film): 3031, 2957, 1583, 1477, 1074, 1058, 738,

-1 + 698 cm . ESI-LRMS [M+Na] Calculated for NaC24H32O3S3 487.14, found 487.00.

2.3.4 Synthesis of Phenyl 3,4-di-O-tert-butyldimethylsilyl-1-thio-D-olivoside 121.

A solution of bis-TBS protected D-Rhamnal 79 (3.05 g, 8.5 mmol) in 28 mL toluene was cooled to 0 ºC, then thiophenol (1.1 mL, 10.2 mmol) was added followed by the addition of ReOCl3(SMe2)(OPPh3) (83 mg, 0.13 mmol). The reaction mixture was slowly warmed up to room temperature and stirred for 5 hours. The mixture was directly

128

concentrated under vacuo and crude residue was purified by flash column chromatography (hexanes:dichloromethane = 10:1 to 5:1) to afford 3.66 g (95% yield) of

22 o glycosyl phenyl sulfide 121 as a mixture of anomers (α/β = 3/1). [α]D = 206.1 (c = 1.0,

1 CHCl3). H NMR (600 MHz, CDCl3) δ 7.49 - 7.54 (ovrlp, 2 H, α and β), 7.24 - 7.36

(ovrlp, 3H, α and β), 5.56 (dd, J=5.2, 2.1 Hz, 1 H, H1 α), 4.84 (dd, J=12.1, 1.8 Hz, 1 H,

H1 β), 4.10 - 4.19 (m, 1 H, H5 α ), 3.95 - 4.02 (m, 1 H, H3 α), 3.72 (ddd, J=11.0, 8.1, 5.0

Hz, 1 H, H3 β ), 3.34 (dd, J=8.8, 6.2Hz, 1H, H5 β), ,3.20 - 3.28 (ovrlp, 1H, H4 α, β), 2.27 -

2.35 (m, 1 H, H2 α, β) 2.08 (ddd, J=13.6, 10.7, 5.4 Hz, 1 H, H2 α), 1.82 (m, 1 H, H2 β),

6 6 1.36 (d, J=6.2 Hz, 3 H, C -CH3 β ), 1.31 (d, J=6.4 Hz, 3 H, C -CH3 α), 0.93 - 1.01 (ovrlp,

t 13 18H, Bu-TBS α, β), 0.12 - 0.21 (ovrlp, 12H, CH3-TBS α, β). C NMR (150 MHz,

CDCl3) δ 135.76, 134.89, 131.34, 131.13, 129.15,129.13,127.40, 127.17, 83.52, 81.96,

78.36, 77.93, 74.79, 71.66, 70.71, 41.41, 40.06, 26.65, 26.58, 26.45, 26.42, 26.10, 19.36,

18.77, 18.64, 18.59, 18.40, -2.37, -2.56, -2.62, -2.88, -3.54, -3.68, -3.84, -4.08. FT-IR

(thin film): 2954, 2929, 1472, 1250, 1099, 831, 773 cm-1. ESI-LRMS [M+Na]+

Calculated for NaC24H44O3SSi2 491.24, found 491.00.

2.3.5 Synthesis of S-linked Disaccharide 123.

Phenyl 3,4-di-O-tert-butyldimethylsilyl-1-thio-D-olivoside 121 (3.39 g, 7.0 mmol) and 1,10-phenanthroline (1 small crystal) was dissolved in 15 mL tetrahydrofuran.

The resulting mixture was cooled down to 0 ºC and n-BuLi (2.5 M) was added dropwise until a dark brown color persisted. This procedure serves to quench proton sources

(moisture). The reaction mixture was cooled down to -78 ºC and 0.4 M LiDBB in THF

129

(38.5 mL, 15.4 mmol) was added dropwise. The resulting mixture was stirred for 15 minute before it was placed in -20 ºC bath and allowed to stand for 45 minutes. The reaction mixture was cooled back to -78 ºC, disulfide acceptor 122 (1.15 g, 3.5 mmol) solution in 7.5 mL dry THF was added. The resulting mixture was stirred overnight before aqueous saturated sodium bicarbonate was added. After THF was removed under reduced pressure, the remaining aqueous mixture was extracted with EtOAc. Combined extracts were washed sequentially with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column (hexanes:methylene chloride = 10:1 to 1:1) to afford 984 mg of S-linked

23 disaccharide 5 (47% yield) and 121 mg of recovered disulfide acceptor 122. [α]D = ‒

o 1 159.1 (c = 1.0, CHCl3). H NMR (600 MHz, CDCl3) δ 7.43 - 7.53 (m, 2 H, aromatic),

7.25 - 7.32 (m, 2 H, aromatic), 7.19 - 7.25 (m, 1 H, aromatic), 5.59 (d, J=4.8 Hz, 1 H,

H1), 4.64 (dd, J=6.4, 2.0 Hz, 1 H, H5), 4.54 (dd, J=11.7, 1.7 Hz, 1 H, H1΄), 3.64 (ddd,

J=11.0, 8.1, 5.0 Hz, 1 H, H3΄), 3.24 (dd, J=8.8, 6.2 Hz, 1 H, H5΄), 3.18 (t, J=8.4 Hz, 1 H,

