Chemical and biological analysis of novel thiazolidine prodrugs of L-cysteine

The therapeutic manipulation of glutathione (GSH) levels is important in cases where toxic agents cause GSH depletion. GSH depletion causes deleterious effects inside cells, as this thiol serves may protective roles. One way to increase intracellular GSH level is to supply cysteine, the rate-limiting precursor in GSH biosynthesis. Cysteine is known to be toxic to cells and therefore, cannot be administered directly. To overcome this toxicity, cysteine can be delivered in a produg form. The development of cysteine prodrugs had been found to be clinically relevant, especially during GSH depletion caused by acetaminophen (APAP). One class of cysteine prodrugs, 2-alkyl-thiazolidines, has proven to be useful delivery agents. Described here are four novel 2-alkylthiazolidines. The amide and ethyl ester of 2(R,S)-D-gluco1',2',3,'4',5'-pentahydroxypentylthiazolidine-4(R)-carboxylic acid (GlcCys amide and GlyCys ethyl ester) and 2(R,S)-D-ribo-1',2',3',4'-tetrahydroxybutylthiazolidine-4(r)-carboxylic and (RibCys amide and RibCys ethyl ester) were synthesized and assayed for their protective capabilities. To test compounds against APAP-induced toxicity, a cell assay using HepG2 cells and the toxin 2,6-dimethyl-N-acetyl-p-benzoquinoneimine (2,6-diMeNAPQI), a synthetic analog of a reactive APAP metabolite, was utilized. GlcCys ethyl ester, RibCys amide, and GlcCys amide were proven to protect against 2,6-diMeNAPQI-induced toxicity. This protection was dependent on intracellular process to activate the prodrugs evidenced by their lack of reaction with 2,6-diMeNAPQI in solution. To further the understanding of these and other cysteine prodrugs and to facilitate the study of biological thiols in general, an HPLC assay that allows the concomitant separation and measurement of cysteine and GSH and their oxidized species was developed. This assay showed that the prodrugs elevated cysteine but not GSH levels in HepG2 cells. These results indicate that the novel prodrugs are attractive lead compounds for further analysis as cysteine delivery vehicles to abate the toxicity of APAP overdose.

THE CHEMICAL AND BIOLOGICAL ANALYSIS OF NOVEL THIAZOLIDINE PRODRUGS OF L-CYSTEINE by PalTIeia K. Dominick A dissertation submitted 10 the faculty of The University of Utah in partial fulfillment of the requirements for the degree Doctor of Philosophy Department of Medicinal Chemistry The University of Utah May 2002 Copyright Pan1ela K. Dominick 2002 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Pamela K. Dominick This dissenation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. LI -2-7-0 I / J Chris M. Ireland /(j '-/ ~i Bradley D. Anderson THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Pamela K. Dominick in its final form and have found that (1) its forma4 citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures. tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. Jean~ te C. Roberts Chair./Srlpervisory Committee I. J ' .. / AP~,~roved ,for thhLeMajor Department :1 \\ r' TI I '.' ~ ': _; GiJi:nlD . Prestwich , Cbair/Dean Approved for the Graduate Council David S. Chapman , Dean of The Graduate School ABSTRACT The therapeutic manipulation of glutathione (GSH) levels is important in cases where toxic agents cause GSH depletion. GSH depletion causes several deleterious effects inside cells~ as this thiol serves many protective roles. One way to increase intracellular GSH levels is to supply cysteine, the rate-limiting precursor in GSH biosynthesis. Cysteine is known to be toxic to cells and therefore, cannot be administered directly. To overcon1C this toxicity, cysteine can be delivered in a prodrug fonn. The development of cysteine prodrugs has been found to be clinically relevant, especially during GSH depletion caused by acetmninophen (APAP). One class of cysteine prodrugs, 2-alkyl-thiazolidines, has proven to be useful delivery agents. Described here are four novel 2-alkylthiazolidines. The amide and ethyl ester of 2(R,S)-D-R/uco- 1',2',3',4',5' -pentahydroxypentyIthiazolidine-4(R)-carboxylic acid (GlcCys amide and GlyCys ethyl ester) and 2(R,S)-D-riho-1 ',2',3',4' -tetrahydroxybutylthiazolidine-4(R)­carboxylic acid (RibCys amide and RibCys ethyl ester) were synthesized and assayed for their protective capabilities. To test con1pounds against APAP-induced toxicity, a cell assay using HepG2 cells and the toxin 2,6-dimethyl-N-acetyl-p-benzoquinoneimine (2,6-diMeNAPQI), a synthetic analog of a reactive APAP n1etabolite, was utilized. GlcCys ethyl ester, RibCys amide, and GlcCys amide were proven to protect against 2,6-diMeNAPQI-induccd toxicity. This protection was dependent on intracellular processes to activate the prodrugs evidenced by their lack of reaction with 2,6-diMeNAPQI in solution. To further the understanding of these and other cysteine prodrugs and to facilitate the study of biological thiols in general, an HPLC assay that allows the concomitant separation and nleaSUrel11ent of cysteine and GSH and their oxidized species was developed. This assay showed that the prodrugs elevated cysteine but not GSH levels in HepG2 cells. These results indicate that the novel prodrugs are attractive lead compounds for further analysis as cysteine delivery vehicles to abate the toxicity of AP AP overdose. v In n1emory of my n10ther, Mitsuko Yanagi TABLE OF CONTENTS ABSTRACT ........................................................................................ IV LIST' OF 'I'ABLES ................................................................................ ix LIST OF ABBREVIATIONS ..................................................................... x ACKNOWLEDGMENTS ......................................................................... xii Chapter 1. OVERVIEW OF GLUTATHIONE FUNCTION AND CYSTEINE PRODRUGS ......................................................... 1 Introduction .......................................................................... 1 OS H: structure determination .................................................... 2 GSH: biosynthesis .................................................................. 3 GSH: biological functions ......................................................... 3 Acetaminophen toxicity ........................................................... 7 Chemicallnethods for increasing intracellular GSf1 levels .................................................................. 9 Thiazohdine esters and amides: a novel approach to cysteine delivery ...................................................... 17 Overview of thesis ............................................................... 19 Referellces ......................................................................... 20 DEVELOPMENT AND VALIDATION OF A NEW HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE DETERMINATION OF BIOLOGICALLY IMPORTANT l'HI()LJl\MINI~S .................................................................. 29 Introduction ..................................................................... 29 Results ............................................................................. 31 IJiscussioll ........................................................................ 45 Conclusions ........................................................................ 47 Experilnental ..................................................................... 48 Refere11ces ........................................................................ 54 3. SYNTHESlS OF NOVEL THIAZOLIDINE PRODRUGS ()F' L-("YS"rEINE ................................................................. 55 Introductiol1 ...................................................................... 55 Rationale ........................................................................... 56 Results and discussion .......................................................... 59 Conclusions ........................................................................ 66 Experilnental ...................................................................... 67 References ......................................................................... 78 4. BIOLOGICAL STUDIES OF CYSTEINE THIAZOLIDINE PRODRUGS ...................................................................... 81 Introduction ........................................................................ 81 Results .............................................................................. 82 Discllssion ........................................................................ 1 00 ('011clusions ...................................................................... 1 07 Experimental ..................................................................... 1 08 References ........................................................................ 119 5. CONCLUSIONS AND FUTURE DIRECTIONS .................................. 122 Appendices A. HPLC CHROI'vlATOGRAMS ......................................................... 127 B. NMR SPECTRA ......................................................................... 137 VIll LIST OF TABLES Table Page 2.1. Retention tinles of DNP derivatives and side products ............................... 37 2.2. Standard curve equations of DNP derivatives in water, HepG2 ceillysates, and nlouse liver honl0genates ............................................................ 38 2.3. Intraday variation in the peak area ratio of DNP derivatives in water, HepG2 celilysates, and lTIOUSe liver homogenates ............................................ .40 2.4. Intraday variation in the slope of DNP derivatives in water, HepG2 cell lysates, and nl0use liver hon10genates ................................................. .41 2.5. Interday variation in the peak area ratio of DNP derivatives in water .............. .43 2.6. Interday variation in the slope of DNP derivatives in water ........................ .44 4.1. Thiollneasurelllents fronl the l-IPLC analyses of HepG2 celllysates from cells treated "vith 1 0 nlM of protector ................................................... 96 4.2. Thiol measuren1ents from the HPLC analyses of residual treatnlent media from 11epG2 cells treated with 10 mM of protector ................................... 97 APAP BNPP BPOS BSO CV 2,6-0iMeNAPQI DNFB DNP EDTA EMEM GlcCys GSH GSSG MTS NAC NAPQI LIST OF ABBREVIA'fIONS Acetaminophen Bis-(p-nitrophenyl)phosphate Buthophenanthrolinedisulfonic acid Buthionine sulfoxinline Coefficient of variation N-Acetyl-2.6-dinlethyl p-benzoquinoneimine Oinitrofluoro benzene Oinitrophenylated Ethylenedianlinetetraacetic acid Eagle nlinin1um essential medium 2(R,5)-D-gluco-1 ',2',3',4',5' -Pentahydroxypentylthiazolidine- 4(R)-carboxylic acid Glutathione Glutathione disulfide 3-( 4,5-Dimethylthiazol-2-yl)-5-(3-carboxynlethoxyphenyl)-2-( 4- suI fopheny 1)-2 H -tetrazo I i urn N-Acetylcysteine lV-Acetyl-p-benzoquinoneimine OTC PBS PCA PES RibCys ROS SD TFA 2-0xothiazolidine-4-carboxylic acid Phosphate buffered saline Perchloric acid Phenazine ethosulfate 2(R,S)-D-rihu-I' ,3' A' ,-Tetrahydroxybutylthiazolidine-4(R)­carboxylic acid Reactive oxygen species Standard deviation Triiluoroacetic acid xi ACKNOWLEDGMENTS I would like to express Iny appreciation to several people for their contributions during my graduate career. First and foremost, this would have not been possible without the guidance, dedication, and patience of nly thesis advisor, Dr. Jeanette Roberts. Also, Dr. Roberts provided rne with the opportunity to teach classes in the College of Pharnlacy, which allowed me to gain valuable experience in this area. I would like to thank nly coworkers. Dr. Pamela Cassidy, Dr. Britta Wilmore and Megan Short, for their many insightful scientific discussions and friendship. Dr. Pamela Cassidy contributed greatly to Inany facets of my project including work with Rachel Jameton in establishing the initial cell study experinlents and with Robyn James on the synthesis of 2,6-dimethyl­NAPQI, which was crucial for several experiments. I anl very grateful to my mother, Mitsuko Yanagi, who provided me with constant love and encouragenlent throughout nly years as a student. And I vvould like to thank nly sister, Dr. Cynthia Woodley, for all her love and support. I am very thankful to them both for helping me achieve my educational goals. I would like to thank nly husband, Dr. Michael Jarstfer, for all his persistence and support. His scientilic discussions were greatly appreciated and contributed to my success. CHAPTER 1 OVERVIEW OF GLUTATHIONE FUNCTION AND CYSTEINE PRODRUGS Introduction Organisms depend on the ability to protect against damaging chenlical insults for their survival. Various toxins originate from nornlal cellular processes, including reactive oxygen species (ROS) resultant from electron transfer reactions, I and from external sources known collectively as xenobiotics. Because of the complexity and variety of potential harm from chenlical insult, cells have evolved an equally cOluplex and varied defense nlechanislu for detoxification.2 One major protective mechanisnl, ubiquitous in nlamlnalian tissues, is the reaction of glutathione (GSlI; L-y-glutaluyl-L­cysteinylglycine) with harInful agents.3 In humans, the liver is the site of clearance of the 111ajority of exogenous toxins and has one of the highest concentrations (5-10 luM) of GSH of all tissues.4 GSH is the 111ajor nonprotein intracellular thiol. GSH is a tripepetide of glutanlic acid, cysteine, and glycine containing an unusual y-peptide linkage between glutamate and cysteine. This y-linkage renders GSH inert to intracellular peptidases.5 Within the intracellular l11ilieu, GSH can react with itself to yield its disulfide fonu (GSSG). 