NOVEL CHEMISTRY AND CHEMICAL TOOLS FOR
HYDROGEN SULFIDE RESEARCH
By
BO PENG
A dissertation submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY Department of Chemistry
MAY 2016
© Copyright by BO PENG, 2016 All Rights Reserved
© Copyright by BO PENG, 2016 All Rights Reserved
To the Faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of
BO PENG find it satisfactory and recommend that it be accepted.
Ming Xian, Ph.D., Chair
Cliff Berkman, Ph.D.
Jeff Jones, Ph.D.
Rob Ronald, Ph. D.
ii ACKNOWLEDGEMENT
I would like to thank Dr. Ming Xian for supervising me during my Ph.D study and research at WSU. He provided guidance in person on both my experimental techniques and scientific writing. Dr. Xian led me to the research fields of hydrogen sulfide, from where I developed my research interest in bioorganic chemistry. I learned not only various research skills from him, but also critical thinking which is important for my entire research career. Dr. Xian also encouraged me to broaden my horizons in research fields by providing opportunities attending to conferences and symposiums.
To my committee members, Dr. Cliff Berkman, Dr. Rob Ronald, and Dr. Jeff
Jones, I will always be thankful for teaching me important core courses and giving all the suggestions and comments on my seminars, research and proposals. I would also like to thank Dr. Hector Aguilar-Carreno for his help in developing my cell culture skills and advising in the cell experiments I have done in his laboratory. I also want to thank Dr. Chulhee Kang for teaching me chemical biology and helping me with my grant applications. I would like to thank Dr. Phil Garner for teaching me synthetic methods.
During my Ph.D. study, I received the assistance and support from every members of Dr. Xian’s group including Dr. Chunrong Liu, Dr. Chung-Min Park,
Dr. Dehui Zhang, Dr. Pei Zhang, Dr. Nelmi Devarie, Dr. Jia Pan, Dr. Wei Chen
Dr. Yu Zhao, Tyler Biggs, Armando Pacheco, Jianming Kang, Shi Xu, and Jake
Day.
iii I would like to thank Nikki Clark, Michael Finnegan, Scot Wherland, and Ryan
Rice for their help in my teaching assistance work. I also want to thank Trent
Amonett, Debbie Arrasmith, Lori Bruce, Stacie Olsen-Wilkes, Molly Spain,
Yoshi Kodama and all the other staffs in the chemistry department who offered me help during these years.
Most importantly, I want to thank my parents for encouraging me to study abroad from the beginning and for offering spiritual support whenever I need. I also want to thank my best friend, my beloved partner Tianjiao for her unconditional love, and support.
Lastly, I would like to thank the Chemistry Department, Graduate School, and
College of Science and Art at WSU for giving me the opportunity to study here and providing all the support these years. Also, I would like to thank all the research funding and scholarship I have received for their financial support.
iv NOVEL CHEMISTRY AND CHEMICAL TOOLS FOR
HYDROGEN SULFIDE RESEARCH
Abstract
by Bo Peng, Ph.D. Washington State University May 2016
Chair: Ming Xian
Hydrogen sulfide (H2S) is a critical cell signaling molecule which has attracted attention recently for its contributions to human health and diseases. H2S has been considered as a cytoprotectant and gasotransmitter in many tissue types, including mediating vascular tone in blood vessels as well as neuromodulation in the brain. It is important, therefore, to understand the fundamental chemistry of H2S and to develop effective and convenient methods for H2S detection.
The thesis will discuss three aspects of H2S: first, the development of fluorescent probes for the detection of H2S; second, the study on the generation of H2S from NADH (reduced nicotinamide adenine dinucleotide) model compounds and sulfane sulfurs; lastly, development of a new method for persulfide generation.
Development of such chemical tools will help study the mechanisms by which
H2S modulates signaling pathways and maintains cellular functions.
v TABLE OF CONTENTS
Page
ACKNOWLEDMENT..………………………………..…….……………….iii
ABSTRACT………………………………………………….………..……….v
LIST OF TABLES...…………………………………………….………...…....x
LIST OF FIGURES...…………………………………………..…………...... xi
LIST OF SCHEMES………………………………………………………...xiii
ABBREVIATIONS………………...…………………………………………xv
CHAPTER 1. INTRODUCTION…………………..…………………………..1
1.1 HYDROGEN SULFIDE…………………………………………...... 1
1.2 TRADITIONAL DETECTION OF HYDROGEN SULFIDE…...….….3
1.3 FLUORESCENT PROBES FOR HYDROGEN SULFIDE…...... ……..4
1.3.1 REDUCTION-BASED H2S FLUORESCENT PROBES………...4
1.3.2 NUCLEOPHILIC REACTION-BASED H2S FLUORESCENT
PROBES………………………………………………………………6
1.3.3 METAL SULFIDE FORMATION-BASED H2S FLUORESCENT
PROBES………………………………………….……………………..9
1.4 ENDOGENOUS GENERATION OF H2S…………………..………….9
1.5 CHEMISTRY OF S-SULFHYDRATION………………..……………11
1.6 REFERENCES…………………..…………………………………….14
CHAPTER 2. DISULFIDE-BASED FLUORESCENT PROBES FOR
HYDROGEN SULFIDE……………………………………………………….26
vi 2.1 ABSTRACT……………………………………………………….…...26
2.2 DESIGN AND SYNTHESIS OF PROBES…..………………………..26
2.3 FLUORESCENCE PROPERTIES AND RESPONSES OF PROBES
WSP1-5 TO H2S………………………………..………………………….29
2.4 LIVING CELL IMAGING STUDIES………..……………………..…39
2.5 CONCLUSION…………………………………………..…………….42
2.6 EXPERIMENTAL SECTION…………………………………………42
2.6.1 SYNTHESIS……………………………………………..………43
2.6.2 FLUORESCENCE ANALYSIS…………………….…………...47
2.7 REFERENCE……….…………………………………………..…....52
CHAPTER 3. DISELENIDE-BASED FLUORESCENT PROBES FOR
HYDROGEN SULFIDE……………………………………………………..54
3.1 ABSTRACT………………..………………………………………..…54
3.2 DESIGN AND SYNTHESIS……………………………………..…54
3.3 FLUORESCENCE ANALYSIS……………………………………….58
3.4 CONCLUSION……………………………….………………………..63
3.5 EXPERIMENTAL SECTION………………………………………....64
3.5.1 CHEMICAL SYNTHESIS…………………………………...... 64
3.5.2 MODEL REACTIONS AND STABILITY STUDIES………...... 67
3.5.3 FLUORESCENCE ANALYSIS…………………………………68
3.6 REFERENCES………………………………….…………………..…71
vii CHAPTER 4. STUDY OF THE REACTIONS BETWEEN SULFANE
SULFURS AND NAD(P)H AS A NON-ENZYMATIC H2S GENERATION
PATHWAY...... 73
4.1 ABSTRACT…………………………………………………………....73
4.2 REACTIONS BETWEEN NADH MODEL COMPOUNDS AND
SULFANE SULFUR...... 73
4.3 DETECTION OF H2S GENERATION…………..……………………78
4.4 CONCLUSION………………………………………………………...80
4.5 EXPERIMENTAL SECTION………………………………………....81
4.5.1 CHEMICAL SYNTHESIS…………...………………………….81
4.5.2 REACTIONS BETWEEN NADH MODEL COMPOUNDS...…84
4.5.3 DETECTION OF H2S GENERATION……...…………………..84
4.6 REFERENCES……………………………………………………...…86
CHAPTER 5. DEVELOPMENT OF A NEW METHOD FOR PERSULFIDE
GENERATION……………………………...... 88
5.1 ABSTRACT……………………………………………………………88
5.2 DESIGN OF THE NEW METHOD…………………………………...88
5.3 TEST THE NEW STRATEGY WITH THIOLS……………………....89
5.4 CONCLUSION………………………………………………………...92
5.5 EXPERIMENTAL SECTION……………………...………………….93
5.6 REFERENCES……………………………………………….………..95
viii APPENDIX……...... 96
ix LIST OF TABLES
Table 2.1 Fluorescent properties of probes WSP1-5………………………....30
Table 2.2 Turn-on fold changes and detection limits of probes WSP1-5…….33
Table 3.1 Fluorescent properties of SeP probes……………………………58
x LIST OF FIGURES
Figure 2.1 Time-dependent fluorescence changes of WSP1………...... 31
Figure 2.2 Time-dependent fluorescence changes of WSP probes…………....32
Figure 2.3. Fluorescence spectra changes of WSP probes……...…..……….33
Figure 2.4 Fluorescence emission spectra of WSP probes with varied concentrations of NaHS……………………………………………..…………34
Figure 2.5 Fluorescence intensity changes of WSP probes at different pH………………………………………………………………………………36
Figure 2.6 Fluorescence intensity of WSP probes in the presence of various reactive sulfur species……………………………….…………………...…….37
Figure 2.7 Fluorescence intensity of WSP probes with esterase…...……….39
Figure 2.8 Fluorescence images of H2S in HeLa cells...... 40
Figure 2.9 Fluorescence images of H2S production from a H2S donor...... 42
Figure 3.1 Time-dependent fluorescence changes of SeP probes……………..59
Figure 3.2 Fluorescence emission spectra of SeP2 in the presence of varied concentrations of Na 2 S……………….……...……………………….60
Figure 3.3 Fluorescence intensity of SeP probes in the presence of various reactive sulfur species………………………………………………………….61
Figure 3.4 Fluorescence images of H2S in HeLa cells using SeP2………..….62
Figure 3.5 Fluorescent detection of in-situ generated H 2 S in human neuroblastoma cell……………………………………………………………63
xi Figure 4.1 UV absorption spectra of methylene blue which indicate the production of H2S……………………………………………………………..80
Figure 4.2 Fluorescence spectra of the samples which indicate the production of
H2S……………………………………………………………………………..80
xii LIST OF SCHEMES
Scheme 1.1 Structures and fluorescence turn-on mechanisms of SF1, SF2, and
DNS-Az……………………………………………………………...………….6
Scheme 1.2 Structures and fluorescence turn-on mechanisms of WSP1, SFP-1 and SFP-2…………………………………………………………….…………8
Scheme 1.3 Structure and mechanism of HSip-1…………….…………………9
Scheme 2.1 General design of the nucleophilic substitution/cyclization based fluorescent probes……………………………………………………...………27
Scheme 2.2 Model reaction between compound 2.1 and H2S………….……...28
Scheme 2.3 Preparation of fluorescent probes WSP1-5……………………….29
Scheme 3.1 Design of diselenide-based probes for H2S…………….………56
Scheme 3.2 Model reactions between compound 3.6 and H2S…………..….57
Scheme 3.3 Structures of SeP probes……………………...…………………..58
Scheme 4.1 Optimization of reaction conditions between Hantzsch ester and cysteine polysulfide…………………………………………………………....74
Scheme 4.2 Structures of various sulfane sulfurs………….…………………..75
Scheme 4.3 Reactions between Hantzsch ester and different sulfane sulfurs…76
Scheme 4.4 Optimization of the reaction conditions between BNAH and cysteine polysulfide……………………………………………………………77
Scheme 4.5 Reactions between BNAH and different sulfane sulfurs…………78
xiii Scheme 4.6 Procedures for detection of H2S generated from the reactions between NADH model compounds and sulfane sulfurs…………….……...….79
Scheme 5.1 Design of the one-step persulfide formation from thiol……..……89
Scheme 5.2 Structures of the thiols and electrophiles…...…………………….90
Scheme 5.3 Results of the reactions between Beaucage reagent and thiols…...91
Scheme 5.4 The reactions between Beaucage reagent and thiols in various solvents…………………...……………………………………………………92
xiv ABBREVIATIONS
ABBREVIATION EQUIVALRNT
AdSH adamantanethiol
BNAH 1-benzyl-1,4-dihydronicotinamide
CAT cystine aminotransferase
CBS cystathionine β-synthase
CH3CN acetonitrile
CO carbon monoxide
CSE cystathionine γ-lyase
CTAB hexadecyltrimethylammonium bromide
Cys cysteine
DCM dichloromethane
DI H2O double deionized water
DMAP 4-dimethylaminopyridine
DMEM Dulbecco's Modified Eagle Medium
DMF dimethylformamide
DMSO dimethyl sulfoxide
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
FDNB 1-fluoro-2,4-dinitrobenzene
FeCl3 ferric chloride
GAPDH glyceraldehyde 3-phosphate dehydrogenase
xv GSH glutathione
H2O water
H2S hydrogen sulfide
H2S2 hydrogen disulfide
HSNO thionitrous acid
IAM iodoacetamide
IR infrared
MB methylene blue
MeOH methanol
3-MP 3-mercaptopyruvate
3-MST 3-mercaptopyruvate sulfurtransferase
NADH nicotinamide adenine dinucleotide (reduced)
NADPH nicotinamide adenine dinucleotide phosphate (reduced)
NMR nuclear magnetic resonance
NO nitric oxide
PBS phosphate buffered saline
RNS reactive nitrogen species
ROS reactive oxygen species
RSS reactive sulfur species
RSSH persulfide rt room temperature
xvi SNO S-nitrosothiol
THF tetrahydronfuran
TLC thin layer chromatography
TrSH triphenylmethyl mercaptan
UV/VIS ultraviolet–visible
WSP Washington State probe
Zn(OAc)2 zinc acetate
xvii CHAPTER ONE
INTRODUCTION
1.1 HYDROGEN SULFIDE
Hydrogen sulfide (H2S) has been known as a gaseous pollutant with the smell of rotten eggs for years. People have been aware of its lethal effects and toxicity
[1.1-1.2] based on the occupational and toxicological studies on H2S. Hydrogen sulfide-induced acute central toxicity includes reversible unconsciousness, eye irritation, and respiratory irritation. However, recent studies have shown that
H2S is naturally occurring in mammalian cells. The endogenous formation of
H2S is mainly attributed to enzymes including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase
[1.3-1.8] (3-MST). These enzymes convert cysteine or cysteine derivatives into H2S in different tissues and organs.