H4΄), 3.14 (br. s., 1 H, H4), 2.38 - 2.47 (m, 1 H, H2), 2.25 - 2.33 (m, 1 H, H3), 2.22 (ddd,

130

J=13.0, 4.9, 1.7 Hz, 1 H, H2΄), 2.11 (dd, J=13.7, 3.8 Hz, 1 H, H3), 1.82 (d, J=13.4 Hz, 1

2 2΄ 6 6΄ H, H ), 1.67 - 1.77 (m, 1 H, H ), 1.23 - 1.30 (m, 6 H, C , C - CH3) 0.86 - 0.97 (m, 18 H, t 13 Bu-TBS) 0.06 - 0.16 (m, 12 H, CH3-TBS ). C NMR (150 MHz, CDCl3) δ 135.93,

130.99, 129.11, 126.93, 84.94, 81.14, 78.02, 74.73, 68.11, 47.94, 41.98, 28.60, 27.05,

26.63, 26.44, 19.29, 19.23, 18.64, 18.38 , -2.40, -2.62, -3.57, -3.83 FT-IR (thin film):

2954, 2928, 1473, 1251, 1095, 1035, 831, 773cm-1. ESI-LRMS [M+Na]+ Calculated for

NaC30H54O4S2Si2 621.29, found 621.30.

2.3.6 Synthesis of S-linked Trisaccharide 125.

S-linked disaccharide 123 (221 mg, 0.37 mmol) and a pinch of 1, 10- phenanthroline were dissolved in 2 mL tetrahydrofuran and cooled to 0 ºC. n-BuLi (2.5

M) was added dropwise until dark color persisted. The mixture was cooled to -78 ºC and

0.4 M LiDBB in THF (2.1 mL, 0.81 mmol) was added dropwise. The resulting mixture

131

was stirred for 15 min and a tetrahydrofuran solution of asymmetric disulfide acceptor

124 (114 mg, 0.245 mmol) was added. The reaction mixture was stirred overnight before aqueous saturated sodium bicarbonate (1 mL) was added. Tetrahydrofuran was removed under reduced pressure and the remaining aqueous portion was extracted with ethyl acetate (3X15 mL). Combined organic extracts were sequentially washed with water (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography

(toluene:dichloromethane = 5:1 to 1:1) to provide 95 mg of S-linked trisaccharide 125

22 o 1 (45% yield). [α]D = ‒99 (c = 1.0, CHCl3). H NMR (600 MHz, CDCl3) δ 7.50 - 7.56

(m, 2 H, aromatic), 7.25 - 7.41 (m, 8 H, aromatic), 5.61 (d, J=5.3 Hz, 1 H, H1΄), 4.93 (d,

J=10.6 Hz, 1 H, benzylic), 4.72 (d, J=10.6 Hz, 1 H, benzylic), 4.57 (d, J=9.5 Hz, 1 H,

H1), 4.44 (dd, J=11.8, 1.6 Hz, 1 H, H1΄΄), 4.34 (dd, J=6.4, 1.7 Hz, 1 H, H5΄), 3.69 (s, 3 H,

2 3΄΄ 5 C -OCH3), 3.59 - 3.65 (m, 1 H, H ), 3.44 (dd, J=8.6, 6.2 Hz, 1 H, H ), 3.15 - 3.26 (m, 3

H, H2 , H4΄΄ , H5΄΄), 2.90 - 2.99 (m, 2 H, H3 , H4), 2.83 (br. s., 1 H, H4΄), 2.30 - 2.37 (m, 1

H, H2΄), 2.16 - 2.23 (m, 2 H, H2΄΄ ,H3΄), 1.96 - 2.01 (m, 1 H, H3΄), 1.60 - 1.72 (m, 2 H, H2΄΄ ,

2΄ 6 6΄΄ H ), 1.39 (d, J=6.2 Hz, 3 H, C -CH3), 1.28 (d, J=6.1 Hz, 3 H, C -CH3), 0.89 - 0.94 (m,

6΄ t 13 21 H, C -CH3, Bu- TBS) 0.08 - 0.13 (m, 12 H, CH3- TBS). C NMR (150 MHz,

CDCl3) δ 138.60, 134.49, 131.77, 129.17, 128.52, 128.16, 127.96, 127.57, 89.03, 85.26,

82.15, 81.53, 81.22, 78.23, 78.04, 77.29, 76.06, 74.73, 67.14, 61.27, 52.91, 48.12, 41.95,

28.24, 26.63, 26.44, 26.18, 19.36, 19.23, 19.06, 18.63, 18.38, -2.40, -2.63, -3.57, -3.82.

FT-IR (thin film): 2928, 2855, 1462, 1360, 1251, 1093, 1036, 831, 775 cm-1. ESI-

+ LRMS [M+Na] Calculated for NaC44H72O7S3Si2 887.39, found 887.60.

