Reprinted from the Journal of the American Chemical Society, 1990,t-I? 1190. Copyright @ 199^0by the American Chemical Society and reprinted by permission of the copyright owner.

Comparisonsof Rate Constantsfor Thiolate-Disulfide Interchangein Water and in Polar Aprotic SolventsUsing Dynamic rH NMR Line ShapeAnalysis

RajeevaSingh and GeorgeM. Whitesides*

Contributionfront theDepartment o.f Chenri.rtrt, Hurrard L nirer.sit.t, Cambridse, ,llassat'husr'tts (): | -l\ Rt',t'itcd .lulr ll. i 919

Abstract:The rateconstants for representativethiolate-disuliide intcrchange rcactions are larger in DMSO andDMF than in waterb1 a iactorof approximately2300 at 24 oC. The logof the rateconstant is directlyproportional to the molefraction ol DrO in mixturesof DMSO and D2O,even at smallmole fractions of D2O. This linearproportionality suggests that thiolate anionis not specificallysolvated by waterand that hydrogenbonding is relativelyunimportant in stabilizingthis species. The valucsof -\Sr for thiolate-disulfideinterchange are approximately-10 cal/(degmol), presumptivelybecause of lossin the entropyof the reactantsin goingfrom groundto transitionstate, partially compensated by a gain in entropyfrom solvent release.Introduction of a hydroxylgroup p to the C-S bondslows the reactionby a factorof 2-15; the introductionof methyl groupsp to the C-S bondslows the rate by factorsof 3-20. A numberof substanceshave been screened as potentialcatalysts ior thiolate-disulfideinterchange in water: noneshowed useful levels of catalyticactivity, although phenylselenol did accelerate the interchangesignificantly.

This paperexamines the influenceof solventon rate constants in proteinsmay be enzvmecatalyzed.e-la Many aspectsof the for thiolate-disulfideinterchange (eq l: nuc = nucleophile,c = mechanismof thiolate-disulfideinterchange are understood'15-22 central,lg = leavinggroup). This reactionis one of broad

Rn*S-+ S-SRls : (8) Saxena,V. P.; Wetlaufer,D. B. Biochemistry1970,9,5015-5022. I (9) Freedman,R. B. Nature 1987,329,196-191. Rc (10) Lang,K.; Schmid,F. X. Nature1988, 331,453-455. --'5*'n -- ( I I Pain. R. TrendsBiochem. Soc. 1987,12, 309-312. ;'n"*3---s lt Rrucs-s + sRrs (1) ) llll (12) Wetzel,R. TrendsBiochem. Soc. 1987, 12,478-482. LR"JRe (13) Pigiet,V. P.;Schuster,B. J. Proc.Natl. Acad.Sci. U.S.A.l9t6,8-?, 7643-7647. importancein biochemistry.r-7Although it is oftennot catalyzed (14) Holmgren,A. Ann. Reu.Biochem. 1985, 54,23'l-271. enzymaticallyin vivo,8the formation of certaincystine linkages (15) Szajewski,R. P.;Whitesides, G. M. "/.Am. Chem.Soc.1980, 102, 20n-2026. (16) Whitesides,G. M.; Lilburn,J. E.; Szajewski,R. P. J Org. Chem. (l) Freedman,R. B.; Hillson,D. A. In The Enzymologyof Post-Trans- 1977,42,332-338. lational Modificationof Proteins;Hawkins, H. C., Freedman,R. B., Eds.; (17) Shaked,Z.;Szajewski, R. P.;Whitesides, G.M. Biochemistry1980, Academic:London. 1980; pp 157-212. r9.4156-4t66. (2) Creighton,T, E. J. Phys.Chem.l985, 89, 2452-2459. ( l8) Whitesides,G. M.; Houk,J.; Patterson,M. A. K. J. Org.Chem. 1983, (3) Creighton,T. E. MethodsEnzymol. 1984, 107,305-329. 48, 112-|5. (4) Ziegler,D. M. Ann. Reu.Biochem. 1985, 54,305-329. ( l9) Houk,J.; Singh, R.; Whitesides,G. M. MethodsEnzymol. 1987, 143, (5) Gilbert,H. F. MethodsEnzymol. 1984, 107, 330-351. 129-140. (6) Buchanan,B, B. Ann. Reu,Plant Physiol.1980, 31,345-361. (20) Hupe,D. J.;Pohl, E. R. /sr.J. Chem.1985,26,395-399. (7) Jocelyn,P. C. Biochemistryof the SH Group; Academic: London, (21) Wilson.J. M.; Bayer,R. J.;Hupe, D. J. J. Am. Chem.Soc.1977,99, 1972. Huxtable,R. J. Biochemistryof Sulfur; Plenum:New York, 1986. 1922-7926. T hio I ate- Di s u lfi de I nt erc hange J. Am. Chem.Soc., Vol. I12, .\'o.3, 1990 llgl (A) EXPERIMENTAL SIMUI^ATED temp (K) (u-1s-11 366 _J1--/1.- -rL-[- 53s 352 _/LJ\-- _I_A-_ 3L2