2 However, under physiological conditions GSH reductase rapidly reduces any GSSG, in a NADPH dependent reaction, so that nlore than 98% of intracellular GSH is in the fully reduced fonn.4 GSH: structure determination The accurate assignment of the chemical structure of GSH and the ability of chenlists to direct its synthesis in the laboratory facilitated further advancelnents in understanding the functions of GSH.6 The structure of GSH was detern1ined by the conlbined efforts of several researchers. ]. Harris determined the equivalent weight of GSH as 307 0.8. Later, Pirie and Pinhey, in a serendipitous discovery, proposed the structure y-glutamylcysteinylglycine from titration curves from which they assigned the following pKa's: SH 9.62, NH2 8.66, COOH 3.53, and COOH 2.12. Finally, several groups working on the degradation and lllanipulation of GSH, established its structure. The first de novo synthesis of GSH was reported in 1935 by Harrington and Mead and confirnled the structure assignment (Figure 1.1). o (SHH H,NyJNl(N,-/CO,H H(\C H 0 Figure 1.1. The structure of GSH. 3 GSH: biosynthesis GSH is synthesized biochelnically in the cytosol of n10st cells, although the major site of synthesis is the liver. Bloch and coworkers first discovered the enzymes responsible for GSII biosynthesis in the early 1950s.7 ,8 GSH is produced in two ATP dependent steps that couple the amino acids L-cysteine, L-glutaInate, and glycine (Figure 1.2). The first step, catalyzed by y-glutamylcysteine synthetase, is specific for the y­carboxylate of glutan1ate and is feedback inhibited by GSH with a Ki 2-3 InM.'uo The second reaction is catalyzed by GSH synthetase, which produces a peptide bond between the cysteinyl carboxylate of y-glutamylcysteine and the glycinyl amine. In the liver, the ll1ajor lin1it on GSH biosynthesis is the bioavailability of L-cysteine. II GSH: biological functions The cellular functions of GSH began to attract considerable interest in the early 1900s. One clue CaIne from the key observation by F. G. Hopkins that total non-protein cellular cysteine was actually contained in GSH and not free cysteine.6 Thus, the first assigned function of GSH, then thought to be a dipeptide, was as a cellular storage facility for cysteine. y-glutamylcysteine synthetase L-glutamic acid + L-cysteine + ATP L-y-glutamylcysteine ADP Pi GSH synthetase L-y-glutamylcysteine + glycine ATP GSH + ADP + Pi Figure 1.2. Biosynthesis of GSII. 4 Today~ GSH is known to function in many important biological pathways. The diverse roles played by GSH can be divided into several n1ajor functions: antioxidation including regulation of cellular sulfuydryl status,5J2 conjugation of electrophiles and n1eta Is , I-,, 14 prOVI' d'l 11g a reserVOI,r f'o r cysteI. ne, l'-i an d mo d u lat'I ng ce 11 u 1a r processes including DNA synthesis~ 16 Inicrotubular-related processes~ and immune functions. 17 The cornerstone of GSH function is the free thiol of its cysteine residue. One of the best-studied functions of GSH is its role in redox homeostasis. Respiration creates a collection of reactive intermediates and side products, including superoxide, hydrogen peroxide, and oxygen radicals. Left unattended~ these reactive oxygen species cause lipid peroxidation and can disrupt n1etabolic processes, 5,1 x The predon1inant defense Inechanisn1 against these reactive oxygen species is the GSH redox systen1~ which includes GSH, GSH reductase, GSH peroxidase, and NADPH.5,IX,19 A GSH synthetase knock-out mouse provides interesting insight into the biological role of GSH.20 Shi et al. found that GSH synthetase was essential for en1bryonic development. Surprisingly, however~ it was found that in cultured cells~ GSH function could be replaced by N-acetylcysteine (NAC). This suggests that n1any of the housekeeping roles of GSH do not require GSH specifically, but rather require reducing cquivalents~ which can be substituted by NAC. Most gern1ane to this thesis is the role of GSH in detoxification of xenobiotics. An enormous array of electrophilic xenobiotics and xenobiotics that are biotransforn1ed into electrophiles are detoxified through conjugation with GSH.21 Systen1ic detoxification occurs in the liver, which has a high concentration of GSH. Because of the high GSH content, conjugation could occur enzymatically or nonenzymatically.s Many 5 xenobiotics are conjugated stereospecifically, indicating that these reactions are mostly catalyzed by GSH S-transferases, which are a broad class of enzymes that catalyze the nucleophilic attack of GSH on varied substrates.22 Consistent with the liver being the site for detoxi fication, this organ has the highest concentration of GSH S-transferases of all organs.23 The GSH S-transferases can be broken down into four groups, alpha, mu, pi, and theta, and are differentiated by their protein prinlary sequences, subunit organization, and substrate specificity. 22,23 Despite the variety of GSII S-transferases, there are C01111110n features amongst the substrates that can be conjugated to GSH:21 they are hydrophobic, they contain an electrophilic reaction center, and they react with GSH nonenzY111atically at SOl11e n1easurable rate. GSH conjugation can occur by two distinct mechanisms depending on the reactivity of the xenobiotic. One reaction involves direct displacement of an electrophilic leaving group, whereas the other involves an addition reaction to activated carbon-carbon double bonds or strained ring systems. Once a xenobiotic is conjugated to GSH, it can be extracted in the bile, or converted to a nlore hydrophilic species via the mercapturic acid biosynthetic pathway to allo\v excretion in the urine (Figure 1.3 ).21 The first two steps of the pathway remove glutanlic acid and glycine, leaving a cysteine conjugate. The final step is the acetyl-CoA dependent iV-acetylation to produce the mercapturic acid moiety. A comI11on 1110del for hepatotoxicity and a major medical concern is the toxicity of acetaminophen (APAP), which is nletabolized in part by the GSH dependent mercapturic pathway as discussed in nlore detail below. RX substrate + cysteine conjugate acetyl-CoA CoA SR GSH S-transferases H2N~N~~'-/C02H + H02C H 0 GSH conj ugate y-glutamyltranspeptidase o HN /'. Jl 2 Y "'-/ 'OH H07C - glutamic acid glycine N-acetyltransferase mercapturic acid execreted ill urine Figure l.3. The mercapturic acid biosynthetic pathway. 6 7 Acetaminophen toxicity APAP, introduced worldwide in the 1950s, is a popular and frequently used antipyretic and analgesic drug. COlTIlTIercially available, AP AP can be used alone, but is also found forn1ulated in cough and cold lTIedicines.24 AP AP is a relatively safe drug at therapeutic doses (lO to 15 lTIg/kg).25 Ho\vever, there has been increased concern regarding APAP toxicity associated with overdose related to suicide,25 and lTIOre recently, APAP taken after large consun1ption of ethanot26 and in accidental poisonings. 27 APAP poisonings accounted for 53% of toxic exposures to analgesic n1edications in 1997, according to the American Association of Poison Control Centers.28 Mitchell and coworkers provided the seminal work that defined the toxicology of AP AP overdose.29-32 At therapeutic doses, ~95-98% of APAP is lTIetabolized by the liver and ~2-50/0 is excreted unchanged in the urine. 33 In the liver, the majority of APAP 95%) is 111etabolized by conjugation to glucuronide or sulfate and is safely excreted. The renlainder « 50/0) is metabolized to the highly toxic and electrophilic n1etabolite N­acetyl- p-benzoquinoneimine (NAPQI) by the P450 mixed function oxidase systenl (Figure 1.4). This intern1ediate is readily detoxified by conjugation to GSFI and is safely excreted as the mercapturate conjugate. Toxic doses of AP AP saturate the glucuronidation and sulfation pathways allowing the P450 mixed function oxidase systenl to don1inate APAP n1etabolisn1 resulting in high levels ofNAPQI. GSH stores beconle depleted resulting in an excess ofNAPQI, which then arylates hepatic cellular proteins leading to hepatic necrosis, and ultimately death. The work of Mitchell and coworkers29-32 suggest that in overdose situations APAP-induced hepatotoxicity is mediated by NAPQI accUlTIulation resulting in the o HN~ PAP PAPS ¢ Phenolsulfotransferase OSO-J APAP Sulfate o II ¢~ OH APAP 8 U DP -gl ucuronosy Itransferase APAP Glucuronide NADPH,O:: Cytochrome P450 o N~ o o NAPQI j GSH o ~ ~SG OH GSH conjugate Bind to hepatotocellular proteins leading to hepatic necrosis Figure 1.4. MetabolislTI of APAP. AP AP metabolism occurs primarily by conjugation with sulfate and glucuronide. A small percent of APAP is metabolized by cytochrome P450 to give the reactive electrophilic intermediate, NAPQI, which is detoxified by GSH conjugation. Under conditions of APAP overdose, NAPQI can bind to cellular proteins in the liver leading to hepatic necrosis. 9 depletion of intracellular levels of GSH. Upon GSH depletion, N APQI is available to covalently bind to cellular proteins in the liver resulting in hepatic necrosis. Several investigators have confirn1ed the covalent binding ofNAPQI to cellular proteins.34 - 40 Further studies since this pivotal work have shown that several specific P450 isoenzYlnes are responsible for bioactivation of APAP to NAPQI. The P450s implicated in the for111ation ofNAQPI include CYP2E 1, CYP IA2, and CYP3A4.26 ,41-44 Of these P450s, CYP2E 1 is thought to be the principal enzyme responsible.26 ,41 Though the structure of NAPQI has been convincingly shown as the responsible agent for APAP toxicity,45,46 the chen1ical mechanisn1 of formation of this reactive intermediate is still being investigated.47 Also, other mechanislTIs of APAP-induced hepatotoxicity fron1 NAPQI fonnation besides protein arylation are becon1ing evident. These include oxidative stress48 involving lipid peroxidation,49 disruption of Ca2 + homeostasis,50 oxidation of protein thio]S,51 and respiratory dysfunction. 52 Although these other mechanisms of APAP-induced hepatotoxicity are suggested, these n1echanisms are usually preceded by depletion of intracellular GSH. Understanding the mechanism of APAP-induced hepatotoxicity has been a research tool for understanding the mechanisms of GSH depletion and the development of ways to restore intracellular GSH levels. Of all the ways to protect against APAP-induced hepatotoxicity during an overdose, restoration of intracellular GSH stores is most clinically relevant. Chemical methods for increasing intracellular GSH levels As described above, depletion of GSH from toxic insult results in severe deleterious effects and sometimes death. Moreover, it has recently been shown that depleted aSH levels in HIV infected subjects is related to decreased survivaL53,54 It follows that the ability to increase cellular aSH levels is an important objective to combat the effects of xenobiotic insult and other Inedical conditions that result in depleted aSH levels. 53-56 The Inost straightforward approach to increasing aSH would be the delivery of aSH or its rate limiting precursor cysteine. However, GSH is not readily transported into cells, but instead is broken down by mel11brane-bound y­g] utanlyltranspeptidase and dipeptidase activities. 57 The amino acid products of these enzylnes are transported into cells where they can serve as substrates for aSH biosynthesis. The direct therapeutic use of cysteine is even more problelnatic. Cysteine has been found to be toxic to rats and Inice,58's9 leads to degeneration of the central nervous systenl in new born nlice60 and 4-day-old rats,61 and is toxic in cell culture.62 Moreover, cysteine in solution rapidly oxidizes to cystine, which is highly insoluble. These problelns have created an entire field of research dedicated to the ability to enrich and replenish intracellular GSl-I. 10 The greatest effort in the direction of aSH enrichment has COIne from the field of snlall molecule chelnistry. These efforts will be catalogued in detail below. An alternative approach, however, warrants Inentioning. Wendel and Jaeschke have shown that GSH can be delivered to cells through carrier liposolnes.63 GSH delivered in this nlanner through the tail vein of mice was shown to maintain hepatic GSH levels during high levels of acetalninophen dosing. Interestingly, the GSH increase observed in these experinlents was inhibited by buthionine sulfoxilnine (BSO), an inhibitor of y­glutanlylcysteine synthetase. This suggests that the liposomally delivered GSH was transforIned to its constitutive parts then resynthesized in the liver. II The approaches to GSH delivery can be broken down into two main divisions including GSH agents and cysteine agents. The search for GSH delivery agents is still an active research goaL and a review of the GSH and cysteine agents developed to date is presented below. GSH delivery agents The use of y-gl utatTIylcysteine and y-glutatnylcystine as vehicles to increase GS H levels has been studied. These agents bypass the feedback inhibition of y­glutamylcysteine synthetase and require an active GSH synthetase. It became apparent through these studies that y-glutan1ylcysteine and y-glutamylcystine are readily taken up by kidney and increase GSH levels.64 Further studies, using C5S]-labeled y­glutamylcystine on both cysteine residues, reveal that the GSH was preferentially increased frOlTI the y-glutatnylcysteine moiety, not cysteine.65 In an atten1pt to make y­glutamylcysteine more alTIenable to cysteine delivery to a broader class of cell types~ the ethyl ester was prepared. y-Glutan1ylcysteine ethyl ester protects the liver against toxic insult. 