132

2.3.7 Synthesis of Thiocyanate 138.

To a solution of triflate 133 (1.49 g, 4.1 mmol) in 20 mL degassed N,N- dimethylformamide was added potassium thiocyanate (1.99 g, 20.5 mmol), and the reaction mixture was stirred at 60 ºC for 14 hours. N,N-dimethylformamide was removed under reduced pressure and the residue was diluted with water. The aqueous solution was extracted with ethyl acetate, and combined extracts were washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl acetate = 5:1) to

22 o afford 777 mg (47% yield for two steps) of thiocyanate 138. [α]D = ‒26.2 (c = 1.0,

1 CHCl3). H NMR (600 MHz, CDCl3) δ 7.56 - 7.60 (m, 2 H, aromatic), 7.39 - 7.44 (m, 4

H, aromatic), 7.31 - 7.38 (m, 4 H, aromatic), 4.94 (d, J=10.6 Hz, 1 H, benzylic), 4.75 (d,

1 2 J=10.5 Hz, 1 H, benzylic), 4.57 (d, J=9.4 Hz, 1 H, H ), 3.77 (s, 3 H, C -OCH3), 3.39 -

3.45 (m, 1 H, H5), 3.32 - 3.37 (m, 2 H, H4 and H2), 2.99 (t, J=10.1 Hz, 1 H, H3), 1.44 (d,

6 13 J=6.1 Hz, 3 H, C -CH3). C NMR (150 MHz, CDCl3) δ 137.13, 133.32, 131.96, 129.13,

128.65, 128.35, 128.24, 127.92, 110.56, 88.83, 80.47, 80.35, 77.67, 75.90, 61.87, 58.82,

18.66. FT-IR (thin film): 3029, 2986, 2898, 2149, 1581, 1479, 1114, 1094, 1077, 1057,

-1 + 742, 686 cm . ESI-LRMS [M+Na] Calculated for NaC21H23NO3S2 424.10, found

423.90.

133

2.3.8 Synthesis of S-linked Trisaccharide 125 from S-linked Disaccharide 123 and

Thiocyanate 138.

S-linked disaccharide 138 (180 mg, 0.3 mmol) and a pinch of 1,10-phenanthroline were dissolved in 1.5 mL tetrahydrofuran and cooled to 0 ºC. n-BuLi (2.5 M) was added dropwise until dark color persisted. The mixture was cooled to -78 ºC and 0.4 M LiDBB in THF (1.7 mL, 0.66 mmol) was added dropwise. The resulting mixture was stirred for

15 min and a tetrahydrofuran solution of thiocyanate 138 (81 mg, 0.2 mmol) was added.

The reaction mixture was stirred overnight before aqueous saturated sodium bicarbonate

(1 mL) was added. Tetrahydrofuran was removed under reduced pressure and the remaining aqueous portion was extracted with ethyl acetate (3X15 mL). Combined organic extracts were sequentially washed with water (20 mL) and brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (toluene:dichloromethane =

5:1 to 1:1) to provide 113 mg of S-linked trisaccharide 4 (65% yield).

2.3.9 Synthesis of S-linked Trisaccharide Glycal 139.

To a solution of S-linked trisaccharide 125 (31 mg, 0.036 mmol) in 0.4 mL dry tetrahydrofuran cooled at -78 ºC was added 0.4 M LiDBB in THF dropwise until dark 134

green color persists. The reaction mixture was stirred for 10 minutes before being quenched with saturated sodium bicarbonate (1 mL). Tetrahydrofuran was removed under reduced pressure and the resulting aqueous solution was extracted with dichloromethane. Combined organic extracts were sequentially washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduce pressure. The crude residue was purified by flash column chromatography (hexanes:ethyl

22 acetate = 10:1) to afford 21 mg (92% yield) of S-linked trisaccharide glycal 139. [α]D =

1 1 ‒85.2 (c = 0.5, CHCl3). H NMR (600 MHz, CDCl3)  6.36 (dd, J=5.9, 2.2 Hz, 1 H, H ),

5.31 (dd, J=5.0, 3.0 Hz, 1 H, H1΄), 4.74 (d, J=1.7 Hz, 1 H, C4-OH), 4.64 (dd, J=6.5, 2.5

Hz, 1 H, H5΄), 4.57 (dd, J=6.0, 2.1 Hz, 1 H, H2), 4.51 (dd, J=11.7, 1.8 Hz, 1 H, H1΄΄), 3.70

- 3.76 (m, 1 H, H5), 3.61 (ddd, J=11.0, 8.0, 5.0 Hz, 1 H, H3΄΄), 3.43 (dt, J=7.7, 2.1 Hz, 1

H, H3), 3.38 (ddd, J=9.3, 7.8, 1.7 Hz, 1 H, H4), 3.18 - 3.23 (m, 1 H, H5΄΄), 3.15 (d, J=8.3

Hz, 1 H, H4΄΄), 3.11 - 3.14 (m, 1 H, H4΄), 2.34 - 2.42 (m, 1 H, H2΄), 2.17 (ddd, J=13.0, 5.0,

1.8 Hz, 1 H, H2΄΄), 2.08 - 2.15 (m, 1 H, H3΄), 1.99 - 2.04 (m, 1 H, H H3΄), 1.78 (dd, J=14.1,

2΄ 2΄΄ 6 3.3 Hz, 1 H, H ), 1.64 - 1.72 (m, 1 H, H ) 1.39 (d, J=6.2 Hz, 3 H, C -CH3), 1.32 (d,

