Synthetic Uses of S-Nitrosothiols in Organic Chemistry

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SYNTHETIC USES OF S-NITROSOTHIOLS IN ORGANIC CHEMISTRY

TYLER DAVID BIGGS

A dissertation submitted in partial fulﬁllment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Chemistry

MAY 2017

The members of the Committee appointed to examine the dissertation of

TYLER DAVID BIGGS ﬁnd it satisfactory and recommend that it be accepted.

Ming Xian, Ph.D., Chair

Cliﬀ E. Berkman, Ph.D.

Aurora Clark, Ph.D.

Jeﬀery Jones, Ph.D.

ii ACKNOWLEDGMENT

I would like to thank Dr. Ming Xian for his supervision, help, and guidance over the course of my Ph.D. study. His challenges and questions kept me on track, and forced me to constantly reevaluate myself critically. I would also like to thank

Dr. Ronald for helping to kindle my interest and passion in chemistry.

I would like to thank my committee members for sharing their time and knowledge. I would also like to thank the Washington State University Chemistry department for the resources and equipment provided, and those in the front oﬃce who keep things running.

Finally I would like to thank my fellow students who have supported me. Ryan

Joseph, for his help and enthusiasm in chemistry and LATEX. Nelmi Devarie, for talks of cooking and science. Laksiri, Hua Wang, and Chung-Min Park for their help in training me in the basics.

Most importantly are my family and friends, with out their support I would have never started this journey, or have a place to go up on ﬁnishing it, without their help and commitment.

iii SYNTHETIC USES OF S-NITROSOTHIOLS IN ORGANIC CHEMISTRY

Abstract

by Tyler David Biggs, Ph.D. Washington State University May 2017

Chair: Ming Xian

S-Nitrosothiols have risen to prominence since their identification as an important post-translational modification of cysteine residues. The full range of chemistry and biological implications of this motif are under exploration. Direct chemical reactions of S-nitrosothiols should reveal clues into their biological effects, and produce new synthetic tools for the construction of sulfur and nitrogen containing compounds.

Here several explorations into synthetic applications of S-Nitrosothiols are explored. The relevant background literature is reviewed. Then a phosphine mediated reductive ligation of aldehydes and S-Nitrosothiols is discussed. Followed the development of a proline-based phosphine reagent and its reactivity with

S-nitrosothiols is explored.

iv TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... iii

ABSTRACT ...... iv

A REVIEW OF S-NITROSOTHIOLS ...... 1

1.1 Introduction...... 1

1.1.1 Historical Context...... 1

1.1.2 Chemical Background...... 2

1.1.3 Biological Signiﬁcance...... 3

1.2 Properties of S-Nitrosothiols...... 4

1.2.1 Structure and Electronic Nature of S-Nitrosothiols...... 5

1.2.2 Isomerization of S-Nitrosothiols...... 6

1.3 Spectra...... 7

1.3.1 Infrared and Raman Spectra...... 7

1.3.2 UV-Vis Spectra...... 8

1.4 S-Nitrosation...... 8

1.4.1 Direct Reaction of Thiols with Nitric Oxide...... 8

1.4.2 Nitrosation by Nitrite in Acidic Media...... 10

1.4.3 Nitrosation by Alkyl Nitrites...... 10

1.4.4 Transnitrosation...... 11

1.5 Decomposition...... 12

1.5.1 Homolytic & Heterolytic Cleavage of the S-N Bond...... 13

1.5.2 Rates of Decomposition of S-Nitrosothiols...... 14

v 1.5.3 Metal-based SNO Decomposition...... 15

1.6 Reactions of S-Nitrosothiols...... 16

1.6.1 Bioorthogonal Reactions of SNO...... 16

1.6.2 Reactions with Phosphines...... 16

1.6.3 Light Induced Reactions...... 28

1.6.4 Reactions with Sulfenic Acids...... 29

1.6.5 Reactions with Carbanions...... 30

1.6.6 Miscellaneous Reactions...... 31

CONJUGATION OF S-NITROSOTHIOLS WITH ALDEHYDES ...... 34

2.1 Introduction...... 34

2.1.1 Hypothesis...... 34

2.2 Results and Discussion...... 35

2.2.1 Experimental Plan...... 35

2.2.2 Preparation of Starting Materials...... 36

2.2.3 Condition Screen...... 37

2.2.4 Intramolecular Substrate Screen...... 38

2.2.5 Intermolecular Substrate Screen...... 38

2.2.6 Application - Synthesis of Benzoisothiazole...... 41

2.3 Conclusion...... 43

2.A Appendix...... 43

2.A.1 Methods...... 43

PROLINE BASED PHOSPHORAMIDITE REDUCTIVE LIGATION REAGENTS 49

3.1 Introduction...... 49

3.1.1 Rationale for S-N bond Formation Methodology Development 50

3.2 Results and Discussion...... 52

3.2.1 Experimental Plan...... 52

3.2.2 Reaction Screening...... 53

3.2.3 Removal of the Phosphine Oxide Moiety...... 55

vi 3.2.4 Use as a Nitroxyl trap...... 55

3.3 Conclusion...... 58

3.A Appendix...... 58

3.A.1 Methods...... 58

3.A.2 Preparation of S-Nitrosothiols...... 60

3.A.3 General Reductive Ligation Procedure...... 60

3.A.4 Deprotection of the Diphenylphosphoryl Group...... 64

BIBLIOGRAPHY ...... 78

vii LIST OF TABLES

Page

1.1 Bond lengths and angles of selected S-nitrosothiols...... 5

1.2 Characteristic IR bands of SNO...... 7

1.3 Characteristic UV/Vis bands S-nitrosothiols...... 8

1.4 Rates of nitrosation of the thiolate anion...... 11

1.5 Rates of transnitrosation by SNAP...... 12

1.6 Half-life values of selected SNO compounds...... 15

1.7 Thioether formation from SNO...... 31

1.8 Generation of polysulﬁdes from copper (II) halides...... 32

2.1 Solvent screen for the intramolecular phosphine mediated conjugation. 38

2.2 Substrate screen of the intramolecular conjugation...... 39

2.3 Aldehyde substrates amenable to the conjugation...... 41

3.1 Substrate screen of the proline-based phosphoramidate coupling.. 56

viii LIST OF FIGURES

Page

1.1 Seminal nitrosation of phenyl mercaptan...... 2

1.2 Resonance description of S-Nitrosothiols...... 5

1.3 The cis-trans isomerization of t-butyl-S-nitrosothiol...... 7 1.4 Mechanism of the Staudinger Reaction...... 18

1.5 Radical TrSNO addition to oleﬁns...... 29

2.1 General form of the aldehyde & SNO coupling...... 34

2.2 Benzoisothiazole containing pharmaceuticals...... 42

3.1 General form of the proline based phosphoramidite coupling..... 49

3.2 S-N linkages in pharmaceuticals...... 50

ix LIST OF SCHEMES

Page

1.1 Glutathione and cysteine SNO...... 4

1.2 Synthesis of a stable S-nitrosothiol iridium complex...... 6

1.3 Formation of a thio-azaylide...... 17

1.4 Trapping of a nitrene derived from TrSNO...... 17

1.5 Proposed nitrene mechanism pathway A...... 18

1.6 Proposed 3-membered ring mechanism pathways B1 & B2...... 19

1.7 Fast reductive ligation...... 19

1.8 Mechanism of the reductive ligation...... 20

1.9 Traceless ligations using phosphine esters/thioesters...... 20

1.10 Mechanism of the traceless ligation...... 21

1.11 Formation of thioimidates from phosphine thioesters...... 22

1.12 One-step reductive disulﬁde formation...... 22

1.13 One-step disulﬁde formation from SNO...... 23

1.14 SNO labeling with the traceless ligation reagent...... 24

1.15 One-pot thioether formation...... 24

1.16 Mechanism of the one-pot thioether formation...... 25

1.17 Bis-ligation of primary SNO...... 26

1.18 Dehydroalanine formation...... 26

1.19 Determination of the intra-molecular elimination...... 27

1.20 A water soluble phosphine...... 27

1.21 One-pot formation of benzoisothiazoles...... 28

1.22 Synthesis of α-tritylthio oximes...... 29

x 1.23 Dimerization of allyl SNO compounds...... 29

1.24 Thiosulfonates from sulfenic acids and S-nitrosothiols...... 30

1.25 Cross-reactivity proﬁle of SNO and sulfenic acids...... 30

1.26 Asymmetrical disulﬁde formation from SNO...... 31

1.27 Amines converted to their corresponding aryl halides...... 32

1.28 Yields of 2-aryl-1-haloethanes...... 33

2.1 Inspiration for aldehyde conjugation from our previous work.... 35

2.2 Proposed mechanism of an intramolecular conjugation of an alde-

hyde and SNO...... 36

2.3 Synthesis of 2-(diphenylphosphaneyl) benzaldehyde...... 37

2.4 Benzaldehyde fails to couple with TrSNO...... 40

2.5 Cinnamaldehyde successfully undergoes the intermolecular conjuga-

tion...... 40

2.6 One-pot synthesis of benzoisothiazole...... 43

3.1 Phosphine based reductive ligation...... 52

3.2 General form of the proline-based ligation...... 53

3.3 Preparation of the proline-phosphoramidate - esteriﬁcation...... 53

3.4 Preparation of the proline-phosphoramidate - phosphoramidite for-

mation...... 54

3.5 Trityl-SNO coupled to the proline-phosphoramidite...... 54

3.6 Dehydroalanine formation with primary SNO...... 57

3.7 Removal of the phosphine moiety under acidic conditions...... 57

3.8 Trapping of HNO with the proline phosphoramidite reagent..... 58

xi Dedication

Without the support I have received this journey would not have been possible.

From my parents, Liz and Dave, who instilled drive and character in me. From my brother, Conner, who’s friendship has sustained me. From my wife, Karin, without her support and belief I surely would not have been successful. Finally to my Son,

Logan, the Sunshine of his Mom’s and Dad’s lives.

I hope to make you all proud.

xii CHAPTER 1. A REVIEW OF S-NITROSOTHIOLS

1.1 INTRODUCTION

1.1.1 Historical Context

Interest in S-Nitrosothiols (SNO) has surged since its identification as a post- translational modification induced by nitric oxide (NO). NO is a gaseous signaling molecule that affects many biological pathways. The process of S-nitrosation in vivo is referred to as S-nitrosylation. The full nature of NO and SNO role in biological systems is under active investigation.[1] The unstable nature of both SNO and NO complicate examination of their physiological roles. The detection of biological

SNO is diﬃcult and error-prone, and new methods are needed to address these issues.[2]

Unlike other post-translational modiﬁcations, protein sequence motifs do not reveal a pattern of S-nitrosated cysteine residues. Analysis of cysteine residue properties, including pKa, exposed surface availability, secondary structure and hy- drophobicity all fail as predictors of SNO sites. Tools to detect SNO are limited and error prone. Despite these issues, modulation of NO levels is used therapeutically.

The longest running example being the use of nitroglycerin to treat heat disease. In fact, amyl nitrite has been used to treat chest pains as early as 1857. Despite the use of these drugs for over 150 years, their mechanisms of action are still poorly understood. With this motivation we set out to examine and explore the synthetic chemistry of SNO.

1 This review will focus speciﬁcally on the known chemistry of SNO. Readers interested in the biological aspects of SNO are invited to read one of the reviews available on biological SNO detection,[3] the physiological eﬀects of the SNO/NO system,[4] or biomedical applications.[5] Two previous reviews have been written,[6,

7] and these cover in great detail the initial exploration of SNO chemistry.

1.1.2 Chemical Background

While chemists have been aware of S-Nitrosothiols for over 100 years, only recently has this functionality been discovered to have biological importance. The ﬁrst reported synthesis of S-Nitrosothiols was reported in 1909 by H. Tasker and H.

Jones[8].

SH Cl N O S O N + HCl

Figure 1.1: Seminal nitrosation of phenyl mercaptan.

A solution of phenyl mercaptan was treated with nitrosyl chloride (Figure 1.1)

Noted in their report is the characteristic color change indicating the formation of the S-nitrosothiol, followed by the evolution of nitric oxide and recovery of the disulfide. Phenyl-SNO and other aryl-SNO compounds are noticeably less stable than their alkyl counterparts. So it should not come as a great surprise that early chemists did not think much of this transient species. SNO was then largely ignored by the synthetic community for the next 60 or so years, until it was identified as a protein post-translational modification. Chemists at the time were far more interested in determining if a azo-dyes could be used as antibiotics. It was common to study sulfur-nitrogen linkages in various oxidation states in an exploratory manner, and much of the initial work on SNO was done in this context.

There is now a renewed interest in this functionality and its chemistry. Our group has been exploring new reactions of this species in an attempt to develop new reactions to label SNO in proteins. This area is still under active investigation.

2 A series of methods have been developed for SNO detection, a full discussion of these methods is outside the scope of this review, those interested should refer to one of several reviews on the subject.[3,9]

1.1.3 Biological Signiﬁcance

In 1981 Ignarro et al. proposed that nitric oxide (or an SNO derivative) was the endothelium derived relaxing factor.[10] Since, S-nitrosylation has been found to be an important post-translational modiﬁcation that not only modulates biological activity of NO species but has also been shown to regulate protein function.[11]

The levels of nitrosylated protein thiols are thought to be highly regulated, and imbalance of these levels is associated with several pathologies. Recently studies have found complexes with NOS isoforms which direct S-nitrosylation to speciﬁc protein targets.[1]

Enzymatic Production of Nitric Oxide Three nitric oxide synthase (NOS) isoforms generate NO in mammals: 1) endothelial; found in endothelium tissue, 2) neuronal; found in nervous tissue and skeletal muscle, and, 3) inducible; found in the cardiovascular & immune systems. These enzymes catalyze the conversion of

L-arginine to citrulline and nitric oxide.

+ NOS + 2L-arginine + 3NADPH + 1H + 4O2 2citrulline + 2NO + 3NADP

The only known NO receptor is soluble guanylate cyclase (sGC). sGC has a heme co- factor to which NO binds and thereby stimulates the production of cyclic guanosine monophosphate. Our knowledge of these processes is incomplete, as NO eﬀects are abundant even in the absence of sGC.

S-nitrosylation of cysteine residues and other small molecule thiols are hypothesized to be the pathways of signal transduction. It is also been proposed that some proteins may be directly nitrosated thought that the NO synthases may directly

3 nitrosylate some proteins. The most common small molecule biological thiols are cysteine and glutathione, present at mM concentrations. Their nitrosated forms are shown in Scheme 1.1

O N S O OO O H N N O S OH HO N OH H NH2 NH2 O cysteine glutathione

Scheme 1.1: Glutathione and cysteine SNO

The complexity of this signaling pathway is compounded by interactions between NO and other small inorganic compounds, such as O2, super-oxide, hydrogen sulfide (and other polysulfides), as well as interactions with metal centers. The accurate detection of S-nitrosation in biological systems is still a challenge due to the labile nature of the SNO moiety. With this problem in mind, our group and others have been working towards bioorthogonal labeling of SNO sites. These projects have forced us to consider SNO under a new light, and to wonder if they might find more applications in the synthetic chemists toolbox.