66 Importantly~ y-glutamylcysteine ethyl ester is converted to GSH in cells in an esterase dependent n1anner as evidenced by the inhibition of GSl1 increase with bis-(p­nitrophenyl) phosphate (BNPP), which is a nonspecific esterase inhibitor.67 An alternative approach that has received more attention is the delivery of GSH analogs that are readily transported and converted into GS}! by cellular processes. To this end, n10no- and diesters of GSH have proven effective. The primary study with GSH lnonoester was perforn1ed by Puri and Meister who utilized GSH methyl and ethyl esters in which the ester moiety is attached to the glycine carboxyl group.68 They found that 12 mice treated with GSH Inonomethyl or n10noethyl ester had elevated GSH levels in both the liver and kidney; n10re importantly, the authors reported that this increase was not inhibited by BSO treatment. This latter result indicated that the esters were transported into the cells intact, the ester n10iety was hydrolyzed, and GSH was delivered without the requiren1ent of GSH synthesis. This contrasts to the situation for the delivery of free aSH, which, as described above, is broken down into its constitutive an1ino acid parts and resynthesized intracellularly. The investigation of aSH monoesters, primarily the ethyl ester and isopropyl ester, is well docun1ented in 111any biological systems. The aSH lnonoesters have been shown to increase intracellular GSH levels69 and have been used for protection against ischemic rat brain damage,70 cataracts,71 cisplatin,72 AP AP, 73 radiation,74 hydrogen peroxide,75 and n1ercuric chloride. 76 The GSH monoesters are able to increase intracellular GSH levels, in contrast to GSH, even in the presence of BSO and are not substrates for y-glutamyltranspeptidase. The phannacokinetics of GSH monoesters as delivery vehicles for intact aSH has recently been examined more closely.77-79 Several authors have reported that the monoesters could be hydrolyzed enzymatically by extracellular esterases, leaving free aSH. The free aSH can be broken down by y-glutamyltranspeptidase and dipeptidase leaving cysteine, which was observed to be increased dramatically by treatment with GSH monoesters. In this case, only the slow hydrolysis of the Inonoester to free aSrI is an improvement over the direct delivery of aSH itself. The diesters of GSH, however, have improved phannacokinetics. 79.8o This is presumably because of the slow hydrolysis of one ester group, allowing the intracellular Inonoester concentration to increase and produce as H after esterase action. 79 13 Cysteine delivery agents The n10st con1mon and clinically advanced approach for the therapeutic elevation of OSII levels is the delivery of cysteine, the lilniting biosynthetic precursor of aSH. As mentioned above, there are several problelns inherent in the direct delivery of cysteine, particularly at the levels required as an antidote for toxic insult. These problen1s have been overcon1e by n1asking cysteine in a variety of prodrug forms. Many of the cysteine prodrugs presented below are described for their ability to deliver cysteine specifically for protection against APAP toxicity. The ability of cysteine esters to function as delivery vehicles for cysteine has been investigated. Phannacokinetic analysis in rats indicates that cysteine ethyl ester is readily distributed throughout all tissues with a high affinity for the lung and is metabolized to inorganic sulfate, taurine, and cysteine. 81 Changes in GSH levels were not examined. A structure activity relationship of cysteine esters revealed that all carboxylate esters tested Inaintained increased intracellular levels of cysteine, though GSH levels were unchanged.82 The most hydrophobic ester tested, cysteine cyclohexyl ester, had the longest sustained increased cysteine level and the greatest cellular uptake. 82 Also, cysteine isopropyl ester was shown to protect against APAP-induced hepatotoxicity in . 83 mIce. A clinically relevant cysteine delivery agent is NAC. NAC is used as a n1ucolytic agent and is also an antidote in the treatment of APAP overdose. 84 It is thought that NAC is converted by an intracellular N-deacetylase to give cysteine, though the exact n1etabolism of this agent is not fully understood. 84 However, studies investigating the n1echanisl11 of protection ofNAC against APAP toxicity have provided supporting 14 evidence that protection is Inediated through liberation of cysteine thereby increasing aSH biosynthesis.85 - 88 Although NAC is the most con11non antidote for APAP toxicity its efficacy suffers frOln several drawbacks. 89 These include the large doses required 89 . 90 (300 n1g/kg)· and adverse sIde effects. An alternative cysteine delivery system is based on the thiazolidine ring structure. Of these, 2-oxothiazolidine-4-carboxylic acid (OTe) has received the most attention,91 and it is currently in phase II trials for the enrichn1ent of GSH levels in HIV -infected individuals.92 OTC was first reported as a potential source of cysteine for the elevation of aSH by Meister and coworkers in the early 1980s.93 ,94 OTC is converted to S-carboxy-cysteine by the action of 5-oxoprolinase. The intermediate then undergoes nonenzymatic decarboxylation to produce cysteine (Figure 1.5). In 1982, Meister and Williamson patented OTe for restoring GSH levels in human tissues, specifical1y the liver, for treatlnent against APAP overdose. OTe is well-known and docun1ented as a cysteine prodrug that is converted intracellularly to cysteine in support of GSH biosynthesis.95 ,96 Another type of thiazolidine prodrug of cysteine is based on the condensation of cysteine with a variety of aldehydes and ketones (Figure 1.6). The simplest of these is the product frOln the condensation of cysteine with formaldehyde, thiazolidine-4(R)- OTC ATP ADP, Pi \ ) .. o 0 A II HO SAOH 5-oxopro 1i nase' II NH~ S-Carboxycysteine o II HSAOll H NH 2 Cysteine Figure 1.5. Conversion of OTe lnediated by 5-oxoprolinase. o 0"- ~ JL II) HS T 'OH R~R' ~~ Cysteine Aldehyde or ketone o r-!OH S NH X R R' Thiazolidine Schiff's base intermediate Figure 1.6. Formation of thiazolidine compounds fron1 cysteine and an aldehyde or ketone. Fornlation and deconlposition of the thiazolidine ring occurs through a Schiffs base internlediate. carboxylic acid. 97,98 This thiazolidine is metabolized by liver nlitochondrial proline oxidase giving N-fonnyl-L-cysteine, which then hydrolyzes to give cysteine.97,98 Thiazolidine-4-carboxylic acid has been reported to protect against a variety of toxins including APAP toxicity in mice. 97,98 However, the thiazolidine itselfhas been shown to be toxicY7,98 A series of2-alkyl- or 2-arylthiazolidine-4-carboxylic acids have been investigated and proven to be prodrugs of cysteine.99 These thiazolidines do not require enzYlnatic activation and release cysteine through nonenyzymatic hydrolytic ring opening. A wide variety of these 2-substituted thiazolidines-4-carboxylic acids have been evaluated for their ability to release cysteine and protect mice against APAP-induced hepatotoxictyl()O and cataracts. IOl 15 16 A Inechanisn1 for the formation of 2-alkylthiazolidines is shown in Figure 1.6. The first step in principle could be nucleophilic attack of the carbonyl carbon of the ketone or aldehyde by either nitrogen or sulfur frOln cysteine. Evidence indicates that the first step is nucleophilic attack by nitrogen to allow fonnation of a Schiff's base and this is followed by ring closure. Release of cysteine fron1 thiazolidine prodrugs follows the reverse reaction. The release of cysteine froln 2-alkylthiazolidines occurs concomitantly with the release of one equivalent of the aldehyde. Roberts et al. realized the potentiallilnitation due to the possible toxicity of the released aldehyde and replaced the aldehyde with aldose Inonosaccharides to produce a new class of cysteine prodrugs. J 02 The most successful of these were 2(R,I..'f}-D-gluco-1 ' ,2',3',4',5' -pentahydroxypentylthiazolidine- 4(R)-carboxylic acid (GlcCys) and the 2(R,k'f}-D-ribo-l' ,2',3',4' -tetrahydroxy­butylthiazolidine- 4(R)-carboxylic acid (RibCys). RibCys has been shown to protect against APAP-induced renal toxicityl(J3 and hepatotoxicity, 104 as well as cyclophosphamide urotoxicity.l0S,J06 The fact that these 2-substituted thiazolidine-4- carboxylic acids protect through release of cysteine providing a precursor for GSH biosynthesis has been suggested. The polyhydroxyalkyl chain found at the C2 position of the thiazolidine ring makes these conlpounds hydrophilic. This was suggested fron1 the results of a biodistribution study using eSS]-RibCys and eSS]-GlcCys in lnice showing rapid excretion of these compounds in the urine. I07 Thus an obvious goal towards inlproving RibCys and GlcCys efficacy is to increase their bioavailability and retention. 17 Thiazolidine esters and amides: a novel approach to cysteine delivery The rapid excretion of RibCys and GlcCys could be a result of the free carboxylate. It was speculated that capping the free carboxylate would increase cellular uptake and decrease the rapid clearance observed in the biological studies. One goal of this thesis was to synthesize and evaluate novel thiazolidine prodrugs of cysteine. Thiazolidine esters and amides were designed to overcome the rapid excretion observed with the thiazolidine carboxylic acids. In this study, the carboxylic acid functional group was replaced with either an ethyl ester or carboxamide to increase the lipophilicity of these cOlnpounds. The rationale for this chen1ical modification was to decrease the rapid excretion of these thiazolidine prodrugs, thereby influencing the efficacy by increasing drug absorption versus drug clearance. Esterification of a carboxylic acid functional group in a drug is a very con1mon n10dification since esterases are widely distributed in the plasma, liver, kidney, and intestine, and are usually substrate nonspecific. This is the san1e for amidation of the carboxylic acid. An1idases are also widely distributed and nonspecific. Therefore, the metabolic hydrolysis of the ester or amide functional group is a facile process in the body thereby allowing generation of the actual drug. In the case of the novel thiazolidine prodrugs, the ester or an1ide derivatives were designed to be '"pro-prodrugs" in the sense that the thiazolidine ring and the carboxylate functionality both must hydrolyze to release cysteine. Two possible mechanisms exist for the hydrolysis of the thiazolidine ester or amide (Figure 1.7). The hydrolysis to release the free carboxylate could occur with the intact thiazolidine ring giving the 18 + sugar Esterase or am idase Esterase or amidase o ~OH HS NH2 + sugar RI R2 -OCH2CH3 ~OH OH OH or HO or OH OH OH OH -NH2 OH ribose glucose Figure 1.7. Possible mechanisms of cysteine release from the thiazolidine ethyl ester and amide. thiazolidine carboxylic acid, which would then undergo nonenzymatic ring opening to release cysteine. Another possible mechanism is that the thiazolidine ester or amide could undergo nonenzymatic ring opening to liberate cysteine ethyl ester or cysteinemnide. Cysteine would then be released by hydrolysis of the ester or an1ide functional group. Results in this thesis assist in elucidating the precise Inechanism. 19 Overvie\v of thesis The overall ainl of this thesis was to synthesize novel thiazolidine prodrugs of cysteine and investigate the protective abilities of these prodrugs against toxicity in vitro. To assist in these and other studies, an HPLC lTIethod for measuring biologically important thiolamines was also developed. Chapter 2 describes the developll1ent and validation of this novel HPLC nlethodology. The ITIethod was validated in three nlatrices including celllysates and mouse liver honl0genates. Important factors in this development were the validation of the HPLC method and the ability to sinlultaneously quantitate both reduced and oxidized thiolamines in the same sample. Chapter 3 describes the synthesis of four novel thiazolidine prodrugs of cysteine as delivery agents for this inlportant precursor in OSH biosynthesis. The thiazolidines were modeled after the known thiazolidine carboxylic acids, RibCys and OlcCys, in which the carboxylic acid functionality was lTIodified with an ethyl ester or amide. These modifications were chosen based on the hypothesis that they would increase the lipophilicity, and hence, the uptake and half-life of these compounds in comparison to the carboxylic acid derivatives. Chapter 4 describes the in vitro investigation of the newly synthesized thiazolidine ethyl esters and amides for their ability to protect against APAP-induced toxicity. 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Intracellular delivery of cysteine. A1ethods Enzymol. 1987, 1-13, 313-325. 97. Harnland, M. F.; Blanquet, P. Pharmacokinetics and metabolisln of CSS]-thiazolidine carboxylic acid in the rat. 1. Elinlination and distribution. Eur. J. Drug Metab. Pharmacol. 1982, 7,323-327. 98. Nagasawa, H. '1'.; Goon, D. J. W.; Zera, R. T.; Yuzon, D. L. Prodrugs of L-cysteine as liver protective agents. 2(R,S)-Methylthiazolidine-4(R)-carboxylic acid, a latent cysteine . .J NJed. Chern. 1982,25,489-491. 99. Nagasawa, H. 'r.; Goon, D. J. W.; Muldoon, W. P.; Zera, R. T. 2-Substituted thiazolidine-4(R)-carboxylic acids as prodrugs of L-cysteine. Protection of mice against ace1alninophen hepatoxicity . .J A1ed. Chern. 