It has been demonstrated that H2S can target different ion channels and modify
[1.9-1.13] various physiological functions. H2S is known as the first gas molecule that can open KATP channels in vascular smooth muscle cells. H2S or
H2S-releasing compounds can also cause negative inotropic and chronotropic actions due to activating mitoKATP and sarcKATP channels and inhibiting the
2+ - L-type Ca channel. What is more, H2S can activate Cl channels, a mechanism
1 that is known to protect neurons from oxytosis. Studies have demonstrated that the endogenous production of H2S and exogenous administration of H2S can exert protective effects in many pathologies.[1.9-1.14] These include regulation of blood pressure, inhibition of leukocyte adherence, and neuromodulation. As a potent antioxidant, H2S can upregulate antioxidant defense under chronic conditions. Inadequate levels of H2S are linked to a variety of diseases including Down syndrome, Alzheimer’s disease, diabetes, and liver
[1.15-1.19] cirrhosis. These studies have established H2S as the third endogenous gaseous transmitter along with nitric oxide (NO) and carbon monoxide (CO).
To date, the exact mechanisms of action of H2S are still under active investigation. As a highly reactive molecule, H2S can react with a number of biological targets, and these reactions may be responsible for the biological functions of H2S. For example, oxidation of H2S by O2 can produce polysulfides, sulfite, thiolsulfate, or sulfate as intermediates or products.
Evidence has been provided that H2S can react with reactive oxygen species quickly such as superoxide, hydrogen peroxide, peroxynitrite, and hypochloric acid. Therefore, H2S is considered as an effective scavenger of those
[1.20] species. Recently it was reported that H2S can promote protein
S-sulfhydration, that is, converting cysteine residues into persulfides. This post-translational modification is critical as it provides a possible mechanism by
[1.21-1.26] which H2S alters the functions of cellular proteins. Moreover, it is likely
2 that H2S can react with protein or small molecule S-nitrosothiols to form thionitrous acid (HSNO). The metabolites of HSNO, including NO+, NO, and
NO-, have distinct but important physiological consequences.[1.27] In addition to these reported reactions, it is possible that other biologically important reactions of H2S will be discovered. The complexity of these reactions and highly reactive nature of H2S make the detection of H2S in biological systems a difficult task. It is important, therefore, to understand the fundamental chemistry of H2S and to develop effective and convenient methods for H2S detection.
1.2 TRADITIONAL DETECTION OF HYDROGEN SULFIDE
As H2S has been considered as a pollutant for years, many methods for its detection in environmental and industrial samples have been developed, which include colorimetric measurements, electrochemical assays, gas chromatography, and polarographic sensors.[1.28-1.30] In general these methods are sensitive and specific. However, these classical measurements do not allow for the temporal and spatial monitoring of reactive and transient H2S in biological samples. They usually require complicated sample destruction which may lead to inconsistent results due to the high reactivity and volatility of
[1.31-1.35] H2S. For example, one of the most often used methods is the methylene blue method. This method calls for strongly acidic conditions. In the presence of
FeCl3 and Zn(OAc)2, H2S reacts with N,N-dimethyl-p-phenylenediamine to
3 give methylene blue as the product, which has a characteristic absorbance at
670 nm. Although this method is selective and sensitive, strong acid pretreatments can liberate H2S from other forms of sulfide, for example, acid-labile pools. Therefore this method may cause erroneous results, making it only suitable for measuring H2S in simple aqueous solutions. Most of these measurements cannot be readily applied in biological studies because of the presence of H2S scavengers, the influence of hemoglobins or other pigment compounds, or redox-balance or pH changes.[1.12] As such, real-time detections of H2S in biological samples are critically needed and have become a hot area in
H2S research. In this regard, fluorescence-based assays have great potential as they are suitable for nondestructive detection in live cells or tissues with readily available instruments. In the past five years (2011-2016), a number of strategies have been reported for H2S fluorescence detection and all of these strategies are
[1.36-1.40] based on H2S-specific reactions.
1.3 FLUORESCENT PROBES FOR HYDROGEN SULFIDE
1.3.1 REDUCTION-BASED H2S FLUORESCENT PROBES
It has been long known that aryl azides (ArN3) can be reduced to amines by hydrogen sulfide.[1.41-1.43] In 2011, Chang and coworkers reported the first
[1.44] azide-reduction-based fluorescent probes for H2S detection. In this work, two azide-caged rhodamine analogues (SF1 and SF2, scheme 1.1) were
4 prepared. The fluorescence properties of these two probes were evaluated in aqueous buffers at physiological pH. Upon treatment of the probes with NaHS
(as the equivalent of H2S), significant fluorescence increases were observed.
The selectivity of both probes for H2S over other reactive sulfur species (RSS), reactive oxygen species (ROS), and reactive nitrogen species (RNS) was found to be good. Moreover, the abilities of SF1 and SF2 in visualizing H2S concentration changes in living cells were demonstrated by exogenous administration of NaHS into cells.
Almost during the same time when SF1/SF2 were reported, another group led by Wang disclosed a structurally different azide-based fluorescent probe dansyl azide (DNSAz, scheme 1.1).[1.45] DNS-Az itself is nonfluorescent, but treatment with H2S led to dramatic fluorescence enhancements in sodium phosphate buffers (containing Tween-20). To study the selectivity of the probe, the responses of DNSAz in the presence of various possible reducing agents, such as bisulfite and thiosulfate and RSS, such as cysteine and benzyl mercaptan were examined. The response of the probe to cysteine was negligible but it gave some fluorescent enhancement in the presence of benzyl mercaptan. It is unclear if DNS-Az can be turned on by other common biothiols, such as glutathione and homocysteine, as those data were not provided.
5 H H R N O N3 R N O NH2 H2S O O O CO2 O O R= t-BuO- , N
SF1 SF2
O O N NH2 O S 3 O S H2S
N N
DNS-Az
Scheme 1.1 Structures and fluorescence turn-on mechanisms of SF1, SF2, and
DNS-Az.
After these reports, many other azide-based fluorescent probes have been developed, which include SS1 (a colorimetric probe),[1.46] P1,[1.47] Eu·2 (a luminescent probe),[1.48] and P2 (a turn-off fluorescent probe with a NIR emission wavelength).[1.49]
1.3.2 NUCLEOPHILIC REACTION-BASED H2S FLUORESCENT
PROBES
A nucleophilicity-based strategy for H2S detection using direct fluorescence imaging was developed by our laboratory.[1.50] In this work, a reactive disulfide-containing probe WSP1 (scheme 1.2) was developed. WSP1 adopts a
6 closed lactone conformation and does not absorb in the visible region. In the presence of H2S, WSP1 yields significant fluorescence signals. Control experiments with cysteine and glutathione did not lead to any fluorescence increase. These results demonstrate that WSP1 was a selective probe for H2S.
Also in 2011, He and co-workers reported another nucleophilic reaction based strategy for H2S detection. This method used a tandem aldehyde addition and intramolecular Michael reaction.[1.51] In this work, two fluorescent probes SFP-1 and SFP-2 (scheme 1.2) containing acrylate methyl ester and a pseudofluorescent aldehyde were synthesized. It is believed H2S first reacts with the aldehyde group to form a hemithioacetal intermediate. The exposed thiol then undergoes a Michael addition with the acrylate to provide a trapped thioacetal as the product. This tandem reaction should affect the photoinduced electron transfer of the molecule, which leads to fluorescence turn-on.
7 S S N HO O O O O O S H2S + O S O O O O O
WSP1
O H HO SH HO O O O S R R H2S R
Fluorophore Fluorophore Fluorophore
H O COOMe H O COOMe
N N N N B F F F F SFP-2 SFP-1
Scheme 1.2 Structures and fluorescence turn-on mechanisms of WSP1, SFP-1 and SFP-2.
In addition to these attempts, other works based on different nucleophilic
[1.52] [1.53] reactions have been reported, including NIR-H2S, Guo’s probe, and
CouMC. [1.54]
8 1.3.3 METAL SULFIDE FORMATION-BASED H2S
FLUORESCENT PROBES
In 2011, Nagano and co-workers developed a series of selective probes. The fluorescein scaffold was conjugated with an azamacrocyclic Cu2+ complex
(HSip-1, scheme 1.3).[1.55] Because azamacrocyclic rings form stable metal
2+ complexes with Cu , excellent selectivity for H2S over glutathione (even under high concentrations) was obtained. In addition, a diacetylated derivative of the probe was prepared and showed good cell permeability. It was used in real-time fluorescence imaging of intracellular H2S in live cells. Several other probes based on metal-sulfide formation have also been reported by the groups of Guo,
Jin, and Long.[1.56-1.58]
N N H O HN O H HN Cu2+ N H N H NH N NH N
H2S COOH COOH
HO O O HO O O
HSip-1
Scheme 1.3 Structure and mechanism of HSip-1.
1.4 ENDOGENOUS GENERATION OF H2S
9 Hydrogen sulfide can be generated from enzymatic pathways and non-enzymatic pathways. For enzymatic pathways, L-cysteine, a sulfur-containing amino acid derived from alimentary sources, is the main substrate synthesized from L-methionine through the so-called “transsulfuration pathway”. There are two major pathways of cysteine catabolism: oxidation and
[1.59] desulfhydration. Desulfhydration of cysteine is the major source of H2S in mammals and is catalyzed by the trans-sulfuration pathway enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and
3-mercaptopyruvate sulfurtransferase (3-MST). Cystathionine can be converted by CSE to form H2S. CBS can form cystathionine from serine and homocysteine, and additionally can form H2S from cysteine. Cysteine, along with alpha-ketoglutarate (alpha-KG), can be converted into 3-mercaptopyruvate
(3-MP) by cysteine aminotransferase (CAT). 3MP can then be broken down by
[1.60] 3-MST to form H2S.
The mechanism of H2S release is not well understood. At least two possibilities exist. One possibility is that H2S is immediately released after its production by enzymes. Another possibility is that H2S produced by enzymes is stored and is released in response to a physiologic signal.[1.61] Two forms of sulfur stores in cells have been identified.[1.62] Acidic conditions release acid-labile sulfur, which is mainly from the iron-sulfur center of enzymes in mitochondria.
Acid-labile sulfur in the brain of rats, humans, and bovines has been measured
10 as brain sulfide.[1.63] Another form of storage is called bound sulfur, which is
[1.64] localized to the cytoplasm and releases H2S under reducing conditions.
Because the activity of reducing substances is increased in alkaline conditions,
H2S can be released from bound sulfur when intracellular conditions become alkaline.