6΄ 6΄΄ t J=6.4 Hz, 3 H, C -CH3) 1.24 (d, J=6.1 Hz, 3 H, C -CH3), 0.87 - 0.92 (m, 18 H, Bu-

13 TBS), 0.05 - 0.10 (m, 12 H, CH3-TBS). C NMR (150 MHz, CDCl3)  145.61, 100.18,

83.07, 80.85, 77.99, 77.43, 76.73, 75.72, 74.68, 69.23, 50.36, 47.23, 41.91, 28.27, 27.69,

26.63, 26.45, 19.23,18.65, 18.44, 18.40, 18.05, -2.38, -2.61, -3.54, -3.81. FT-IR (thin film): 3387, 2956, 2928, 2149, 1641, 1472, 1256, 1095, 1058, 742 cm-1. ESI-LRMS

+ [M+Na] Calculated for NaC30H58O6S2Si2 657.31, found 657.50.

135

2.3.10 Synthesis of S-linked Trisaccharide glycal of Derhodinosylurdamycin A

(120).

To a solution of S-linked trisaccharide glycal 20 (20.7 mg, 0.033 mmol) in 0.37 mL tetrahydrofuran was added 1 M tetra-n-butylammonium fluoride in THF (0.1 mL, 0.1 mmol). The resulting mixture was stirred overnight. The reaction mixture was quenched with a pinch of solid sodium bicarbonate and purified via flash column chromatography

(hexanes:ethyl acetate = 1:1) to produce 11.7 mg (87% yield) of desired S-linked

22 1 trisaccharide glycal of derhodinosylurdamycin A. [α]D = ‒128.1 (c = 1.0, CHCl3). H

1 NMR (600 MHz, CD3OD)  6.31 (dd, J=6.0, 1.9 Hz, 1 H, H ), 5.37 (d, J=4.6 Hz, 1 H,

H1΄), 4.71 (dd, J=6.1, 2.2 Hz, 1 H, H2), 4.59 - 4.65 (m, 2 H, H5΄, H1΄΄), 3.64 - 3.70 (m, 1

H, H5), 3.49 (ddd, J=11.2, 8.9, 5.2 Hz, 1 H, H3΄΄), 3.35 (dt, J=8.1, 2.1 Hz, 1 H, H3), 3.21 -

3.28 (m, 2 H, H4, H5΄΄), 3.12 (d, J=2.4 Hz, 1 H, H4΄), 2.89 (t, J=9.0 Hz, 1 H, H4΄΄) 2.35 -

2.43 (m, 1 H, H2΄) 2.12 - 2.20 (m, 2 H, H3΄, H2΄΄), 1.99 - 2.05 (m, 1 H, H3΄), 1.61 - 1.67

2 2 6 (m, 1 H, H ΄), 1.52 - 1.59 (m, 1 H, H ΄΄), 1.32 (d, J=6.2 Hz, 3 H, C -CH3), 1.24 (dd,

6΄ 6΄΄ 13 J=6.2, 2.0 Hz, 6 H, C , C -CH3). C NMR (150 MHz, CD3OD)  145.08, 102.42,

82.51, 82.34, 78.27, 77.37, 76.85, 75.61, 73.38, 69.05, 49.38, 47.79, 41.42, 29.10, 27.51,

19.05, 18.43, 18.09 FT-IR (thin film): 3379, 2922, 2851, 1737, 1640, 1238, 1080, 1058,

-1 + 770 cm . ESI-LRMS [M+Na] Calculated for NaC18H30O6S2 429.14, found 429.10.

136

NMR Spectra for Selected Compounds

1H NMR for Compound 132

WORKED UP HNMR.ESP

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

137

13C NMR for Compound 132

WORKED UP CNMR.ESP

136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 Chemical Shift (ppm)

138

1H NMR for Compound 124

WORKED UP HNMR.ESP

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

139

13C NMR for Compound 124

WORKED UP CNMR.ESP

136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 Chemical Shift (ppm)

140

1H NMR for Compound 121

WORKED UP HNMR.ESP

0.45

0.40

0.35

0.30

0.25

0.20 Normalized Intensity Normalized

0.15

0.10

0.05

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

141

13C NMR for Compound 121

WORKED UP CNMR.ESP

140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

142

1H NMR for Compound 123

WORKED UP HNMR.ESP 0.40

0.35

0.30

0.25

0.20

Normalized Intensity Normalized 0.15

0.10

0.05

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

143

13C NMR for Compound 123

1.0 Worked up CNMR.esp

0.9

0.8

0.7

0.6

0.5

0.4 Normalized Intensity Normalized

0.3

0.2

0.1

130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Chemical Shift (ppm)

144

1H NMR for Compound 125

WORKED UP NMR.ESP

0.45

0.40

0.35

0.30

0.25

0.20 Normalized Intensity Normalized

0.15

0.10

0.05

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

145

13C NMR for Compound 125

WORKED UP CNMR.ESP

140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

146

1H NMR for Compound 138

WORKED UP HNMR.ESP

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

147

13C NMR for Compound 138

WORKED UP CNMR.ESP

136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 Chemical Shift (ppm)