1984,27,591-596. 28 100. Srinivasan, C.~ Willian1s, W. M.; Ray, M. B.; Chen, T. S. Prevention of acetaminophen-induced toxicity by 2(R,Sj-n-propylthiazolidine-4-(R)-carboxylic acid in ll1ice. Biochem. Pharmacol. 2001, 61, 245-252. 101. Rathbun, W. B.; Nagasawa, H. T.; Killen, C. E. Prevention of naphthalene-induced cataract and hepatic glutathione loss by the L-cysteine prodrugs, MTCA and PTCA. E~'(p. Eye Res. 1996, 62,433-441. 102. Roberts, J. C; Nagasawa, H. T.; Zera, R. T.; Fricke, R. . Goon, D. J. W. Prod rugs of L-cysteine as protective agents against acetaminophen-induced hepatotoxicity. 2- (Polyhydroxyalkyl)- and 2-(polyacetoxyalkyl)thiazolidine-4(R)-carboxylic acids. J Med. Chem. 1987.30,1891-1896. 103. Lucas, A. M.; Henning, G.; Dominick, P. K.; Whiteley, H. E.; Roberts, J. C.; Cohen. S. D. Ribose cysteine protects against acetaminophen-induced hepatic and renal toxicity. Toxicol. Pa/hol. 2000, 5, 697-704. 104. Roberts. J. C.; Charyulu, R. L.; Zera, R. T.; Nagasawa, H. T. Protection against acetan1inophen hepatotoxicity by ribose-cysteine (RibCys). Pharamcol. Toxicol. 1992, 7(), 281-285. 105. Roberts, J. C.; Francetic, D. J.; Zera, R. T. L-Cysteine prodrug protects against cyclophosphamide urotoxicity without comprolnising therapeutic activity. Cancer Chemother. Pharmacol. 1991. 28, 166-170. 106. Roberts, J. C.; Francetic, D. J.; R. T. Chemoprotection against cyclophosphan1ide-induced urotoxicity: comparison of nine thiol protective agents. Anticancer Res. 1994, J 4, 389-396. 107. Roberts, J. C.; Phaneuf, II. L.; Dominick, P. K.; Wilmore, B. H.; Cassidy, P. B. Biodistribution of r35S]-cysteine and cysteine prodrugs: potential impact on chemoprotection strategies. J Labelled Cpd. Radiopharm. 1999,42,485-495. CHAPTER 2 DEVELOPMENT AND VALIDATION OF A NEW HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE DETERMINATION OF BIOLOGICALLY IMPORTANT THIOLAMINES Introduction The ability to accurately n1easure reduced and oxidized biological thiols is in1portant to understanding the roles these con1pounds play in cellular functions. A variety of HPLC n1ethods are available for the detection and quantitation of thiol and disulfide containing compounds. 12 Many of these methods utilize thiol-specific reagents that attach a chron10phore to the thiol group thereby assisting in separation and detection. The more COlTIlnonly used HPLC lnethods for the determination of thiols and disulfides are based on the formation of fluorescent derivatives of thiols using reagents such as n10nobron10bimane, o-phthalaldehyde, or N-substituted malein1ides. Fluorometric detection provides good sensitivity for thiol meaSUrelTIent but lacks the ability to simultaneously measure thiols and disulfides. 1 Disulfides are detected only as their free thiol forn1 following a separate reduction step. Another commonly used HPLC method in 30 which thiols and disulfides can be measured in the same sample uses electrochemical detection. However, this n1ethod suffers from a severe problem related to the sensitivity of the detector to interference frOln oxidizable contanlinants. I Another widely used HPLC nlethod was developed by Reed and coworkers and depends on trapping free thio] groups with iodoacetic acid followed by the formation of N-dinitrophenylated (DNP) derivatives by reaction with dinitrofluorobenzene (DNFB), which is also known as Sanger's reagent.3A The derivatives are then separated on an ion exchange colunln and observed at 365 nm. This procedure has been successful in the simultaneous n1easurement of glutathione (GSH) and GSH derivatives, as well as other thiols and disulfides, in the same sample. Although UV -VIS detection is not as sensitive as other detection nlethods, the sensitivity of the Reed method has been reported in the nanonl0le range.3 The primary disadvantage of this procedure is the inability to analyze thiol cOlnpounds of neutral charge (i.e., no free carboxyl group). Also, the high salt concentration necessary for the elution of the derivatives is damaging to the HPLC instrumentation, and the colunln itselfbeconles derivatized by Sanger's reagent leading to a rapid reduction of performance over time. This chapter describes a new method for determining concentrations of GSH, and oxidized GSH (GSSG), cysteine, and cystine in a single analysis. In the method described here, both free thiols and amino groups are dinitrophenylated with Sanger's reagent and the derivatives are separated on a CI8 reversed-phase column. This method allows the sinlultaneous determination of both reduced and oxidized thiol compounds. The development and validation of this method is presented. 3 J Results Preliminary studies Initial HPLC studies were performed using the Reed protoco1.3 ,4 Standard solutions of GSH, GSSG, cysteine, cystine, and 2-atnino-3-mercapto-3-methylbutanoic acid (penicillanline) were derivatized and injected onto a 3-aminopropyl-spherisorb colun1n according to the literature procedure.4 The free thiols were carboxyn1ethylated with iodoacetic acid followed by derivatization of amino groups with DNFB under basic conditions to give S-carboxymethylated-N-DNP derivatives of GSH, cysteine, and penicillanline. Cystine, GSSG, and y-glutan1yl-glutmnate (the internal standard), which lack a free thiol moiety, were derivatized with DNFB to give N,N'-di-DNP derivatives. I.Jysine was added to react with excess DNFB, as suggested, to extend the life of the column.4 The elution profile of the above thiolan1ines was the same as the literature with a slnall variation in retention time, which, according to Reed et a1.,4 varies depending on the age of the HPLC colun1n. Subsequent injection of derivatized standards on the 3-aminopropyl-spherisorb colul11n after column storage resulted in no observable peaks, which indicated loss of colun1n function. An attempt was n1ade to recover function by following a colUlnn regeneration protocol (column wash with 0.5 M sodium acetate in 640/0 methanol for 3 h at 1.5 mL/min).4 After regeneration, injection of analytes showed significant decrease in retention tilnes 5 min) compared to initial injections. Changing the gradient conditions to increase retention tin1es was not successful. The column required regeneration after each colUlnn storage and retention tiInes continued to progressively decrease upon regeneration. After < 40 injections, the column was discarded. 32 Due to the observed rapid degeneration of the 3-anlinopropyl-spherisorb column, another cation exchange column was investigated. The thiols, GSH and cysteine, were deriva1ized using conditions described by Reed followed by separation on a quaternary an1ine colun1n. Several variant mobile phase conditions were investigated in order to detennine optimal conditions for elution and separation of the analytes. Initially, 0.05 M phosphate buffer, pH 7.5, and 0.5 M phosphate buffer, pH 7.5, were used for solvent A and solvent B, respectively. The analytes did not elute with various gradients using this lnobile phase and subsequent injections resulted in complete retention of all analytes. Elution of the analytes required 100% solvent B with the addition of 2 M NaCI for several hours. Under these conditions, the derivatives eluted as one broad peak. Other elution profiles investigated with this column included the addition of 20% methanol to solvent B and the solvent system described by Reed.4 None of these protocols were successful in eluting the analytes. The analytes continued to be retained on the column and elution required extensive column washes with 1000/0 solvent B with 2 M NaCl. Experin1entation with this column was discontinued and further HPLC was performed using a C 18 reversed-phase colunln. C 18 reversed-phase column The HPLC method described here is a modification of the Reed method.3A The derivatization chemistry, column, and internal standard have been changed. DNFB was used to derivatize both thiol and mnino groups under basic conditions to give N,S-di-DNP derivatives of cysteine and GSH (Figure 2.1 ).5 NE-1\1ethyl-lysine (the internal standard), GSSG, and cystine, which lack a free thiol moiety, were derivatized with DNFB to give O,N SONO, i\',S-di-DN P-GSI I N,N'-di-DNP-Cystinc N,N'-di-DNP-GSSG N, N'-di-DNP-NE-Mcthyl-Iysine 33 Figure 2.1. DNP derivatives of cysteine, cystine, aSH, ossa, and NE-methyl-lysine. N, N '-di-DNP derivatives (Figure 2.1). A time course analysis revealed that maxinlunl derivatization required a 12 h reaction. Importantly, the quality of the DNFB dictated the efficacy of the derivatization reaction. quality of the DNFB was found to be batch, not vendor, dependent. A C 18 reversed-phase coltunn was used for separation of DNP analytes. Experiments in method development with the C 18 reversed-phase colUlnn included identifying peaks, optimizing gradient conditions, finding a suitable internal 34 standard, and performing n1ethod validation in three different matrices: water, cell lysates and mouse liver hOlnogenates. Analytes were identified by mass spectrometry and cOlnparison to retention times of synthesized standards. Two peaks were identified by n1ass spectron1etry as 2,4-dinitrophenol and 2A-dinitrophenyl ethyl ether, which are by-products of the derivatization reaction (Figure 2.2). Derivatized analytes were eluted with a water/acetonitrile gradient containing 0.1 % TF A. The gradient conditions and tin1e were optimized to allow for baseline separation of the DNP derivatives of cysteine, cystine, GSH, GSSG, and Nc-n1ethyl-lysine. Optin1ization of the gradient included investigating initial solvent conditions and steepness of the gradient. The gradient conditions in the first 20 min were specifically designed to give sufficient retention of N,N '-di-DNP-GSSG. Sub-optimum gradients were particularly poor in their ability to separate N,N '-di-DNP-GSSG from neighboring peaks originating fron1 the derivatization reaction. The overall time for analysis of the analytes exan1ined was 55 n1in. This time included a 10 min colUlnn equilibration. An internal standard was added to monitor the chemical derivatization process in each san1ple. Fariss and Reed4 used y-glutamyl-glutamate as the internal standard, but it was not used here due to the early retention tin1e and broadness of the peak on the C 18 2,4-Dinitrophenol 2,4-Dinitrophenyl ethyl ether Figure 2.2. By-products of the derivatization reaction. 35 reversed-phase column. Penicillan1ine was then chosen as the internal standard as suggested by Reed et al. as an alternative standard.3 However, penicillamine was shown to react with GSSG in a concentration dependent manner to fonn a mixed disulfide that interfered with quantitation (peak identified by Inass spectrometry, mlz 785 M - H). This was the only mixed disulfide observed. One possible mechanisln for the exchange reaction is illustrated in Figure 2.3. Other possible internal standards investigated included ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, and NE-Inethyl-lysine. NE-Methyl-Iysine was chosen since it cannot participate in artifactual thiol-disulfide exchange, is not found in HepG2 celllysates or lnouse liver homogenates, does Figure 1 o (SIIII H02C~N~N NFl H 0 2 m/2 785 (M - H) Possible Inechanism of the disulfide exchange reaction between penicillamine and GSSG. 36 not interfere in the elution of other peaks, and possesses two functional groups that can be chelnically modified by DNFB to allow attachment of two chromophores. HPLC method using the C 18 reversed-phase cO}U111n was validated by several criteria in three different matrices: water, HepG2 celllysates, and mouse liver hOlnogenates. Linearity, intraday and interday variability, recovery, and limits of detection and quantitation were determined as described below. Statistical analysis Linear regression analysis was performed on all standard curves generated. Results were expressed as mean ± standard deviation (SD) and coefficient of variation (CV). Statistical differences were detennined by ANOV A followed by Tukey Kramer 111ultiple comparisons tests. Means were considered significantly different if P < 0.05. Sample preparation During sample preparation, cell and tissue samples were homogenized in 100/0 perchloric acid (PCA) in the presence of the n1etal ion chelator, bathophenanthrolinedisulfonic acid (BPDS), to prevent auto-oxidation of thiols and to n1inin1ize thiol-disulfide exchange. PCA was chosen for sample homogenization in this procedure in order to facilitate subsequent precipitation of the acid as the potassium salt. This prevented the separation of the reaction mixture into aqueous and organic phases. Previously, salicylic acid, which did not precipitate as a salt, was used, and a biphasic reaction l11ixture resulted preventing complete san1ple derivatization. 