Nevertheless, it has been overlooked that H2S can also be produced through non-enzymatic pathways from glucose, glutathione, inorganic and organic
[1.65] polysulfides and elemental sulfur. H2S can be generated from glucose either via glycolysis or from phosphogluconate via NADPH oxidase. Glucose reacts with methionine, homocysteine or cysteine to produce gaseous sulfur compounds – methanethiol and hydrogen sulfide. H2S is also produced through direct reduction of glutathione and elemental sulfur. For example, reduction of elemental sulfur to H2S is mediated through reducing equivalents of the glucose oxidation pathway such as NADH, or NADPH.[1.66]
1.5 CHEMISTRY OF S-SULFHYDRATION
S-sulfhydration or persulfidation refers to the post-translational modification of cysteine residues on target proteins by H2S, wherein the -SH groups are converted to persulfide (-SSH) groups.[1.21] This is similar to nitrosation in which NO targets cysteine residues to form -SNO groups.[1.67] Generally,
S-sulfhydration increases the reactivity of the cysteine residues being modified,
11 while nitrosylation decreases the reactivity. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions.[1.21]
Because H2S cannot react with cysteine thiols directly, before the modification
[1.68] cysteines must be oxidized by reactive oxygen species (ROS). Low pKa cysteines are more susceptible to S-sulfhydration, because they exist as thiolate anions (RS-) under physiological conditions.[1.69] S-sulfhydration can occur in several ways: (a) H2S can attack as a nucleophile to oxidized cysteine residues including cysteine sulfenic acid (Cys-SOH) or cysteine disulfides; (b) Oxidized sulfide species such as polysulfide may react with cysteine thiols to generate persulfides; (c) Cysteine thiol residues can react with hydrogen disulfide (H2S2) to form persulfides.[1.70-1.71]
To date, the chemical foundation of S-sulfhydration is still unclear. The chemistry and chemical biology of the S-sulfhydration products, i.e. persulfides
(RSSH), are still poorly understood.[1.72] Methods that allow easy and reliable access to RSSH (both in small molecules and proteins) are critical for the study of RSSH and S-sulfhydration. For biologically important RSSH (such as GSSH and protein-SSH), the common preparation method is the reaction between a
[1.25] disulfide (RSSR) and H2S. However, this reaction requires high concentrations of the reactants, which are not biologically relevant. In addition the reaction is under equilibrium, thus H2S and RSH always remain in the
12 solutions. This makes it very difficult (unless the remaining H2S and RSH can be completely removed) to analyze the property of RSSH exclusively (as H2S and thiol may have similar reactivity). For small molecule RSSH, currently the standard method is to convert thiols to acylated disulfides and then hydrolysis
(by HCl/MeOH) to produce RSSH. In this method, the hydrolysis appears to be slow (usually needs >12 h). This long process of RSSH formation is not suitable for the study of RSSH. For example, although RSSH are believed to be stronger nucleophiles than their thiol analogues (RSH), the reactions between RSSH and thiol blocking reagents rarely give informative results.[1.73]
Recently, our lab reported a novel functional disulfide compound which could effectively convert small molecule and protein thiols (-SH) to form -S-S-Fm adducts, and in turn to form persulfides (-S-SH) under mild conditions. This method allows for a H2S-free protocol to generate highly reactive persulfides in their anionic forms. The high nucleophilicity of persulfides toward a number of thiol-blocking reagents is also demonstrated.[1.74]
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25 CHAPTER TWO
DISULFIDE-BASED FLUORESCENT PROBES FOR HYDROGEN SULFIDE[2.1]
2.1 ABSTRACT
Fluorescence methods are suitable for non-destructive detection of bio-targets in live cells or tissues with readily available instruments. In 2011 our laboratory and several other groups reported the first reaction-based fluorescent probes for
[2.2-2.6] H2S detection. One of the main challenges for current fluorescent probes is the high selectivity for H2S versus other biothiols. The strategy used in the design of our first generation H2S probe was based on a unique H2S-mediated nucleophilic addition followed by an intramolecular cyclization to turn on the
[2.3] fluorescence signals. This method is highly specific for H2S. We have expanded this strategy and a series of new probes have been prepared and evaluated.
2.2 DESIGN AND SYNTHESIS OF PROBES
H2S is the simplest thiol. It can undergo a nucleophilic reaction twice whereas other biothiols, that is, cysteine and glutathione can only undergo a nucleophilic reaction once. As shown in scheme 2.1, it is likely that H2S can react with the electrophilic component of the probe to form an intermediate I-1 containing a
26 free SH group. If a pseudofluorescent group is present at the same molecule, such as the fluorophore through an ester linkage, the SH group should undergo a fast cyclization to release the fluorophore. This strategy should allow direct visualization of H2S signals by convenient and sensitive fluorescence measurements. It is possible that such a probe can react with biothiols (RSH).
However, the intermediate I-2 cannot undergo the cyclization to release the fluorophore and product P. In addition, if appropriate electrophiles are used, the first reaction between the electrophile and biothiols can be reversible or nonproductive so that the probes will not be consumed by biothiols.
Electrophile SH Linker O H S 2 Fluorophore O Electrophile Electrophile I-1 Linker Linker S O P O Fluorophore O Electrophile + SR Linker Probe RSH O Fluorophore Fluorophore non-fluorescent O strong fluorescent I-2
Scheme 2.1 General design of the nucleophilic substitution/cyclization based fluorescent probes.
To test this idea, we first prepared a model compound 2.1 and tried the reaction with H2S (using NaHS as the equivalent in a buffer system; scheme 2.2). In this
27 model compound we envisioned 2,2’-dipyridyl disulfide to be an effective electrophile for trapping H2S and benzene to be an appropriate linker. The rigidity of benzene 1,2-substitutions should facilitate the proposed intramolecular cyclization. As expected, the reaction went well and the desired cyclization product 2.2 and phenol were obtained in good yield (83%).
CH3CN/PBS buffer S S OH S N (10mM, pH 7.4, 1:1, v/v) + H S S + O 2 83% O O 2.1 2.2
Scheme 2.2 Model reaction between compound 2.1 and H2S.
Based on the structure of the model compound, we believed the introduction of pseudo fluorophores through the ester linkage should result in selective fluorescent probes for H2S. Therefore a series of probes were synthesized
(shown in scheme 2.3). The common intermediate,
2-(2-pyridinyldithio)-benzoic acid 2.3, was readily prepared from a mixed-disulfide formation between 2-mercaptobenzoic acid and 2,2’-dipyridyl disulfide. Compound 2.3 was then coupled with OH-containing fluorophores to give the probes WSP1-5 (WSP=Washington State probes). We chose methoxy fluorescein, 7-hydroxycoumarin, resorufin, and 2-methyl TokyoGreen as the fluorophores because of their readily availability, excellent fluorescence properties, and easy fluorescence quenching by hydroxy group substitution. For
28 WSP5, two reaction centers were introduced to the core structure of fluorescein.
Upon reaction with H2S, it should produce highly fluorescent species.
SH N S S S 2 S N HO Fluorophore S N O CO2H CHCl3 CO2H EDC, DMAP, DCM Fluorophore yield: 85% 2.3 yield: 45%-92% O WSP
S S N S S O O O S N S N O O O O O O O O O O N O WSP1 WSP2 WSP3
S S S S N S N N S O O O O O O
O O O O
O WSP4 WSP5
Scheme 2.3 Preparation of fluorescent probes WSP1-5.
2.3 FLUORESCENCE PROPERTIES AND RESPONSES OF
PROBES WSP1-5 TO H2S
With these probes in hand, we tested their fluorescent properties. As expected, these probes exhibited very weak fluorescence with low quantum yields (Φf<0.1, as shown in table 2.1) owing to the esterification of the hydroxy group of
29 fluorophores. This low background fluorescence is critical for highly sensitive detection of H2S.
Table 2.1 Fluorescent properties of probes WSP1-5.
[a] [b] Probes λex (nm) λem (nm) Φf
WSP1 476 516 0.003
WSP2 385 456 0.003
WSP3 550 586 0.014
WSP4 512 531 0.088
WSP5 502 525 0.020
[a] The maximal emission of the probes. [b] The fluorescence quantum yield.
We then tested their fluorescence responses to H2S and optimized the fluorescence measurement conditions. WSP1 was used as the representative species in these studies. As shown in figure 2.1, the fluorescence intensity of
WSP1 increased dramatically when H2S was present in the solution. We also found that media plays an important role in this process. When a mixed
30 CH3CN/PBS buffer (10 mM, pH 7.4, 1:1, v/v) solution was used (owing to poor water solubility of the probe), the fluorescence turn-on rate was somewhat slow.
The intensity could reach the maximum in about 30 min (36-fold increase).
However, when a small amount of surfactant hexadecyltrimethylammonium bromide (CTAB) was added into detection system, the turn on rate was significantly increased and the fluorescence intensity was also significantly enhanced (110-fold). The effects of CTAB may be attributed to 1) CTAB can increase the solubility of the probe in aqueous buffers; and 2) CTAB is a cationic surfactant, which may absorb sulfide anion (HS-) and facilitate the reaction between sulfide anion and the probe. These effects were also noted in previous work on fluorescent probes.[2.7]
Figure 2.1 Time-dependent fluorescence changes of 10 μM WSP1 in the presence (■ and ▲) or absence (●) of 50 μM NaHS. (▲) and (●) data were obtained in 10 mM PBS buffer (pH 7.4) containing 1 mM CTAB. (■) data were
31 obtained in CH3CN/PBS buffer (10 mM, pH 7.4, 1:1, v/v). Data were acquired at 516 nm with excitation at 476 nm.
We next applied this optimized condition to other probes. As shown in figure
2.2 and 2.3, all of them (WSP2, WSP3, WSP4, and WSP5) exhibited fast fluorescence turn-on toward H2S and usually the fluorescence signals can reach a steady state in a few minutes, the intensities increased 275-, 68-, 20-, and
60-fold (for WSP2, WSP3, WSP4, and WSP5, respectively). This may be very favorable for fast detection of H2S. Upon gradual introduction of H2S, the fluorescence intensity at the maximal emission wavelength of the probes increased drastically. As can be seen in figure 2.4, good linear relationships were obtained between the fluorescence intensity at the maximal emission wavelength of the probes and different hydrogen sulfide concentrations. The detection limits were determined to be 60, 79, 47, 266 and 47 nM for WSP1,
WSP2, WSP3, WSP4, and WSP5, respectively (table 2.2).
Figure 2.2 Time-dependent fluorescence changes of WSP probes (10 μM) in the presence of NaHS (50 μM), The reactions were carried out for 30 min at
32 room temperature in PBS buffer (10 mM, pH 7.4) with 1 mM CTAB. Data were acquired at 456 nm with excitation at 385 nm for WSP2 (■); at 586 nm with excitation at 550 nm for WSP3 (●); at 531 nm with excitation at 512 nm for
WSP4 (▲); at 525 nm with excitation at 502 nm for WSP5 (▼).
Figure 2.3 Fluorescence spectra changes of WSP probes (10 μM) in the absence (dot line) or presence (solid line) of NaHS (50 μM), The reactions were carried out for 5 min at room temperature in PBS buffer (10 mM, pH 7.4) with 1 mM CTAB. Data were acquired with excitation at 385 nm for WSP2 (a); with excitation at 550 nm for WSP3 (b); with excitation at 512 nm for WSP4 (c); with excitation at 502 nm for WSP5 (d).
33
Figure 2.4 Fluorescence emission spectra of WSP probes (10 μM) with varied concentrations of NaHS. The reactions were carried out for 5 min at room temperature in PBS buffer (10 mM, pH 7.4) with 1mM CTAB. Data were acquired at 456 nm with excitation at 385 nm for WSP2 (a); at 586 nm with excitation at 550 nm for WSP3 (b); at 531 nm with excitation at 512 nm for
WSP4 (c); at 525 nm with excitation at 502 nm for WSP5 (d).
Table 2.2 Turn-on fold changes and detection limits (DL) of probes WSP1-5.
Probes Turn-on folds DL[a]/nM
WSP1 130 60
WSP2 275 79
34 WSP3 68 47
WSP4 20 266
WSP5 60 47
[a] DL is the detection limit (3S/m, in which S is the standard deviation of blank measurements, n = 11, and m is the slope of the linear equation).
To evaluate the potential applications of the probes in different biological environments, we studied pH effects for all of the five probes (figure 2.5). In general these probes worked well under normal biological pH ranges (6 ~ 9).
However, WSP3 and WSP4 showed increased background fluorescence under pH 10, which might be due to the hydrolysis of the ester linkage under this pH.
We next investigated the probes’ selectivity for H2S, which is the most important property to justify the efficiency of the probes. In these experiments, each probe (10 μM) was treated with different reactive sulfur species including
2- 2- Cys, GSH, Hcy, SO3 , S2O3 (all at 200 μM) under the optimized conditions and fluorescence signals were recorded after mixing for 5 min. As shown in figure 2.5, these sulfur-containing species did not lead to any significant fluorescence responses while NaHS (50 μM) gave very strong signals for all
35 probes. Therefore all the probes are proved to have good selectivity for H2S over
Figure 2.5 Fluorescence intensity changes of WSP probes (10 μM) at different pH values in the absence (▲) or presence (■) of NaHS (50 μM), The reactions were carried out for 5 min at room temperature in 10 mM PBS solution with 1 mM CTAB. WSP1 (a), WSP2 (b), WSP3 (c), WSP4 (d), and WSP5 (e).
36
Figure 2.6 Fluorescence intensity of WSP probes (10 μM) in the presence of various reactive sulfur species: 1) control; 2) 50 μM NaHS; 3) 200 μM Cys; 4)
200 μM GSH; 5) 200 μM Hcy; 6) 200 μM Na2SO3; 7) 200 μM Na2S2O3; 8) 50
μM NaHS + 200μM Cys; 9) 50 μM NaHS + 200μM GSH. WSP1 (a), WSP2
(b), WSP3 (c), WSP4 (d), and WSP5 (e). other reactive sulfur species including Cys and GSH. As one can imagine, these disulfide-based probes may also react with biothiols in biological systems.
Although such reactions won’t turn on fluorescence, it could consume the probes and higher loading of the probes may be needed. This is a potential
37 problem for this type of probes. However we realized that the fluorescence turn-on rates of our probes under the optimized conditions were quite fast
(within a few minutes). Therefore it is possible that the probes can give significant fluorescence signals even when H2S co-exists with biothiols. To prove this, a solution of NaHS (50 μM) and Cys or GSH (200 μM) was prepared and the probe (10 μM) was then loaded. Pleasantly we still observed significant fluorescence signals for each probe (figure 2.6), although at decreased levels compared to NaHS only. These results suggested that the probes were effective for H2S detection in the presence of biothiols.