1.2 PROPERTIES OF S-NITROSOTHIOLS

Small molecule organic S-nitrosothiols are characteristically colored a vivid red or green. The red and green colors correspond to the substitution nature of the alkyl group, primary and secondary as red, and tertiary as green. Few SNO compounds are stable in a pure form, instead they decompose thermally and photochemically to give the corresponding disulﬁde and nitric oxide. The rates of decomposition are substrate and condition dependent.

Some tertiary and bulky compounds like trityl-S-nitrosothiol (TrSNO, where

– trityl = Ph3C ) and S-nitroso-N-acetyl-penicillamine (SNAP) can be isolated as stable solids. Solid SNAP must to be heated to ca. 150 ◦C before decomposition occurs[12]. However most small molecule SNO are unstable with half-lives measured

4 in hours, and one should expect to have to prepare them freshly prior to use.

1.2.1 Structure and Electronic Nature of S-Nitrosothiols

The ﬁrst X-ray crystal structure of an SNO, S-nitroso-penicillamine was published in 1978, further studies of this type did not occur until 1999. This can be attributed to the use of N2O4 based nitrosative conditions being overly oxidative, preventing the isolation of clean SNO samples. Arulsamy[13] et. al used a system of aqueous acidiﬁed nitrite to generate the SNO, then extracted the compound cleanly into an organic layer. This method cleanly prepares TrSNO suitable for examination by

X-ray crystallography. This X-ray data is summarized in Table 1.1.

bond lengths, Å bond angles, ° z }| { z }| { N–OS–NC–SO–N–SN–S–CC–S–N–O

Ph3C – SNO[13] 1. 777(6) 1 . 795(5) 1 . 867(3) 114 . 0(4) 102 .1(2) 175 . 7 SNAP[14] 1.206(3) 1.762(3) 1.833(1) 113.99(11) 100.80(7) 176.3

Table 1.1: Bond lengths and angles of trityl S-nitrosothiol and S-nitroso-N-acyl- penicillamine.

The dihedral angles of SNO are indicative of a double bond along the S–N axis.

Typical S – N double bonds are reported to be 1.5 Å,[15] slightly shorter than those observed in SNO, which range from 1.76 Å to 1.85 Å.[16]

Timerghazin et al. have proposed that SNOs are better understood by a com- bination of resonance structures.[17] PBE0/aug-pc-1 density functional theory calculations and natural resonance theory calculations were used to generate the resonance description outlined in Figure 1.2. The NRT theory used predicts the structures shown and their normalized contributions.

O S S O S O R N R N R N SDI

Figure 1.2: The resonance structures are designated S, D, I; for standard, double-bond and ionic, respectively.

5 These are: (S) the standard R – SNO lewis structure, (D) a double bond between along the S–N bond, and (I) an ionic resonance. Resonance structure S is dominant

(70 % to 80 %), followed by D ( 15 % to 25 %) and lastly I ( 6 % to 10 %). ∼ ∼ Lewis acid interaction with at the oxygen position favors the D resonance.

While interaction with at the nitrogen favors the standard (S) resonance. Lewis acid interaction with the sulfur atom favors the ionic resonance structure (I). These resonance structures provide satisfactory predictive explanations of some of the properties observed of SNO.

The S–N bond can be signiﬁcantly altered upon interactions with metals.[18] It has been shown that coordination with CuI weakens the S–N bond, both computa- tionally[19] and experimentally[20]. Coordination with N is predicted to strengthen the S – N bond[21].

O N N S S O K[IrCl5NO] Cl Cl Ir Cl CH3CN N Cl 1a 2

Scheme 1.2: Synthesis of a stable S-nitrosothiol iridium complex.

Perissinotti et al. found[22] that benzyl-SNO (1a) forms a (surprisingly) stable complex. Treatment (Scheme 1.2) of benzyl-SNO (1a) with K[IrCl5(NO)] in acetonitrile at room temperature produces the complex 2, which crystallizes from solution.

X-ray crystallography conﬁrmed the structure of 2.

1.2.2 Isomerization of S-Nitrosothiols

The SNO motif contains some double bond character along the S–N bond. This imparts a cis-trans property, as shown in Figure 1.3, which is responsible for the color shift between the (cis-) red primary and secondary SNO, and the (trans-) green tertiary SNO. Bartberger et al. noted that the visible absorption by SNO in the 520 nm to 590 nm range corresponds to a n π* transition.[23] The maximum absorption of the trans conformers are red-shifted by ca 30 nm. This absorption

6 band is responsible for the change in color.

S S O N N O cis trans

1 Figure 1.3: Cis-trans isomerization of t-butyl-SNO, 10.0(77) kcalmol−

The rotational energy barrier for t-butyl-SNO (tBuSNO)has been determined experimentally by Arulsamy et al. by synthesizing 15N tBuSNOand performing variable temperature 15N NMR. [13] The structures and value are shown in Fig- ure 1.3.

1.3 SPECTRA

1.3.1 Infrared and Raman Spectra

Infra-red (IR) and Raman spectra are not commonly used techniques for charac- terizing SNO. Some reports indicate that acquisition of these spectra can induce decomposition. In IR spectra, two shifts are attributed to SNO at 1500 cm 1 and ∼ − 650 cm 1. These peaks appear in the same regions in Raman spectra, they are at- ∼ − tributed to the VNO and VNS vibrations. A useful feature to note is the complete loss 1 of the VSH stretching band in infra-red spectra at 2600 cm− . Upon S-nitrosation, the S–H vibration of GSNO is visible at up to 5 % thiol contamination.[13] Typical ranges for these absorptions are given in Table 1.2.

1 1 Alkyl Group VNO, cm− VNS, cm− 1°, 2° 1500 - 1530 610 - 650 3° 1450 - 1500 650 - 685 aromatic 1430 - 1710 1000 - 1170

Table 1.2: Characteristic IR bands of SNO.

7 1.3.2 UV-Vis Spectra

Some SNO compounds can be observed by UV/vis spectrometry in aqueous solutions. Drawbacks to this method include a required puriﬁcation before measurement, either by HPLC or electrophoresis. The sensitivity of this method is relatively low, due to the poor molar absorptivity () of SNO compounds. UV/Vis spectra of SNOs show three bands, their assignments and typical ranges are shown in Table 1.3.

range transition 3 1 1 (nm) (dm mol− cm− ) π π* 255 261 10,000 20,000 − ∼ nO π* 340 1000 n π* 550∼ 600 10 20 N − ∼

Table 1.3: Characteristic UV/Vis bands SNO. The nN π* transition is responsible for the red or green color of SNO.

1.4 S-NITROSATION

The two most facile methods for the synthesis of SNO compounds involve the treatment of the starting thiol with either: an aqueous acidiﬁed nitrite solution[24], or the addition of an alkyl nitrite in an organic solvent.[25] Both of these methods easily convert most thiols to the corresponding SNO within 5 to 15 minutes. Lower temperatures may be required, depending on the stability of the target SNO.

1.4.1 Direct Reaction of Thiols with Nitric Oxide

Historically a range of N-oxides (NOCl, N2O4, N2O3, NO2, HNO2) were used in inert solvents (eg. CCl4) to generate SNO from the corresponding thiol.[6] When these reactions are carried out at low temperatures (<263 K) SNOs can be obtained in high yields. Care must be taken, as this system is sensitive to disulﬁde formation.

Sundquist et al.[26] determined that the active nitrosation reagent in these systems as N2O3. The mechanism of nitrosation for these N-oxides appears to be a

8 series of disproportionation and redox reactions. More importantly, SNO are often not the ‘ﬁnal’ product of these reaction mixtures, sulfonic anhydrides and other oxidation products of the starting thiol are often obtained if the build up of nitric acid is not controlled.[27] Oae et al. did identify the intermediate presence of SNO in such reaction mixtures by the characteristic color change.

Under anaerobic conditions, direct reactions between thiols and nitric oxide

(NO) do not produce SNO. Instead small molecule thiols are oxidized to their corresponding disulfide[28]. Oxidation of a R–S–N – O• radical by oxygen is thought to be pathway required for nitrosation to occur directly from NO. Conversely, under aerobic conditions SNOs are formed. Kinetic studies of such systems reveal first order oxygen concentration, and second order NO concentration dependence. Thiol concentrations show effects only at low concentrations.

The generation of SNO by NO and O2 follows the reactions outlined in below.

2NO• + O2 2NO2

NO2 + NO• N2O3

N2O3 2NO2•

NO• + NO2• N2O3

+ N2O3 + H2O 2H + 2NO2−

+ N2O3 + RSH H + NO2− + RSNO

At higher concentrations of thiols, the above rate equation simpliﬁes to:

d[RSNO] = k [NO]2[O ] (1.1) dt NO 2

These equations and empirical results obtained by Goldstein et al. support the equations outlined.[29] The rate-determining step of the auto-oxidation chain has been shown to be the formation of N2O3.

9 1.4.2 Nitrosation by Nitrite in Acidic Media

Addition of acidiﬁed nitrite cleanly and quickly generates SNO from the thiol starting material. This is a very convenient method to synthesize water soluble thiols, especially cysteine and other small alkyl thiols (eg t-butyl mercaptan). Treatment of the parent thiol in aqueous acid (typically 1N HCl) with 5 equivalents of sodium

1 nitrite (NaNO2) generates the desired SNO in 5 to 15 minutes. Methanol can be added to the reaction mixture to assist with solubility. After completion of the nitrosation the products can typically be extracted from the aqueous solution with diethyl ether or other organic solvents. The acidic conditions favor the protonated state of amines (like those in amino acids) and this helps deter unwanted

N-nitrosation. H+ RSH + HNO2 RSNO + H2O (1.2) keq k for these reactions range from 1 103 mol 1 s 1 to 1 105 mol 1 s 1.[30] This is eq × − − × − − far more favored than the analogous nitrosation of alcohols, this is attributed to the

O and S atoms diﬀerences in nucleophilicity and pKa values.

1.4.3 Nitrosation by Alkyl Nitrites

Conversion to SNO proceeds cleanly and nearly to completion at room temperature.

Examination of the reaction kinetics on pH dependence reveals that this reaction is thiolate dependent. A series of papers[25, 31–35] explored nitrosation of small molecule thiols with a series of alkyl nitrites. Williams et al. measured the reaction rates of a series of thiols with some simple alkyl nitrites. This reaction is dependent on total thiolate concentration, and reaches a maximum rate just below a pH of 10.

A series of alkyl nitrites and their nitrosation rates are shown in Table 1.4.

1 Occasionally 0.1 equivalents of H2SO4 is added.

10 S O O S O R + R N R N

Base Thiols R – SNO Cys CysOMe CysOEt AcNHCys Glu

(CH3)3CONO 1.7 1.6 1.5 1.8 1.8 (CH3)3CHONO 11 12 12 12 11 CH3CH2ONO 28 24 25 31 28 (CH3)2CH(CH2)2ONO 27 25 26 30 27 C2H5O(CH2)2ONO 169 150 165 169 159 CI(CH2)2ONO 1045 1050 1100 1010 1070 Br(CH2)2ONO 1055 1055 1085 1030 1055 I(CH2)2ONO 1060 1060 1080 1020 1060

1 1 Table 1.4: Rates (mol− s− ) of nitrosation of the thiolate anion at 25 ◦C.

1.4.4 Transnitrosation

Analogous to the alkyl nitrite transfer discussed previously. Transnitrosation between two thiols is typically an equilibrium reaction. Three forms of the thiol can be considered for a trans-nitrosation reaction: 1) The neutral thiol R – SH. 2) The radical thiyl R – S . 3) The anionic thiolate R – S – . · Of these equilibrium species, the neutral and anionic forms are the most pre- dominant, while the radical thiol concentration is typically very low. Li et al. used CBS-QB3 level of theory to explore possible mechanistic pathways.[36] They found the radical thiyl to have the lowest energy barrier to trans-nitrosation, followed by the thiolate anion and the neutral form. Since the thiolate is expected to dominate the equilibrium between the species, trans-nitrosation incidents are likely to occur via the thiolate.

SNAP has a very strong tendency to donate NO, which has been attributed to steric repulsion from the methyl groups. Transnitrosation can be monitored by UV- vis detection if the λmax gap between the two species is large enough. SNAP has a UV absorption at 590 nm. Primary SNO have a λmax around 540 nm. The UV-vis band gap between SNAP and primary SNOs is large enough to allow monitoring both species simultaneously. The system was modeled as shown in Table 1.5.Accordingly

Wang et al. used SNAP as a reference to gauge the transnitrosative properties of a

11 series of thiols.[37]

O O H k1 H N N SH S O OH + R R N + OH O N k2 O S O SH [NAP][RSNO] keq = k1/k2 = [SNAP][RSH]

1 Thiol pKa k1 keq keq− L-cysteine ethyl ester 6.50 6.45 10 1 4.99 0.20 × − L-cysteine 8.30 6.12 10 2 3.63 0.28 × − D-penicillamine 8.53 3.01 10 2 × − glutathione 8.75 1.68 10 2 3.34 0.30 × − N-acetyl cysteine 9.52 8.41 10 3 1.47 0.68 × − N-acetyl-D-penicillamine 9.90 9.90 10 3 × − Table 1.5: Rates of transnitrosation by SNAP.

1.5 DECOMPOSITION

S-nitrosothiols are generally unstable, and can decompose through a range of processes, dependent on the alkyl substituent and the experimental conditions.

Condition sensitivities include pH, light, temperature, as well as the presence of heavy-metals. The study of SNO stability is diﬃcult, as seemingly minor changes in reaction conditions yield dramatically diﬀerent pathways of decomposition. The stability of SNO varies greatly with the nature of the alkyl substituent.

Some are unstable oils that decompose over the course of minutes to hours, while others (such as TrSNO and SNAP) can be isolated as crystal solids that can be stored for months. Early literature reports tertiary SNO compounds are more thermally stable than their primary and secondary counterparts. However careful study has revealed that primary and secondary SNO are more thermally stable than their tertiary counterparts.[38] It was shown that n-butyl-SNO was fairly stable for 20 hours at 70 ◦C in a solution of deoxygenated solvent saturated with gaseous NO. An explanation comes from the reversibility of the homolytic cleavage, as shown

12 below.

R SNO R S• + NO• − −

R S• R S S R − − − −

Initial homolysis of the S–N bond gives two radical species, R–S• and NO•. Ac- cordingly, the mostly irreversible 2 decomposition to the disulfide then depends on the rate of disulfide formation from the thiyl. Tertiary radicals are more stable, and more sterically hindered from combining to the disulfide. Constant argon flow, commonly used to protect reactions from oxygen, forces out the gaseous NO and pushes the equilibrium to the disulfide.