37 HPLC analysis Typical chrolnatograms in each of the three Inatrices are shown in Appendix A. The retention tinles of the DNP analytes and the major side products are shown in Table 2.1. Retention tinles were reproducible with CY 1 % for all analytes. The retention tinles did not decrease with> 100 injections on the same C 18 column. HPLC method validation Linearity in different matrices. The standard curve equations for .NE-methyl­lysine, GSH/GSSG, and cysteine/cystine in each 111atrix are shown in Table 2.2. The standard curve of lYE-methyl-lysine \vas linear in the concentration range investigated (5- 15 nnl0llmL) in all three matrices with an r2 ~ 1.00. Based on this result, a concentration Table 2.1. Retention times of DNP derivatives and side products. Conlpound Peak Number Retention time (min)a n 2A-Dinitrophenol 21.0 ± 0.2 (10/0) 108 GSSG 2 22.8 0.2 (10/0) 27 GSH 4 25.8 0.2 (10/0) 27 2A-Dinitrophenyl ethyl ether 5 28.4 0.2 (10/0) 108 Cystine 6 30.9 ± 0.2 (10/0) 27 Cysteine 7 32.1 ± 0.2 (10/0) 27 NE-nlethyl-lysine 3 36.9 ± 0.4 (10/0) 99 aYalues are expressed as nlean SD (CY) Table 2.2. Standard curve equations of DNP derivatives in HepG2 lysates, and mouse liver homogenates. Compound Standard curves (n = 3) water In HepG2 cells In mouse liver NE-methyl-lysine y = 5.67 X 10I4X + 3.01 X 104 y=5 1014X 2.03xlO'+ y = 4.58xl0 1'+x + 2.15xl r2 = 1.000 r2 = 0.9987 r2 = 0.9950 GSH y 5.62x 108x + 4.48x 1 0-1 Y 8.09x 1 08x + 1.48x 10-1 y 6.62x108x + 7.97xl r 2 0.9984 r2 = 0.9953 r2 0.9998 GSSG y 5.78x108x - 1.97xl y = 8.78x108x 3.08x10- 1 y 6.92x10ox + 1.803xl r2 0.9956 r 2 1.000 r2 0.9791 Cysteine y = 6.63x 108x + 4.28x 10-2 y = 7.69x108x + 5.81xl0-2 y = 6.14xl08x + 1.40xl r2 = 0.9998 r2 = 0.9986 r2 = 0.9970 Cystine y = 8.73x108x + 1 10-1 Y 9.55x108x + 1.20xl0-1 y 9.86x 100x + 8.89x 1 ") 0.9997 r2 1.000 2 c r 0.9948 w 00 39 of 10 nmolhnL was chosen for the internal standard since this value was within the linear range of detection for NE-methyl-Iysine and is in the range expected for endogenous thiols from tissue and cell culture samples. All standard curves generated for the analytes of interest in all three nlatrices were linear in the concentration range investigated (l0-50 nnl01lmL) with r2;:::; 1.00 (except for GSSG in mouse liver, r2;:::; 0.98). Variability. The intraday variability of the DNP derivatives of GSH, GSSG, cysteine, and cystine was investigated at three concentrations in all three nlatrices. Areas of each analyte \vere normalized to the internal standard and therefore are reported as peak area ratios. Standard curves were made in triplicate on the sanle day to assess variability in peak area ratios and slopes for each analyte. Intraday variability in the peak area ratio for each analyte was reproducible within each matrix with CV :::; 170/0 in all cases except for GSSG in cells and liver at 10 nmol/nlL Crable 2.3). The CV for the intraday variability in the slopes of each analyte within each matrix was:::; 13% (Table 2.4). Tukey Kramer nlltltiple cOlnparisons tests were applied to compare the slopes between each matrix to evaluate if matrix effects \vere present (Table 2.4). The slopes were significantly different between the matrices for GSH (P < 0.05), but not cystine (P > 0.05). For GSSG and cysteine no consistent trend was observed. This variability observed in the slopes between matrices for some of the analytes, but not all, indicates that the lllatrix can affect the derivatization reaction. Therefore, matrix effects need to be considered for the accurate deternlination of thiolamines in various nlatrices. The interday variability of the DNP derivatives of GSH, GSSG, cysteine, and cystine was investigated at three concentrations in water. Standard curves of each analyte werc perfornlcd in triplicatc over 3 days and thc variability in the peak area ratio and Table 2.3. Intraday variation in the peak area ratio of DNP derivatives in water, HepG2 celllysates, and mouse liver honl0genates. Conlpound Peak area ratio (n = 3t nnlol/mL In water In HepG2 cells In mouse liver GSH 10 1.03 ± 0.10 (9%) l.04 ± 0.10 (10%) 0.71 ± 0.1 1 (15°~) 30 2.07 ± 0.02 (1 %) 2.49 ± 0.24 (9%) 1.92 ± 0.13 (7%) 50 3.26 ± 0.10 (3%) 4.33 0.23 (5%) 3.19 ± 0.21 (7%) GSSG 10 0.50 ± 0.02 (3%) 0.60 ± 0.24 (41 %) 0.74 0.23 (31 %) 30 l.74 ± 0.13 (7%) 2.42 ± 0.19 (80/0) 2.41 ± 0.34 (140/0) 50 2.73 ± 0.28 (10%) 4.23 ± 0.34 (8%) 3.41 ± 0.17 (5%) Cysteine 10 0.73 ± 0.02 (2%) 1.09 ± 0.06 (6%) 0.69 ± 0.03 (4%) 30 2.04 ± 0.06 (3%) 2.95 ± 0.18 (6%) l.96 ± 0.30 (15%) 50 3.43 ± 0.17 (5°~) 5.06 ± 0.34 (7%) 3.02 ± 0.03 (1 %) Cystine 10 1.00 ± 0.03 (3%) 1.04 ± 0.08 (80/0) l.11 ± 0.19 (17%) 30 2.85 ± 0.02 (1 %) 2.87 ± 0.19 (7%) 2.88 ± 0.08 (3%) 50 4.59 ± 0.03 (1 %) 4.70 ± 0.07 (1 %) 5.16 ± 0.21 (4%) aYalues are expressed as nlean ± SO (CY). 40 Table 2.4. Intraday variation in the slope of DNP derivatives in water~ HepG2 celllysates, and mouse liver homogenates. Compound Slope (n = 3t Tukey Kramer analysis In water In HepG2 cells In mouse liver P values GSH (5.62 ± 0.44)x108 (8%) (8.09 ± 0.32)x108 (40/0) (6.62 ± 0.33)x108 (5%) Water vs cells P < 0.001 Water vs liver P < 0.05 GSSG Cysteine Cells vs liver P < 0.01 (5.78 ± 0.70)x 108 (12%) (8.78 ± 1.15)x 108 (130/0) (6.92 ± 0.62)x1 08 (9%) Water vs cells P < 0.05 Water vs liver P> 0.05 Cells vs liver P > 0.05 (6.63 ± 0.29)x108 (40/0) (7.69 ± 0.59)x108 (80/0) (6.14 ± 0.02)x108 (0.3%) Water vs cells P < 0.05 Water vs liver P> 0.05 Cells vs liver P < 0.01 Cystine (8.73±0.01)x108 (0.10/0) (9.55 ±0.32)x108 (3%) (9.86 ± 0.82)x108 (8%) Watervscells P>0.05 aYalues are expressed as mean ± SD (CY). Water vs liver P > 0.05 Cells vs liver P > 0.05 ~ 42 slope was assessed. Table 2.5 shows that the interday variability in the peak area ratio for each analyte was reproducible with CV :::; 12%. The interday variability in the slopes was also reproducible with CV ~ 140/0 for each analyte (Table 2.6). Tukey Kramer multiple comparisons tests comparing the interday variability in slopes showed that slopes were the sanle for GSH and GSSG between the 3 days (P 0.05). However~ the slopes for cysteine were different between the 3 days (P < 0.05) and cystine showed inconsistent behavior (Table 2.6). Recovery. Recovery of each analyte was analyzed in duplicate in each matrix. Recovery was defined as the percent of observed analyte compared to the expected value based on the known amount added. For analysis in celllysates and liver homogenates, the expected value is the amount in excess of the endogenous thio!. In water, the recoveries were 95% for GSH, 940/0 for GSSG, 103% for cysteine, and 102% for cystine. In HepG2 cells, the recoveries were 120% for GSH, 119% for cysteine, and 1030/0 for cystine. The recovery of GSSG could not be measured reproducibly in the HepG2 cells due to an unidentified contanlinating peak in certain (not all) samples. This unidentified contanlinant was analyzed by nlass spectrometry to reveal a compound with mlz 803.1. The identity of this contaminant was not further investigated. In mouse livers, the recoveries were 100% for GSH, 92% for GSSG, 1040/0 for cysteine, and 107% for cystine. Limits of detection and quantitation. The limit of detection and quantitation was deternlined in triplicate for GSH and GSSG analytes in water. The linlit of detection and quantitatiol1 for GS11 was 0.01 nn101ln1L and 0.10 nmollmL (CV 13% ), Table 2.5. lnterday variation in the peak area ratio of DNP derivatives in water. Compound Peak area ratio (n = 6)3 nmol/mL In water GSH 10 0.90 ± 0.02 (2%) 30 2.00 ± 0.06 (30/0) 50 3.20 0.15 (50/0) GSSG 10 0.54 ± 0.02 (4%) 30 1.71 ±0.11 (6%) 50 2.81 ± 0.11 (4%) Cysteine 10 0.76 0.09 (12%) 30 2.25 ± 0.22 (100/0) 50 3.74 ± 0.40 (11 %) Cystine 10 0.96 ± 0.07 (70/0) 30 2.60 ± 0.18 (7%) 50 4.20 ± 0.30 (70/0) aYalues are expressed as mean ± SD (CY). 43 Table 2.6. Interday variation in the slope of DNP derivatives in water. Conlpound Slope (n = 6t Tukey Kramer Analysis In water P values GSH (5.85 ± 0.13)xl08 (2%) Day 1 vs day 2 P> 0.05 Day 1 vs day 3 P> 0.05 Day 2 vs day 3 P 0.05 GSSG (5.75 ± 0.20)xl08 (30/0) Day 1 vs day 2 P> 0.05 Day 1 vs day 3 P > 0.05 Day 2 vs day 3 P >0.05 Cysteine (6.00 ± 0.84)x 1 08 (140/0) Day 1 vs day 2 P < 0.01 Day 1 vs day 3 P < 0.05 Day 2 vs day 3 P < 0.01 Cystine (8.33 ± 0.57)xl08 (7%) Day 1 vs day 2 P < 0.05 Day 1 vs day 3 P> 0.05 Day 2 vs day 3 P> 0.05 aYalues are expressed as l11ean SD (CY). 44 45 respectively. The limit of detection and quantitation for GSSG was 0.1 nlTIollnlL and 1 nnl01/mL (CV = 7%), respectively. Discussion Many HPLC methods are available for the detection and quantitation ofthiol and disulfide containing compounds. L2 Separation and detection in several of these methods is achieved by the attachment of a chromophore to the thiol group using thiol specific reagents. Though separation and detection of fluorescent derivatives of thiols is the most widely used HPLC method, other HPLC nlethods including electrochemical detection and UV detection are known. Each of the previously reported methods for detection sufTers froll1 specific inherent problems described above. The protocol described here successfully circumnavigates these problems. A versatile method for the simultaneous measurement of biological thiols and disulfides including GSH, GSSG, cysteine, and cystine is reported. This method is a modification of the techniques described by Mertens et a1. 6 and Reed et a1. The reactivity of DNFB with both thiol and amino groups was exploited to produce N,S-di­DNP derivatives of GSH and cysteine and N,N )-di-DNP derivatives of Ns-methyl-Iysine, GSSG, and cystine.7 These derivatives were separated by HPLC using a C 18 reversed­phase colunln. Because both oxidized and reduced thiols were converted to UV -active derivatives, the SilTIultaneous observation of both the oxidized and reduced forms of these inlportant nletabolites could be investigated. Several benefits in the use of C 18 reversed-phase chromatography for this application have been realized. Reversed-phase columns are c01TImercially available and 46 chen1ically inert to the derivatization conditions. ColUlnns containing a free amine, as previously employed, are susceptible to chemical n10dification by derivatization reagents, DNFB in the case described here, which decreases column life. Furthern10re, the buffers for elution in the protocol described here contain no salts, thereby preventing unnecessary wear on the HPLC systen1. Many of the published HPLC methods do not utilize an internal standard. It is important to monitor an HPLC system during use as changes can occur that cannot be readily detected without a proper internal control. The internal standard used here, Nf.­methyl- lysine, served to n10nitor both the derivatization reaction in each sample preparation and the properties of the HPLC systeln including injection volume, column perforn1ance, and detector response. Peak areas were normalized to an internal standard. An in1portant observation made was the unexpected reaction of penicillamine with GSSG to give a mixed disulfide. This highlights the importance of careful selection of an internal standard for analyses of redox active metabolites. Surprisingly there are no reports of this type of thiol-exchange reaction in the literature. Perhaps, previous results utilizing penicillatnine as an internal standard are erroneous. To validate the n1ethod, linearity, variability, recovery, and limits of detection and quantitation of the HPLC system was examined. A linear response was shown over the concentration range investigated for all of the analytes. The intraday variability in the peak area ratio in all matrices was reproducible for each analyte except for GSSG. In HepG2 cells and n10use livers, GSSG gave a high CV for peak area ratios (41 o/r) and 31 %, respectively). One possible explanation for this result in the HepG2 cells was the presence of an unidentified contan1inating peak, which eluted at a similar retention time. 47 The source of this contaminant has yet to be defined, but is not a facet of the derivatization reaction or sample preparation~ since it was only observed occasionally and only with HepG2 celllysates and never with mouse liver hon10genates. The interday variability in the peak area ratio for each analyte in water was also low and reproducible. The presence of the contmninating eluent in HepG2 cells affected the ability to assess percent recovery of GSSG in HepG2 cells. Percent recoveries of the other analytes were comparable to established protocols.6 The limit of quantitation for GSH and GSSG was tested in water and found to be 0.01 nmol/n1L and 0.1 nlnolimL, respectively. By comparison, the Inethod of Reed allows detection in the nn101/mL range.3A Similarly, Mertens et a1. reported detection between 0.5-1.0 nmol/mL in cultured rat hepatocytes.6 The method reported here appears to have lower lilnits of detection and quantitation. This is expected since two chron10phores are added per analyte. By cOlnparison, protocols described by Reed3A and Mertens6 rely on the incorporation of a single chron10phore per molecule for the reduced thiols. Tukey Kran1er analyses of the slopes showed that in some cases an effect of the matrix on the standard curves of the analytes was observed. Also, the standard curves for some analytes varied to a small extent on a day-to-day basis. These inconsistencies in the results indicate the necessity to produce standard curves for each n1etabolite in the relevant n1atrix and on the day of analysis for the most accurate results. Conclusions This chapter reports the investigation and validation of a new and versatile method for the detern1ination of ilnportant biological thiols. Important advances by 48 comparison to previous tnethods include the ability to simultaneously determine both reduced and oxidized thiols and the mild elution conditions on a commercially available HPLe colunln. Although not presented in this chapter, another iluportant advance of this HPLC nlethod in cOlnparison to Reed is the ability to detect thiolmnines that do not contain a carboxylate group. The luethod described by Reed3 ,4 is based on anion exchange chroluatography and this requires every analyte to contain a charged group, i.e. carboxylate group. Conlpounds lacking a negatively charged functional group cannot be resolved with the Reed method, but can be analyzed with the method described here. This technique will be useful for the routine determination of numerous thiolamines in a variety of biological systems. Experimental Chemicals and materials GSH, GSSG, L-cysteine, L-cystine, NE-methyl-L-Iysine hydrochloride, L­ornithine, D-penicillamine, 2,3-diatninopropionic acid, 2,4-diaminobutyric acid, sodiunl chloride (NaCl), BPDS, trit1uoroacetic acid (TFA), 700/0 PCA, DNFB, Eagle minimum essential mediunl (EMEM), antibiotic/antilnycotic solution (10,000 units penicillin, 10 mg streptomycin, ~lg amphotericin B; 100X), Hank's balanced salt solution (without or Mg2+), phosphate buffered saline (PBS; 10 lnM phosphate, 138 mM NaCl, lnM KCI, pH 7.4), trypsin (1 :250), and ethylenedianline tetraacetic acid (EDT A) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Fetal bovine serunl (Fetal Clone I) was purchased fronl Hyclone Laboratories (Logan, UT). HPLC grade acetonitrile, ethanol, potassium hydroxide (KOH), and potassium bicarbonate (KHC03) were purchased from Fisher Scientific (Fair Lawn, NJ). Water was purified with an E­pure Barnstead purifier (Fisher Scientific). All chemicals purchased were of analytical grade and were used without further purification. 