After proving the sensitivity and selectivity of this series of probes for H2S in aqueous buffers, we decided to explore their applications in imaging H2S in living cells. WSP4 and WSP5 were selected for this study because of their strong fluorescent intensity observed when treating with H2S. Before we conducted cell imagine experiments, we realized that our probes contain the fluorophores through an ester linkage. One concern is that such a linkage may be labile in the presence of cellular esterases. In order to address this question, we tested the stability of the probes (WSP1, WSP4 and WSP5) in the presence of esterase (esterase E-0887, from rabbit liver). As shown in figure 2.7, the probes were incubated with the enzyme for 30 min and no significant fluorescence increase was observed. After that NaHS was added into the
38 mixture and a strong fluorescence increase was observed. These results suggested that the probes were stable to esterases.
Figure 2.7 Fluorescence intensity of the probes (10 μM). 1) control, without the esterase; 2) incubate with esterase (0.06 U/mL) at room temperature for 30 min;
3) incubate with 50 μM NaHS for 5 min after 2). WSP1 (a), WSP4 (b), WSP5
(c).
2.4 LIVING CELL IMAGING STUDIES
Having demonstrated the stability of the probes, we then used them in monitoring H2S in live cells. In brief, freshly cultured Hela cells were incubated with probe WSP4 for 30 min. Then the cells were washed by medium buffer to remove excess probe and treated with different concentrations of NaHS. As shown in figure 2.8, we did not observe significant fluorescent cells when NaHS
39 was absent. However, strong fluorescence in the cells was observed after treating with NaHS for 30 min. Cells treated with 60 μM NaHS displayed obviously stronger fluorescence than cells treated with 30 μM NaHS. In addition, WSP5 was also tested by using the same protocol and similar results were observed. Thus we concluded that these probes can be used for the detection of H2S in living cells.
(a) (b) (g) (h)
(c) (d) (i) (j)
(e) (f) (k) (l)
Figure 2.8 Fluorescence images of H2S in HeLa cells using WSP4 and WSP5.
(a-f) Cells on 24-well plate were incubated with WSP4 (30 μM) for 30 min, then washed and subjected to different treatments. (a and b) control (no NaHS was added); (c and d) treated with 30 μM NaHS; (e and f) treated with 60 μM
NaHS. (g-l) Cells on 24-well plate were incubated with WSP5 (50 μM) for 30 min, then washed and subjected to different treatments. (g and h) control (no
40 NaHS was added); (i and j) treated with 50 μM NaHS; (k and l) treated with 100
μM NaHS. (Scale bar: 100 nm)
To further explore the applications of the probes in H2S study we used them in the evaluation of novel H2S donors. H2S donors are another type of important research tools in this field and our laboratory has recently developed several different controllable H2S donors. Among these the perthiol-based donors are of particularly interest because they have exhibited promising activity against
[2.8] myocardial ischemia-reperfusion injury. To monitor H2S generation in cells from this type of donors, we applied our newly developed fluorescent probes.
Briefly, Hela cells were first incubated with YZ-4-074, a perthiol-based donor, for 30min. After that, the exo-cellular donor was removed by washing with buffers. The cells were then incubated with WSP4. As shown in figure 2.9, donor-treated cells showed much enhanced fluorescent signals compared to vehicle-treated cells. We also applied WSP5 in the same protocol and similar results were observed. These results demonstrated that these probes can be used to evaluate synthetic H2S donor in cells.
41
Figure 2.9 Fluorescence images of H2S production from a H2S donor YZ-4-074 in HeLa cells. Cells on 24-well plate were incubated with or without YZ-4-074 for 30 min, then washed and incubated with 30 μM WSP4 for 30 min. (a and b) controls (no donor was added); (c and d) treated with 100 μM YZ-4-074. (Scale bar: 100 nm)
2.5 CONCLUSION
In summary, we reported in this study the development of a series of nucleophilic substitution-cyclization based fluorescent probes for H2S. Five probes (WSP1-5) were prepared and evaluated. These probes proved to be selective for H2S over other sulfur-containing species including Cys and GSH.
Moreover, fluorescence ‘turn-on’ are fast. The efficiencies of these probes were demonstrated in aqueous solution and in cell imaging. Further development of nucleophilic substitution-cyclization based probes and application of these probes in H2S studies are currently ongoing in our laboratory.
2.6 EXPERIMENTAL SECTION
42 Materials and Methods: All solvents were reagent grade. All the reagents were purchased from ACROS ORGANICS. Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with 0.25mm pre-coated silica gel plates. Flash chromatography was performed with silica gel 60
(particle size 0.040-0.062mm). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Proton NMR spectra were recorded on a 300 MHz spectrometer and carbon-13 NMR spectra were recorded on a 75 MHz spectrometer. Chemical shifts are reported relative
1 13 to CDCl3 (δ 7.26) for H NMR and CDCl3 (δ 77.0) for C NMR.
PBS buffer was prepared in nanopure water. Fluorescence excitation and emission spectra were measured on a fluorescence spectrophotometer.
Stock solutions: Probes were dissolved in DMSO to prepare 2.5 mM stock solutions. NaHS was freshly dissolved in PBS to prepare a 10 mM stock solution. CTAB was dissolved in ethanol to prepare a 100 mM stock solution.
Cysteine, glutathione and other RSS were dissolved in PBS to prepare 100 mM stock solutions. All of the solutions were freshly prepared before use.
2.6.1 SYNTHESIS
Synthesis of the probes:
43 Compound 2.3 was prepared using the protocol described previously[2.4].
General procedure for probes synthesis: to a mixture of compound 3 (262 mg,
1.0 mmol), OH-containing fluorophore (1.0 mmol), EDC (192 mg, 1.0 mmol), and DMAP (12.2 mg, 0.1 mmol) in a 50 mL round bottom flask was added dry
CH2Cl2 (25 mL) at room temperature. The mixture was stirred for 12 h. Then solvent was removed under reduced pressure and resulted crude product was purified by fresh column chromatography to provide the desired product.
OH S EDC, DMAP S N S N S + DCM O CO2H O 2.3 2.1
Compound 2.1 was obtained as a white solid (273 mg, 80.5% yield). M.p.
1 113 °C-115 °C; H NMR (300 MHz, [D1]CDCl3, 25 °C, TMS) δ= 7.06-7.11 (m,
1H, HAr), 7.24-7.36 (m, 4H, HAr), 7.41-7.47 (m, 2H, HAr), 7.50-7.57 (m, 3H,
HAr), 7.97 (dd, J1=8.1 Hz, J2=2.4 Hz, 1H, HAr), 8.30 (dd, J= 7.8, 1.5 Hz, 1H,
13 HAr), 8.45-8.47 (m, 1H, HAr); C NMR (75 MHz, CDCl3, 25 °C, TMS) δ=119.7,
121.0, 121.7, 125.8, 126.0, 126.2, 126.4, 129.8, 132.0, 133.8, 137.4, 141.4,
149.6, 150.5, 159.0, 164.9; IR (thin film, cm-1) 3999.0, 3072.0, 3041.0, 1707.0,
+ 1562.5; MS (ESI) [M+Na] calcd for C18H13NO2S2Na, 362.0; found, 362.0;
+ HRMS [M+H] calcd for C18H14NO2S2, 340.0466; found, 340.0468.
WSP1: Data was the same as previously reported.[2.4]
44 S N S HO O O S S N EDC, DMAP + O O O DCM CO2H O
2.3 WSP2
WSP2 was obtained as a white solid (374 mg, 92% yield). M.p.
1 165 °C-167 °C; H NMR (300 MHz, [D1]CDCl3, 25 °C, TMS) δ= 6.44 (d,
J=9.3 Hz, 1H, HAr), 7.09-7.14 (m, 1H, HAr), 7.23 (d, J=2.1 Hz, 1H, HAr),
7.28-7.29 (m, 1H, HAr), 7.34-7.39 (m, 1H, HAr), 7.52-7.61 (m, 4H, HAr),7.74 (d,
J=9.9 Hz, 1H, HAr), 7.99-8.01 (m, 1H, HAr), 8.31 (dd, J1=7.8 Hz, J2=1.2 Hz, 1H,
13 HAr), 8.48 (dd, J=4.2, 0.9 Hz, 1H, HAr); C NMR (75 MHz, CDCl3, 25 °C,
TMS) δ=110.9, 116.5, 117.2, 118.8, 120.0, 121.4, 125.8, 126.2, 126.5, 129.0,
132.4, 134.5, 137.6, 142.2, 143.1, 149.9, 153.3, 155.0, 158.9, 160.5, 164.4; IR
(thin film, cm-1) 3098.6, 3064.4, 3034.0, 1733.6, 1707.0; MS (ESI) [2M+Na]+
+ calcd for C42H26N2O8S4Na, 837.0; found, 836.7; HRMS [M+H] calcd for
C21H14NO4S2, 408.0364; found, 408.0362.
S S N S S N HO O O EDC, DMAP O O O + O CO2H N DCM N
2.3 WSP3
WSP3 was obtained as an orange solid (266 mg, 58% yield). M.p.
1 139 °C-141 °C; H NMR (300 MHz, [D1]CDCl3, 25 °C, TMS) δ=6.37 (d, J=1.8
45 Hz, 1H, HAr), 6.90 (dd, J1=9.6, J2=1.8 Hz, 1H, HAr), 7.11-7.15 (m, 1H, HAr),
7.31-7.41 (m, 3H, HAr), 7.48 (d, J=9.9 Hz, 1H, HAr), 7.54-7.63 (m, 3H, HAr),
7.89 (d, J=8.1 Hz, 1H, HAr), 8.02 (d, J=7.5 Hz, 1H, HAr), 8.32 (dd, J1=7.8 Hz,
13 J2=0.9 Hz, 1H, HAr), 8.50 (d, J=3.9 Hz, 1H, HAr); C NMR (75 MHz, CDCl3,
25 °C, TMS) δ=107.2, 109.9, 119.4, 119.9, 121.2, 125.4, 126.0, 126.3, 131.2,
131.4, 132.1, 134.3, 134.8, 135.2, 137.5, 141.9, 144.4, 148.4, 149.2, 149.5,
153.2, 158.5, 163.9, 186.2; IR (thin film, cm-1) 3100.1, 3064.4, 3040.6, 1704.4,
+ 1624.0; MS (ESI) [2M+Na] calcd for C48H28N4O8S4Na, 939.1; found, 938.8;
+ HRMS [M+H] calcd for C24H15N2O4S2, 459.0473; found, 459.0487.
S HO O O S N S O O O S N EDC, DMAP + O CO2H DCM
2.3 WSP4
WSP4 was obtained as an orange solid (301mg, 55% yield). M.p.
1 114 °C-117 °C; H NMR (300 MHz, [D1]CDCl3, 25 °C, TMS) δ=2.12 (s, 3H;
CH3) , 6.47 (d, J=1.8 Hz, 1H, HAr), 6.60 (dd, J1=9.9 Hz, J2=1.8 Hz, 1H, HAr),
6.99 (d, J=9.9 Hz, 1H, HAr), 7.10-7.14 (m, 3H, HAr), 7.19-7.21 (m, 1H, HAr),
7.34-7.47 (m, 4H, HAr), 7.50 (d, J=8.4 Hz, 1H, HAr), 7.54-7.62 (m, 3H, HAr),
8.00 (d, J= 8.1 Hz, 1H, HAr), 8.30 (dd, J1=7.8 Hz, J2=1.8 Hz, 1H, HAr),
46 13 8.47-8.50 (m, 1H, HAr); C NMR (75 MHz, CDCl3, 25 °C, TMS) δ=19.6(s;
CH3), 106.1, 110.4, 118.4, 118.6, 119.6, 120.4, 121.0, 125.3, 125.9, 126.1, 126.2,
129.0, 129.1, 129.6, 130.5, 130.6, 130.9, 131.9, 132.0, 134.2, 136.1, 137.3,
141.9, 148.0, 149.6, 153.0, 154.0, 158.4, 158.4, 163.8, 185.8; IR (thin film, cm-1)
3053.0, 2946.6, 2916.1, 1726.0, 1596.7; MS (ESI) [2M+Na]+ calcd for
+ C64H42N2O8S4Na, 1117.2; found, 1116.9; HRMS [M+H] calcd for
C32H22NO4S2, 548.0990; found, 548.0974.
S S HO O OH S N N S O O O EDC, DMAP S + S N O O O DCM O CO2H O O
2.3 WSP5
WSP5 was obtained as a light yellow solid (370 mg, 45% yield). M.p.
1 125 °C-128 °C; H NMR (300 MHz, [D1]CDCl3, 25 °C, TMS) δ=6.92-6.95 (m,
2H, HAr), 7.00-7.03 (m, 2H, HAr), 7.10-7.12 (m, 2H, HAr), 7.23-7.38 (m, 5H,
HAr), 7.52-7.61 (m, 6H, HAr), 7.64-7.75 (m, 2H, HAr), 7.99 (d, J=7.8 Hz, 2H,
HAr), 8.07 (d, J=7.2 Hz, 1H, HAr), 8.30 (d, J=7.2 Hz, 2H, HAr), 8.47 (d, J=4.2
13 Hz, 2H, HAr); C NMR (75 MHz, CDCl3, 25 °C, TMS) δ 81.6, 110.7, 116.8,
118.0, 119.8, 121.1, 124.1, 125.3, 125.8, 126.0, 126.2, 129.2, 130.2, 132.1,
134.1, 135.4, 137.4, 141.8, 149.6, 151.6, 151.9, 153.0, 158.8, 164.3, 169.2; IR
(thin film, cm-1) 3060.6, 2954.2, 2923.7, 1771.6, 1718.4; MS (ESI) [M+Na]+
47 + calcd for C44H26N2O7S4Na, 845.1; found, 845.1; HRMS [M+H] calcd for
C44H27N2O7S4, 823.0701; found, 823.0714.