148

1H NMR for Compound 139

WORKED UP NMR.ESP

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

149

13C NMR for Compound 139

WORKED UP CNMR.ESP

140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

150

1H NMR for Compound 120

1.0 WORKED UP HNMR.ESP

0.9

0.8

0.7

0.6

0.5

0.4 Normalized Intensity Normalized

0.3

0.2

0.1

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

151

13C NMR for Compound 120

WORKED UP CNMR.ESP 0.075

0.070

0.065

0.060

0.055

0.050

0.045

0.040

0.035

Normalized Intensity Normalized 0.030

0.025

0.020

0.015

0.010

0.005

140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

152

COSY for Compound 120

5 F1 Chemical Shift (ppm) Shift Chemical F1

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F2 Chemical Shift (ppm)

153

HSQC for Compound 120

100

120 (ppm) Shift Chemical F1

140

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 F2 Chemical Shift (ppm)

154

References

1. Rohr, J.; Thiericke, R. "Angucycline Group Antibiotics," Nat. Prod.

Rep. 1992, 9, 103-137.

2. Elshahawi, S. I.; Shaaban, K. A.; Kharel, M. K.; Thorson, J. S. "A

comprehensive review of glycosylated bacterial natural products,"

Chem. Soc. Rev. 2015, 44, 7591-7697.

3. Dann, M.; Lefemine, D. V.; Barbatschi, F.; Shu, P.; Kunstmann, M.

P.; Mitscher, L. A.; Bohonos, N. "Tetrangomycin, a new quinone

antibiotic," Antimicrob. Agents Chemother. 1965, 5, 832-835.

4. Kuntsmann, M. P.; Mitscher, L. A. "The structural characterization of

tetrangomycin and tetrangulol," J. Org. Chem. 1966, 31, 2920-2925.

5. Krohn, K.; Rohr, J. "Angucyclines: Total syntheses, new structures,

and biosynthetic studies of an emerging new class of antibiotics," Top.

Curr. Chem. 1997, 188, 127-195.

155

6. Kharel, M. K.; Pahari, P.; Shepherd, M. D.; Tibrewal, N.; Nybo, S. E.;

Shaaban, K. A.; Rohr, J. "Angucyclines: Biosynthesis, mode-of-

action, new natural products, and synthesis," Nat. Prod. Rep. 2012,

29, 264-325.

7. Henkel, T.; Rohr, J.; Beale, J. M.; Schwenen, L. "Landomycins, new

angucycline antibiotics from Streptomyces sp. I. Structural studies on

landomycins A-D," The Journal of Antibiotics 1990, 43, 492-503.

8. Uchida, T.; Imoto, M.; Watanabe, Y.; Miura, K.; Dobashi, T.;

Matsuda, N.; Sawa, T.; Naganawa, H.; Hamada, M.; Takeuchi, T.

"Saquayamycins, new aquayamycin-group antibiotics," The Journal

of antibiotics 1985, 38, 1171-1181.

9. Takahashi, K.; Yoshida, M.; Tomita, F.; Shirahata, K. "Gilvocarcins,

new antitumor antibiotics. 2. Structural elucidation," The Journal of

antibiotics 1981, 34, 271-275.

10. Rix, U.; Zheng, J.; Remsing Rix, L. L.; Greenwell, L.; Yang, K.;

Rohr, J. "The dynamic structure of jadomycin B and the amino acid

incorporation step of its biosynthesis," J. Am. Chem. Soc. 2004, 126,

4496-4497.

11. Rasmussen, R. R.; Nuss, M. E.; Scherr, M. H.; Mueller, S. L.;

McAlpine, J. B.; Mitscher, L. A. "Benzanthrins A and B, a new class 156

of quinone antibiotics. II. Isolation, elucidation of structure and

potential antitumor activity," The Journal of antibiotics 1986, 39,

1515-1526.

12. Drautz, H.; Zahner, H.; Rohr, J.; Zeeck, A. "Metabolic products of

microorganisms. 234. Urdamycins, new angucycline antibiotics from

Streptomyces fradiae. I. Isolation, characterization and biological

properties," The Journal of Antibiotics 1986, 39, 1657-1669.

13. Rohr, J.; Zeeck, A. "Metabolic products of microorganisms. 240.

Urdamycins, new angucycline antibiotics from Streptomyces fradiae.

II. Structural studies of urdamycins B to F," The Journal of Antibiotics

1987, 40, 459-467.

14. Rohr, J.; Beale, J. M.; Floss, H. G. "Urdamycins, new angucycline

antibiotics from Streptomyces fradiae. IV. Biosynthetic studies of

urdamycins A-D," The Journal of Antibiotics 1989, 42, 1151-1157.

15. Kunzel, E.; Faust, B.; Oelkers, C.; Weissbach, U.; Bearden, D. W.;

Weitnauer, G.; Westrich, L.; Bechthold, A.; Rohr, J. "Inactivation of

the urdGT2 gene, which encodes a glycosyltransferase responsible for

the C-glycosyltransfer of activated D-olivose, leads to formation of

the novel urdamycins I, J, and K," J. Am. Chem. Soc. 1999, 121,

11058-11062. 157

16. Rix, U.; Remsing, L. L.; Hoffmeister, D.; Bechthold, A.; Rohr, J.

"Urdamycin L: A novel metabolic shunt product that provides

evidence for the role of the urdM gene in the urdamycin A

biosynthetic pathway of Streptomyces fradiae TU 2717,"

Chembiochem 2003, 4, 109-111.