49 WhatInan PVDF syringe filters, sterile syringes, pipets, centrifuge tubes, and tissue culture flasks were purchased from Fisher Scientific. Male Swiss Webster mice were purchased from Charles River Laboratories (Wihnington, MA). HepG2 cells were purchased froln American Type Culture Collection (A TCC HB-8065, Manassas, V A). Sterile lnanipulations were conducted in a Forma Scientific Class II Type AB3 Model 1186 biological safety cabinet (Marietta, OH). Cell number was determined using a Coulter counter (Coulter Corporation, Miami, FL). Cells were grown at 37°C in a hunlidified 5% CO2 atmosphere in an Ultra-Tech Model WJ 301 D incubator (Fisher Scientific ). F ABMS and ElMS data were collected from a Finnegan MAT 95 mass spectron1eter (San Jose, CA). ESIMS data was collected from a Micromass Quattro II Triple Quadrupole mass spectrOlneter (Beverly, MA). Mass spectral analyses were obtained at the Departlnent of Chemistry at the University of Utah. HPLC instrumentation and conditions Samples were separated on a Rainin Dynamax 5 ~lln, 4.6 x 250 mIn C 18 colun1n fitted with a C 18 guard column (Rainin, Emeryville, CA). The chrolnatographic system consisted of a Hitachi Model L-6200A pump equipped with a 4250 UV -VIS detector and an AS-2000 autosanlpler with a Rheodyne injection valve and a 100 I-lL sample loop 50 (Hitachi, San Jose, CA). Hitachi Model D6000 version 2, revision 06 software was used to control systenl operation and facilitate data collection. Sanlples were eluted with a mobile phase consisting of solvent A (acetonitrile/O.l % TFA) and solvent B (water/0.1 % TF A). The elution profile was as follows: 0-5 nlin, 20% A 800/0 B isocratic; 5-20 min, 500/0 A 500/0 B linear gradient; 20-34 111in, 500/0 A 500/0 B isocratic; 34-37 min, 100% A linear gradient; 37-40 min, 100% A isocratic; 40-45 min, 200/0 A 80% B linear gradient; 45-55 min, 200/0 A 800/0 B isocratic (column equilibration). All HPLC solvents were filtered through a 0.45 ~Lln nylon filter. Analyses of 100 /J.L of sanlple were performed at a flow rate of 1.0 mL/min at ambient tenlperature vvith UV -VIS detection at 365 nn1. Sample preparation Cell culture. Tissue culture flasks (75 cm2 ) containing 25 mL of EMEM (supplelnented with 100/0 fetal bovine serum and 1 % antibiotic/antimycotic) were seeded with 6 106 HepG2 cells. The flasks were incubated at 37 DC in a humidified 50/0 CO2 atmosphere for 7 days (I11edium was replaced every 3 days), at which time cells were confluent. Mediunl was renl0ved and the cells were rinsed with Hank's balanced salt solution. Cells were trypsinized with 5 InL oftrypsiniEDT A for 2 min at 37°C followed by further incubation in the absence of trypsin for 11 nlin at 37°C. The trypsin was quenched with 5 InL of EMEM. Cell clun1ps were broken up by striking the flask sharply against the palm of the hand 10 tilnes followed by passage of the cell suspension four times through a gauge needle. Cell suspensions were transferred to 50 mL centrifuge tubes and cell nmnber was determined. The cell suspension was centrifuged at 5 J 3400 g for 5 min~ and the pellet was suspended in 5 mL of PBS and centrifuged again at 3400 g for 5 n1in. The pellet was resuspended in 2.14 mL of 0.9% NaClIl mM BPDS and 0.36 n1L of 700/0 PCA was added. Samples were sonicated in a sonic bath for 5 min followed by centrifugation at 3400 g for 5 min. Supernatants were transferred to fresh centrifuge tubes and stored at -70°C until analysis. Tissue samples. Sanlples of male Swiss Webster murine liver tissue (50-100 mg wet weight) were flash frozen in liquid nitrogen and stored at -70°C. At the tin1e of assay ~ frozen smnples were homogenized in 3 IT1L of 10% w/v PCAIl mM BPDS using a tissue hon10genizer and centrifuged at 1400 g for 10 Inin at 4 °C. Stock solutions Stock solutions were prepared for standard curves as follows: 0.48 nlg/nlL of NE­Inethyl- lysine (diluted 1 :5), 0.15 Ing/nlL of cysteine, 0.29 mg/nlL of cystine, 0.37 IngitY1L ofOSH, and 0.74 mg/nlL of OS SO. All stock solutions were prepared in \vater except for cystine, which was prepared in 100/0 PCAII n1M BPDS due to insolubility in \vater. A separate stock solution of the internal standard, NE-nlethyl-Iysine, was prepared at a concentration of 0.05 mg/mL in water. All stock solutions were freshly prepared on the day of analysis. Standard curves Triplicate standard curves of lVE-nlethyl-lysine, OSH/OSSO, and cysteine/cystine were generated in three Inatrices: water (10% PCAII mM BPDS), Hep02 celllysates, and nlouse liver honlogenates. Standard curves were prepared by taking a 0.5 nlL aliquot 52 of san1ple n1atrix and adding varying an10unts (20-100 J.lL) of the stock solutions of NE-n1ethyl- lysine, GSH/GSSG, and cysteine/cystine followed by 0.48 mL of 2 M KOH-2.4 M KHC03 and] n1L of 1 % DNFB in ethanol. The standard curves of GSH/GSSG and cysteine/cystine were spiked with 0.1 lnL of the internal standard, iVE-methyl-Iysine. Samples were derivatized overnight at room temperature in the dark. Prior to injection, salnples were acidified with 0.15 mL 700/0 PCA, clarified by centrifugation at 5600 g for 1 n1in, and filtered through a 0.45 f.lm PVDF syringe filter. Standard curves were generated by plotting the ratio of the peak area of analyte to internal standard versus analyte concentration. The standard curve of NE-nlethyl-lysine \vas generated by plotting the peak area versus analyte concentration. Intraday and interday variation For intraday variation, both the slope and the ratio of the peak area of analyte to internal standard were compared fron1 the triplicate standard curves in all three matrices prepared above. For interday variation, duplicate standard curves of GSH/GSSG and cysteine/cystine were generated for 3 consecutive days at the same tinle each day. Standard curves were prepared in water as described above. Interday variability of both the slopes and the ratio of peak area of analyte to internal standard from the standard curves was analyzed. Recovery Duplicate standard curves of GSH, GSSG, cysteine, and cystine were prepared individually in water as described above. The three sanlple matrices were spiked 53 separately with a known amount of GSH, GSSG, cysteine, and cystine. Blank samples were also prepared in the sample lnatrices by adding equivalent amounts of water. Recovery was defined as the percent of observed analyte compared to the expected value based on the known amount added. For analysis in celllysates and liver homogenates, the expected value is the aITIount in excess of the endogenous thiol. Limit of detection and quantitation Triplicate standard curves of GSH and GSSG were prepared individually in water as described above. Concentrations of GSH and GSSG for the standard curves were 0.01, 0.1, 1, and 10 nmol/mL. The limit of detection was defined as the lo\vest analyte concentration resulting in an observable peak. The limit of quantitation was defined as the analyte concentration resulting in the lowest measurable peak height with acceptable precision (CV :s; 150/0). Synthesis of DNP derivatives and mass spectral analysis N,S-di-DNP-GSH, N,N '-di-DNP-GSSG, N,S-di-DNP-cysteine and N,N'-di-DNP­cystine were prepared by known procedures.3j ,7-9 The follo\ving compounds were collected fron1 an HPLC run and identified by mass spectrometry: 2,4-dinitrophenol LRMS (EI 80 e V) m/z 184 (M+); 2A-dinitrophenyl ethyl ether LRMS (F AB) m/z 212 (M+); N,S-di-DNP-GSH LRMS (FAB) m/z 638 (M H); N,N'-di-DNP-GSSG LRMS (FAB) m/z 943 (M - H)~ and N,N'-di-DNP-NE-n1ethyl-lysine LRMS (ES) m/z 493 (M + H). 54 References 1. Fahey, R. C. Methods for deternlination of glutathione and derivatives using high­perfornlance liquid chromatography. In Glutathione: Chemical, Biochemical and JvJedical Aspects Part A; Dolphin, D., Poulson R., Avranlovic, O. Eds.; John Wiley & Sons: New York, 1989; pp 303-337. 2. Shin1ada, K.; Mitamura, K. Derivatization of thiol-containing compounds. J Chromatogr. 13 1994, 659, 227-241. 3. Reed, D . .1.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Ellis W. W.; Potter, D. W. High-perforn1ance liquid chromatography analysis of nanOlllole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 1980, 106, 55-62. 4. Fariss, M. W.; Reed, D. J. High-performance liquid chromatography ofthiols and disulfides: dinitrophenol derivatives. Methods Enzymol. 1987, 143, 101-109. 5. Porter, R. R.; Sanger, F. The free amino groups ofhaemoglobins. Biochem. J 1948, ,/,2,287-294. 6. Mertens, K.; Rogiers, V.; Sonck, W.; Vercruysse, A. Measurement of reduced and oxidized glutathione in cultures of adult rat hepatocytes. J Chromatogr. 13 1991, 565, 149-157. 7. Sokolovsky, M.; Sadeh T.; Patchornik, A. Nonenzymatic cleavages of peptide chains at the cysteine and serine residues through their conversion to dehydroalanine (DHAL). II. The specific chen1ical cleavage of cysteinyl peptides. JAm. Chem. Soc. 1964, 86, 1212-1217. 8. Levy A. L.; Chung, D. A simplified procedure for the synthesis of 2,4-dintrophenyl (DNP)-anlino acids. JAm. Chem. Soc. 1955, 77, 2899-2900. 9. Vinson J. A.; Pepper, D. Preparation of 2,4-dintrophenyl derivatives of amino acids in dipolar aprotic solvents. Anal. Chim. Acta 1972, 58, 245-247. CHAPTER 3 SYNTHESIS OF NOVEL THIAZOLIDINE PRODRUGS OF L-CYSTEINE Introduction There are several therapeutic approaches for replenishing glutathione (GSH) after its loss from toxic insult. l The most obvious approach is the direct replacement of GSH. However, GSH has poor bioavailability denlanding alternative methods? One such nlethod is the delivery of cysteine, the limiting reagent in the biosynthesis of GSH. However, cysteine oxidizes easily to the insoluble cystine3 and is reported to be toxic. 4 - 7 Prodrugs of cysteine have therefore been investigated as cysteine delivery agents.8 These prodrugs nlask the sulfhydryl group of cysteine to increase oxidative stability and facilitate cellular uptake. Several prodrug approaches for the delivery of cysteine have been successful. Currently, the most common clinically used compound for the delivery of cysteine is N­acetylcysteine (NAC), which delivers cysteine after metabolism by an N-deacetylase.9 Therc are several drawbacks ofNAC, as discussed in Chapter 1, that make researching alternative cysteine prodrugs an important endeavor. A promising alternative approach was reported by Nagasawa et al. and involves masking cysteine as a prodrug in a 56 t hI· azo I1' d 'm e n'n.g structure1.0' I] Th'I S c h apter d ocun1ents t I1 e syntheSI.S and chelnical characterization of several novel cysteine thiazolidine prodrugs. Rationale The thiazolidine prodrugs described here are designed as cysteine delivery agents for GSH biosynthesis during conditions of GSH depletion. The prodrugs are lnodeled after two known thiazolidine carboxylic acids, 2(R,S)-D-ribo-l ',2',3',4'- tetrahydroxybutylthiazolidine-4(R)-carboxylic acid (RibCys) and 2(R,S)-D-gluco- 1 ' ,2',3',4',5' -pentahydroxypentylthiazolidine-4(R)-carboxylic acid (GlcCys), which have been extensively studied as protective agents designed as cysteine prodrugs. 12 - 15 RibCys and GlcCys are products of the condensation between L-cysteine and the aldose sugars, D-ribose and D-glucose, respectively. 12, 13 'The tethering of a sugar moiety to the cysteine prodrugs increases the hydrophilicity of RibCys and GlcCys cOlnpared to other lhiazolidines. The hydrophilic nature of these compounds leads to rapid urinary excretion as suggested by biodistribution studies using eSS]-RibCys and eSS]-GlcCys in the presence and absence of acetatninophen (APAP).] 6 Although the hydrophilic nature of RibCys may be beneticial in cases such as APAP-induced nephrotoxicity, 17 decreasing excretion to Inaintain RibCys and GlcCys concentrations in inflicted tissues should afford greater protection in certain cases, especially in cases of hepatotoxicity. To exan1ine the possibility that decreasing the water solubility of the cysteine prodrugs will increase their ability to deliver cysteine, analogs of RibCys and GlcCys with increased lipophilicity were investigated. 57 This chapter documents the synthesis and chemical properties of the carboxylic acid ethyl esters and carboxanlides of both RibCys and G1cCys (Figure 3.1). These new C0111pounds should increase the lipophilicity of the cysteine delivery agents by masking the charged carboxylate group with an uncharged ester and anlide functionality, respectively. Cysteine release should be dependent on common cellular esterases or a111idases 18 making the compounds nlore stable and lipophilic than other thiazolidine prodrugs. As mentioned in Chapter 1, two possible Dlechanisms exist for the hydrolysis of the thiazolidine ester or amide (Figure 3.2). RibCys o ~R S NH LOf-! f-OH LOH LOH R OH RibCys ethyl ester RibCys amide o ~R S NH OH HO G1cCys GlcCys ethyl ester GlcCys amide OH OH OH R OH Figure 3.1. Structures of thiazolidine prodrugs of cysteine. 