1.5.1 Homolytic & Heterolytic Cleavage of the S-N Bond

Whether a given SNO decomposes in a homolytic or heterolytic fashion depends on a number of factors. Under aerobic conditions primary and secondary SNO are less stable. These empirical observations match early reports that the rates of decomposition depend on oxygen concentration and the bulkiness of the alkyl group.

These reaction traits are indicative of an auto-catalytic process, and this theory has empirical support. [38] Under aerobic conditions the S–N bond scission to form

NO• occurs, then reacts with oxygen to form N2O3. This compound is a potent oxidizing agent, and is responsible for further decomposition of SNO. Addition of antioxidants inhibits this decomposition pathway, as well as the removal of endoge- nous NO. Additional NO speeds up the decomposition under aerobic conditions, due to the increased formation of N2O3.

2An equilibrium exists between the disulﬁde, thiyl and persulﬁde.

13 Homolytic bond cleavage

Detailed studies on the electronic nature of the S–N – O bonding motif are compli- cated by its instability. Jian-Ming Lü[39] et al. were the ﬁrst to report experimental results of the S–N bond homolytic cleavage. Both alkyl- and aryl-SNO were examined, aryl-SNO display a BDE on the order of 20 kcalmol 1, alkyl-SNO are ∼ − 1 1 slightly more stable at 25 kcalmol− to 30 kcalmol− . The authors also calculated

∆HOMO using DFT at the B3LYP/6-31+G* energy level. Such calculations were in good agreement with the values obtained experimentally.

Heterolytic bond cleavage

Heterolytic cleavage energies can be compared to some analogous compounds, and a relative ranking of bond strength can be inferred: RO – NO < RS – NO < RN – NO. Calculations of the energy required for heterolytic cleavage reveal them to be higher in energy than the homolytic pathways. Heterolytic cleavage is not thought to be relevant for biological SNO.[40]

1.5.2 Rates of Decomposition of S-Nitrosothiols

The prototypical decomposition of SNO is the formation of the corresponding disulﬁde and release of NO. The general form of this decomposition is given in

Equation 1.3. This reaction can be induced thermally and photo-chemically to give the disulﬁde and NO.

R SNO RSSR + 2NO• (1.3) −

Biologically relevant SNO decompositions A detailed decomposition study of

S-nitroso-cysteine was preformed by Gu and Lewis[18]. In controlling the pH with buﬀers, and the heavy metal ion concentration through either high-purity materials or the addition of chelating agents revealed a strong dependency on metal ion concentration. They found the decomposition rate to be highly pH dependent, and decomposed most readily near physiological pH (7.4). In alkaline and acidic media

14 Parent Thiol t1/2 (h) N-Acetyl-L-cysteine 14.14 Glutathione 8.74 Cysteine 0.64 (N-mercaptoethyl)-1-3-diaminopropane 1.51

Table 1.6: Half-life values of selected SNO compounds. cysteine-SNO was found to be more stable, with the most stable condition tested having been a pH of 5.85 with chelating agents.

Whiteside et al. examined the decomposition rates of some common nitrosated thiols.[41] Measurement by HPLC silica column (50 nM phosphate buﬀer containing 10 % methanol, 5.2 pH, 0.5 mL/ min), and detection at 334 nm. There is a large range in half-life values, as seen in Table 1.6

1.5.3 Metal-based SNO Decomposition

The same general decomposition reaction occurs in the presence of Cu2+ and Cu+.

There is often enough Cu2+ in distiled water to bring about the decomposition of SNO. The addition of metal chelators, such as EDTA almost completely stops the decomposition. It is thought that the ‘true’ decomposition reagent is Cu+, that forms from reduction with thiolate, show below.

2+ + Cu + 2RS− 2Cu + RSSR (1.4)

+ 2+ Cu + RSNO Cu + RS− + NO (1.5)

Both the Cu2+ and RS – are regenerated, and are likely present in catalytic quantities.

The metal induced decomposition rates of SNO are structure dependent. The most vulnerable groups are those that can complex to the Cu+ bidentately. Swift also notes that apart from some indication of reaction with Ag2+, Fe2+ and Mg2+, no other metal ions tested (Zn2+, Ca2+, Ni2+, Co2+, Mn2+, Cr3+, or Fe3+) were eﬀective in the decomposition of SNO.[42]

15 1.6 REACTIONS OF S-NITROSOTHIOLS

1.6.1 Bioorthogonal Reactions of SNO

Due to the large number of ill-deﬁned roles NO, and by extension, SNO play in biological systems there is a strong need to develop bioorthogonal labeling techniques. Such tools would allow for the exploration of the physiological eﬀects at a needed resolution.

Many methods rely on indirect measurement of NO products released by induc- ing SNO decomposition. Such methods include ozone oxidation of NO, photolysis, triiodide base chemiluminescence, copper/cysteine and copper/ascorbate reagent mixtures. These methods are diﬃcult due to the large abundance of nitrate, which if not carefully controlled will give rise to artifacts. With enough care these techniques can give concentrations of SNO, but no structural information.

The most widely used site labeling technique for SNO is the biotin-switch as- say. The biotin-switch method has four steps: 1) block free thiols with S-methyl methanethiosulfonate, 2) selectively reduce remaining SNO to thiols with ascorbate, 3) label resulting thiols with biotin, 4) detection. Incomplete or uncontrolled reactivity at each step easily generates artifacts. Unblocked thiols from the first stage will give false positives. Under-reduced SNO will not show, and over reduced disulfides will give even more false positives. Current methods are difficult and artifact-prone, and reported values for the same sample can span orders of magnitude.

1.6.2 Reactions with Phosphines

S-Nitrosothiols react quickly with two equivalents of tri-aryl phosphines to give a phosphine oxide and an thioaza-ylide. This reaction class has the most synthetic promise, as it can allow for the construction of multiple bonds in a one-pot procedure. The exact nature of this mechanism is not clear.

Two molar equivalents of the phosphine reagent is required per SNO, one is

16 converted to the corresponding phosphine oxide, and the other forms the aza- ylide. The nature of the formed ylide is relatively well established. More stable examples, such as Ph3C – SN – PPh3 (3) can be isolated as stable solids by ﬂash chromatography.[43] These ylides eventually decompose or react with water, and are further reduced to the corresponding phosphine oxide and thioamine. The aza-ylides themselves are analogous to Wittig reagents, the nitrogen is nucleophilic and can react with carbonyl centers and other electrophiles.

Seminal Report

In 1972 Haake reported that treatment of TrSNO (1b) with two equivalents of triphenyl phosphine PPh3 (4) gave good yields of (Tr– SN – PPh3 (3) and O – PPh3 (5),[44] as show in Scheme 1.3.

Ph Ph Ph Ph O Ph Ph N Ph P N + P Ph S P + Ph Ph S O Ph Ph Ph Ph 2 eq. Ph 1b 4 3 5

Scheme 1.3: Formation of a thio-azaylide.

Also proposed in Haake’s paper was the potential of a nitrene intermediate.

Dimethyl sulfoxide (DMSO) is a known nitrene trap.[45] By treating TrSNO (1b) with only one equivalent of PPh3 (4) in a DMSO/benzene mixture the expected conjugate 6 with DMSO was recovered in 11 %, and the ylide in 30 % (Scheme 1.4).

O Ph Ph Ph Ph Ph Ph S N Ph Ph N + P Ph S P + N Ph S O Ph Ph Ph Ph S S Ph O 1 eq. benzene 30% 11% 1b 4 3 6 Scheme 1.4: Trapping of a nitrene derived from TrSNO.

Mechanism of Thio-Aza-Ylide Formation

This ylide formed from this reaction is similar to those formed from the Staudinger

Reaction,[46] in that the nitrogen is nucleophilic, and undergoes similar reactions.

17 A major diﬀerence between these reactions is the requirement of two equivalents of a phosphine to form the thio aza-ylide(Figure 1.4). The exact nature of this mechanism remains unclear, but two pathways seem likely.

N R N N Ph Ph P Ph Ph Ph Ph Ph P Ph Ph N Ph P N R P N N N Ph N R N R N Ph R N N

Figure 1.4: Mechanism of the Staudinger Reaction.

The ﬁrst to consider is the possibility of a nitrene intermediate. There is some evidence that this nitrene species exists from Haake’s seminal paper (Scheme 1.4).

It is not clear if this trapped species represents the primary pathway, as general concerns of the chemistry of nitrenes would seem to preclude much of the observed chemistry. Nitrenes are known to rapidly react with a wide range of substrates almost instantly. They can insert into C–H, O–H, S–H and other bonding motifs.

Moreover, other synthetic nitrene uses require careful reagent and condition control, typically a heavy metal ’nitrene trap’ is used. These nitrene traps are typically ruthenium complexes that are activated to release a nitrene upon heating. It is diﬃcult to rationalize the stark diﬀerences in chemistry between our hypothetical, SNO-derived nitrene, and more typical nitrenes. A possible explanation is whether the adjacent sulfur atom imparts some stability to the nitrene, but the nature of these sulfenyl nitrenes remains unexplored.

PPh PPh R N Ph S O 3 S 3 S P R N R N Ph Ph 1 O PPh 7 3 8

Scheme 1.5: Proposed nitrene mechanism pathway A.

The proposed mechanisms of this process are shown in Scheme 1.5 and Scheme 1.6.

In pathway A, the phosphine reagent is thought to abstract the oxygen from SNO in a concerted fashion to give the phosphine oxide and the nitrene intermediate (7).

18 The second equivalent of phosphine is expected to then act as a nitrene trap, which forms the thio-aza-ylide (8).

B1 S O R N P Ph Ph Ph Ph Ph 9 S O + P R N Ph

S O S PPh R N R N O 3 R N Ph B2 S P P Ph P Ph Ph Ph Ph Ph Ph Ph O PPh3 10 11 8 Scheme 1.6: Proposed 3-membered ring mechanism pathways B1 & B2.

A second possibility is a more traditional reaction mechanism, and shown in

Scheme 1.6. Where there is some initial attack by the phosphorous on either the oxygen (pathway B1) or nitrogen (pathway B2) atom of SNO. In either case the formed intermediates (9 and 10), are expected to collapse to the same 3-membered ring 11. The second equivalent of phosphine then pushes the reaction to completion.

It is not clear which phosphine (temporally) ends up as the oxide or the ylide.

Attempts to observe these intermediates by 31P nmr have not been productive, only the ylide and oxide can be clearly seen.

Reductive Ligation

In 2008 Xian et al. showed that when an electrophile is positioned appropriately the formed ylide can be trapped[43]. A series of tri-aryl phosphines were synthesized, with esters positioned ortho to the phosphine substituent. The general form of this reaction is shown in Scheme 1.7.

O O S Ph R N S O + O R N H O P Ph2P Ph Ph 1 12 13

Scheme 1.7: Fast reductive ligation.

19 An ortho-phenyl ester (12) proved to be a suitable trapping group for the aza- ylide intermediate (14). The formed electrophilic nitrogen displaces the phenol group, to form the intermediate 15. Hydrolysis completes the reaction to give the sulfenamide (13), as shown in Scheme 1.8. The reaction was found to proceed quickly, and under mild and aqueous conditions, and the phosphine reagent was able to trap a range of biologically relevant small-molecule SNO compounds.

O O R R N R O S O + O N R O P S 1 PPh2 R Ph Ph 2 eq. O 14 12 PPh2 O 16

O O O S R R N R H2O O H N S N R P O P P S Ph Ph Ph Ph Ph Ph 13 15 14

Scheme 1.8: Mechanism of the reductive ligation.

Traceless Reductive Ligation of S-Nitrosothiols

The analogous Staudinger ligation of azides can be modiﬁed to a traceless version, in that the phosphine core will not be present in the ﬁnal product. This is a directly analogous to the ligations explored by Raines and Bertozzi.[47–50]

O O

S S O S N H N N PPh2 S O PPh2 S PPh 18 19 2 1c O O 20 17

Scheme 1.9: Traceless ligations using phosphine esters/thioesters.

As shown in Scheme 1.9 the two electrophiles used for this work, an ester (19) and thioester (18), gave unexpectedly diﬀerent products: the expected traceless

20 ligation product (20) from the ester, and the unexpected, stable thioimidate 17 from the thioester.

The traceless ligation proceeds through the mechanism outlined in Scheme 1.10.

The formed aza-ylide (21) attacks the carbonyl position on the ester, and displaces the tri-aryl phosphine, which is then oxidized further to the phosphine oxide (22).

No stable products were obtained when primary SNO substrates were used.

11 11 R O R O O R N O S O + 11 N R R O P S PPh 1 2 Ph Ph 2 eq. O 21 23 O P Ph2

24 11 R O

OH H 11 O R N R H2O + S O N R P O P S Ph Ph 25 Ph Ph 22 21

Scheme 1.10: Mechanism of the traceless ligation.

When thioester 18 was used the expected traceless ligation products are observed in minor yields, the major products are instead thioimidates. It was determined that the traceless ligation products observed when 18 was used were not the result of the hydrolysis of the thioimidates. Instead of displacing the aryl-thiol to form a traceless ligation product, the Ar – C – S bonds remain intact. Instead the intermediate 26 undergoes an intra-molecular aza-Wittig reaction to give 27 as depicted in Scheme 1.11.

21 11 11 11 R O R O R O S R N S S S O + 11 N R R O P S N R 1 PPh P S 2 Ph Ph 2 eq. S Ph Ph 26 26 28 O P Ph2 29 11 11 R R S N R S S S O O R11 N R N R P P S P S O Ph Ph Ph Ph Ph Ph 27 31 30

Scheme 1.11: Formation of thioimidates from phosphine thioesters.

One-Step Disulﬁde Formation of S-Nitrosothiols

Based on the unexpected formation of stable thioimidates discussed previously it was hypothesized that a sulfur homologue (32) of the ester used in the seminal reductive ligation should lead to a traceless ligation with an asymmetrical disulﬁde as the product (Scheme 1.12).

PPh2 O S O S S Ph + R N R S Ph THF/PBS (7.4) 3:1 32 1 33 Scheme 1.12: One-step reductive disulﬁde formation.

This mechanism is shown in Scheme 1.13. The key diﬀerence here is the dis- placement of a thiolate equivalent from the 5-membered ring intermediate (34).

The formed thioamide (35) is vulnerable to attack by the thiolate, this reaction forms the disulﬁde 36 and the amide by-product (37).