17. Sezaki, M.; Kondo, S.; Maeda, K.; Umezawa, H.; Ono, M. "The

structure of aquayamycin," Tetrahedron 1970, 26, 5171-5190.

18. Omura, S.; Tanaka, H.; Oiwa, R.; Awaya, J.; Masuma, R.; Tanaka, K.

"New antitumor antibiotics, OS-4742 A1, A2, B1 and B2 produced by

a strain of Streptomyces," The Journal of Antibiotics 1977, 30, 908-

916.

19. Kawashima, A.; Kishimura, Y.; Tamai, M.; Hanada, K. "New platelet

aggregation inhibitors," Chem. Pharm. Bull. 1989, 37, 3429-3431.

20. Henkel, T.; Ciesiolka, T.; Rohr, J.; Zeeck, A. "URDAMYCINS, NEW

ANGUCYCLINE ANTIBIOTICS FROM STREPTOMYCES

FRADIAE. DERIVATIVES OF URDAMYCINA," The Journal of

Antibiotics 1989.

21. Sezaki, M.; Hara, T.; Ayukawa, S.; Takeuchi, T.; Okami, Y.; Hamada,

M.; Nagatsu, T.; Umezawa, H. "Studies on a New Antibiotic Pigment

Aquayamycin," The Journal of Antibiotics 1968, 21, 91-&. 158

22. Krohn, K.; Frese, P.; Florke, U. "Biomimetic synthesis of the racemic

angucyclinones of the aquayamycin and WP 3688-2 types," Chem.

Eur. J. 2000, 6, 3887-3896.

23. Yamaguchi, M.; Okuma, T.; Horiguchi, A.; Ikeura, C.; Minami, T.

"Total Synthesis of (-)-Urdamycinone-B through Polyketide

Condensation," J. Org. Chem. 1992, 57, 1647-1649.

24. Boyd, V. A.; Sulikowski, G. A. "Total Synthesis of the Angucycline

Antibiotics Urdamycinone-B and 104-2 Via a Common Synthetic

Intermediate," J. Am. Chem. Soc. 1995, 117, 8472-8473.

25. Kim, K.; Boyd, V. A.; Sobti, A.; Sulikowski, G. A. "A unified

strategy for the total synthesis of the angucycline antibiotics SF

2315A, urdamycinone B, and the shunt metabolite 104-2," Isr. J.

Chem. 1997, 37, 3-22.

26. Matsuo, G.; Miki, Y.; Nakata, M.; Matsumura, S.; Toshima, K. "Total

synthesis of urdamycinone B via C-glycosidation of an unprotected

sugar and Diels-Alder reaction of C-glycosyl juglone," Chem.

Commun. 1996, 225-226.

27. Matsuo, G.; Miki, Y.; Nakata, M.; Matsumura, S.; Toshima, K. "Total

synthesis of C-glycosylangucycline, urdamycinone B, using an

unprotected sugar," J. Org. Chem. 1999, 64, 7101-7106. 159

28. Krohn, K.; Agocs, A.; Bauerlein, C. "Total synthesis of angucyclines.

XVII. First synthesis of antibiotic 100-1, a deoxydisaccharide

angucycline antibiotic of the urdamycinone B-type," J. Carbohydr.

Chem. 2003, 22, 579-592.

29. Kirschning, A.; Chen, G. W.; Drager, G.; Schuberth, I.; Tietze, L. F.

"Syntheses and biological evaluation of new glyco-modified

angucyclin-antibiotics," Bioorg. Med. Chem. 2000, 8, 2347-2354.

30. Kusumi, S.; Nakayama, H.; Kobayashi, T.; Kuriki, H.; Matsumoto,

Y.; Takahashi, D.; Toshima, K. "Total Synthesis of Aquayamycin,"

Chem. Eur. J. 2016, 22, 18733-18736.

31. Matsumoto, T.; Yamaguchi, H.; Hamura, T.; Tanabe, M.; Kuriyama,

Y.; Suzuki, K. "Synthetic study of aquayamycin. Part 1: Synthesis of

3-(phenylsulfonyl)phthalides possessing a beta-C-olivoside,"

Tetrahedron Lett. 2000, 41, 8383-8387.

32. Yamaguchi, H.; Konegawa, T.; Tanabe, M.; Nakamura, T.;

Matsumoto, T.; Suzuki, K. "Synthetic study of aquayamycin. Part 2.

Synthesis of the AB ring fragment," Tetrahedron Lett. 2000, 41,

8389-8392.

160

33. Matsumoto, T.; Yamaguchi, H.; Tanabe, M.; Yasui, Y.; Suzuki, K.

"Synthetic study of aquayamycin. Part 3. First total synthesis,"

Tetrahedron Lett. 2000, 41, 8393-8396.

34. Khatri, H. R.; Nguyen, H.; Dunaway, J. K.; Zhu, J. "Total Synthesis

of Antitumor Antibiotic Derhodinosylurdamycin A," Chem. Eur. J.