58 + sugar Esterase or am idase Esterase or amidase o r-10H HS NH2 + sugar RI ~OH R2 -OCH2CH3 ~OH Oli or H01 or OH OH OH OH -NH2 OH ribose glucose Figure 3.2. Possible mechanisms of cysteine release from the thiazolidine ethyl ester and amide. 59 Results and discussion Ethyl esters 2(R,S)-o-ribo-l' ,2',3',4' -Tetrahydroxybutylthiazolidine-4(R)-carboxylic acid ethyl ester (RibCys ethyl ester) and 2(R,S)-o-gluco-l ',2',3',4' -pentahydroxy-pentylthiazolidine- 4(R)-carboxylic acid ethyl ester (GlcCys ethyl ester) were synthesized by the condensation of L-cysteine ethyl ester and o-ribose and o-glucose, respectively, in the presence of potassiUln acetate (Figure 3.3). Both RibCys ethyl ester and GlyCys ethyl ester were readily purified by crystallization frOln ethanol and were produced in good yields, 83% for RibCys ethyl ester and 88% for GlcCys ethyl ester. NMR data of RibCys ethyl ester and GlcCys ethyl ester revealed the characteristic downfield diastereomeric C2 protons providing evidence for the forn1ation of the thiazolidine ring. As indicated in Figure 3.3, two epimers at C-2 can form. Both epimers of RibCys ethyl ester were observed and were produced in a 5:2 ratio based on integration of the signals for the C2 and both C5 protons for each diastereomer, which are well resolved in the lH NMR spectrum of RibCys ethyl ester. The corresponding signal for the C2 proton o r ~OCH,CH' S NH II~OH 011 110 1', -OH OH GlcCys ethyl ester, 88% glucose KOAc ribose KOAc Cysteine ethyl ester o ,---/OCH,CfI) S NH H OH OH OH Oll RibCys ethyl ester, 83% Figure 3.3. Synthesis of GlcCys ethyl ester and RibCys ethyl ester. 60 in the GlcCys ethyl ester IH NMR spectrum was hidden underneath the water peak, but the C5 signals showed that GlcCys also formed in a 5:2 ratio of diastereomers. Radomski and Temeriusz have shown that, in thiazolidine compounds, the C-2(Sj proton is shifted further downfield than the C-2(R) proton for mixtures of 2(S),4(R) and 2(R),4(R) epimers. 19 Inference based on the literature therefore indicates that RibCys ethyl ester fornled predominantly the C-2(S) epimer. Comparison of the GlcCys and RibCys ethyl ester II-I NMR spectra, in particular the magnitude of the signals for each proton at C5, indicate that GlcCys ethyl ester was also formed predon1inantly as the C-2(S) epimer. Carboxamides The syntheses of 2(R,S)-o-ribo-l ',2',3',4' -tetrahydroxybutylthiazolidine-4(R)- carboxaIllide (RibCys amide) and 2(R,S)-o-gluco-l ',2'J\4',5'-pentahydroxypentyl-thiazolidine- 4(R)-carboxamide (GlcCys amide) required the synthesis of the parent thiol, cysteineamide. Amidation was initially attempted by anln10nolysis of cysteine ethyl ester,20 which resulted in unrecoverable product. This probably resulted from the unstable nature of the free base of cysteineamide, which is known to decompose slowly at 0 °C?O An1idation of N-Boc-L-cystine using N,N '-dicyclohexylcarbodiimide (DCC) or ethyl chloroformate and anlmonium hydroxide was then attempted. Because the use of DCC or ethyl chloroforn1ate required that both the thiol and amino groups be protected, N-Boc-L-cystine was chosen since the thiol is protected in its oxidized form. The cysteineaInide product could then be isolated by deprotection of the amino group followed by reduction of the disulfide. Unfortunately, deprotection of the amino group 61 resulted in products difficult to isolate in pure enough form to proceed to the reduction step. Successful anlidation was finally achieved by the initial treatnlent of S-benzyl-N-Boc- L-cysteine with ethyl chlorofornlate followed by addition of ammonium hydroxide (Figure 3.4).21 S-Benzyl-N-Boc-L-cysteineanlide was synthesized in 75% yield. III NMR of S-benzyl-N-Boc-L-cysteineanlide revealed broad singlets at 6.30 ppm and 5.66 ppm, indicative of the amide functionality. In this synthesis, optinlal product yield was dependent on the purity of solvents and reagents and the exclusion of moisture. Deprotection of the amino group in S-benzyl-N-Boc-L-cysteineamide was performed with 3 M HCl-ethyl acetate and resulted in 75% yield. 22 Prior to debenzylation of the thiol group, S-benzyl-L-cysteine was washed with acetone, which renloved any impurities, and was dried in a drying thimble under hexanes. Debenzylation was perfornled with freshly distilled liquid NH3 and Na metal.23 This reaction was performed under an inert ~NH'J'-- C6HsCH2S ~-Oy \ o 1. 3 M HCI-EtOAc 2. liquid NHlNa '1 cysteineamide Figure 3.4. Synthesis of cysteineamide. 62 atlTIosphere with careful exclusion of moisture. Excess reactant was quenched with DOWEX SOW x 8-400 (NH4 + form) following the procedure of Adang et al.,24 which exchanged the Na + with NH4 +. This quenching procedure was an improvement over the use ofNH4CI, which created a purification problem due to the excess salts that formed with NH4Cl. After the NH3 was evaporated under a stream of argon, the reaction was dried in vacuo. The product was then washed fron1 the DOWEX by vacuum filtration. Cysteineamide was isolated as the HCI salt and the yield was consistently> 1 000/0 suggesting the contamination by a salt despite the care taken to prevent excess salt with the use of DOWEX. The salt was relTIoved and the product purified by column chron1atography on a DOWEX SOW x 8-400 (H+ forn1) column. Elution was performed with a HC} step gradient, and cysteinean1ide was found to elute with 2 M HCl. Cysteineamide was routinely synthesized in 70-800/0 yield. Importantly, the cysteineamide produced here was the reduced forn1 as indicated by elemental analysis, F ABMS ( m/z 121.1 for M + H) and by the reaction with lV-acetyl-2,6-dimethyl-p­benzoquinoneimine (2,6-diMeNAPQI), as described below. RibCys amide and GlcCys amide were synthesized by the condensation of cysteineamide and D-ribose and D-glucose, respectively (Figure 3.S). Several bases were investigated to catalyze the condensation of cysteineamide with the linear sugars. Of these, only potassium acetate was productive. RibCys amide was obtained by crystallization from ethanol to yield 8S% product. GlcCys amide did not crystallize successfully frOll1 ethanol and was purified by trituration with ethanol and subsequent isolation by repeated washes with ethanol and centrifugation. This purification resulted in a lower yield (3S%) than that found for RibCys amide. Both RibCys amide and o ~NH2 S NH H HO OH OH OH OH GIcCys amide, 35% glucose ribose KOAc KOAc Cysteineamide RibCys amide, 85% Figure 3.5. Synthesis of RibCys amide and GlcCys amide. GlcCys amide commonly contained inlpurities of potassium acetate and ethanol as indicated by NMR data. These ilnpurities were difficult to remove even after extensive purification procedures including recrystallization. tIl NMR of RibCys amide revealed the characteristic downfield diastereonleric C2 protons providing evidence for the formation of the thiazolidine ring. The diastereomers were formed in a 5:2 ratio for RibCys atnide based on integration of the C2 proton signal in a I H NMR spectrum. The C5 protons for G1cCys amide were used to deternline that this compound was also 63 produced in a 5:2 ratio of disastereomers. By comparison to literature as described above for RibCys ethyl ester, RibCys amide was predominately the C2(S) epinler. Interestingly RibCys ethyl ester and RibCys mnide were produced in a 5:2 ratio of diastereomers in contrast to the case of RibCys for which the C2(S) epilner is formed with a 5:4 preference. In the case of G1cCys, the ratio of diastereomers increases from 1 :] for the acid to 5:2 for the amide and ester derivatives. Evidence in the literature suggests that solvent conditions can int1uence the outcome of thiazolidine ring formation and that thiazolidine rings can epimerize at C2 through a ring opening mechanism. 19 Therefore, 64 the results for the amides and esters indicate that the carboxylate group inf1uences the stereochenlical outcome of ring closure in thiazolidine fornlation or the thernl0dynamic equilibrium of the two epinlers. Chemical stability of the thiazolidine ring of the ester and amide prod rugs compared to RibCys and GlyCys The ability of the new thiazolidine compounds to release L-cysteine in solution was determined by monitoring their ability to form thiol conjugation products with 2,6- diMeNAPQl. In this assay, the stability tested is that of the thiazolidine ring. Compounds containing a free thiol are known to react with 2,6-diMeN APQ I via nucleophilic addition. 25 2,6-DiMeNAPQI has a diagnostic UV Amax at 255 nm. When thiol cornpounds react with the electrophile, 2,6-diMeNAPQI, a new signal at 296 mn is observed (Figure 3.6).25 The ability of the prodrugs to produce the 296 nm species from 2,6-diMeNAPQI is therefore a simple test of the ability to form a free thiol. Compounds were incubated with 2,6-diMeNAPQI at 37°C and pH 7.0. Table 3.1 shows the results fron1 this experiment. First, the reactions between 2,6-diMeNAPQI and con1pounds o N"~ V o Amax = 255 2,6-DiMeNAPQI RSH OH \mlx 296 Figure 3.6. Reaction of 2,6-diMeNAPQI with a free thiol. Table 3.1. Results from the incubation of thiol containing compounds with 2,6- diMeNAPQI. Conlpound Reactivity with 2,6-diMeNAPQI Cysteine Rapid fornlation of 296 nm species RibCys Rapid fornlation of 296 nin species GlcCys Rapid formation of296 nm species Cysteine ethyl ester Rapid fornlation of 296 nm species RibCys ethyl ester No change GlcCys ethyl ester No change Cysteineamide Rapid formation of 296 nnl species RibCys amide No change GlcCys anlide No change NAC Rapid formation of296 nm species OTC No change 65 containing a free thiol were monitored. Cysteine, cysteine ethyl ester, cysteineamide, and NAC reacted with 2,6-diMeNAPQI in less than 30 s to fonn the conjugation product observed at 296 nm. To control for reaction with a trapped thiol, 2-oxothiazolidine carboxylic acid (OTC), which lacks a free thio!, was incubated with 2,6-diMeNAPQI. No reaction occurred after 10 111in. This is not surprising since OTC is known to require enzymatic activation to form a free thiol. Addition of cysteine to an incubation of OTC and 2,6-diMeNAPQI quickly resulted in the formation of the 296 nm species indicating 66 that the presence of OTC did not inhibit the conjugation reaction. To test the stability of the cysteine prodrugs, in particular their ability to release a free thiol, compounds were incubated with 2,6-diMeNAPQI. Notably, only RibCys and GlcCys formed the 296 nm species, whereas the amide and ester prodrugs did not. The lack of reactivity with 2,6-diMeNAPQI suggests that esterase and amidase activity will be required in vivo for these amide and ester prodrugs to undergo ring opening. This also suggests that the thiazolidine esters and mnides do not undergo epill1erization in solution as do RibCys and GlcCys, which contain a free carboxylate. This fact was suggested by polarimetry studies with RibCys and GlcCys ethyl esters. In these experilnents, the optical rotation of a solution of RibCys or GlcCys ethyl ester did not change over tilne. By comparison, RibCys and GlcCys underwent mutarotation, indicative of rapid epimerization. 19 Ring opening cannot be ruled out for the esters and anlides and it may be that ring closure is faster than hydrolysis to produce the aldose sugar. It is tenlpting to speculate that the free carboxylate plays a role in ring opening at prJ 7. For exanlple, the free carboxylate could act anchimerically as a base to promote . . nng opemng. Conclusions This chapter describes the synthesis of new thiazolidine prodrugs of L-cysteine. These thiazolidines were designed to act as "pro-prodrugs." These novel compounds could decompose intracellularly to produce first cysteinemnide or cysteine ethyl ester. or the carboxylic capping functionality could be cleaved to give RibCys or GlyCys, depending on the prodrug. The order of ring opening and ester or amide cleavage of the 67 prodrugs is not known, but the UV assay of chemical reactivity indicates that the thiazolidine rings were stable in the assay conditions since no thiol conjugation was observed. This suggests that, in vivo, anlidases and esterases will have to catalyze hydrolysis of the amide or ester functionalities before ring opening can occur. This may serve to increase the lifetime of the produgs. The ethyl ester and amide prodrugs are designed to increase lipophilicity in cOlnparison to the parent compounds, RibCys and GlcCys. Increased lipophilicity should assist in more rapid transport across the cell menlbrane and decrease rapid urinary excretion, thereby affording better drug efficacy. This hypothesis is tested in the following chapter. Experimental General methods Melting points were obtained on a Laboratory Devices USA Mel-Temp II melting point apparatus in open capillary tubes and are uncorrected. Nuclear magnetic resonance spectra (NMR) were obtained on Bruker 200 MHz or Varian Unity 400 or 500 MHz FTNMR spectrometers, as indicated in the text. Chemical shifts for J H NMR are referenced to the residual proton in the NMR solvent (CDCh 7.26 ppm, DMSO-d6 2.50 ppm, D20 4.67 ppIn). Chelnical shifts for J3C NMR are referenced to an external reference with DSS and D20 to 0 ppm. Mass spectral analyses were carried out on a Finnegan MAT 95 through the Departnlent of Chemistry at the University of Utah, with accurate mass nleasurelnent acquired using peak matching techniques. UV spectra were obtained on a Hewlett Packard 8452A Diode Array Spectrophotometer. IR spectra were 68 obtained on a JASCO FT/IR-420. Optical rotations were obtained on a JASCO OIP-370 digital polarimeter. IH, l3C, and 20 IH_1H COSY spectra are provided in Appendix B. The 20 spectra were used to assign the IH NMR spectra of the novel thiazolidine compounds. Chemicals o-Ribose, o-glucose, L-cysteine, L-cysteine ethyl ester hydrochloride, S-benzyl­N- Boc-L-cysteine, OTC, NAC, ethyl chloroformate, and OOWEX 50W x 8-400 (l-t form) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Ethanol, methanol, pyridine, potassium acetate, ethyl acetate, triethylamine, tetrahydrofuran (THF), lnethylene chloride (CH2Cb), n1agnesiun1 sulfate (MgS04), concentrated ammoniUln hydroxide, concentrated hydrochloric acid, and NH3 were purchased fron1 Fisher Scientific (Fair Lawn, NJ). Water was purified with an E-pure Barnstead purifier (Fisher Scientific). 