22 11 PPh2 O 11 11 R R R S 22 S Ph S R N S P Ph N O O PPh2 O O 11 R 1 S 38

11 11 R R 22 S S R 22 S Ph R S H2O O N O N P Ph P Ph Ph

11 R

S 22 O NH R S S 2 O NH R S R + O O 11 22 P P Ph Ph Ph Ph

35 36 37

Scheme 1.13: One-step disulﬁde formation from SNO

This traceless ligation was applied to the detection of S-nitrosated proteins, shown in Scheme 1.14. COS-7 cells were treated with CysNO to effect nitrosation of the proteins within. Afterwards the cells were washed, fixed and permeabilized, then free thiols were blocked with N-ethylmaleimide (NEM). Then the cells were incubated with a modified traceless ligation reagent with a biotin linker (39). Using this technique S-nitrosated proteins were visualized by Western Blot. The attached biotin linkers are affixed to the proteins through a disulfide bond, and it is possible to cleave these bonds with dithiothreitol (DTT).

23 S O S O S biotin N N 39 S

NEM = SH S S O S S NEM S NEM S S S N

phosphine core 39 O PPh2 O O HN O S N NH 3 H S

S Biotin

Scheme 1.14: SNO labeling with the traceless ligation reagent

One-Pot Thioether Formation from S-Nitrosothiols

Based on the previously discussed disulﬁde formation, Xian et al. developed a one-pot thioether formation, shown in Scheme 1.15. This work was inspired by a phosphine-mediated allyl disulﬁde rearrangement.[51, 52]

SNO O S R R S MeO + S NHAc MeO NHAc O PPh2 O 1d 2 eq. 40 41

S R PPh3 MeO NHAc O 42

Scheme 1.15: One-pot thioether formation. CH3CN/PBS buﬀer: (3:1), 25 ◦C, 15 min, 50 ◦C, 2.5 h

The formation of the aza-ylide (43), and the formation of the disulﬁde (44) proceeds as previously described in Scheme 1.13. The mechanism of the one-pot thioether formation is shown in Scheme 1.16. In the thioether case, the displaced

24 thiol 45 is an allyl disulﬁde. Upon the formation of 44, which rearranges to 46.

This divalent sulfur species is vulnerable to sulfur extraction by phosphines. The phosphine reagent 47 or additional PPh3 can be used in large excess to push the reaction to the stable thioether, 42.

22 O R 22 O R H2O S O S 11 R N S P N S 11 PPh2 R Ph Ph 1 47 43

O 22 S 11 N R R O 11 S 22 H + R S R P O S NH2 Ph Ph P O 48 45 Ph Ph 44

excess of S 47 22 22 S 11 S R 11 S R 11R S R 22 R R 44 46 49

Scheme 1.16: Mechanism of the one-pot thioether formation

Bis-ligation of S-Nitrosothiols

In an attempt to develop a traceless ligation an unexpected bis-ligation was discovered. While tertiary SNO compounds undergo the desired traceless ligation, it was found when primary SNOs were used an unexpected disulﬁde was the major product[53]. When an R – SNO is treated with a tri-aryl phosphine with an internal thioester an unexpected N-acyl transfer occurs as shown in Scheme 1.17. The ylide intermediate (50) undergoes an S to N acyl transfer to give the aryl thiolate (51).

The N–P bond survives this process, and the resulting thiolate attacks the sulfur bonded to the nitrogen to give a stabilized Ac – N – P ylide (52).

25 Ph Ph R S P 11 Ph 22 N S O S R P Ph N + 22 R S R O 11 O

53 54 50

22 R O O 22 R Ph Ph N NP Ph R S P Ph 11 S S S

11 R 51 52

Scheme 1.17: Bis-ligation of primary SNO

Dehydroalanine Formation

Subsequently, the same chemistry was observed to induce an unexpected product through an elimination reaction.[54] As shown in Scheme 1.18. When the same phosphine reagents were used with amide amino acid derivatives the aza-ylide intermediate can abstract the relatively acidic α-proton (55). This intra-molecular reaction results in the elimination of the phosphine substituent to give the corresponding dehydroalanine ().

R R P R N H 2 eq. S H N N H N R R R S O 55 H R P R R RHN N R + O N R O N HN H O H S 1e 55 56 57

R R R P H2O 57 O=PPh3 + HSNH2 S NH 5 59 58

Scheme 1.18: Dehydroalanine formation

Mechanistic studies were preformed to determine if the elimination occurs intra or inter molecularly, as shown in Scheme 1.19.

26 2 equiv. NHBz MeO C NHBz MeO C NHBz H 2 2 PR3 or CO2Me SNO or 2 equiv. ZE 1f P(OEt)3 60 61

Scheme 1.19: Determination of the intra-molecular elimination

A chiral derivative of cysteine-SNO (1f) was synthesized and treated with phosphines. If the reaction proceeds intra-molecularly only the Z isomer (60) should be observed. While the tri-aryl substrate showed both Z (60) and E (61) isomers. We attributed this to the large molecular weight of the triaryl phosphine lowering the inversion barrier. The lower weight P(OEt)3 phosphine provided only the Z isomer in 33 % yield.

A Water Soluble Phosphine Reagent

A limitation of this phosphine chemistry with respect to labeling biological SNO comes from their poor solubility in aqueous solutions. King[55] et al. addressed this by using a tri-aryl phosphine with sulfonate groups (62) to improve solubility (Scheme 1.20). The ylide is not clearly formed from 62 at a pH of 7.1.

Instead it seems that this phosphine directly displaces NO to form a charged sulfur- phosphorous adduct. The displaced NO is protonated to HNO and subsequently trapped by 62. These complexes are stable enough to be detected by LC-MS.

HO HO A O O H2N H2N

O P O HN HN SO3Na A 3 O S N 62 O S P A NH O NH A

O O HO HO

Scheme 1.20: A water soluble phosphine

27 Benzoisothiazole Synthesis

Xian et al. have also demonstrated a route to benzoisothiazoles, a pharmaceutically relevant heterocyclic core.[56–59] Nitrosation of an aryl thiol with an aldehyde or ketone in the ortho position, followed by treatment with phosphine, produces benzoisothiazoles neatly in one pot (Scheme 1.21).[60]

O i-pentyl-ONO (3 eq.) R

R EtPPh2 (2.1 eq.) N SH S

Scheme 1.21: One-pot formation of benzoisothiazoles.

Aryl thiols have a lower pKa then their alkyl counterparts. Accordingly aryl- SNO compounds show lower stability, and are more diﬃcult to handle then most alkyl SNO. In this procedure, handling and puriﬁcation of this delicate intermediate is omitted, as the nitrosating agent used is inert to the phosphine reagents within the time-frame of the reaction. In contrast, the NaNO2/HCl system completely oxidizes triphenyl phosphine to the oxide in under one minute.[60]

A series of alkyl nitrites were tested to gage their eﬀect. i-pentyl nitrite was found to be the most eﬀective substituent, and preformed best in tetrahydrofuran.

A series of phosphines were also tested, phosphines such as nBuPPh2 and EtPPh2 worked well. While those with smaller alkyl groups were found to be ineﬀective.

1.6.3 Light Induced Reactions

Cavero et. al. explored light and thermally induced radical reactions of some SNO compounds. Either a 500 W tungsten lamp or a temperature of 65 ◦C was used to initialize the reaction. The proposed intermediates are shown in Scheme 1.22, and the proposed radical catalytic cycle is shown in Figure 1.5.

28 O 65°C N S O + R Tr N 11 or hv S (6 eq.) Tr R 11 1b 63 64

OH S O- N Tr 11 N+ R S + Tr R 11 11 R N - Tr O S 66 65

Scheme 1.22: Synthesis of α-tritylthio oximes

R 11

∆ S O S S Ph3C N Ph3C Ph3C R or hv 11

N CPh3 O S O O S N N CPh3 S S Ph3C R Ph3C R 11 11

Figure 1.5: Radical TrSNO addition to oleﬁns

Cavero also explored radical chemistry with an internal alkene trap.[61] Instead of the expected thiirane, only disulﬁdes (67) and nitroso dimers (68) were isolated

(Scheme 1.23).

22 11 O R R 22 N 33 - 33 11 R R O R R 33 S R + 33 22 N + S 11 R R + S S R N 33 R R S O- R R 11 22 11 R 22 69 68 67 Scheme 1.23: Dimerization of allyl SNO compounds

1.6.4 Reactions with Sulfenic Acids

Hart reported the coupling of sulfenic acids and SNO to give thiolsulphonate derivatives as shown in Scheme 1.24.[62, 63]

29 O 22 S 22 N2O4 R OH 11 S R 11 SH 11 S O R R N R S O O 1 70 71 TMP4 Scheme 1.24: Thiosulfonates from sulfenic acids and S-nitrosothiols.

It was later found that this reaction is robust enough to label protein SNO.[64]

Indeed, both S-sulﬁnation (the formation of a sulfenic acid) and S-nitrosated thiol residues are detectable by this method. Biotin linked to either an SNO or SO2H group reacts with either protein SNO or SO2H to form fairly stable thiosulfonate bonds. This reaction proceeds at a very low pH, so much so that it seems that the lone pair on the SO2H appears to be the nucleophilic site. An overview of the protein labeling experiments is shown in Scheme 1.25. Free thiols must still be blocked, as the thiosulfonates are vulnerable to thiol exchanges.

O S S O OH N

O S O S biotin N biotin OH

O S S biotin S biotin O S O O

Scheme 1.25: Cross-reactivity proﬁle of SNO and sulfenic acids.

1.6.5 Reactions with Carbanions

The labile nature of the S–N bond makes SNO vulnerable to attack by strong nucleophiles. Shinhama et al. showed that reaction of 1 with carbanions (72 eg. Grignard or alkyl-lithium reagents) quickly displace the NO group to give thioethers (73 as shown in Table 1.7).[65]

30 11 11 22 R N + 22 R Y R R S O S Et2O 1 72 < 5 min 73

R1 R2 Y Y:SNO ◦C % Yield of 73 p-Tol Ph MgBr 3 70 57 p-Tol CH MgI 3 −70 43 3 − p-Tol CH3 Li 1 70 57 t-Bu Ph MgBr 3 0− to 5 72 p-Tol PhCH Li 3 70 32 2 − Table 1.7: Thioether formation from SNO

1.6.6 Miscellaneous Reactions

This section contains reactions in which SNO plays a critical role, either as a nitrosation reagent or some other role. However, in these reactions few, if any, of the

S, N or O atoms that begin in the SNO starting material are in the ﬁnal products.

Disulfide Formation Often disulfides are unwanted by-products when working with SNO. Obviously, one can exploit this trait if disulfides are desired

(Scheme 1.26). Nitrosation of one thiol, followed by the addition of another gives unsymmetrical disulﬁdes in decent to excellent yield.

22 SH N2O4 R 22 11 SH 11 S O 11 S R R R N R S

Scheme 1.26: Asymmetrical disulﬁde formation from SNO[66]

Trisulfide Formation Although SNO slowly decomposes to their corresponding disulfides in solution, at a rate dependent on the alkyl substituent, some reagents can speed up this process.[27, 67] Most notable among these is the copper (II) containing salts, as well as the mercury (II) salts, due to their well known affinity for thiols. When SNO are treated with copper(II) chloride trisulfides are obtained.

Trisulfides, especially asymmetrical examples, are unusual species, and few reactions are available for their synthesis. Curiously enough trisulfides are formed in relatively high yields and purity (relative to the disulfide content) from this method,

31 as outlined in Table 1.8.

Substrate Halide Halide : Substrate ◦C min. % Yield

R2S2 R2S3 R2S4

t-BuSNO CuCl2 1 25 20 trace 81 3 t-C5H11SNO CuCl2 1.5 25 10 9 59 8 t-C9H19SNO CuCl2 1 25 20 trace 77 4 s-BuSNO CuCl2 1 25 5 91 0 0

Table 1.8: Generation of polysulﬁdes from copper (II) halides.

Reactions with Amines Amines can be nitrosated by SNO. This is analogous to the Sandmeyer reaction, in that a diazomium species is formed, then displaced.[66]

The reactions in this section use SNO in this way, while it is conceivable that a use case exists in which a sulfur-based nitrosating reagent would preform better than

NaNO2, it is not clear when this would be the case. Regardless, the nitrosation of amines results in the generation of a very reactive species, and from that perspective these reactions play an important role in SNO based synthetic design. The yields are rates of this reaction vary, and are likely substrate dependent.

Kim et al. found[68] that arylamines (74) react with t-BuSNO (1g) in the presence of copper(II) halides to give aryl halides (Scheme 1.27). The starting SNO (1g) are converted into the di- and tri-sulﬁde with a ratio of ca. 0.35 to 2.0. The same products can be obtained using t-BuSNO2.

CuX2 N + Ar NH2 Ar X S O CH3CN 0-25°C 1c 74 -N2 75

Scheme 1.27: Amines converted to their corresponding aryl halides.

Oae et al. expanded[67] this chemistry to synthesize 2-aryl-1-haloethanes (76). Surmising that the likely intermediate was a diazonium salt, and that in the presence of oleﬁns, a Meerwein arylation should occur (Scheme 1.28).

32 CN CN CuX2 N + Ar NH2 + Ar S O X CH3CN Ar, 25°C, 2 hr 1c 74 77 76

Scheme 1.28: Yields of 2-aryl-1-haloethanes

33 CHAPTER 2. CONJUGATION OF S-NITROSOTHIOLS WITH ALDEHYDES

2.1 INTRODUCTION

S-Nitrosothiols (SNO) and their implications as a nitric oxide (NO) induced post- translational modiﬁcation are under active investigation.[69–71] In our work on bioorthogonal reactions of protein SNO we have uncovered chemistry of this functionality that shows synthetic promise. Herein is reported a phosphine mediated reaction between SNO and aldehydes to form thioimines. This reaction was found to precede both intramolecularly and intermolecularly. A simple synthesis of benzoisothiazole based on this reaction is presented. The general form of the reaction is shown in Figure 2.1.

H PR3, i-pentyl-ONO H + 22 SH 11 R S R O 11R N R22 CH2Cl2 or THF/H2O

Figure 2.1: General form of the aldehyde & SNO coupling.

2.1.1 Hypothesis

In recent work on SNO bioorthogonal reactions, aimed at the development of detection methods for S-nitrosylated proteins, we have uncovered some unique reactivity of SNO.[3,9] The reaction between SNO ( 1c) and two equivalents of a triaryl phosphine (eg 78) generates reactive thio-aza-ylides in high yields under mild conditions.

34 R O R N S + S S SNO O PPh2 P 1c Ph Ph 78 17

Scheme 2.1: Inspiration for aldehyde conjugation from our previous work

The formed thio-aza-ylides are potent nucleophilic species. Upon manipulating the electrophilic group on the phosphine reagent, we found that the thio-aza-ylides can be trapped as stable products.[53] In one example, we trapped a relatively stable tertiary SNO substrates with thioesters to form N-alkyl-thio-imidothioates

(eg. 79, Scheme 2.1). We wondered if similar reactions with other electrophiles could ﬁnd unique synthetic applications. This chapter will cover reactions between

SNO-derived thio-aza-ylides and selected aldehydes.