2015, 21, 13553-13557.

35. Adhikari, S.; Baryal, K. N.; Zhu, D.; Li, X.; Zhu, J. "Gold-Catalyzed

Synthesis of 2-Deoxy Glycosides Using S-But-3-ynyl Thioglycoside

Donors," ACS Catalysis 2012, 3, 57-60.

36. Sparks, S. M.; Chen, C. L.; Martin, S. F. "Tandem Intramolecular

Benzyne-Furan Cycloadditions. Total Synthesis of Vineomycinone

B(2) Methyl Ester," Tetrahedron 2007, 63, 8619-8635.

37. Kowalski, C. J.; Weber, A. E.; Fields, K. W. "Alpha-Keto Dianion

Precursors Via Conjugate Additions to Cyclic Alpha-Bromo Enones,"

J. Org. Chem. 1982, 47, 5088-5093.

38. Kobayashi, Y.; Feng, C.; Ikoma, A.; Ogawa, N.; Hirotsu, T.

"Synthesis of trans-2,6-disubstituted cyclohexanones through allylic

substitution," Org. Lett. 2014, 16, 760-763.

161

39. Eames, J.; Mitchell, H. J.; Nelson, A.; O'Brien, P.; Warren, S.; Wyatt,

P. "An efficient protocol for Sharpless-style racemic dihydroxylation,"

J Chem Soc Perk T 1 1999, 1095-1103.

40. Shimizu, I.; Minami, I.; Tsuji, J. "Palladium-Catalyzed Synthesis of

Alpha,Beta-Unsaturated Ketones from Ketones Via Allyl Enol

Carbonates," Tetrahedron Lett. 1983, 24, 1797-1800.

41. Ueberschaar, N.; Dahse, H. M.; Bretschneider, T.; Hertweck, C.

"Rational design of an apoptosis-inducing photoreactive DNA

intercalator," Angew. Chem. Int. Ed. Engl. 2013, 52, 6185-6189.

42. Kim, S. G.; Kee, I. S.; Park, Y. H.; Park, J. H. "Selective Cleavage of

Acetal-Type Ethers with Magnesium Bromide and Butyl Mercaptan in

Diethyl-Ether," Synlett 1991, 183-184.

43. Schmidt, B.; Krehl, S.; Kelling, A.; Schilde, U. "Synthesis of 8-aryl-

substituted coumarins based on ring-closing metathesis and Suzuki-

Miyaura coupling: synthesis of a furyl coumarin natural product from

Galipea panamensis," J. Org. Chem. 2012, 77, 2360-2367.

44. Okazaki, K.; Nomura, K.; Yoshii, E. "An Efficient Synthesis of 3-

Cyano-1(3h)-Isobenzofuranones," Synth. Commun. 1987, 17, 1021-

1027.

162

45. Mal, D.; Pahari, P. "Recent advances in the Hauser annulation,"

Chem. Rev. 2007, 107, 1892-1918.

46. Raw, A. S.; Pedersen, S. F. "Synthesis of Cis-1,2-Cyclobutanediols

Via Intramolecular Pinacol Coupling of 4-Oxo Aldehydes," J. Org.

Chem. 1991, 56, 830-833.

47. Tius, M. A.; Gomezgaleno, J.; Gu, X. Q.; Zaidi, J. H. "C-

Glycosylanthraquinone Synthesis - Total Synthesis of

Vineomycinone-B2 Methyl-Ester," J. Am. Chem. Soc. 1991, 113,

5775-5783.

48. Acharya, P. P.; Khatri, H. R.; Janda, S.; Zhu, J. "Synthesis and

antitumor activities of aquayamycin and analogues of

derhodinosylurdamycin A," Organic & biomolecular chemistry 2019,

17, 2691-2704.

49. Toshima, K.; Nagai, H.; Ushiki, Y.; Matsumura, S. "Novel

glycosidations of glycals using BCl3 or BBr3 as a promoter for

catalytic and stereoselective syntheses of 2-deoxy-alpha-glycosides,"

Synlett 1998, 1007-+.

50. De Lederkremer, R. M.; Marino, C. "Deoxy sugars: Occurrence and

synthesis," Adv Carbohyd Chem Bi 2008, 61, 143-216.

163

51. Kirschning, A.; Bechthold, A. F. W.; Rohr, J. "Chemical and

biochemical aspects of deoxysugars and deoxysugar

oligosaccharides," Top. Curr. Chem. 1997, 188, 1-84.

52. Kongkathip, N.; Kongkathip, B.; Siripong, P.; Sangma, C.;

Luangkamin, S.; Niyomdecha, M.; Pattanapa, S.; Piyaviriyagul, S.;

Kongsaeree, P. "Potent antitumor activity of synthetic 1,2-

Naphthoquinones and 1,4-Naphthoquinones," Biorg. Med. Chem.

2003, 11, 3179-3191.

53. Trefzer, A.; Hoffmeister, D.; Kunzel, E.; Stockert, S.; Weitnauer, G.;

Westrich, L.; Rix, U.; Fuchser, J.; Bindseil, K. U.; Rohr, J.;

Bechthold, A. "Function of glycosyltransferase genes involved in

urdamycin A biosynthesis," Chem Biol 2000, 7, 133-142.