2.6.2 FLUORESCENCE ANALYSIS
General procedure for H2S determination: Unless otherwise noted, all the measurements were carried out at room temperature for 5 min in 10 mM PBS buffer (pH 7.4) containing 1 mM CTAB according to the following procedure: in a test tube, 3.5 mL of 10 mM PBS buffer (pH 7.4) and 40 μL of the stock solution of CTAB were mixed. To this mixture was then added 16 μL of the stock solution of the probe. The resulting solution was well-mixed, followed by the addition of the requisite volume of testing species solution. The final volume of the solution was adjusted to 4 mL with 10 mM PBS buffer (pH 7.4). After mixing and standing for 5 min at room temperature, 3 mL of the solution was transferred into a 1 cm quartz cell and fluorescence signal was recorded.
Quantum Yields: The quantum yield was calculated according to the equation:
sample standard sample standard standard sample sample standard 2 Φf = Φf × (I / I ) × (A / A ) × (n / n )
Φf denotes the quantum yield; I denotes the area under the fluorescence band; A denotes the absorbance at the excitation wavelength; n denotes the refractive
48 index of the solvent. Dilute solutions (0.01 < A < 0.05) are used tominimize reabsorption effects.
The fluorescence quantum yields for WSP1 and WSP5 were calculated with fluorescein (Φf = 0.85, excited at 490 nm in 0.1 N NaOH) as the standard. The fluorescence quantum yields for WSP2 were calculated with
7-hydroxycoumarin (Φf = 0.76, excited at 330 nm in 0.1 M pH 7.4 sodium phosphate buffer) as the standard. The fluorescence quantum yields for WSP3 were calculated with resorufin (Φf = 0.74, excited at 572 nm in pH 9.5 water) as the standard.[2.1cs] The fluorescence quantum yields for WSP4 were calculated with 2-Me TokyoGreen (Φf = 0.85, excited at 491 nm in 0.1N NaOH) as the standard.
Esterase activity assay: The protocol provided by Sigma-Aldrich was followed(http://www.sigmaaldrich.com/technical-documents/protocols/biology/ enzymatic-assay-of-esterase.html). Briefly, a 0.62 mg/mL solution of boric acid in purified water was prepared. pH was adjusted to 8.0 with 1.0 N NaOH to result in 10 mM borate buffer. Then 5 mg esterase (E-0887 from Sigma) was dissolved in 10 mL borate buffer (10 mM , pH=8.0) (Con. = 0.5 mg/mL). To 25 mL of borate buffer was added 0.1 mL of ethyl butyrate. Then several drops of
0.01 N NaOH was added into the solution till pH reached 8.1. Next 0.1 mL of esterase solution was added in and after 2 min, pH was changed to 8.0. After that, 0.1 mL of 0.01 N NaOH was added in and pH was changed to 8.2 and then
49 decreased to 8.0 by esterase hydrolysis. These steps were repeated for 8 times in
5 min which demonstrated the activity of the enzyme.
Stability assay of the probes: 16 μL of the probe stock solution, 16 μL of esterase stock solution, and 40 μL CTAB solution were added into 3.5 mL PBS.
The resultant mixture was diluted to 4mL with PBS. The fluorescence intensity was measured after the mixture was kept at room temperature for 30min. And then to the mixture was added 20 μL of 10 mM NaHS stock solution. The fluorescence intensity was measured after 5 min.
Cell imaging experiments with H2S: Hela cells were cultured in Dulbecco's modified Eagle's Medium (DMEM, Cellgro company) supplemented with 10% fetal bovine serum, 4 mM glutamine, 100 IU/mL penicillin, and 100 ug/mL streptomycin at 37 °C and with 5% CO2 for two days. One day before imaging, cells were transferred to 24-well plates. Before use, the adherent cells were washed one time with FBS-free DMEM. For intracellular H2S imaging, the cells were incubated with the probe in FBS-free DMEM at 37 °C for 30 min.
After removal of excess probe and washed with PBS (pH 7.4), the cells were incubated with NaHS for 30 min in PBS buffer (pH 7.4, containing 100 μM
CTAB). Cell imaging was carried out after washing the cells three times with
PBS (pH 7.4). All microscopy images were taken on a fluorescence microscope with excitation at 490 nm (green channel).
50 Cell imaging experiments with H2S donor: Medium was removed from the cell plate. The cells were washed by FBS free medium once before they were incubated with or without the donor in FBS free medium. After 30 min, the medium was removed and the cells were washed by FBS free medium. Then the cells were incubated in FBS free medium with the probe (containing 100 μM
CTAB) for 30 min. After washing by PBS twice, the cells were ready for imaging. Fluorescence imaging was performed with fluorescence microscope with GFP light cube for fluorescence channel and 40 × objectives.
51 2.7 REFERENCE
2.1 Peng, B.; Chen, W.; Liu, C.; Rosser, E. W.; Pacheco, A.; Zhao, Y.;
Aguilar, H. C.; Xian, M. Fluorescent probes based on nucleophilic substitution-cyclization for hydrogen sulfide detection and bioimaging. Chem.
Eur. J. 2014, 20, 1010-1016.
2.2 Lippert, A. R.; New, E. J.; Chang, C. J. Reaction-based fluorescent probes for selective imaging of hydrogen sulfide in living cells. J. Am. Chem.
Soc. 2011, 133, 10078-10080.
2.3 Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer, D. J.;
Wang, B. A fluorescent probe for fast and quantitative detection of hydrogen sulfide in blood. Angew. Chem. Int. Ed. 2011, 50, 9672-9675.
2.4 Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.; Whorton, A.
R.; Xian, M. Capture and visualization of hydrogen sulfide by a fluorescent probe. Angew. Chem. Int. Ed. 2011, 50, 10327 -10329.
2.5 Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S. Y.; Zhu, H. L.; Banerjee, R.;
Zhao, J.; He, C. Selective fluorescent probes for live-cell monitoring of sulphide.
Nat. Commun. 2011, 2, 495-501.
2.6 Sasakura, K.; Hanaoka, K.; Shibuya, N.; Mikami, Y.; Kimura, Y.;
Komatsu, T.; Ueno, T.; Terai, T.; Kimura, H.; Nagano, T. Development of a
52 highly sensitive fluorescence probe for hydrogen peroxide. J. Am. Chem. Soc.
2011, 133, 10629-10637.
2.7 Guo, Y.; Yang, X.; Hakuna, L.; Barve, A.; Escobedo, J. O.; Lowry, M.;
Strongin, R. M. A fast response highly selective probe for the detection of glutathione in human blood plasma. Sensors 2012, 12, 5940-5950.
2.8 Y. Zhao, S. Bhushan, C. Yang, H. Otsuka, J. D. Stein, A. Pacheco, B.
Peng, N. O. Devarie-Baez, H. C. Aguilar, D. J. Lefer, M. Xian, Controllable hydrogen sulfide donors and their activity against myocardial ischemia-reperfusion injury. ACS Chem. Biol. 2013, 8, 1283-1290.
53 CHAPTER THREE
DISELENIDE-BASED FLUORESCENT PROBES FOR HYDROGEN SULFIDE [3.1]
3.1 ABSTRACT
WSP probes have been proved to be selective and sensitive for H2S. Even in the presence of high concentrations of biothiols, a fast reaction between the probes and H2S allows effective detection of H2S. However, the fluorescence signals were significantly decreased which indicated the possible consumption of WSP probes by biothiols. To solve this problem, we developed a unique reaction between phenyl diselenide-ester substrates and H2S to form
1,2-benzothiaselenol-3-one. This reaction can trap H2S effectively and the presence of thiols does not affect the process. Based on this reaction two fluorescent probes are prepared and proved to be specific for H2S.
3.2 DESIGN AND SYNTHESIS
To solve the problems of WSP probes, two strategies may be applied: 1) a
H2S-specific ‘trapper’ should be used to replace the pyridyl-disulfide. Such a trapper should only react with H2S, not react with biothiols. This might be difficult as intracellular concentrations of biothiols are much higher than H2S; 2) a non-consumptive trapper with biothiols should be identified and used. Such a
54 trapper may react with biothiols, but the reaction should not lead to the release of fluorophore. Moreover, ideally the reaction product or intermediate should maintain high reactivity toward H2S therefore can lead to fluorescence ‘turn-on’ by H2S. Herein we report our progress in pursuing the latter strategy.
Diselenide-based substrates were found to be suitable for this goal.
Our design of the diselenide-based strategy for the selective trapping H2S (not consumed by biothiols) is shown in scheme 3.1. It is known that diselenide bonds can be cleaved by sulfur-based nucleophiles very effectively (about 5
[3.2-3.3] orders of magnitudes faster than disulphide bonds). H2S (pKa 6.8) is a stronger nucleophile than common biothiols such as Cys and GSH. Therefore we expected diselenide-based reagents like 3.1 should react with H2S very effectively. As such two products should be formed: a thio-benzeneselenol derivative 3.2 and a benzeneselenol derivative 3.3. As an extremely unstable intermediate, benzeneselenol 3.3 should be rapidly oxidized to re-form the probe 3.1.[3. 4] In contrast, 3.2 should undergo a fast and spontaneous cyclization to form 1,2-benzothiaselenol-3-one 3.4 and release the fluorophore. We also expected the diselenide bond should be quite reactive to biothiols (RSH). Such reactions should lead to two products: 3.6 and a S-Se conjugate 3.5. Again, 3.3 should be oxidized to re-generate the probe. As for 3.5, previous studies revealed that nucleophilic attack by thiols at selenium is both kinetically much faster and thermodynamically more favourable.[3.2] As such, the reaction
55 between 3.5 and other biothiols should not change the probe’s structural template (i.e. maintaining the –S-Se- conjugation). However, if H2S is present, the reaction should lead to intermediate 3.2, and the following cyclization should produce 3.4 and the fluorophore. Overall we expected the probe would specifically react with H2S to release the fluorophore and the presence of biothiols should not interfere with the process.
[O] HSe O Fluorophore O O Fluorophore Se H S O Se 2 3.3 O + Fluorophore O 3.1 O Fluorophore Diselenide-based probes O
Se SH O R-SH H2S 3.2 fast [O] S O Se Fluorophore 3.4 O 3.3 + HO Se S-R Fluorophore 3.5
Scheme 3.1 Design of diselenide-based probes for H2S.
With this idea in mind, a model compound 3.6 was prepared and tested. As shown in scheme 3.2, the reaction between compound 3.6 and H2S (5 eq, using
Na2S as the equivalent) was found to be fast, which completed in 10 min. The desired products 3.4 and phenol were obtained in excellent yields. The yield of
3.4 was calculated based on two selenium moieties in the starting material. We
56 did not observe the formation of benzeneselenol product in the reaction. When
Cys (10 eq) co-existed with H2S (5 eq) in the reaction, we obtained the same products in high yields. More interesting, even if Cys (5 eq) was treated with
3.6 first for 1 h, the addition of H2S (2.5 eq) still provided the desired products in similar yields. These results supported our hypothesis that diselenide-based substrates could selectively and effectively react with H2S to form the cyclized product 3.4 and this reaction was not affected by thiols.
Scheme 3.2 Model reactions between compound 3.6 and H2S.
Based on this unique reaction we synthesized two fluorescent probes SeP1 and
SeP2 (shown in scheme 3.3). 7-Hydroxycoumarin and 2-methyl TokyoGreen were selected as the fluorophores as they have excellent fluorescence properties and their fluorescence can be easily quenched upon acylation on hydroxy groups.
57 O O
O O O O O O Se Se Se Se O O O O O O
O O SeP1 SeP2
Scheme 3.3 Structures of SeP probes.
3.3 FLUORESCENCE ANALYSIS
With these two probes in hand, we tested their fluorescent properties. As shown in table 3.1, both probes exhibited very weak fluorescence with low quantum yields (Φ < 0.1), due to esterification of the fluorophores.
Table 3.1. Fluorescent properties of SeP probes
Probe λex (nm) λem (nm) Φf
SeP1 340 455 0.011
SeP2 498 521 0.035
We then tested the probes’ fluorescence responses to H2S in different solvent systems and a mixed CH3CN/PBS buffer (10 mM, pH 7.4, 1:1) solution was
58 found to be the optimum system for the measurement. In this system the fluorescence intensity of the probe (10 μM) could reach the maximum in less than 2 min upon treatment of H2S (using 50 μM Na2S) as shown in figure 3.1, demonstrating this was a fast process. Fluorescence increases were also found to be significant. Intensities increased 35- and 14-fold for probe SeP1 and SeP2 respectively. When a series of different concentrations of Na2S were treated with the probes, we observed fluorescence intensities increased in almost a linear fashion in the range of 0 ~ 15 μM. Data obtained with SeP2 were shown in figure 3.2. The detection limit of SeP2 was calculated to be 0.06 μM.