2.2 RESULTS AND DISCUSSION

2.2.1 Experimental Plan

Aldehydes are highly reactive electrophiles and are expected to react with thio-aza- ylides. The intramolecular and intermolecular versions of this reaction should both be feasible. The intramolecular version is shown in Scheme 2.2. We chose to explore the reaction conditions using this substrate, over using PPh3 and an aldehyde in an intermolecular reaction. This set up should reduce the experiment complexity by limiting the number of reactants in the ﬂask to two, rather than four (an aldehyde, thiol, phosphine and a nitrosating reagent). The close proximity of the electrophilic trap and the formed ylide should quickly tell us if this reaction is feasible. This turned out to be a prudent step, as several ligations one would expect to succeed were found to fail.

35 Ph Ph Ph P P S Ph Ph Ph Ph N + N Ph S O Ph Ph 81 O 1h O 80

Ph Ph Ph Ph Ph Ph P P P O Ph N S O N S Ph N Ph Ph S Ph O Ph Ph Ph Ph 84a 82 83

Scheme 2.2: Proposed mechanism of an intramolecular conjugation of an aldehyde and SNO.

The proposed mechanism for this reaction is shown in Scheme 2.2. One equivalent of the phosphine reagent is sacrificial, in the sense that it will be oxidized and will not proceed to the final product. The formed thio-aza-ylide 81 is then expected to attack the aldehyde to form the five membered ring betaine 82. This ring then closes to release the desired N-thioimine 84a and triphenyl phosphine oxide (O – PPh3). O – PPh3 is quite stable, and its formation is likely the driving force behind this reaction.

Trityl S-nitrosothiol (TrSNO, 1h) makes a convenient platform for the exploration of SNO chemistry. As it can be easily prepared, and then stored as a relatively stable solid for months in the dark at 0 ◦C. Placing the aldehyde on the SNO compound was determined to be ill-advised. Although the six-membered ring intermediate could proceed, nitrosative conditions are slightly oxidative, and a potential reaction between aldehydes and thiols before their nitrosation. While both aldehydes and phosphines are known to be vulnerable to oxidation, there should be no cross reactivity.

2.2.2 Preparation of Starting Materials

A coupling reaction between commercially available o-bromobenzaldehyde and diphenyl phosphine was preformed. The reaction scheme is shown in Scheme 2.3.

Aldehyde 85 and phosphine 86 where reﬂuxed under argon in the presence of

36 palladium tetrakis and triethyl amine. The target compound was isolated by column chromatography in an 84 % yield.

H O O Pd(PPh3)4, Et3N + P Ph P Br toluene Ph 85 86 80 Scheme 2.3: Synthesis of 2-(diphenylphosphaneyl) benzaldehyde

Trityl S-nitrosothiol (TrSNO) is easily synthesized by treating triphenyl methyl thiol with isoamyl nitrite in dichloromethane. After the nitrosation is well on its way (ca. 30 minutes) methanol is added, and the S-nitrosated product crystallizes from solution upon standing overnight at 15 C − ◦

2.2.3 Condition Screen

We chose to screen a series of solvents ﬁrst, as needed the solvating power for PPh3 and TrSNO limited in our choices to begin with. In this study, two equivalents of phosphine 80 were treated with trityl-SNO (1h). The results are summarized in

Table 2.1. Dichloromethane and chloroform were found to be the best solvents, and gave the desired product 84a with yields of 57 %. The progress of the reaction was monitored by thin-layer chromatography. Consumption of the SNO by phosphine

80 to form the thio-aza-ylide 81 was complete within three hours by TLC. The subsequent intra-molecular aza-Wittig reaction was somewhat slow and required overnight reaction times.

37 Ph Ph Ph Ph Ph O S + N Ph SNO PPh2 O P 1h 80 Ph Ph 84a

entry solvent % yield of 84a

1 tetrahydrofuran 20 2 dichloromethane 43 3 dioxane 57 4 toluene 12 5 chloroform 57

Table 2.1: Solvent screen for the intramolecular phosphine mediated conjugation.

2.2.4 Intramolecular Substrate Screen

Next we tested the reactions with a range of SNO substrates; the results are summarized in Table 2.1. In general the reaction worked well for tertiary SNOs (entries

1-4); the corresponding products were stable and could be purified by flash column chromatography. Some products derived from secondary SNOs (entries 5 and 6) were stable enough for purification and characterization. The products from the secondary SNO 1i, and the primary SNO 1j were found to be unstable. Crude 1H

NMR analysis showed the presence of the products in the reaction mixture, but attempts to isolate resulted in decomposition. Disulﬁdes, thiols, and the oxide of starting phosphine 80 were recovered. Presumably thioimines are vulnerable to decomposition by the acidic column conditions. These results indicate a correlation between the stability of the starting SNO, and the stability of the ﬁnal product.

2.2.5 Intermolecular Substrate Screen

Having demonstrated the intramolecular coupling between thio-aza-ylides and aldehydes, we set out to study the application of this reaction in intermolecular bond formation. Initial attempts to couple a series of benzaldehyde derivatives

(Scheme 2.4) with the TrSNO-derived thio-aza-ylide produced none of the antici-

38 S O N R S O + + O R N CH Cl , 25°C P 1 PPh2 2 2 80 overnight Ph Ph 84

% yield of entry RSNO product 84

S Ph Ph Ph N 1 O Ph Ph 57 84 Ph SNO P 84 Ph Ph 1h 84a NHAc O S OMe AcHN N 2 OMe 58 O O P SNO 84 84 Ph Ph 1k 84b S N 3 O 46 84 SNO P 84 Ph Ph 1g 84c

S 4 SNO N 55 O 84 P 1l 84 Ph Ph 84d S SNO N 5 O 46 84 P 84 Ph Ph 1m 84e S SNO N 6 O 39 84 P 84 Ph Ph 1i 84f S N 7 O 31 84 SNO P 84 Ph Ph 1j 84g

Table 2.2: Substrate screen of the intramolecular conjugation

39 pated product. Heating this reaction proved unproductive, TrSNH2, a decomposition product from thio-aza-ylide, was observed by electro-spray ionization mass spectrometry but no trace of the product was observed. We ascribe the lack of any observed product to the excessive steric bulk of the phenyl rings in close proximity.

S Ph Ph S O O N N + Ph Ph Ph Ph R CH2Cl2, 25°C R 1h 87 overnight 88 not formed

87: a: R = H, b: R = OMe, c: R = CF3

Scheme 2.4: Benzaldehyde fails to couple with TrSNO.

We then turned to α, β-unsaturated aldehyde substrates for this reaction as such substrates were found to react well in the analogous Wittig cross couplings.[72,

73] Cinnamaldehyde (89) proved to be a more suitable substrate for this reaction, and under the same conditions produced the desired product (90a) in a modest yield (57%, Scheme 2.5). t-Butyl SNO was also coupled with cinnamaldehyde to produce the desired product 7 in a similar yield (64%). We also found that the use of excess of SNO (1.5 equivalents) could improve the yield to 89%. Presumably the unavoidable decomposition of the S-nitrosothiols into their corresponding disulﬁde should occur over the reaction times used.

O S Ph PPh3 S Ph O + N N Ph Ph Ph Ph CH2Cl2, o/n 89 1h 90a

O S PPh3 S O + N N CH2Cl2, o/n 89 1c 90b

Scheme 2.5: Cinnamaldehyde successfully undergoes the intermolecular conjugation.

These disulﬁdes may react with, and consume the phosphine reagent. However simply adding more SNO and phosphine should force the reaction to completion. Under these conditions, t-butyl SNO (1g) even reacted successfully with

40 p-methoxybenzaldehyde to produce the corresponding product 90, albeit the yield was not high (18 %). Employing a more nucleophilic phosphine, i.e. EtPPh2, led to a signiﬁcantly improved yield (54 %). With this optimized protocol, benzaldehyde and 4-(triﬂuoromethyl) benzaldehyde were successfully coupled with t-butyl SNO.

S O 22 phosphine S 11R N + R O 11R N R22 CH2Cl2, 25°C 1 91 overnight 90

eq. entry R – SNO aldehyde phosphine product % yield RSNO

S Ph Ph Ph O N 1 1.0 PPh3 57 90 Ph SNO 90 90 Ph Ph 1b 89 90a S O N 2 1.0 PPh3 64 90 SNO 90 90 1g 89 90b S O N 3 1.5 PPh3 89 90 SNO 90 90 1g 89 90b S O N 4 1.5 PPh3 18 SNO 90 90 MeO 90 MeO 1g 91a 90c S O N 5 1.5 PPh2Et 54 SNO 90 90 MeO 90 MeO 1g 91a 90c S O N 6 1.5 PPh2Et 68 90 SNO 90 90 1g 91b 90d S S N N 7 1.5 PPh2Et 44 90 SNO 90 F3C 90 F3C 1g 91c 90e

Table 2.3: Aldehyde substrates amenable to the conjugation.

2.2.6 Application - Synthesis of Benzoisothiazole

Finally we set out to explore the application of this chemistry in making S–N containing molecules. Benzoisothiazole (92) is a fused, two-ring heterocycle with a unique S–N bond. Benzoisothiazole derivatives have been reported to act as antibacterials[59], anti-HIV[59] as well as some other activities.[57–59] For example, the anti-psychotics Ziprasidone and Lurasidone contain benzoisothiazole rings.

Lurasidone, also has the trade name Latuda, this compound was approved in 2010

41 for the treatment of bipolar disorders. Sales for Lurasidone in North America and

China in 2014, and 2015, where $752, and $1.002 million, respectively.[74]

H S N N Cl O N N O N N N S N O lurasidone ziprasidone

Figure 2.2: Benzoisothiazole containing pharmaceuticals.

Current synthetic methods for this heterocycle are either low yielding and ineﬃcient, or require the insertion of an amine at the 3-position.[57–59] Starting from 1,2-benzoisothiazole-3(2H)-one (13) is undesirable as this compound and its 3-chlorinated derivative (not shown) are both strong dermal, ocular, and nasal irritants which require special containment and handling.[57, 58]

We envisioned that our SNO-mediated aldehyde condensation could be used to access benzoisothiazole. Retro-synthetically, the target molecule 92 could be synthesized from SNO-mediated intramolecular aza-Wittig reaction. As such o- mercaptobenzaldehyde 93 should be the starting material. We tested this idea by starting from the commercially available o-mercaptobenzoic acid 94, which was reduced to the alcohol 95 upon reduction with lithium aluminum hydride

(LAH). An attempted oxidation of 95 to the aldehyde 93 caused the formation of disulﬁde-aldehyde 96.

Disulﬁde formation is often an unavoidable consequence of working with thiols, especially under oxidative conditions. Our initial plan called for the reduction of 96 to aldehyde 93, and then S-nitrosation and ring closure by the phosphine-mediated aza-Witting reaction. We recognized that PPh3 would be used in step 1 and 3 in this 3-step process; and S-nitrosation by organic nitrites should proceed in the presence of PPh3. As such it might be possible to achieve the 3-step transformation in one-pot with all the reagents presented. This was found to be the case.

42 O O OH OH [O] LAH S S SH SH O 94 95 96

PPh3, R-ONO THF, H2O PPh3, H2O

S PPh O R-ONO O N 3 SNO SH 92 97 93

Scheme 2.6: One-pot synthesis of benzoisothiazole.

Simply dissolving the disulﬁde-aldehyde 93 (1.0 equiv) in a THF/H2O system with excess phosphine (6.0 equiv) and isoamyl nitrite (2.5 equiv) led to the complete conversion to benzoisothiazole (92) in an excellent yield. The lower isolated yield of

81%, as compared to almost quantitative conversion observed by NMR, is ascribed to the fact that benzoisothiazole easily evaporates during solvent removal. This route is highly preferable to handling the free thiols, as their odor, propensity to act as a nucleophile or oxidize to their corresponding disulﬁdes can be painful problems to deal with.

2.3 CONCLUSION

To conclude, we have presented a simple method for the synthesis of thioimine bonds from aldehydes and S-nitroso compounds, readily obtained via nitrosation of thiols. This reaction proceeds under mild conditions and should ﬁnd applications in the preparation of some pharmaceutically important molecules.

2.A APPENDIX

2.A.1 Methods

1H NMR spectra, 13C nmr and 31P nmr were recorded at 300 MHz (VX 300, Varian,

Palo Alto, CA, USA) and are reported in p.p.m. on the δ scale relative to residual

43 1 13 CHCl3 (δ 7.25 for H and δ 77.0 for C). These experiments were performed at room temperature. Mass spectra were recorded using an electrospray ionization mass spectrometry (esi, Thermo Finnigan LCQ Advantage, San Jose, CA, USA) or MALDI-TOF (matrix-assisted laser desorption ionization time-of-ﬂight) mass spectrometry. Mass data were reported in units of m/z for [M+H]+ or [M+Na]+.

Compound 80 A ﬂame dried, 50 mL, two neck round bottom

ﬂask was equipped with a stir bar, reﬂuxing condenser and rubber O

P septum. 2-bromobenzaldehyde (1.0 g, 5.4 mmol) and palladium tetrakis (cat. 0.6 mol%, 37 mg) were then added and the ﬂask

was then evacuated and flushed with argon 3x. Toluene (20 mL), triethylamine (0.75 mL, 5.4 mmol), and diphenylphosphine (1.3 g, 7.0 mmol) were added via syringe. Reaction was then brought to reflux and monitored by TLC (20% ethyl acetate in hexanes, rf = 0.45). Upon completion the mixture was then washed with saturated NH4Cl (3x, 20 mL), brine (20 mL), then dried over MgSO4 and filtered. Solvent was then removed by reduced pressure (20 mmHg) and the product was either recrystallized from MeOH, or purified by flash column chromatography

(5%-9% MeOH in DCM), 80 % isolated yield. Data matches previous reports.

1 NMR H (300 MHz, CDCl3) δ 10.50 (s, 1H), 7.97 (m. 1H), 7.47 (m, 2H), 7.38 – 7.23 31 NMR (m, 11H), 6.97 (dd, J = 6.6, 4.6, 1H); P (122 MHz, CDCl3) δ = -11.72.

Compound 84a In a small scintillation vial equipped with a stir O

Ph2P bar, phosphine (2) (26 mg 0.10 mmol) was dissolved chloroform Ph Ph N Ph S (3 mL). Trityl S-nitrosothiol (1) was then added (30 mg, 0.05 mmol), the flask covered in aluminum foil and let stir over night. Purification by flash chromatography, elution with 1% MeOH in CH2Cl2 gives compound 4 in 57%.