54. Bililign, T.; Griffith, B. R.; Thorson, J. S. "Structure, activity,

synthesis and biosynthesis of aryl-C-glycosides," Nat. Prod. Rep.

2005, 22, 742-760.

55. Hou, D.; Lowary, T. L. "Recent advances in the synthesis of 2-deoxy-

glycosides," Carbohydr. Res. 2009, 344, 1911-1940.

56. Lown, J. W. "Discovery and Development of Anthracycline

Antitumor Antibiotics," Chem. Soc. Rev. 1993, 22, 165-176.

164

57. Newman, R. A.; Yang, P. Y.; Pawlus, A. D.; Block, K. I. "Cardiac

glycosides as novel cancer therapeutic agents," Mol. Interventions

2008, 8, 36-49.

58. Weymouth-Wilson, A. C. "The role of carbohydrates in biologically

active natural products," Nat. Prod. Rep. 1997, 14, 99-110.

59. Driguez, H. "Thiooligosaccharides in glycobiology," Top. Curr.

Chem. 1997, 187, 85-116.

60. Szilagyi, L.; Varela, O. "Non-conventional glycosidic linkages:

Syntheses and structures of thiooligosaccharides and carbohydrates

with three-bond glycosidic connections," Curr. Org. Chem. 2006, 10,

1745-1770.

61. Driguez, H. "Thiooligosaccharides as tools for structural biology,"

Chembiochem 2001, 2, 311-318.

62. Baryal, K. N.; Zhu, D.; Li, X.; Zhu, J. "Umpolung reactivity in the

stereoselective synthesis of S-linked 2-deoxyglycosides," Angew.

Chem. Int. Ed. Engl. 2013, 52, 8012-8016.

63. Baryal, K. N.; Zhu, J. L. "Sterioselective Synthesis of S-Linked 2-

Deoxy Sugars," Synlett 2014, 25, 308-312.

165

64. Crich, D.; Ritchie, T. J. "Preparation of 2-Deoxy-Beta-D-Lyxo-

Hexosides (2-Deoxy-Beta-D-Galactosides)," Carbohydr. Res. 1989,

190, C3-C6.

65. Sherry, B. D.; Loy, R. N.; Toste, F. D. "Rhenium(V)-catalyzed

synthesis of 2-deoxy-alpha-glycosides," J. Am. Chem. Soc. 2004, 126,

4510-4511.

66. Issa, J. P.; Lloyd, D.; Steliotes, E.; Bennett, C. S. "Reagent controlled

beta-specific dehydrative glycosylation reactions with 2-deoxy-

sugars," Org. Lett. 2013, 15, 4170-4173.

67. Baryal, K. N.; Zhu, J. "Stereoselective Synthesis of S-Linked

Hexasaccharide of Landomycin A via Umpolung S-Glycosylation,"

Org. Lett. 2015, 17, 4530-4533.

68. Acharya, P. P.; Baryal, K. N.; Reno, C. E.; Zhu, J. "Synthesis of S-

linked trisaccharide glycal of derhodinosylurdamycin A: Discovery of

alkyl thiocyanate as an efficient electrophile for stereoselective

sulfenylation of 2-deoxy glycosyl lithium," Carbohydr. Res. 2017,

448, 103-109.

69. Baryal, K. N.; Adhikari, S.; Zhu, J. "Catalytic stereoselective

synthesis of beta-digitoxosides: direct synthesis of digitoxin and C1'-

epi-digitoxin," J. Org. Chem. 2013, 78, 12469-12476. 166

70. Li, X.; Woodward, J.; Hourani, A.; Zhu, D.; Ayoub, S.; Zhu, J.

"Synthesis of the 2-deoxy trisaccharide glycal of antitumor antibiotics

landomycins A and E," Carbohydr. Res. 2016, 430, 54-58.

71. Shing, T. K. M.; Leung, G. Y. C. "Asymmetric epoxidation catalyzed

by D-glucose-derived uloses," Tetrahedron 2002, 58, 7545-7552.

72. Dick, W. E.; Weisleder, D.; Hodge, J. E. "Dioxolane and Dioxane

Acetal Derivatives of D-Allose - Condensation of 3-O-Benzyl-D-

Allose with Acetaldehyde," Carbohydr. Res. 1975, 42, 55-63.

73. Lee, D.; Williamson, C. L.; Chan, L.; Taylor, M. S. "Regioselective,

borinic acid-catalyzed monoacylation, sulfonylation and alkylation of

diols and carbohydrates: expansion of substrate scope and mechanistic

studies," J. Am. Chem. Soc. 2012, 134, 8260-8267.

74. Boons, G. J.; Isles, S. "Vinyl glycosides in oligosaccharide synthesis

.2. The use of allyl and vinyl glycosides in oligosaccharide synthesis,"

J. Org. Chem. 1996, 61, 4262-4271.

75. Roush, W. R.; Bennett, C. E. "A highly stereoselective synthesis of 2-

deoxy-beta-glycosides using 2-deoxy-2-iodo-glucopyranosyl acetate

donors," J. Am. Chem. Soc. 1999, 121, 3541-3542.

167

168