Figure 3.1 Time-dependent fluorescence changes of SeP probes (10 μM) in the presence of Na2S (50 μM). The reactions were carried out for 30 min at room temperature in CH3CN/PBS buffer (10 mM, pH 7.4, 1:1, v/v). Data were acquired at 455 nm with excitation at 340 nm for SeP1 (■); at 521 nm with excitation at 498 nm for SeP2 (●).
59
Figure 3.2 Fluorescence emission spectra of SeP2 (10 μM) in the presence of varied concentrations of Na2S (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 μM). The reactions were carried out for 5 min at room temperature in CH3CN/PBS buffer
(10 mM, pH 7.4, 1:1, v/v). Data were acquired with excitation with excitation at
498 nm for SeP2.
We also examined the selectivity of the probes for H2S over other reactive
2- sulfur species, including cysteine (Cys), glutathione (GSH), sulfite (SO3 ),
2- 2- sulfate (SO4 ), and thiosulfate (S2O3 ). As shown in figure 3.3, all of these species did not show significant fluorescence enhancements even under much higher concentrations (up to mM). In addition, when H2S (50 μM) coexisted with Cys or GSH (1 mM), we observed strong fluorescence responses that were comparable to the signals obtained with H2S only. This was a significant improvement from our pyridyl disulfide based probes.[3.5]
60
Figure 3.3 Fluorescence intensity of SeP probes (10 μM) in the presence of various reactive sulfur species: 1) control; 2) 50 μM Na2S; 3) 200μM Na2SO3;
4) 200 μM Na2S2O3; 5) 200 μM Na2SO4; 6) 1 mM Cys; 7) 1 mM GSH; 8) 50
μM Na2S + 1 mM Cys; 9) 50 μM Na2S + 1 mM GSH. SeP1 (A), SeP2 (B).
Next we tested SeP2 in imaging H2S in cells. Freshly cultured HeLa cells were first incubated with SeP2 (50 μM) for 30 min and then washed with DMEM to remove excess probe. We did not observe significant fluorescent cells (figure
3.4). However, strong fluorescence in the cells was observed after treating with
61 Na2S (100 μM) for 30 min. These results demonstrated that SeP2 could be used for cell imaging.
Figure 3.4 Fluorescence images of H2S in HeLa cells using SeP2. Cells were incubated with the probe (50 μM) for 30 min, then washed and subjected to different treatments. (a) & (b) control (no Na2S was added); (c) & (d) treated with 100 μM Na2S. (Scale bar: 100 nm)
Finally we wondered if SeP2 could be used to measure endogenous H2S concentrations changes. To this end, human neuroblastoma cells (SH-SY5Y) were separately treated with L- and D-cysteine (which are H2S biosynthestic substrates), S-adenosylmethyonine (SAM, a CBS activator), and aminooxyacetic acid (AOAA, a CBS inhibitor). SeP2 was loaded into each experiment before or after treatments (see detailed protocols in the supporting information). Fluorescence intensities were measured by a plate-reader and compared to both negative and positive controls. As shown in figure 3.5, cells
62 treated with H2S substrates or CBS activator showed clearly enhanced fluorescence while cells treated with CBS inhibitor showed decreased fluorescence. Not surprisingly cells treated with Na2S showed the strongest fluorescence. These results suggest that SeP2 can be used in determining endogenous H2S changes.
Figure 3.5 Fluorescent detection of in-situ generated H2S in human neuroblastoma cells. (*P<0.05, **P<0.01, ***P<0.001 vs. control; N = 5 each)
3.4 CONCLUSION
In conclusion, we reported herein a unique reaction between phenyl diselenide and H2S to form 1,2-benzothiaselenol-3-one. The presence of thiols did not affect this process. Based on this reaction two fluorescent probes for the
63 detection of H2S were prepared and evaluated. The probes showed excellent sensitivity and selectivity.
3.5 EXPERIMENTAL SECTION
Materials and Methods: All solvents were reagent grade. Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with
0.25 mm pre-coated silica gel plates. Flash chromatography was performed with silica gel 60 (particle size 0.040-0.062 mm). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Proton and carbon-13 NMR spectra were recorded on a 300 MHz spectrometer. Chemical shifts are reported relative to chloroform (δ 7.26) for 1H NMR and chloroform
(δ 77.0) for 13C NMR. Absorption spectra were recorded on a Lambda 20
UV/VIS spectrophotometer using 1 cm quartz cells. Fluorescence excitation and emission spectra were measured on a Cary Eclipse fluorescence spectrophotometer.
3.5.1 CHEMICAL SYNTHESIS
O
COOH OH O EDC, DMAP Se Se Se + Se DCM O HOOC O 3.7 3.6
64 Model compound 3.6: To a solution of compound 3.7[3.6] (500 mg, 1.25 mmol) in CH2Cl2 (30 ml) was added EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (576 mg, 3 mmol), DMAP (4-dimethylaminopyridine) (60 mg,
0.5 mmol) and phenol (470 mg, 5 mmol). The mixture was allowed to stir at rt overnight. The mixture was then washed with saturated NH4Cl solution and brine and was extracted with CH2Cl2 (20 ml × 2). The combined organic layers were dried (by MgSO4) and concentrated under reduced pressure. The resulted crude material was purified by flash column chromatography. Compound 9 was
1 obtained as a white solid (560 mg, 80 %). H NMR (300 MHz, CD3Cl) δ
7.18-7.30 (m, 5H), 7.32-7.37 (m, 3H), 7.42-7.46 (m, 6H), 7.90 (d, J=7.8 Hz,
13 2H), 8.31-8.34 (m, 2H); C NMR (75 MHz, CD3Cl) δ 166.06, 150.77, 136.10,
134.19, 132.18, 130.84, 129.77, 129.72, 127.49, 126.40, 121.86; MS (ESI+) m/z
+ + 577.0 (M+Na ); HRMS m/z 576.9444 [M+Na] ; calcd for C26H18NaO4Se2
576.9433. IR (cm-1) 1700, 1485, 1268, 1183, 1023, 919, 744. m. p. 114-115 °C.
O
COOH O O O O O OH EDC, DMAP Se Se Se Se + DCM O O O HOOC O
3.7 SeP1
Probe SeP1 was prepared using the same method for compound 3.6. 1H NMR
(300 MHz, CD3Cl) δ 6.45 (d, J=9.6 Hz, 2H), 7.24-7.25 (m, 1H), 7.27-7.28 (m,
65 1H), 7.30-7.31 (m, 2H), 7.36-7.41 (m, 2H), 7.45-7.51 (m, 2H), 7.59 (d, J= 8.7
Hz, 2H), 7.75 (d, J= 9.3 Hz, 2H), 7.91 (d, J= 7.8 Hz, 2H), 8.34 (dd, J= 7.5, 1.5
Hz, 2H); MS (ESI+) m/z 712.9 (M+Na+); HRMS m/z 712.9220 [M+Na]+; calcd
-1 for C32H18NaO8Se2 712.9230. IR (cm ) 1702, 1618, 1454, 1242, 1119, 1020,
984, 741. m. p. 275-276 °C.
O
COOH O O OH O O O EDC, DMAP Se Se Se Se + DCM O O O HOOC O
3.7 SeP2
Probe SeP2 was prepared using the same method as for compound 9. 1H NMR
(300 MHz, CD3Cl) δ 2.12 (s, 6H), 6.46 (d, 2H), 6.60 (dd, 2H), 6.97-7.00 (m,
2H), 7.14 (m, 4H), 7.19-7.21 (m, 2H), 7.39-7.49 (m, 12H), 7.90 (dd, J=8.1, 0.9
13 Hz, 2H), 8.33 (dd, J=7.5, 1.5 Hz, 2H); C NMR (75 MHz, CD3Cl) δ 186.1,
165.2, 158.6, 154.2, 153.2, 148.1, 136.5, 136.4, 134.7, 132.4, 132.2, 131.2,
130.9, 130.8, 130.7, 129.9, 129.4, 129.3, 126.6, 126.4, 120.7, 118.9, 118.6,
110.6, 106.5, 19.8; MS (ESI+) m/z 993.1 (M+Na+); HRMS m/z 971.0657
+ -1 [M+H] ; calcd for C54H35O8Se2 971.0662. IR (cm ) 1702, 1618, 1454, 1242,
1119, 1020, 984, 741.. m.p. 270-271 °C.
66 3.5.2 MODEL REACTIONS AND STABILITY STUDIES
To the solution of 3.6 (55.4 mg, 0.1 mmol) in THF (4 mL) and PBS buffer (4 mL, 10 mM, pH 7.4) was added Na2S∙9H2O (96 mg, 0.5 mmol). The mixture was stirred for 0.5 hour at rt and then diluted with CH2Cl2. The organic layer was separated and dried by MgSO4, and concentrated. Purification by flash column chromatography afforded compound 3.4 as yellow solid (39 mg, 88% yield). In another two reactions cysteine was added.
To the solution of 3.4 (21.6 mg, 0.1 mmol) in THF (2 mL) and H2O (2 mL) was added alanine (44.5 mg, 0.5 mmol) or glycine (37.5 mg, 0.5 mmol) or lysine (73 mg, 0.5 mmol). The mixture was stirred for 45 min at rt and no reaction was observed. The reaction was diluted with CH2Cl2. The organic layer was separated and dried by MgSO4, and concentrated. Compound 3.4 could be recovered by flash column chromatography (90-95%).
67 3.5.3 FLUORESCENCE ANALYSIS
Quantum Yields: The quantum yield was calculated according to the equation:
2 Φsample = Φstandard × (Isample/Istandard) × (Astandard/Asample) × (nsample/nstandard)
Φ denotes the quantum yield; I denotes the area under the fluorescence band; A denotes the absorbance at the excitation wavelength; n denotes the refractive index of the solvent. For quantum yield of SeP1, it was determined using
7-hydroxycoumarin as a standard by comparing the area under the corrected emission spectrum of the test sample with that of a solution of
7-hydroxycoumarin excited at 330 nm in sodium phosphate buffer (0.1 M; pH
7.4), which has a quantum efficiency of 0.76. For quantum yield of SeP2,
Quantum yield was determined using 2-Methyl TokyoGreen as a standard by comparing the area under the corrected emission spectrum of the test sample with that of a solution of fluorescein excited at 491 nm in 0.1 N NaOH, which has a quantum efficiency of 0.85.
Preparation of the solutions and fluorescence measurements: The stock solutions of SeP1 (0.7 mM) and SeP2 (2 mM) were prepared in DMSO, respectively. The solutions of various testing species were prepared from Cys,
GSH, Na2S·9H2O, Na2S2O3, Na2SO3, Na2SO4, in 10 mM PBS buffer. All the test solutions need to be freshly prepared.
68 Unless otherwise noted, all the measurements were carried out for 5 min at room temperature in CH3CN/PBS buffer (10 mM, pH 7.4, 1:1, v/v) according to the following procedure. In a test tube, 1.5 mL of PBS buffer (pH 7.4) and 1.5 mL of acetonitrile were mixed, and then 42 μL of the stock solution of SeP1 or
15 μL of the stock solution of SeP2 were added. The resulted solution was mixed well, followed by the addition of a requisite volume of testing species sample solution. After mixing and then standing for 5 min at room temperature, the reaction solution was transferred into a 1-cm quartz cell to measure fluorescence with λex = 340 nm (for SeP1) or 498 nm (for SeP2). PMT detector voltage = 400V. In the meantime, a blank solution containing no testing species sample was prepared and measured under the same conditions for comparison.
Cell culture and fluorescence imaging: HeLa cells were grown on glass-bottom culture dishes (Corning Inc.) in DMEM supplemented with 10%
(v/v) FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C under a humidified atmosphere containing 5% CO2. Before use, the adherent cells were washed one time with FBS-free DMEM. For intracellular H2S imaging, the cells were incubated with 50 μM SeP2 in FBS-free DMEM at 37 °C for 30 min. After removal of excess probe and washed with PBS (pH 7.4), the cells were incubated with 100 μM Na2S for 30 min in PBS buffer (pH 7.4, containing
100 μM CTAB). Cell imaging was carried out after washing the cells three
69 times with PBS (pH 7.4). All microscopy images were taken on an EVOS fl fluorescence microscope from Advanced Microscopy Group (AMG).
Measurement of endogenous H2S changes: Human neuroblastoma cells
(SH-SY5Y) were cultured in Eagle’s medium/Ham’s F-12 50/50 Mix
(DMEM/F12, Cellgro) supplemented with 10 % fetal bovine serum (FBS,
ATCC) and penicillin (100 Units/ml)/streptomycin (100 µg/ml) as described previously. Cells were seeded into a 96 well-plate (2 x 104 cells/well) and medium was replaced every 2 days. Cells were used after getting 80-90 % confluent.