1 NMR H (300 MHz, CDCl3) δ 9.14 (s, 1H), 7.68 – 7.43 (m, 11H), 7.36 (s, 1H), 7.31 –

44 13 NMR 7.17 (m, 16H), 7.07 (d, J = 13.9 Hz, 1H). C (75 MHz, CDCl3) δ 191.3, 153.6, 144.6, 133.5, 132.2, 132.1, 130.3, 128.9, 128.8, 128.5, 127.9, 127.1, 70.5. 31P NMR

+ + (122 MHz, CDCl3) δ 32.4. ESI-MS calcd for C38H30NNaOPS; found 602.2 [M Na]

General Procedure for Reaction Screen 1 mmol of thiol in a 25 mL round bottom

ﬂask was charged with 1N HCl (1 mL), H2SO4 (0.2 mL), MeOH (1 mL) and THF (1 mL). The vial as then covered in aluminum foil and, in the dark, a solution of 2M

NaNO2 in water was added. An immediate color change was observed and the reaction was allowed to run for ca. 15 min or until complete by TLC (1:4, ethyl acetate: hexanes). The resulting solution was diluted with water (5 mL) and extracted with diethyl ether (10 mL), dried over MgSO4, filtered through a pad of silica gel (1 cm), and the solvent removed under reduced pressure at room temperature (24 ◦C). The resulting red oil was over 95% nitrosated by 1H NMR and was immediately added to a solution of 2-(diphenylphosphanyl) benzaldehyde (2 equiv.) in chloroform and stirred over night (foil covered). Solvent was removed under reduced pressure and the crude mixture was purified by flash column chromatography (0.5% MeOH in

CH2Cl2, rf = 0.25).

1 NMR Compound 84b H (300 MHz, CDCl3) δ 9.17 (s, O Ph P 1H), 8.10 – 8.01 (m, 1H), 7.67 – 7.43 (m, 10H), 7.31 (dd, O 2 N MeO S J = 2.4, 1.3 Hz, 1H), 7.08 (ddd, J = 14.1, 7.7, 1.3 Hz, 1H), NHAc 6.75 (d, J = 8.4 Hz, 1H), 4.76 (d, J = 8.4 Hz, 1H), 3.63 13 NMR (s, 3H), 2.00 (s, 3H), 1.48 (s, 3H), 1.41 (s, 3H). C (75 MHz, CDCl3) δ 170.31, 169.8, 155.4, 133.5, 132.1, 132.0, 131.98, 131.9, 129.0, 128.9, 128.8, 128.6,

127.1, 127.0, 60.6, 52.0, 51.1, 25.6, 24.2, 23.1. ESI-MS calcd for C27H29N2NaO4PS [M+Na]+ 531.2; found 531.2.

45 1 NMR Compound 84c H (300 MHz, CDCl3) δ 9.15 (s, 1H), 8.26 – O

Ph2P 8.12 (m, 1H), 7.71 – 7.43 (m, 11H), 7.27 (s, 1H), 7.09 (ddd, J = 14.0,

N 13 NMR S 7.7, 1.3 Hz, 1H), 1.37 (s, 9H). C (75 MHz, CDCl3) δ 153.5, 153.4, 141.1, 133.6, 133.5, 133.4, 132.3, 132.2, 132.1, 131.0, 123.0, 31 NMR 129.0, 128.8, 128.6, 128.4, 127.3, 127.2, 47.3, 29.4. P (122 MHz, CDCl3) + + 32.6. HRMS (MALDI) calcd for C23H25NOPS [M H] 394.1394; found 394.1386.

1 NMR Compound 84d H (300 MHz, CDCl3) δ 9.12 (s, 1H), O 8.10 (ddd, J = 8.0, 4.0, 1.2 Hz, 1H), 7.69 – 7.40 (m, 11H), Ph2P

N S 7.39 – 7.19 (m, 6H), 7.07 (ddd, J = 14.0, 7.7, 1.3 Hz, 1H), 4.51 (q, J = 7.0 Hz, 1H), 1.67 (d, J = 7.0 Hz, 3H). 13C NMR

(75 MHz, CDCl3) δ 153.8, 141.3, 140.7, 133.5, 132.3, 132.1, 131.0, 129.7, 129.0, 128.8, 128.6, 127.9, 127.4, 49.3, 20.5; 31P NMR (122 MHz,

+ + CDCl3) 32.8. HRMS (MALDI) calcd for C27H25NOPS [M H] 442.1394; found 442.1435.

1 NMR Compound 84e H (300 MHz, CDCl3) δ 9.12 (s, 1H), O

Ph2P 8.14 (ddd, J = 8.0, 4.0, 1.2 Hz, 1H), 7.70 – 7.41 (m, 11H), 7.33

N S – 7.20 (m, 1H), 7.14 – 7.00 (m, 1H), 3.17 (m, 1H), 1.96 (m, 2H), 31 NMR 1.79 – 1.57 (m, 2H), 1.35 (m, 6H). P (122 MHz, CDCl3) + + 32.70 (P=O). ESI-MS: calcd for C25H27NOPS [M H] 442.1; found 442.1.

General Procedure for SNO-aldehyde coupling To a solution of starting thiol

(1.0 equiv) in dichloromethane [0.05 - 0.1 M] was added isoamyl nitrite (1.5 equiv), the reaction was let stir under dark for 5 minutes, usually a suﬃcient enough time for the starting thiol to be over 95% nitrosated. 2.2 equivalents of the phosphine reagent was (relative to the molar equivalents of the SNO used) then added and the red or green color of the SNO solution fades and was replaced with a yellow to orange red solution. ca. 5 minutes later the aldehyde was added and the reaction let stir over night, monitored by TLC and crude 1H NMR . After 16 hours the solvent was removed under reduced pressure and the resulting crude oil separated by ﬂash

46 column chromatography.

Compound 90a as mixture of tautomers: 1H NMR (300 S Ph N MHz, CDCl3) δ 8.26 (d, J = 8.8 Hz, 0.65H), 7.98 (d, J = 8.9 Ph Ph Hz, 0.25H), 7.52 – 7.42 (m, 2H), 7.37 – 7.26 (m, 3H), 7.00

(dd, J = 15.7, 8.9 Hz, 0.25H), 6.92 – 6.82 (m, 1H), 6.74 (d, J = 16.0 Hz, 0.75H), 13 NMR 1.42 (s, 9H). C (75 MHz, CDCl3) δ 157.9, 155.1, 140.7, 137.7, 136.1, 129.3, 128.8, 128.7, 128.6, 128.5, 127.4, 127.0, 121.6, 46.8, 29.0. HRMS (MALDI) calcd for

+ + C13H18NS [M H] 220.1160; found 220.1151.

1 NMR Compound 90c H (300 MHz, CDCl3) δ 8.40 (s, 1H), S N 7.62 – 7.53 (m, 2H), 6.94 – 6.85 (m, 2H), 3.83 (s, 3H), 1.45 (s, MeO 13 NMR 9H). C (75 MHz, CDCl3) δ 160.8, 154.9, 130.3, 128.3, + + 113.9, 55.3, 46.7, 29.2. HRMS (MALDI) calcd for C12H18NOS [M H] 224.1103; found 224.1109.

1 NMR Compound 90d H (300 MHz, CDCl3) δ 8.47 (s, 1H), 7.68 S N – 7.58 (m, 2H), 7.41 – 7.32 (m, 3H), 1.46 (s, 9H). 13C NMR (75

MHz, CDCl3) δ 155.2, 136.9, 129.6, 128.5, 126.8, 47.0, 30.5, 29.2. + + HRMS (MALDI) calcd for C11H16NS [M H] 194.0998; found 194.0995.

1 NMR Compound 90e H (300 MHz, CDCl3) δ 8.49 (s, 1H), S N 7.76 – 7.70 (m, 1H), 7.71 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H). 13C F3C NMR (75 MHz, CDCl3) δ 153.3, 139.7, 131.0, 126.8, 125.5, + + 122.7, 47.4, 29.2. HRMS (MALDI) calcd for C12H15F3NS [M H] 261.0799; found 261.0796.

47 Compound 92 To a round bottom ﬂask equipped with a magnetic stir S N bar was charged with 3 mL tetrahydrofuran, ca. 200 uL water, and

disulfide 20 (50 mg, 0.18 mmol). To this stirring solution was added triphenyl phosphine (310 mg, 1.17 mmol), followed by isoamyl nitrite (55 mg, 0.46 mmol). Reaction monitored by TLC rf = 0.45 (20% ethyl acetate in hexanes). Upon completion reaction mixture was washed with brine (1 mL), dried over MgSO4, solvent was removed under reduced pressure and the crude mixture was purified by flash column chromatography (5% ethyl acetate in hexanes).

1 NMR H (300 MHz, CDCl3) δ 8.92 (d, J = 0.9 Hz, 1H), 8.07 (dt, J = 8.0, 1.0 Hz, 1H), 7.97 (dq, J = 8.2, 0.9 Hz, 1H), 7.53 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 7.48 – 7.39 (m, 13 NMR 1H). C (75 MHz, CDCl3) δ 154.9, 151.6, 136.0, 127.7, 124.8, 124.0, 119.5. + + HRMS (MALDI) calcd for C7H6NS [M H] 136.0221; found 136.0230.

48 CHAPTER 3. PROLINE BASED PHOSPHORAMIDITE REDUCTIVE LIGATION REAGENTS

3.1 INTRODUCTION

S-Nitrosothiols have many biological implications, but are rarely used in organic synthesis. In this chapter the development of proline based phosphoramidite substrates that can eﬀectively convert S-nitrosothiols to proline-based sulfenamides through a reductive ligation process is reported. A unique property of this method is that the phosphine oxide moiety on the ligation products can be readily removed under acidic conditions. In conjugation with the easy preparation of S-nitrosothiols

(SNO) from the corresponding thiols (RSH), this method provides a new way to prepare proline-based sulfenamides from simple thiol starting materials. An overview of this reaction is shown in Figure 3.1.

O N O PPh OPh 10% H O in TFA O R SNO 2 N 2 HN S N Ph P H HN S 1c 98 O R Ph R 99 100

Figure 3.1: General form of the proline based phosphoramidite coupling.

49 3.1.1 Rationale for S-N bond Formation Methodology Develop- ment

Sulfur-nitrogen (S–N) bonds are unique chemical moieties, with a rich history in chemistry and medicine. These structures often show interesting biological activities (Figure 3.2). For example, sulfonylureas are a class of important herbicides.[75,

76] Sulfanilamides have been used as common drugs for infections.[77, 78] N-

Thiolated β-lactams are found to have antibacterial, anti-fungal, and anticancer eﬀects.[79–86] Sulfenamides exhibit antimicrobial activities against various infec- tious pathogens.[87] Proline-based sulfonamides have been recognized as potential anti-proliferative and anti-infective agents.[88, 89]

O O O S R O O O NH R S 2 N N N H H H2N R S R R sulfanilamide N-thiolated β-lactam sulfonylurea R

R O H O HN S O N N HN S H R O proline sulfonamide O R

sulfenamide

Figure 3.2: S-N linkages in pharmaceuticals.

Because of these activities, the preparation of these molecules, especially the formation of the S–N linkages, has become an active area in organic synthesis. So far, many methods have been developed for the construction of the S-N bonds, which include:

1. Sulfenylation between sulfenyl chlorides and amines.[90, 91]

2. Amination of thiols with N-halo compounds or amines in the presence of

oxidizing reagents to give unsubstituted and N-substituted sulfenamides.[92]

3. Treatment of disulﬁdes with ammonia or amines in the presence of silver or

50 mercuric salts in alkaline medium.[93, 94]

4. Sulfenylation of primary and secondary amines with the esters of sulfenic

acids.[95]

5. Derivations of N-chlorothio compounds.[96, 97]

6. [2,3]-sigmatropic rearrangement of S-allylsulfynimines.[98]

7. Reductive cleavage of sulﬁnimidic acids in the presence of thiophenols.[99]

8. Electrolysis of 2-mercapto benzothiazole amine or bis-benzothiazole-2-yldisulﬁde-

-amine.[100, 101]

9. Preparation of sulﬁn- or sulfon-amides by the oxidation of sulfenamides in

the presence of the oxidants.[102, 103]

The formation of S-nitrosothiols in biological systems is an important post- translational modiﬁcation elicited by nitric oxide (NO).[69–71] S-Nitrosothiols include protein-based adducts (through cysteine residues) and small molecules

(such as S-nitroso glutathione and S-nitroso cysteine). Small organic thiols (RSH) can also be easily converted to the corresponding S-nitrosothiols (SNO) under mild nitrosation conditions. Normally SNOs are unstable species, which makes the detection of S-nitrosothiols in biological systems very challenging. Moreover, the instability of SNO makes these compounds unattractive for synthetic chemists. As a result, the application of SNO in synthesis has been rarely reported. Recent work on SNO bioorthogonal reactions (aiming at the development of novel detection methods for protein SNO formation), we have discovered some unusual reactivity and properties of SNO.

In our opinion, SNO are powerful synthons that can be used to introduce S,

N, and/or O atoms into molecular structures. Herein is reported a method to prepare proline-based sulfenamides from readily available RSH substrates via SNO intermediates. The novel reactions and synthetic strategies introduced in this article could be applied for an eﬀective synthesis of biologically active molecules including

51 a proline moiety.

3.2 RESULTS AND DISCUSSION

In our previous work, we discovered that SNO and triarylphosphines (101) can rapidly react to generate reactive thioazaylide intermediates in high yields under mild conditions (Scheme 3.1).[43] Thioazaylides are potent nucleophilic species.

Upon manipulating the electrophilic groups attached to the phosphine reagents, thioazaylides can be trapped as stable products.

O O S Ph R N S O + O R N H O P Ph2P Ph Ph 1 101 102

Scheme 3.1: Phosphine based reductive ligation.

This reductive ligation process provides a way for capturing unstable biological

SNO as stable and detectable conjugates. In our opinion, this reaction, in conjunc- tion with easy SNO formation from RSH, would be also useful for the synthesis of sulfenamide derivatives from simple RSH starting materials. However, the use of phosphine substrates like 101 would lead to products like 102, which contain an unnecessary triphenylphosphine oxide moiety. The removal of this bulky group from the ﬁnal products would require harsh conditions. Therefore, seeking phosphine substrates that can undergo the reductive ligation while leading to a readily removable phosphine oxide moiety from the products is desirable.

3.2.1 Experimental Plan

As shown in Scheme 3.2, the reaction between SNO and 98 should still follow the reductive ligation process. Two equivalents of the phosphine reagent are still required, one is lost as the phosphine oxide. The other forms the thio-aza-ylide and undergoes the intramolecular reaction shown. The resultant phosphoramidate

52 moiety can be considered as a NH-protecting group, removable under acidic conditions. Given this, proline-linked sulfenamides could be prepared from simple RSH and phosphoramidite 98.