2,6-0iMeNAPQI was synthesized by Dr. Pamela Cassidy and Robyn James. 2(R,S)-o-riho-l ',2',3' ,4' -Tetrahydroxybutylthiazolidine- 4(R)-carboxylic acid (RibCys) RibCys was prepared by a modification of a previously reported synthesis. 12.l3 0- Ribose (6.03 g, 0.04 mol) was dissolved in degassed H20 (20 mL) under a N2 atmosphere. To this solution was added L-cysteine (4.83 g, 0.04 Inol). After the solution was stirred for 24 h, the reaction mixture was filtered. Ethanol (100 n1L) was added to the filtrate until the solution turned cloudy. The solution was heated to redissolve the 69 precipitate, allowed to cool to room temperature, and crystallized at 4 °C overnight. The white precipitate was collected by vacuun1 filtration and dried in vacuo to yield a white solid (6.22 g, 620/0): [U]20[) = -118 (c = 0.5, I-hO); mp 136-139 °C (lit. 12 nlp 149-151 °C); IH NMR (D20, 200 MHz, ppm, 5:4 mixture of diastereomers) diastereon1er A: 5.09 (d, .1 3.0 Hz, 1H, C2-H), 4.45 (t, .1= 6.1 Hz, 1H, C4-H), 4.] 3-4.04 (n1, 1H, C1 '-H), 3.73-3.47 (m, 41-1, C2' -H, C3' -H, and C4' -Cfh), 3.35-3.22 (m, 2H, C5-CH2), diastereomer 8: 4.95 (d, .1 4.4 Hz, 1 H, C2-H), 4.29 (t, .1 6.8 Hz, 1 H, C4-H), 4.13-4.04 (n1, 1 H, C l' -H), 3.73-3.47 (m, 4H, C2'-H, C3'-H, and C4'-CH2), 3.35-3.22 (m, 2H, C5-CH2)' 2(R,S)-o-gluco-l',2',3' ,4' ,5'-Pentahydroxypentylthiazolidine- 4(R)-carboxylic acid (GlcCys) GlcCys was prepared by a modification of a previously reported synthesis. 12,13 D­Glucose (10.80 g, 0.06 mol) was dissolved in hot methanol (600 n1L). To this solution were added L-cysteine (4.84 g, 0.04 mol) and pyridine (4 mL, 0.05 mol). The reaction l11ixture was refluxed for 4 h during which time a white precipitate formed. The reaction mixture was cooled to room temperature and refrigerated at 4 °C overnight. The white precipitate was collected by vaCUUl11 filtration and dried in vacuo to yield a white solid (9.70 g, 860/0): [ufO!) -108 (c 0.5, H20); mp 168-171 °C (lit. 12 mp 165°C); IHNMR (D20, 200 MHz, ppnl, 1: 1 n1ixture of diastereon1ers) diastereon1er A: 5.00 (d, .1 3.5 Hz, IH, C2-H), 4.46 (t, .1= 5.5 Hz, IH, C4-H), 4.13 (dd, .1= 6.4 Hz, 3.6 Hz, 1H, C1 '-H), 3.80-3.21 (n1, 7H, C2'-H, C3'-H, C4'-H, C5'-Cfh, and C5-CH2), diastereomer 8: 4.88 (d, .1 6.0 Hz, IH, C2-H), 4.35 (t, .1= 7.1 Hz, 1H, C4-H), 4.03 (dd, .1= 5.9 Hz, 2.4 Hz, 1H, Cl '-H), 3.80-3.21, (111, 7H, C2'-H, C3'-H, C4'-H, C5'-CH2 , and C5-Cfh). 70 2(R,S)-D-ribo-l' ,2',3' ,4'-Tetrahydroxybutylthiazolidine- 4(R)-carboxylic acid ethyl ester (RibCys ethyl ester) L-Cysteine ethyl ester hydrochloride (9.48 g, 0.051 mol) was dissolved in degassed H20 (75 n1L) under a N2 atmosphere. To this solution were added potassium acetate (5.40 g~ 0.055 mol) and D-ribose (7.50 g, 0.051 mol). This solution was stirred for 24 h then was filtered, concentrated by rotary evaporation, and dried in vacuo. The resulting solid was crystallized from warm ethanol to yield a white solid (11.60 g, 83%): [a]20[) -90 (c 0.5, H20); mp 124-127 °C; III NMR (D20, 400 MHz, ppm, 5:2 mixture of diastereon1ers) diastereomer A: 4.82 (d, J 3.2 Hz, IH, C2-H), 4.40 (dd, J 6.6 Hz, 3.4 Hz, IH, C4-H), 4.19-4.10 (n1, 2H, C4-C02CH2CH3), 3.91-3.88 (m, 1H, CI '-H), 3.83- 3.78 (m, 211, C4' -CH2), 3.72-3.67 (m, 1 H, C3' -H), 3.59-3.55 (m, 1 H, C2' -H), 3.15 (dd, J 10.9 Hz, 3.5 Hz, IIi C5-CH2 ), 3.00 (dd,J 10.9 Hz, 6.7 Hz, IH, C5-CH2), 1.14 (t,J 8.0 Hz, 3H, C4-C02CH2Cfi]); diastereomer B: 4.74 (d,.1= 3.7 Hz, IH, C2-H), 4.40 (dd, J= 6.6 Hz, 3.4 Hz, IH, C4-H), 4.19-4.10 (m, 21-1, C4-C02CH2CH3), 3.91-3.88 (m, IH Cl '-H), 3.83-3.78 (m, 2H, C4'-CH2), 3.72-3.67 (m, IH, C3'-H), 3.59-3.55 (m, IH, C2'- H), 3.23 (dd, J 10.5 Hz, 6.8 Hz, 1 H~ C5-Cfh), 2.86 (dd, J = 10.6 Hz, 9.2 Hz, 1 H, C5- I" Cfh)~ 1.17 (t, J 7.2 lIz, 3H, C4-C02CH2Cfi]); .JC NMR (D20, 100 MHz, PPll1) 175.61, 76.95,75.26,73.39,72.66,66.97,65.20,64.58, 38.18,15.81; Anal. Calcd. for CtoHl9N06S + 0.25 H20: C, 42.02; H, 6.88; N, 4.90. Found: C, 42.17; H, 6.62; N, 4.92. 2(R,S)-D-gluco-l ' ,2',3' ,4' ,5'-PentahydroxypentyIthiazolidine- 4(R)-carboxylic acid ethyl ester (GlcCys ethyl ester) 71 L-Cysteine ethyl ester hydrochloride (9.28 g, 0.05 mol) was dissolved in degassed l<bO (75 mL) under a N2 atmosphere. To this solution were added potassium acetate (5.40 g, 0.055 mol) and D-glucose (9.00 g, 0.05 mol). This solution was stirred for 24 h then \vas filtered, concentrated by rotary evaporation, and dried in vacuo. The resulting solid was crystallized from warm ethanol to yield a white solid (13.65 g, 88%): [a]20l) = -78 (c 0.5, H20); nlp 130-133 °C; IH NMR (D20, 400 MHz, ppm, 5:2 mixture of diastereomers) diastereomer A 4.38 (dd, .1 6.8 Hz, 3.7 Hz, 1 H, C4-H), 4.18-4.10 (m, 2H, C4-C02CH2CH3), 3.89 (dd, .1= 6.3 Hz, 4.1 Hz, 1 H, C l' -H), 3.81 .80 (m, 1 H, C2'­H), 3.74-3.64 (m, 2H, C3'-H and C4'-H) 3.57-3.47 (m, 2H, C5'-CH2), 3.16 (dd, .1= 10.9 Hz, 3.5 Hz, 1 H, C5-CH2), 3.05 (dd, .1 = 11.0 Hz, 6.8 Hz, lH, C5-Clh), 1.17 (t, .1 7.2 Hz, 3H, C4-C02CH2ClfJ); diastereomer B 4.65 (d, .1 4.9 Hz, 1 H, C2-H), 4.38 (dd, .1 6.8 Hz, 3.7 Hz, 1H, C4-H), 4.18-4.10 (nl, 2H, C4-C02CH]CH3), 3.96 (dd, .1 8.8 Hz, 6.8 Hz, 1H, C1'-H) 3.81-3.80 (m, 1H, C2'-H), 3.74-3.64 (m, 2H, C3'-H and C4'-H), 3.57- 3.47 (nl, 2H, C5' -Clh), 3.24 (dd, .1 = 10.5 Hz, 6.8 Hz, 1H, C5-Clf2), 2.91 (dd, .1 10.5 Hz, 8.8 Hz, 1H, C5-CH2), 1.17 (t,.J 7.2 Hz, 3H, C4-C02CH2CHJ ); l3C NMR (D20, 100 MHz, ppIll) 175.61,75.21,74.43,73.51 (2 C), 72.09,66.73,65.43, 65.22, 38.45,15.81; Anal. Calcd. for CIIH21N07S + 0.25 H20: C, 41.83; H, 6.86; N, 4.43. Found: C,41.52; H, 6.76; N, 4.39. 72 S-Benzyl-N-Boc-L-cysteineamide Freshly distilled triethylanline (3.1 l11L, 0.022 mol) was added to a solution of s­benzyl-,"'- Boc-L-cysteine in freshly distilled THF (30 mL), and the mixture was cooled to 0-5 °C. To this solution was added a solution of ethyl chloroformate (2.39 g, 0.022 mol) in THF (2.5 InL,), and the mixture was stirred for 15 nlin. Concentrated ammonium hydroxide (1lnL) in THF (2.5 111L) was added dropwise over 5 min to produce a white precipitate. After 15 l11in, the reaction mixture was warnled to room temperature and stirred for 18 h. The reaction mixture was filtered and the filtrate was concentrated by rotary evaporation to give a white solid. The white solid was dissolved in CI-hCh (50 l11L,) and washed with l-hO (3 x 50 ml..,). The organic layer was dried with MgS04 and concentrated by rotary evaporation to yield a white solid (2.24 g, 750/0): IR (KBr, thin film, cnl- I) 3450 (NH), 3400 (NH), 1680 (C=O); IH NMR (CDCI3 , 200 MHz, ppm) 7.37- 7.24 (m, 5H, C3-aromatic H), 6.30 (br s, 1H, C1-C(O)NHH), 5.66 (br s, IH, Cl­C( O)NHH), 5.32 (br d, J = 8.0, 1H, C2-NH), 4.26 (nl, 1H, C2-H), 3.77 (s, 2H, C3- benzylic H), 2.94-2.67 (m, 2H, C3-CH2), 1.45 (s, 9H, C2-t-butyl H). S-Benzyl-L-cysteineamide S-Benzyl-N-Boc-L-cysteineamide (1 g, 0.003 11101) was dissolved in 3 M 11Cl­ethyl acetate (11 mL) and was stirred for 30 min. The reaction l11ixture was concentrated by rotary evaporation and the remaining residue was dissolved in hot ethanol and filtered. The filtrate was concentrated by rotary evaporation, and the residue was washed with acetone and dried in vacuo to yield a white solid (0.51 g, 75%): IH NMR (DMSO-d6, 200 MHz, ppnl) 8.46 (br s, 211, C2-NH2), 8.18 (br s, lH, C1-C(O)NHH), 7.68 (br s, lH, C1- 73 C(O)NIi1l), 7.41-7.21 (m, 5H, C3-aromatic H), 3.99 (t, J 6.0 Hz, 1H, C2-H), 3.84 (s, 2H, C3-benzylic 1-1), 2.96-2.86 (nl, 2H, C3-Cfh). L-Cysteineamide Prior to debenzylation, S-benzyl-L-cysteineanlide was dried overnight in a drying thimble under hexanes. All steps of debenzylatioll were carried out under argon and precaution was taken to exclude nl0isture. S-Benzyl-L-cysteineanlide (3.0 g, 0.012 mol) was dissolved in approximately 75 mL of freshly distilled liquid NH3. To this solution was added Na nletal (~0.3 to 0.5 g) in small portions. After the blue color persisted for 40 nlin, DOWEX SOW x 8-400 (NH4 fornl) was added to quench the excess Na, then the NH3 was allowed to evaporate under a streaIn of argon.24 The residue was dried in vacuo, washed with methanol, and the filtrate was acidified with concentrated HCL The acidified solution was concentrated by rotary evaporation facilitated by adding ethanol and evaporating the solvent as an azeotrope. The residue was purified by column chrolnatography on a DOWEX SOW x 8-400 (H+ form) column as follows. The crude residue was dissolved in H20 (8 nlL) and applied to a 60 InL DOWEX SOW x 8-400 (It forn1) column. A peristaltic pump was used to deliver the following solvents: I-hO (112 mL), 1 N HCl (112 mL), 2 N HCl (156 11lL), 3 N HCI (32 mL), and 6 N HCl (32 InL) and fractions were collected (4 mL). Fractions that stained yellow with ninhydrin were pooled (fractions 78-90) and concentrated by rotary evaporation facilitated by adding ethanol and evaporating the solvent as an azeotrope to produce a white solid (1.5 g, 79%): nlp 196-200 °C dec Oi1.23 191-192 DC); [H NMR (D20, 400 MHz, ppnl) 4.12 (dd, J 74 = 5.0 lIz, 5.0 Hz, 1 H, C2-H), 3.02-2.92 (In, 2H, C3-CH2); Anal. Caled. for C3HgN2SOCl: C, 23.08; H, 5.77, N, 17.95. Found: C, 23.07, H, 5.93, N, 17.85. 2(R,S)-D-ribo-l ' ,2' ,3' ,4' -Tetrahydroxybutylthiazolidine- 4(R)-carboxamide (RibCys amide) L-Cysteinean1ide hydrochloride (0.50 g, 0.0032 mol) and D-ribose (0.48 0.0032) were dissolved in H20 (10 mL). Potassium acetate (0.31 g, 0.0032 mol) was added to the solution and the reaction n1ixture was stirred for 24 h. The reaction mixture was concentrated by rotary evaporation facilitated by adding ethanol and evaporating the solvent as an azeotrope. The resulting solid was crystallized fron1 wann ethanol to yield a white solid (0.69 g, 85% yield): mp 154°C dec; IH NMR (D20, 400 MHz, ppm, 5:2 Inixture of diastereon1ers) diastereomer A: 4.81 (d, .1 = 3.4 Hz, 1 H,C2-H), 4.27 (dd, J = 6.8 Hz, 4.1 Hz, 1H, C4-H), 3.90 (dd, J 7.8 lIz, 3.4 Hz, 1H, C1 '-11),3.84-3.80 (m, 2H, C4'-CH2), 3.72-3.68 (m, 1H, C3'-H), 3.60-3.55 (m, 1H, C2'-H), 3.11 (dd, J 10.9 Hz, 4.3 Hz, 1H, C5-CH2), 2.99 (dd, J 10.9 Hz, 7.0 Hz, 1H, C5-C112), diastereomer B: 4.77 (d, J 3.4 Hz, 1H, C2-H), 4.27 (dd, .1 6.8 Hz, 4.1 Hz, 1H, C4-H), 3.90 (dd, J 7.8 Hz, 3.4 Hz, 1H, C1 '-H), 3.84-3.80 (m, 2H, C4'-CH2), 3.72-3.68 (m, lH, C3'-H), 3.60-3 (111, III, C2'-H), 3.20 (dd, .1= 10.6 Hz, 6.7 Hz, 1H, C5-CH2), 2.81 (dd, J 10.5 lIz, 9.0 1H, C5-CH2); 13C NMR (D20, 100 MHz, ppm) 178.81,76.84,75.18,73.75,73.10, 67.37,64.67,37.86; Anal. Caled. for CSH16N205S 0.25 H20: C, 37.42; H, 6.48; N, ]0.91. Found: C, 37.52~ H, 6.62; N, 10.81. 2 (R,S)-o-gluco-l ',2',3' ,4' ,5'-Pentahydroxypentylthiazolidine- 4(R)-carboxamide (GlcCys amide) 75 L-Cysteineamide hydrochloride (0.40 g, 0.0026 mol), D-glucose (0.46 0.0026 mol), and potassium acetate (0.25 0.0026 tnol) were dissolved in H20 (10 mL). The reaction nlixture was stirred for 24 h and concentrated by rotary evaporation facilitated by adding ethanol and evaporating the solvent as an azeotrope. The resulting solid was triturated with ethanol and refrigerated at 4°C. The white solid was isolated by filtration and washed several times with ethanol using the following procedure. The solid was transferred to a 50 mL centrifuge tube, ethanol (15 mL) was added, and the solid was resuspended by vortexing. The white solid was precipitated by centrifugation, and the liquid was discarded. This procedure was repeated five times. The precipitate was redissolved in H20 (10 mL) and lyophilized to yield a white solid (0.25 g, 35%): mp 146- 155°C dec; lH NMR (D20, 400 MHz, ppm, 5:2 mixture of diastereomers) diastereonler A: 4.65 (d, J= 4.4 Hz. 1H, C2-H), 4.24 (dd, J 7.0 Hz, 4.3 Hz, 1H, C4-H), 3.91-3.88 (nl, IH, C1 '-1-1),3.81-3.64 (m, 3H, C2'-H, C3'-H, and C4'-H), 3.57-3.50 (nl, 2H, C5'-CH2), 3.12 (dd,J 10.9 Hz, 4.1 Hz, 1H, C5-CH2), 3.02 (dd,J= 10.8 Hz, 7.0 Hz, 1H, C5-CH2), diastereomer 8: 4.24 (dd, J= 7.0 Hz, 4.3 Hz, IH, C4-H), 3.91-3.88 (m, 1H, C1'-H), 3.81- 3.64 (m, 3H, C2'-H, C3'-H, and C4'-H), 3.57-3.50 (m, 2H, C5'-CH2), 3.18 (dd,J= 10.6 Hz, 6.8 Hz, 1 H, C5-Cfh), 2.88 (dd, J 10.6 Hz, 8.6 Hz, 1 H, C5-CH2); I3C NMR (D20, 100 MHz, ppm) 178.86,75.47, 74.31, 73.63, 73.56, 72.65, 67.17, 65.43, 38.14; HRMS (FAB) Caled. for C9Hl8N206S (MH+) 283.0964. Found 283.0964. 76 4-Amino-3,5-dimethylphenol Sulfanilic acid (20 g, 0.116 n10l) was diazotized then reacted with 3,5- dimethylphenol (14.17 g, 0.116 mol) following the known procedure.26 The solution was stirred for 1 h, during which time a red precipitate formed. The solution was heated to 65-70 DC and sodiunl dithionate (51 g) was adde