For L-cysteine or D-cysteine experiment, cells were incubated with SeP2 (10
µM, with 0.1 % DMSO) in Hank's Balanced Salt Solution (HBSS) at 37°C for
30 min, washed twice with warmed HBSS, and incubated with L-cysteine (10 mM), D-cysteine (10 mM), or Na2S (0.1 mM) in DMEM/F12 (0.1 % FBS) at
37°C for 2 h. Fluorescent intensity was measured by a plate-reader (SpectraMax
M5, Molecular Devices) after washing cells twice with HBSS. For a CBS activator S-adenosylmethyonine (SAM, AK Scientific) or a CBS inhibitor aminooxyacetic acid (AOAA) experiment, cells were incubated with SAM (2 mM) or AOAA (2 mM) in DMEM/F12 (0.1 % FBS) for 24 h, washed with
HBSS, and loaded SeP2. Fluorescent intensity was measured by a plate-reader
(SpectraMax M5, Molecular Devices) after washing cells twice with HBSS.
70 3.6 REFERENCES
3.1 Peng, B.; Zhang, C.; Marutani, E.; Pacheco, A.; Chen, W.; Ichinose, F.; Xian,
M. Trapping hydrogen sulfide (H2S) with diselenides: the application in the design of fluorescent probes. Org Lett 2015, 17, 1541-1544.
3.2 Steinmann, D.; Nauser, T.; Koppenol, W. H. Selenium and sulfur in exchange reactions: a comparative study. J. Org. Chem. 2010, 75, 6696-6699.
3.3 Bachrach, S. M.; Demoin, D. W.; Luk, M.; Miller, J. V. Nucleophilic attack at selenium in diselenides and selenosulfides. A computational study. J. Phys.
Chem. A 2004, 108, 4040-4046.
3.4 Rasmussen, B.; Sørensen, A.; Gotfredsen, H.; Pittelkow, M. Dynamic combinatorial chemistry with diselenides and disulfides in water. Chem.
Commun. 2014, 50, 3716-3718.
3.5 Peng, B.; Chen, W.; Liu, C.; Rosser, E. W.; Pacheco, A.; Zhao, Y.; Aguilar,
H. C.; Xian, M. Fluorescent probes based on nucleophilic substitution-cyclization for hydrogen sulfide detection and bioimaging. Chem.
Eur. J. 2014, 20, 1010-1016.
3.6 He, J.; Li, D.; Xiong, K.; Ge, Y.; Jin, H.; Zhang, G.; Hong, M.; Tian, Y.; Yin,
J.; Zeng, H. Inhibition of thioredoxin reductase by a novel series of
71 bis-1,2-benzisoselenazol-3(2H)-ones: Organoselenium compounds for cancer therapy. Bioorg. Med. Chem. 2012, 20, 3816-3827.
72 CHAPTER FOUR
STUDY OF THE REACTIONS BETWEEN SULFANE SULFURS AND NAD(P)H AS A NON-ENZYMATIC
H2S GENERATION PATHWAY
4.1 ABSTRACT
Sulfane sulfurs have been suggested to be a H2S pool in biological systems.
However, how H2S is generated from sulfane sulfurs is still unclear. We hypothesized that sulfane sulfurs could be reduced by in vivo reductants such as
NAD(P)H. As far as we know, there is no study of the reactions between sulfane sulfurs and NAD(P)H yet. In this work, we studied the reactions between various sulfane sulfurs and NAD(P)H model compounds. This work should provide informative results regarding non-enzymatically H2S generation in biological systems.
4.2 REACTIONS BETWEEN NADH MODEL COMPOUNDS AND SULFANE SULFUR
In this study, we first chose N-Ac-cysteine methyl ester polysulfide 4.2a as a model compound of sulfane sulfurs because its properties have been studied by our group.[4.1] We used ethyl Hantzsch ester 4.1 as an model of NAD(P)H.
Hantzsch ester is widely used as a H-donor in hydride-transfer reactions.[4.2] We
73 first studied this reaction by monitoring the yields of the oxidized product 4.3 by 1H-NMR as shown in scheme 4.1. We tried different reaction conditions with varying temperatures and solvents. As shown in scheme 4.1, ethyl Hantzsch ester was stable in the absence of polysulfide 4.2a since control experiments gave very low yield of the product 4.3. While in the presence of 4.2a, different amounts of the oxidized product were observed, which proved the occurrence of the reaction. The highest yield was obtained in ethanol at 37 °C. In the following experiments, we applied this condition to all the reactions.
Scheme 4.1 Optimization of reaction conditions between Hantzsch ester and cysteine polysulfide.
EtO2C CO2Et MeO2C NHAc 20 h EtO2C CO2Et +
N S Sx N H 2 4.1 4.2a 4.3
Entry Equiv. of Polysulfide Solvent Temperature Yield
1 0 EtOH r.t. <5% 2 2 EtOH r.t. 26% 3 0 EtOH 37°C <5% 4 2 EtOH 37°C 96% 5 0 ACN r.t. <5% 6 2 ACN r.t. 12% 7 0 ACN 37°C <5% 8 2 ACN 37°C 18%
74 Having proved that ethyl Hantzsch ester can be oxidized by polysulfide 4.2a, we then employed other sulfane sulfurs including N-acetyl-penicillamine methyl ester polysulfide 4.2b, N-benzyl-cysteine methyl ester polysulfide 4.2c, triphenylmethanethiol polysulfide 4.2d, dimethyl trisulfide 4.2e, and elemental sulfur 4.2f (scheme 4.2). Compounds 4.2a-d were synthesized following a known procedure[4.3] and 4.2e-f were purchased from Sigma-Alderich. We expected that different kinds of sulfane sulfurs could react with NAD(P)H model compounds.
Scheme 4.2 Structures of various sulfane sulfurs.
O O O H H H N N N O O O O O O S Sx S S Sx S S Sx S O O O O O O N N N H H H O O O 4.2a 4.2b 4.2c
S Sx S S S S S8
4.2d 4.2e 4.2f
Having these different sulfane sulfur compounds in hands, we then conducted their reactions with ethyl Hantzsch ester under the optimized conditions. From the results shown in scheme 4.3, we found that except for dimethyl trisulfide
75 4.2e, all the sulfane sulfur compounds could react with Hantzsch ester and gave the oxidized product in high yields (≥ 90%).
Scheme 4.3 Reactions between Hantzsch ester and different sulfane sulfurs.
EtO2C CO2Et EtOH EtO2C CO2Et + Sulfane Sulfur N (2 equiv.) 37°C, 20h N H 4.1 4.2 4.3
Entry Sulfane sulfurs Yield
1 4.2a 96%
2 4.2b 94%
3 4.2c 90%
4 4.2d 94%
5 4.2e 14%
6 4.2f 90%
Besides Hantzsch ester, 1-benzyl-1,4-dihydronicotinamide (BNAH) is another widely used NAD(P)H model compound. To further understand the reactions between NAD(P)H and sulfane sulfurs, we also studied the reactions using
BNAH as shown in scheme 4.4. Compared with Hantzsch ester, the reactions with BNAH were about three times faster. We also found that the reactions were faster in acetonitrile than in ethanol. However, since the solubility of polysulfides in ethanol is much better, we used ethanol as the solvent in the
76 following reactions in which higher concentrations of sulfane sulfurs were required.
Scheme 4.4 Optimization of the reaction conditions between BNAH and cysteine polysulfide.
CONH CONH 2 MeO2C NHAc 5 h 2 +
N S Sx N 2 Bn Bn 4.4 4.2a 4.5
Entry Equiv. of Polysulfide Solvent Temperature Yield
1 0 EtOH r.t. <5%
2 2 EtOH r.t. 60%
3 0 EtOH 37°C <5%
4 2 EtOH 37°C 98%
5 0 ACN r.t. <5%
6 2 ACN r.t. 96%
7 0 ACN 37°C <5%
8 2 ACN 37°C 98%
Having proved the reactivity between BNAH and polysulfide 4.2a, we then applied the optimized condition to the reactions with other sulfane sulfurs
(scheme 4.5). The results showed that all the polysulfides 4.2a-d could give
77 high yields of the oxidized product in 5 h while dimethyl trisulfide and elemental sulfur were less reactive.
Scheme 4.5 Reactions between BNAH and different sulfane sulfurs.
CONH2 EtOH CONH2 + Sulfane Sulfur N 37°C, 5h N Bn Bn 4.4 4.2 4.5
Entry Sulfane sulfurs Yield
1 4.2a 93%
2 4.2b 95%
3 4.2c 98%
4 4.2d 99%
5 4.2e 10%
6 4.2f 17%
4.3 DETECTION OF H2S GENERATION
After confirming the reactions between NAD(P)H model compounds and various sulfane sulfurs, we tested whether H2S was a product in these reactions.
In this study, we tried to identify the generation of H2S by both methylene blue method and fluorescence assays. The measurement we used was optimized from previously described method.[4.4] As shown in scheme 4.6, the reaction between
78 NADH models and sulfane sulfurs were conducted in a 20-mL vial (A) while the H2S trapping solution (zinc acetate for methylene blue method or DI water for fluorescence assays) was placed in a centrifuge tube B along with a piece of filter paper. After flushing with argon, the vial was placed at 37 °C water bath to let the reaction complete. After the reaction was done, the trapping solution was mixed with H2S detection solution (N,N-dimethyl-p-phenylenediamine sulphate and ferric chloride for methylene blue method and probe SeP2 for fluorescence assays). The UV signal or fluorescent intensity was measured to confirm the generation of H2S (figure 4.1 & 4.2). After proving H2S as one of the products of the reactions, we tried to identify the other products generated from sulfane sulfurs. We studied the products of the reaction between 4.1 and 4.2a. After comparing the 1H-NMR spectra with the corresponding disulfide of 4.2a, we believed that the polysulfides were reduced to disulfides by NADH analogues.
Trap the produced Mixed the Zn(Ac)2 Measure the UV and absorbance of the sample H2S by Zn(Ac)2 with DMPD FeCl3
37°C NADH analogues and polysulfides were mixed
Mixed the H O Measure the fluorescent Trap the produced 2 intensity of the sample H2S by H2O with SeP2
H2S traping solutions
Reactions between NADH model compounds and sulfane sulfurs
Scheme 4.6 Procedure for detection of H2S generated from the reactions between NAD(P)H model compounds and sulfane sulfurs.
79
Figure 4.1 UV absorption spectra of methylene blue which indicate the production of H2S. Red line: 4.1 and 4.2a; Blue line: 4.4 and 4.2a. Black line: control.
Figure 4.2 Fluorescence spectra of the samples which indicate the production of
H2S. Red line: 4.1 and 4.2a; Blue line: 4.4 and 4.2a. Black line: control.
4.4 CONCLUSION
In conclusion, the reactions between two NAD(P)H model compounds and various sulfane sulfurs were studied in this work. The reactions conditions were optimized and the products were confirmed as H2S and corresponding disulfides.
This work suggested a new pathway for the production of H2S in biological systems.
80 4.5 EXPERIMENTAL SECTION
Materials and Methods: All solvents were reagent grade. Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with
0.25 mm pre-coated silica gel plates. Flash chromatography was performed with silica gel 60 (particle size 0.040-0.062 mm). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Proton and carbon-13 NMR spectra were recorded on a 300 MHz spectrometer. Chemical shifts are reported relative to chloroform (δ 7.26) for 1H NMR and chloroform
(δ 77.0) for 13C NMR. Absorption spectra were recorded on a Lambda 20
UV/VIS spectrophotometer using 1 cm quartz cells. Fluorescence excitation and emission spectra were measured on a Cary Eclipse fluorescence spectrophotometer.
4.5.1 CHEMICAL SYNTHESIS
EtO C CO Et O O O water 2 2 + + Et NH4OAc O H H reflux N H 4.1
Hantzsch Ester 4.1 was prepared according to known procedure.[4.5] A mixture of paraformaldehyde (2.5 mmol), ethyl acetoacetate (10 mmol), and ammonium acetate (5 mmol) in 5 mL of water was vigorously stirred at refluxing temperature for 2h. The reaction was monitored by TLC. After the reaction was
81 completed, the reaction mixture was cooled to room temperature, then filtrated and washed with 5mL of water twice. The obtained yellow solid product (0.472 g, 75%) was nearly pure and used in the next step without further purification.
NMR data matches literature data.
Et3N R + R Sx R SH S2Cl2 S S DCM 4.2
Compounds 4.2a-d were prepared according to known procedure.[4.3] To a stirred solution of equimolar concentrations of the selected thiol and triethylamine in dry dichloromethane at -20°C was added dropwise a solution of sulfur monochloride (0.5 eq) in dichloromethane. The reaction was brought to room temperature after the addition was completed and stirring was continued for about 1 h. The reaction was worked up by the addition of about 100 mL of ice-cold water. This was stirred for a few minutes, transferred to a separatory funnel, and the aqueous phase was removed by draining the organic layer. The organic layer was further washed twice with 50 mL of ice-cold water to remove the triethylamine hydrochloride, and washed with 50 mL of brine to dry the organic layer. The dichloromethane layer was further dried by the addition of anhydrous magnesium sulfate, filtered and concentrated in vacuum. The product was purified by silica gel chromatography.