O O N O OR N PPh2 R SNO Ph P OR N N HN S 1 98 Ph Ph P S Ph O R R 99 103 98: a R = Me, b: R = t-Butyl, c: R = Ph

Scheme 3.2: General form of the proline-based ligation.

With this idea in mind, we proposed that proline-based phosphoramidites like

98a–c would be suitable substrates. Commercially available L-proline, or N-Boc-

L-proline were converted to the esters (104a–c) by either sulfonyl chloride based methanol solvolysis, or carbodiimide based amide formation.

SOCl2 O MeOH O N N H OH H O 105 104a

O Ph OH 1. DCC, DMAP O N 2. HCl OH or N + H O O O R 106 OH 104b,c

Scheme 3.3: Preparation of the proline-phosphoramidate - esteriﬁcation.

3.2.2 Reaction Screening

Following this we formed the nitrogen-phosphorous bond by treating the esters

((104a–c) with triethyl amine, followed by diphenyl phosphine chloride to generate our desired phosphoramidites Scheme 3.4, 98a–c in good yields (ca 80 %).

53 O O Et3N N + PPh2Cl N H CH2Cl2 O O R PPh2 R 104 98

98: a R = Me, b: R = t-Butyl, c: R = Ph

Scheme 3.4: Preparation of the proline-phosphoramidate - phosphoramidite formation.

These substrates were treated with TrSNO (1b) to explore their reactivity. The selection of TrSNO as the SNO model compound was due to its remarkable stability and ease of synthesis. We found the characteristic green color of TrSNO disap- peared immediately when treated with all three phosphoramidites, suggesting the formation of thio-aza-ylides were fast. For 98a and 98b, the reactions stopped at this stage as no desired ligation product was obtained, even when the reaction time was extended to 24 hours. With 98c, however, we obtained the desired ligation product 99a. This can be explained by the factor that phenyl ester is a better leaving group than the methyl and t-butyl esters. We tested a series of diﬀerent solvents for this reaction. The best found was a mixture of THF/phosphate buﬀer (pH 7.4, 20 mM, 3:1), which gave 99a in 84% yield.

O O O N Ph Ph S Ph OR Ph P OR N + 2 N H Ph N Ph SNO N Ph PPh2 S Ph P O Ph 98 1b Ph Ph Ph 99a 103a 98: a R = Me, b: R = t-Butyl, c: R = Ph

Scheme 3.5: Trityl-SNO coupled to the proline-phosphoramidite.

As phosphoramidite 98c was found to be the most reactive substrate for this ligation, it was applied to other SNO substrates to test the generality of the reaction.

A series of primary, secondary, and tertiary SNOs were freshly prepared from RSH and then used in this study without any puriﬁcation. The results were summarized in Table 3.1. In all cases, the desired ligation products were obtained. For relatively stable tertiary SNO substrates, the corresponding ligation products were isolated

54 in modest to high yields (entries 1-4). Secondary and primary SNO substrates gave slightly lower yields (entries 5-9). The puriﬁcation of secondary SNO products

(entries 5 and 6) were found to be very diﬃcult because they overlapped with the phosphine oxide byproduct. Nevertheless the formation of the products was clearly conﬁrmed by NMR and mass spectroscopy analysis. These results demonstrated that RSH can be readily converted to proline-based sulfenamides by this two-step

SNO-ligation process.

While phosphoramidite 98a did not show good reactivity in converting SNO to the desired sulfenamides, we did observe some unique reactivity of 98a. As shown in Scheme 3.6, the treatment of a cysteine SNO derivative 1p with 98a gave dehydroalanine 107 in a modest yield (37%) together with the disulﬁde product (46%).

These results indicated that the reaction indeed proceeded to form the thioazaylide intermediate 108a. Presumably the methyl ester was not reactive enough. So the acyl transfer was slow and non-productive. Instead, the azaylide underwent an intramolecular β-elimination on the cysteine substrate to form dehydroalanine 107.

3.2.3 Removal of the Phosphine Oxide Moiety

In the proline-based sulfenamide products, the diphenylphosphoryl moiety could be considered as the protecting group of proline. We expected it could be removed under acidic conditions.[104] We then tested the deprotection of 99g (Scheme 3.7).

Indeed a cleavage cocktail of 10% water in TFA provided sulfenamide 109 almost quantitatively. This result revealed that the S–N bond on the sulfenamide compounds was quite stable under acidic conditions. Given the eﬃciency of this protocol, it can ﬁnd applications in making water-soluble proline-based sulfenamides.

3.2.4 Use as a Nitroxyl trap

HNO is the one-electron reduced/protonated from of NO. It shows distinct physi- ology and pharmacology from NO.[105] The reductive ligation was also found to

55 O O S N R S O + R N N OPh H THF/buffer N O 1 PPh2 P Ph2 98c 99 entry SNO product 99 % yield

O Ph Ph N 1 HN S 84 Ph SNO Ph P 99 Ph O Ph 1b 99 Ph Ph 99a O O H BzHN N 2 NHBn N S NHBn 92 Ph P O NHBz 99 SNO 99 Ph O 1n 99b O O 3 SNO N 54 Ph P HN S 99 O Ph O 1o 99 99c O 4 N 57 99 SNO Ph P HN S Ph O 1g 99 99d

SNO O 5 N 82 99 Ph P HN S 1m 99 Ph O 99e O N 6 SNO 40 Ph P HN S 99 Ph O 1l 99 99f O O N BzHN 7 OMe Ph P HN S NHBz 62 Ph O 99 SNO OMe 1p 99 O 99g O O NHAc NHAc O H H N SNO S N MeO N OMe 8 N H 42 O P O O 99 Ph Ph Ph 1q 99 Ph 99h O NHAc O O NHAc H H S N N SNO N OMe 9 MeO N H 40 P O O 99 O Ph 1r 99 Ph 99i

Table 3.1: Substrate screen of the proline-based phosphoramidate coupling.

56 Ph N SNO Ph P OMe OMe THF, H2O N + OMe N O BzHN S Ph2P O H O 98a 1p BzHN OMe O 108a

Ph N Ph P O O O N OMe N S Ph P HN S OMe H Ph O BzHN OMe 99 NHBz O 108a

Ph N Ph P OMe N O OMe S BzHN H O OMe BzHN 37% O 107 108a Scheme 3.6: Dehydroalanine formation with primary SNO.

O 10% H O in TFA H 2 O N 30 min, 0 oC H N S OMe N N S OMe Ph2P O NHBz >95% H O O NHBz 99g 109

Scheme 3.7: Removal of the phosphine moiety under acidic conditions. be effective for HNO.[106] Several specific fluorescent probes for HNO detection have been developed based on the triarylphosphine template.[107–109] We next wondered if the proline-based phosphoramidite would work for HNO and tested the reaction between 98c and HNO (generated from Angeli’s salt 110). As shown in Scheme 3.8, the reaction proved to be fast and efficient. The desired ligation product 111 was obtained in a comparable yield (50%) as the one obtained from triarylphosphines,[106] suggesting the proline-based phosphoramidite may be useful for the design of novel HNO sensors.

57 O N OPh O PPh2 O Angeli's salt 98c N HNO OPh N Na N O Ph P NH 2 2 3 CH CN/H O NH Ph P 2 3 2 Ph O 110 Ph 112 111

Scheme 3.8: Trapping of HNO with the proline phosphoramidite reagent.

3.3 CONCLUSION

We developed a reductive ligation of Snitrosothiols using Ndiphenylphosphine proline ester substrates. This reaction was found to be eﬀective for all small molecule S-nitrosothiols (primary, secondary, and tertiary), as well as for HNO.

The ligation products bear a removable phosphine oxide moiety on the proline residue. In conjugation with the facile preparation of S-nitrosothiols (SNO) from the corresponding thiols (RSH), this novel method provides a unique way to prepare proline-based bioactive sulfenamides from simple thiol starting materials.

3.A APPENDIX

3.A.1 Methods

1H NMR spectra, 13C nmr and 31P nmr were recorded at 300 MHz (VX 300, Varian,

Palo Alto, CA, USA) and are reported in p.p.m. on the δ scale relative to residual

1 13 CHCl3 (δ 7.25 for H and δ 77.0 for C). These experiments were performed at room temperature. Mass spectra were recorded using an electrospray ionization mass spectrometry (esi, Thermo Finnigan LCQ Advantage, San Jose, CA, USA) or MALDI-TOF (matrix-assisted laser desorption ionization time-of-ﬂight) mass spectrometry. Mass data were reported in units of m/z for [M+H]+ or [M+Na]+.

Preparation of proline-based phosphoramidites To a solution of the proline ester HCl salt in CH2Cl2 (total c = 0.30 M) was added freshly distilled 2.5 equiv. of triethylamine followed by PPh2Cl (1 eq) at 0 ◦C. The mixture was slowly warmed

58 to room temperature and stirred for 3-4 h. Upon completion (monitored by TLC), the reaction mixture was filtered to remove the triethylammonium salt. The filtrate was concentrated and diluted with EtOAc, and washed with sat. NaHCO3, water, and brine. The organic layer was dried with anhydrous MgSO4, filtered, and concentrated. The crude product was purified by flash column chromatography

(with pre-neutralized silica gel by 3% TEA in hexanes).

1 NMR Compound 98a H (300 MHz, CDCl3) δ 7.59 – 7.27 (m, 10H), O 4.22 (ddd, J = 8.8, 6.0, 3.5 Hz, 1H), 3.63 (s, 3H), 3.17 – 3.06 (m, 1H), O 31 NMR N 2.88 (m, 1H), 2.22 – 1.70 (m, 4H); P (122 MHz, CDCl3) δ P 13 NMR 49.31; C (75 MHz, CDCl3) δ 176.00, 138.93, 132.88, 132.61, 132.23, 131.98, 128.90, 128.40, 128.33, 128.27, 128.19, 65.28, 64.87,

51.96, 47.64, 47.57, 31.80, 31.71, 25.90; MS (ESI) m/z calcd for C18H21NO2P [M+H]+ 314.1, found 314.1.

1 NMR 98b H (300 MHz, CDCl3) δ 7.49 (ddd, J = 8.1, 5.4, 1.5 Hz, 2H), 7.39 – 7.28 (m, 8H), 4.07 (ddd, J = 8.6, 6.6, 3.4 Hz, 1H), 3.17 O

O – 3.06 (m, 1H), 2.83 (dtd, J = 9.2, 6.8, 2.7 Hz, 1H), 2.07 (dq, J = N P 12.0, 8.3 Hz, 1H), 1.99 – 1.82 (m, 2H), 1.76 – 1.64 (m, 1H), 1.40 (s, 13 NMR 9H). C (75 MHz, CDCl3) δ 174.76, 139.27, 132.93, 132.66, 132.26, 132.01, 128.81, 128.38, 128.29, 128.19, 128.12, 80.76, 66.37, 31 NMR 65.95, 47.72, 47.65, 31.83, 31.74, 28.24, 25.87. P (122 MHz, CDCl3) δ 49.64.

59 1 NMR 98c H (300 MHz, CDCl3) δ 7.59 – 7.15 (m, 13H), 7.07 – 6.95 (m, 2H), 4.47 (ddd, J = 8.3, 6.3, 3.8 Hz, 1H), 3.21 (dddd, J = 8.7,

O 7.5, 5.1, 1.0 Hz, 1H), 2.95 (dtd, J = 9.4, 7.0, 2.4 Hz, 1H), 2.36 – 2.14 O N (m, 2H), 2.09 – 1.93 (m, 1H), 1.83 (m, 1H); 31P NMR (122 MHz, P 13 NMR CDCl3) δ 49.99; C (75 MHz, CDCl3) δ 174.0, 150.9, 138.8, 138.7, 138.6, 132.9, 132.7, 132.2, 131.9, 129.5, 129.0, 128.5, 128.3,

128.2, 125.9, 121.6, 65.6, 65.1, 47.8, 47.7, 32.0, 31.9, 25.9; MS (ESI) m/z calcd for

+ + C23H23NO2P [M H] 376.1, found 376.2.

3.A.2 Preparation of S-Nitrosothiols

The thiol starting material (RSH, 0.2 mmol) was dissolved in 1 mL of MeOH followed by the addition of 1 N HCl (1 mL) at room temperature. To this solution was then added freshly prepared 1 N NaNO2 (1 mL) in water in dark (total c = 0.07 M). The color of the reaction was immediately turned to red (for primary and secondary RSNO) or green (for tertiary RSNO). The mixture was stirred for 10-15 min at room temperature. Upon completion (monitored by TLC), the RSNO product was directly extracted with cold diethyl ether (1 mL x 3) in dark. The organic layers were collected and dried. The solvent was removed to provide the RSNO product, which was then used for the ligation reaction without further puriﬁcation.

3.A.3 General Reductive Ligation Procedure

To the freshly prepared RSNO product was added a solution of 2 equiv. of 98c in 3:1 THF-aqueous buﬀer (pH 7.4, de-gassed by bubbling with argon). The ﬁnal concentration was ca. .1 M. The reaction was monitored by TLC and it was usually completed within 15 to 30 min at room temperature. he reaction mixture was extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried by anhydrous Na2SO4, and concentrated. The crude product was

60 puriﬁed by ﬂash column chromatography.

1 NMR 103a H (300 MHz, CDCl3) δ 7.81 – 7.55 (m, 4H), 7.47 – 7.17 O (m, 23H), 6.94 – 6.80 (m, 3H), 4.35 (td, J = 8.8, 3.5 Hz, 1H), 3.14 (qd, N Ph P O Ph 2 N J = 6.5, 2.8 Hz, 2H), 2.30 (m, 1H), 2.13 – 1.99 (m, 1H), 1.99 – 1.78 (m, S Ph 31 NMR Ph 2H); P (122 MHz, CDCl3) δ 18.3; FT-IR (thin ﬁlm) 3025.4, Ph 2985.3, 1728.8 (strong, C=O, carbonyl group), 1601.3, 1450.0, 1372.3,

1 + + 1268.0, 1247.1, 1070.2 cm− MS (ESI) m/z calcd for C42H37N2NaO2PS [M Na] 687.2, found 687.1.

1 NMR 99a H (300 MHz, CDCl3) δ 7.81 (s, 1H), 7.76 – 7.24 (m, O 25H), 3.95 (dt, J = 9.2, 5.5 Hz, 1H), 3.01 (p, J = 8.1 Hz, 1H), 2.66 N HN S Ph P O Ph – 2.51 (m, 1H), 1.92 (m, 2H), 1.68 – 1.61 (m, 1H), 1.34 – 1.26 (m, Ph Ph Ph 31 NMR 13 NMR 1H); P (122 MHz, CDCl3) δ 30.2; C (75 MHz,

CDCl3) δ 178.99, 150.71, 145.25, 133.24, 133.12, 132.87, 132.74, 132.01, 131.59, 130.54, 130.04, 129.81, 129.51, 128.59, 128.41, 128.22, 128.15, 127.56, 126.42,

126.00, 121.61, 120.73, 115.57, 63.10, 60.11, 49.03, 32.18, 25.76; MS (ESI) m/z

+ + calcd for C36H33N2NaO2PS [M Na] 611.2, found 611.1.

1 NMR 99b H (300 MHz, CDCl3) δ 9.55 (t, 5.6 Hz, 1H, O NHBz S NHBn NH)), 9.12 (d, J = 9.5 Hz, 1H, NH)), 8.88 (s, 1H, NH), N H N O 8.12 – 6.82 (m, 20H), 5.18 (d, J = 9.0 Hz, 1H), 4.46 – 4.33 P O Ph Ph (m, 2H), 4.20 – 4.02 (m, 1H), 3.44 (m, 1H), 3.31 (m, 1H),

2.39 (m, 1H), 2.17 (m, 3H), 1.36 (d, J = 10.8 Hz, 6H); 31P NMR 13 NMR (122 MHz, CDCl3) δ 30.2; C (75 MHz, CDCl3) δ 173.13, 169.58, 168.43, 157.53, 150.52, 133.19, 133.06, 133.03, 132.99, 132.53, 132.39, 132.21,

132.06, 131.05, 130.90, 130.49, 129.75, 129.68, 129.62, 129.33, 129.16, 129.01,

128.93, 128.85, 128.61, 128.23, 128.12, 127.42, 126.29, 121.54, 121.42, 119.59,

115.85, 62.20, 60.24, 55.76, 47.78, 43.79, 32.61, 25.91; MS (ESI) m/z calcd for

+ + C36H39N4NaO4PS [M Na] 677.2, found 677.1.

61 1 NMR 99c H (400 MHz, CDCl3) δ 8.72 (s, 1H), 7.82 (m, O 4H), 7.58 – 7.40 (m, 6H), 4.18 (t, J = 7.5 Hz, 1H), 3.19 (dd, S N H N O J = 12.2, 6.0 Hz, 2H), 2.65 – 2.25 (m, 5H), 2.03 (t, J = 11.8 P O Ph Ph Hz, 2H), 1.90 (dd, J = 13.6, 10.1 Hz, 5H), 1.42 (d, J = 7.3 Hz,

3H), 1.25 (d, J = 8.4 Hz, 3H), 1.00 (d, J = 6.0 Hz, 2.5H), 0.93 31 NMR 13 NMR (d, J = 7.1 Hz, 0.5H); P (122 MHz, CDCl3) δ 28.70; C (101 MHz,

CDCl3) δ 211.43, 175.80, 132.34, 132.25, 132.15, 131.96, 131.86, 128.89, 128.79, 128.66, 77.32, 77.00, 76.68, 62.25, 57.80, 52.67, 52.24, 48.20, 36.73, 34.47, 31.11,

31.05, 30.90, 29.74, 25.64, 25.16, 25.10, 22.28, 22.19; MS (Maldi) m/z calcd for

+ + C27H36N2O3PS [M H] 499.2178, found 499.2201.

1 NMR 99d H (300 MHz, CDCl3) δ 8.64 (br-s, 1H, NH), 7.92 – 7.72 O S (m, 4H), 7.52 (dddd, J = 12.8, 9.3, 7.3, 2.3 Hz, 6H), 4.21 (ddd, J N H N = 8.5, 6.5, 2.2 Hz, 1H), 3.20 (m, 2H), 2.43 (dd, J = 12.7, 5.9 Hz, P O Ph Ph 1H), 2.13 – 2.02 (m, 1H), 1.91 (m, 2H), 1.28 (s, 9H); 31P NMR 13 NMR (122 MHz, CDCl3) δ 30.0; C (75 MHz, CDCl3) δ 175.98, 132.67, 132.52, 132.39, 132.32, 132.19, 129.65, 129.25, 129.13, 129.08, 128.97,

121.40, 62.58, 48.91, 48.35, 31.49, 28.92, 25.42, 25.33; MS (Maldi) m/z calcd for

+ + C21H28N2O2PS [M H] 403.1609, found 403.1604.

1 NMR 99g H (300 MHz, CDCl3) δ 8.97 (s, 1H), 8.30 (d, O NHBz S OMe J = 8.3 Hz, 1H), 8.09 – 7.97 (m, 2H), 7.90 – 7.67 (m, 4H), N H N O 7.62 – 7.36 (m, 9H), 5.02 (ddd, J = 8.3, 6.3, 4.3 Hz, 1H), P O Ph Ph 4.27 – 4.02 (m, 1H), 3.74 (s, 3H), 3.32 (dd, J = 14.8, 6.4 Hz,

1H), 3.04 (m, 3H), 2.35 – 2.17 (m, 1H), 2.01 – 1.84 (m, 1H), 31 NMR 13 NMR 1.72 (m, 2H); P (122 MHz, CDCl3) δ 30.1; C (75 MHz, CDCl3) δ 176.84, 171.32, 167.73, 133.84, 132.72, 132.50, 132.37, 132.21, 132.07, 131.97,

129.21, 129.10, 128.93, 128.65, 127.91, 61.90, 52.96, 51.28, 48.72, 42.00, 30.95,

+ + 25.32; MS (ESI) m/z calcd for C28H30N3NaO5PS [M Na] 574.3, found 574.3.

62 1 NMR 99h H (300 MHz, CDCl3) δ 8.92 (d, J = 7.6 O Hz, 1H), 8.44 (s, 1H), 7.75 (tdd, J = 12.0, 8.3, 1.4 Hz, O NH O H S N N OMe 4H), 7.67 – 7.38 (m, 6H), 7.01 (d, J = 7.2 Hz, 3H), H N O P O Ph 6.97 – 6.85 (m, 3H), 5.38 (td, J = 9.5, 3.2 Hz, 1H), Ph Ph 4.63 (td, J = 8.3, 5.0 Hz, 1H), 4.07 (td, J = 7.0, 5.0 Hz,

1H), 3.67 (s, 3H), 3.42 (m, 1H), 3.27 – 2.99 (m, 3H), 2.84 (dd, J = 13.9, 8.9 Hz, 1H),

2.49 (dd, J = 14.1, 9.9 Hz, 1H), 2.23 – 2.10 (m, 2H), 2.08 (s, 3H), 2.02 – 1.76 (m, 3H); 31 NMR 13 NMR P (122 MHz, CDCl3) δ 30.4; C (75 MHz, CDCl3) δ 177.22, 171.75, 171.46, 171.04, 136.73, 132.65, 132.52, 132.33, 132.19, 129.31, 129.20, 129.10,

129.04, 128.93, 128.41, 126.81, 62.41, 54.36, 52.55, 49.93, 43.67, 37.95, 25.70,

+ + 23.40; MS (ESI) m/z calcd for C32H37N4NaO6PS [M Na] 659.2, found 659.3.

1 NMR 99i H (300 MHz, CDCl3) δ 8.21 (d, J = 7.4 O Hz, 1H), 7.80 (m, 5H), 7.45 (m, 6H), 7.19 (d, J = 8.1 O NH O H S N N OMe Hz, 1H), 4.93 (m, 1H), 4.75 (m, 1H), 4.50 (m, 1H), H N O P O 4.01 (ddd, J = 8.2, 5.9, 2.3 Hz, 2H), 3.75 – 3.57 (m, Ph Ph 3H), 3.29 – 3.11 (m, 2H), 2.40 – 2.24 (m, 1H), 2.10 –

2.03 (m, 1H), 1.98 (m, 3H), 1.92 – 1.81 (m, 4H), 1.47 – 1.29 (m, 3H); 31P NMR (122 13 NMR MHz, CDCl3) δ 30.4; C (75 MHz, CDCl3) δ 171.22, 170.56, 170.56, 170.08, 132.61, 132.57, 132.54, 132.51, 132.42, 132.37, 132.30, 132.24, 131.76, 131.52,

130.05, 129.84, 129.19, 129.10, 129.03, 128.93, 62.06, 62.02, 53.70, 52.63, 48.47,

48.06, 48.01, 42.04, 31.27, 31.19, 25.26, 25.17, 23.24, 17.97, 17.57; MS (ESI) m/z

+ + calcd for C26H35N4NaO5PS [M Na] 569.2, found 569.3.

1 NMR Dehydroalanine (Dha) 107 H (300 MHz, CDCl3) δ 8.54 O O (br, 1H), 7.85-7.81 (m, 2H), 7.57-7.43 (m, 3H), 6.79 (s, 1H), 5.98 N H 13 NMR O (d, 1H, J = 1.4 Hz), 3.88 (s, 3H); C (75 MHz, CDCl3) δ 166.0, 165.0, 134.4, 132.3, 131.2, 129.0, 127.1, 109.1, 53.3; MS

+ + (ESI) m/z calcd for C11H12NO3 [M H] 206.1, found 206.2.

63 3.A.4 Deprotection of the Diphenylphosphoryl Group

A sulfenamide product (0.05 mmol) was treated with 1 mL of cold 10% water in

TFA at 0 ◦C (total c = 0.05 M). The resulted solution was stirred for 30 min at 0 ◦C. Upon completion, the excess TFA was removed with hexanes as the co-solvent under vacuum. To the remaining mixture was added cold diethyl ether to solidify the proline-sulfenamide TFA salt. The solid product was further washed with cold diethyl ether (2 mL x 5) and dried to provide the ﬁnal product.

1 NMR 109 H (300 MHz, CDCl3) δ 10.31 (br-s, 1H), 9.70 O H N (br-s, 1H), 9.09 (s, 1H), 7.57 – 7.35 (m, 5H), 4.91 (m, 1H), N S OMe H O NHBz 4.64 (m, 1H), 3.74 (s, 3H), 3.38 (m, 2H), 3.23 (m, 2H), 13 NMR 2.32 (m, 1H), 2.01 (m, 3H); C (75 MHz, CDCl3) δ 172.40, 171.21, 168.61, 132.96, 128.96, 128.70, 127.61, 60.54, 53.21, 51.71, 47.05,

+ + 40.52, 29.64, 24.59; MS (Maldi) m/z calcd for C16H22N3O4S [M H] 352.1331, found 352.1320.

The reaction between 98c and HNO

To an argon sparged mixture of acetonitrile and water was added 11 (0.21 mmol), to this stirring mixture was added freshly prepared Angeli’s salt (0.1 mmol, Na2N2O3). The resulting solution was let stir until the reaction was completed (by TLC, or 20 minutes). The product 21 (50% yield) was isolated by extraction and ﬂash column chromatography.

64 1 NMR 111 H (300 MHz, CDCl3) δ 10.04 (br-s, 1H, NH), 7.96 – 7.68 O (m, 5H), 7.64 – 7.39 (m, 5H), 5.85 (br-s, 1H, NH), 4.03 (ddd, J = 8.1, N Ph P NH2 Ph O 5.7, 2.3 Hz, 1H), 3.18 (m, 2H), 2.46 – 2.18 (m, 1H), 2.18 – 1.99 (m, 1H), 31 NMR 1.89 (ddt, J = 13.5, 8.9, 4.2 Hz, 2H); P (122 MHz, CDCl3) δ 13 NMR 28.6; C (75 MHz, CDCl3) δ 176.32, 132.66, 132.62, 132.59, 132.55, 132.44, 132.41, 132.31, 132.28, 131.65, 131.39, 129.92, 129.71, 129.22, 129.13, 129.05,

128.96, 62.12, 62.08, 60.45, 48.07, 48.02, 31.29, 31.21, 25.27, 25.18; MS (ESI) m/z

+ + calcd for C17H19N2NaO2P [M Na] 337.1, found 337.1.

65 APPENDICES P

O 2 4 2 2 8 2 . 9 0 1 0 . . . 0 . 0 1 2 1 1

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Spectra .1: 80

67 S N

O P 1 2 0 6 3 1 . . 0 2 . 1 9 . 1 1 1 1

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

Spectra .2: 84a

68 P O

N O S NH

CH3 H C 3 CH3 O CH O 3 8 1 8 8 9 0 6 1 3 8 2 5 . 0 0 0 1 0 1 3 2 4 6 . . 2 ...... 1 1 1 1 1 1 1 3 3 3 3

11.5 11.0 10.5 10.0 9.5 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 0.0 f1 (ppm)

P O

N O S NH

CH3 H C 3 CH3 O CH O 3

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Spectra .3: 84b

69 P O

N S 0 9 6 5 7 8 . 9 1 6 1 . . 3 . . 0 1 1 1 1 H3C CH3 CH3 9.0 8.5 8.0 7.5 7.0 f1 (ppm) 0 5 9 6 5 7 8 2 . . 9 1 6 1 . . 3 . . 1 0 1 1 1 1 1

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

P O

N S

CH3 H3C CH3

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

Spectra .4: 84c

70 P O

N S

CH3 0 7 7 5 1 3 9 2 . 9 9 5 0 9 0 . . 1 . . . . 0 0 1 6 1 0 3

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3 f1 (ppm)

P O

N S

CH3

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

Spectra .5: 84d

71 P O

N S 2 0 0 9 1 7 7 6 8 1 5 . 0 0 5 0 0 1 0 2 1 . . 1 ...... 1 1 1 1 1 1 2 2 1 6

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

Spectra .6: 84e

72 S CH3 N CH3 CH3 6 0 8 5 9 5 4 2 1 1 2 9 7 7 ...... 0 0 2 3 0 0 0

8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 f1 (ppm) 6 0 8 5 9 0 5 4 2 1 1 2 9 0 7 7 ...... 0 0 2 3 0 0 0 9

10.5 10.0 9.5 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 0.0 -0.5 f1 (ppm)

S CH3 N CH3 CH3

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Spectra .7: 90a

73 O H3C CH3 CH3 N S CH3 0 4 1 0 4 0 0 0 0 3 . . . . . 1 2 2 3 9

11.5 11.0 10.5 10.0 9.5 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 0.0 f1 (ppm)

O H3C CH3 CH3 N S CH3

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Spectra .8: 90f

74 S CH3 N

H3C CH3 0 3 8 0 0 9 . . . 1 2 2

8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 f1 (ppm) 0 3 8 0 0 0 9 0 . . . . 1 2 2 9

12.0 11.5 11.0 10.5 10.0 9.5 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 0.0 -0.5 -1.0 f1 (ppm)

S CH3 N

H3C CH3

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Spectra .9: 90d

75 S CH3 N F CH3 CH3 F F 1 0 0 0 0 0 . . . 1 2 2

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 f1 (ppm)

230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 f1 (ppm)

Spectra .10: 90e

76 S N 0 9 7 0 9 0 9 9 0 9 . . . . . 1 0 0 1 0

11.5 11.0 10.5 10.0 9.5 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 0.0 -0.5 -1.0 f1 (ppm)

S N

250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 f1 (ppm)

Spectra .11: 92

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