82 N-acetyl cysteine methyl ester polysulfide 4.2a was obtained in 85% yield.
NMR data matches literature data.[4.1]
BNAH 4.4 was prepared according to known procedure.[4.6] Nicotinamide (1.22 g, 10 mmol) was dissolved in acetonitrile (10 mL) and benzyl bromide (1.2 mL,
10 mmol) was added. The reaction mixture was refluxed for 15 h, after which time a precipitate was observed. The solution was cooled and diethyl ether (12 mL) was added to further precipitate the final product. After filtering and washing with diethyl ether (3 × 5 mL), the bromide salt
1-benzyl-3-carbamoylpyridium bromide was obtained as a white powder. Under nitrogen atmosphere, 1-benzyl-3-carbamoylpyridinium bromide (147 mg, 0.5 mmol) was dissolved in distilled H2O (3 mL) and NaHCO3 (210 mg, 2.5 mmol) was added. Sodium dithionite Na2S2O4 (436 mg, 2.5 mmol) was then added in small portions and the reaction mixture was stirred at room temperature for 3 h in the dark, during which time the solution turned from orange to yellow as the yellow product precipitated. The solid was filtered, washed with cold water (3 ×
5 mL) and dried under vacuum to afford product
83 1-benzyl-1,4-dihydronicotinamide 4.4 as a bright yellow powder (40 mg, 37%).
NMR data matches literature data.
4.5.2 REACTIONS BETWEEN NADH MODEL COMPOUNDS AND
SULFANE SULFURS
The stock solutions of NAD(P)H model compounds (4.1 or 4.4) were prepared in organic solvent (DCM or ethanol) with concentration of 50 mM. The stock solutions of sulfane sulfurs were prepared in corresponding organic solvent with concentration of 100 mM. After mixing together and flushing with argon, the reaction was placed in 37 °C water bath under dark for 20 h (for 4.1) or 5 h (for
4.4). Then the solvent was removed by vacuum. Yield was calculated by 1HNMR.
4.5.3 DETECTION OF H2S GENERATION
H2S production was measured as described previously with modifications. The reaction mixture contained: 10 mM Hantzsch ester 4.1 or BNAH 4.4 and 20 mM sulfane sulfur 4.2a. 1.5 mL centrifuge tubes were used as the centre wells each contained 0.5 ml 1% Zn(OAc)2 (for methylene blue method) or DI H2O
(for fluorescence assays) as trapping solution and a filter paper of 2 * 2.5 cm2 to increase the air/liquid contacting surface. The reactions were performed in a 20 ml disposable scintillation vials. The vials containing reaction mixture and
84 centre wells were flushed with argon before being sealed with a double layer of parafilm. Reaction was placed in a 37°C water bath.
For methylene blue method, after incubating 20 h (for Hantzsch ester 4.1) or 5 h
(for BNAH 4.4), the contents of the centre wells were then transferred to test tubes each containing 3.5 ml of water. Subsequently, 0.5 ml of 20 mM
N,N-dimethyl-p-phenylenediamine sulphate in 7.2 M HCl was added immediately followed by addition of 0.4 ml 30 mM FeCl3 in 1.2 M HCl. The absorbance spectra of the resulting solution from 400 nm to 700 nm were measured 20 min later with a spectrophotometer.
For fluorescence assays, after incubating 20 h (for Hantzsch ester 4.1) or 5 h
(for BNAH 4.4), the contents of the centre wells were then transferred to test tubes each containing 1 mL PBS, 1.5 mL acetonitrile, and 15 μL 2 mM SeP2.
The fluorescence spectra of the resulting solution from 510 nm to 600 nm were measured 5 min later with a fluorescence spectrophotometer.
85 4.6 REFERENCES
4.1 Chen, W.; Liu, C.; Peng, B.; Zhao, Y.; Pacheco, A.; Xian, M. New fluorescent probe for sulfane sulfurs and the application in bioimaging.
Chemical Science 2013, 4, 2892-2896.
4.2 Maharjan, B.; Boroujeni, M. R.; Lefton, J.; White, O. R.; Razzaghi, M.;
Hammann, B. A.; Derakhshani-Molayousefi, M.; Eilers, J. E.; Lu, Y. Steric
Effects on the Primary Isotope Dependence of Secondary Kinetic Isotope
Effects in hydride transfer reactions in solution: caused by the isotopically different tunneling ready state conformations? J. Am. Chem. Soc. 2015, 137,
6653-6661.
4.3 Ramaraju, P.; Gergeres, D.; Turos, E.; Dickey, S.; Lim, D. V.; Thomas, J.;
Anderson, B. Synthesis and antimicrobial activities of structurally novel
S,S0-bis(heterosubstituted) disulfides. Bioorg. Med. Chems. Lett. 2012, 22,
3623-3631.
4.4 Zhao, W.; Zhang, J.; Lu, Y.; Wang, R. The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. the EMBO Journal 2001, 20,
6008-6016.
86 4.5 Wang, Y.; Ji, K.; Lan, S.; Zhang, L. Rapid access to chroman-3-ones through gold-catalyzed oxidation of propargyl aryl ethers. Angew. Chem. Int. Ed.
2012, 51, 1915-1918.
4.6 Paul, C. E.; Gargiulo, S.; Opperman, D. J.; Lavandera, I.;
Gotor-Fernandez, V.; Gotor, V.; Taglieber, A.; Arends, I. W. C. E.; Hollmann, F.
Mimicking nature: synthetic nicotinamide cofactors for CdC bioreduction using enoate reductases. Organ. Lett. 2013, 15, 1180-1183.
87 CHAPTER FIVE
DEVELOPMENT OF A NEW METHOD FOR PERSULFIDE GENERATION
5.1 ABSTRACT
The fundamental chemistry and chemical biology of persulfides are poorly understood because (1) the biological significance of RSSH has only been recently discovered; and (2) persulfides are unstable species, especially in aqueous buffers, which make them difficult to study. In this work, we developed a new method to generate persulfide which utilized the reaction between thiol and Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 5.1). It can effectively convert small molecule thiols to persulfide and the generated persulfide can be trapped by electrophiles. The reactions were found to be fast and not be interfered by water or oxygen. This method should be useful for future research on persulfides and S-sulfhydration.
5.2 DESIGN OF THE NEW METHOD
Although persulfides are believed to be stronger nucleophile compared with thiols, thiols could still interfere the trapping or labeling of persulfides with electrophiles. To solve this problem, it is desired to develop a fast method to form persulfides from thiols efficiently.
88 Our method is shown in scheme 5.1. Beaucage reagent 5.1 should react with thiol to form intermediate A which contains an acylated disulfide structure.[5.1]
The sulfenic acid (-SO2H) in A is expected to undergo a fast intramolecular cyclization to attack the carbonyl group, after which the corresponding persulfide C is released. The generated persulfide should be trapped by electrophiles to form isolable product D.
O
O S O O O R SH S B S R S R S SH OH S S C O O O Electrophile Beaucage reagent A 5.1 R S S E D
Scheme 5.1 Design of the one-step persulfide formation from thiols.
5.3 TEST THE NEW STRATEGY WITH THIOLS
We first tested this idea with tertiary thiols because tertiary persulfides are reported to be more stable.[5.2] The nucleophilicity of persulfides towards thiol blocking reagents have been studied previously.[5.3] In this study, we tried to capture the corresponding persulfides of TrSH (triphenylmethanethiol) and
AdSH (adamantanethiol) with IAM (iodoacetamide) and FDNB
(1-fluoro-2,4-dinitrobenzene) (scheme 5.2).
89 O SH H N Thiols SH O SH O SH
TrSH AdSH Bz-Cys-OMe Cyclohexanethiol
O F NO2 Persulfides trapping reagents I NH2 O2N
IAM FDNB
Scheme 5.2 Structures of the thiols and electrophiles.
The desired products were isolated with different yields as shown in scheme 5.3.
The optimized reaction conditions were the following: compound 5.1 was dissolved in DCM first. Then 1 equiv of thiol in DCM was added. Immediately
2 equiv of thiol trapping reagent was added to the reaction. The reactions normally completed in 5 min and the desired products (5.2-5.5) were isolated and characterized. Primary and secondary thiols were also employed in this study. However, we did not observe any desired products. We isolated polysulfides as the main byproducts, indicated the formation of extremely unstable primary and secondary persulfides. It should be noted that we did not observe any thiol trapping products (R-S-E) in the reactions which suggested the conversion of thiols to persulfides was effective.
90 O Electrophile
S + RSH R S SH RSSE S O O 5.1 C D
O2N
O2N S S NO O S S 2 NH2 S S NH S NO2 O 2 S
5.2 5.3 5.4 5.5
RSH Electrophile Product Yield
TrSH IAM 5.2 65%
TrSH FDNB 5.3 32%
AdSH IAM 5.4 23%
AdSH FDNB 5.5 27%
Bz-Cys-OMe IAM N/A 58% polysulfide
Cyclohexanethiol IAM N/A 65% polysulfide
Scheme 5.3 Results of the reactions between Beaucage reagent and thiols.
Having demonstrated this method in DCM, we explored whether it could be done in other solvents. And we were also interested to see if this reaction could occur in the presence of water. By using TrSH and IAM, we found this reaction worked well in different solvents including dioxane, THF, DMF, and CH3CN.
Then we conducted the reaction in mixed solvents of THF/PBS or CH3CN/PBS.
91 High yield of products were still observed which concluded that water or air should not interfere with the generation of persulfides in this method.
O O I O NH2 + TrSSxSTr S + TrSH TrSSH Tr S S NH2 S O O 5.1 5.2 5.6
Solvent Yield of 5.2 Yield of 5.6
DCM 65% 31%
THF 58% 31%
Dioxane 58% 17%
DMF 52% 21%
CH3CN 44% 20%
THF/PBS (1:1, v/v) 66% 21%
CH3CN/PBS (1:1, v/v) 41% 24%
Scheme 5.4 The reactions between Beaucage reagent and thiols in various solvents.
5.4 CONCLUSION
In summary, here we reported a novel method for persulfide formation. We found Beaucage reagent could effectively convert small molecule thiols (−SH)
92 to persulfides (-SSH) under very mild conditions. This allows for a H2S-free protocol to generate highly reactive persulfides. We also demonstrated the high nucleophilicity of persulfides toward thiol-blocking reagents such as IAM and
FDNB.
5.5 EXPERIMENTAL SECTION
Materials and Methods: All solvents were reagent grade. Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with
0.25 mm pre-coated silica gel plates. Flash chromatography was performed with silica gel 60 (particle size 0.040-0.062 mm). Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Proton and carbon-13 NMR spectra were recorded on a 300 MHz spectrometer. Chemical shifts are reported relative to chloroform (δ 7.26) for 1H NMR and chloroform
(δ 77.0) for 13C NMR. Absorption spectra were recorded on a Lambda 20
UV/VIS spectrophotometer using 1 cm quartz cells. Fluorescence excitation and emission spectra were measured on a Cary Eclipse fluorescence spectrophotometer.
General procedure of persulfides generation: In a round bottom flask,
Beaucage reagent 5.1 was dissolved in 1 mL DCM (concentration = 0.1 M). To this solution was slowly added 1 equiv of thiol in 1 mL DCM with HCl contained ethanol. At this time, 1 equiv of thiol blocking reagent with
93 triethylamine was added to the reaction after the addition of thiol. The residue was washed with ammounium chloride, water and brine. The solution was dried over magnesium sulfate and concentrated under reduce pressure. Purification by flash column chromatography using a mixture of hexane /ethyl acetate (for
FDNB trapping product) or DCM/methanol (for IAM trapping product) afforded the corresponding products.
94 5.6 REFERENCES
5.1 Breydo, L.; Gates, K. S. Thiol-dependent DNA cleavage by
3H-1,2-Benzodithiol-3-one 1,1-dioxide. Bioorg. Med. Chem. Lett. 2000, 10,
885-889.
5.2 Bailey, T. S.; Zakharov, L. N.; Pluth, M. D. Understanding hydrogen sulfide storage: probing conditions for sulfide release from hydrodisulfides. J.
Am. Chem. Soc. 2014, 136, 10573-10576.
5.3 Pan, J.; Carroll, K. S. Persulfide reactivity in the detection of protein S‑ sulfhydration. ACS Chem. Biol. 2013, 8, 1110-1116.
95
Appendix
1 Compound 2.1, HNMR, 300 MHz, CDCl3
97 13 Compound 2.1, CNMR, 75 MHz, CDCl3
98 1 Compound WSP2, HNMR, 300 MHz, CDCl3
99 13 Compound WSP2, CNMR, 75 MHz, CDCl3
100 1 Compound WSP3, HNMR, 300 MHz, CDCl3
101 13 Compound WSP3, CNMR, 75 MHz, CDCl3
102 1 Compound WSP4, HNMR, 300 MHz, CDCl3
103 13 Compound WSP4, CNMR, 75 MHz, CDCl3
104 1 Compound WSP5, HNMR, 300 MHz, CDCl3
105 13 Compound WSP5, CNMR, 75 MHz, CDCl3
106 1 Compound 3.6, HNMR, 300 MHz, CDCl3
107 13 Compound 3.6, CNMR, 75 MHz, CDCl3
108 1 Compound SeP1, HNMR, 300 MHz, CDCl3
109 1 Compound SeP2, HNMR, 300 MHz, CDCl3
110 13 Compound SeP2, CNMR, 75 MHz, CDCl3
111