339 -nu,AA-- -^j\_ 145

333 J\l\ JU\- 93 0.0 0.2 0.4 0.6 0.8 1.0 327 J AA-_\-./ J\jL 64 x uro M\J /\,{\- jrJ\/\- 32]- ) 42 Figure2. Eifectof changingthe molefraction oi D,O on thesecond- /\,{AI orderrate constant (k) of thiolate-disulfideinterchangc of potassium 315 __/"'-J "'_ _/Ju\,t_ 24 l-hrdroxyethanethiolateandits disulfide in mixruresoi D\1SO-d,5and ilillt D.O at 197K. Therate constant has units of N'l-rs-r 309 ---]v\J\Jl- _,1\_/U_ IY j,L,u- Methods 302 -,Ltljt- 13 We haveinvestigated self-exchange betueen RS and RSSR rH Hz with useof dynamic N MR spectroscop\:-r:- for four reasons: J.260 540 L260 540 (i) The ratesof representativethiolate-disuliide inrcrchange re- actionsin polar,aprotic solvents are too fast to be studiedcon- (B) venientlyby conventionalkinetic techniques. (ii) Thiolarcanions areeasily oxidized, and exclusion of atmosphericoxrgen is com- parativelyeasy in sealedNMR tubesbut moredifficult in rhc 294 ri- 77 042 typesof apparatusused in classicalkinetic techniques. (iii) The intcrchangereaction is dcgenerateand can be crunrincdat 288 -rrL _/._ L2862 equilibrium. Determinationof ratesof' rcactionrbr \\IR spectroscoprinvolvcd prcparation of'onlr .r :rnulcr.rlnplc of thiolateand disull'ide : it \\J\ n()tnccc\\.ir\ t\) \\ tlhdrrru .ilrquots .rybu 283 -JL ()rtr) lrcpitrc nrultrplc:rrrn[r]c-: rr,, r Thc :rcthricncpcak.: adjacent ttrthc thi,rl.rtc,rntidr.ull'rtic nr(rrclrC:.rrc.cpiii'i.lted br approxi- nrltclr0.-r-1 ; rl00 [lz.rl .100\ll{zr. th:..cprtr.itionmade the 277 Jl. 1 235 analrsis ol- thc lrnc:h.rpc\ rcl.rtr\ .1;.1rghti',.rrrr clr ard. Representativeexpcrrntcntal 5pc!tr.i .trc :ho\\ n rn Figurel. Estimationof rateconstants 272 -ilu -r^r-- 5306 from the'ccrro:rintcntal sDectra was rl accomplishedby visualcomparison of r-\pcnnrcntalspectra and spectrasimulated with the programD\MR+.:o The tcmperature 267 ,_-,lr\_ _/\_ 38s9 dependenceof the chemicalshifts ol the thiorareand disurfide speciescould be measuredindependently': the Erpcrintental Sectioncontains details. Solutionsof thiolatesin poraraororic 260 -,tL _^1_ 25't2 solventswere prepared by reactionof thiolswith I equir ttf po- tassium/erl-butoxidc. The natureof the counterionhad no in- fluenceon the rcaction. Further,addition of l8-crown-6(l equir/equirthiollte) to the reactionsystem involving potassium 250 _Jiii_ 1508 l- hrd rorr Hz etha ncr h iolate a nd bis(2-h1 drox_v-ethyl ) disulfi de did not influencethe rareof thiolate-disuliideinterchanse. 950 bbu vbu oou Results Figurel. Experimentaland calculated line shapes for _100\,tHz rH NMR spectraat severaltemperatures oi (A) sodiuml-hrdrorrethane- Rateconstants and ActivationParameters. Table I summarizes (3.8 thiolate M) andbis(2-hydroxyethyl) disulfide (1.9 \,I; in D2O;(B) rate constantsobtained in this work and valuesof relevant potassium 2-hydroxyethanethiolate(66mM) andbis(2-hrdroxyethyl) thermodynamicparameters derived from variable-temperature disulfide(31 mM) in DMF-d7.The peak assignmenrs are m = (HO-C- work.2e The most importantobservation i12CH2S)2;n = HOCH2CH2S-;1 = (HOCHTCH2S)2;s = HOCHzC- in this tableis that the /12S-;u,v=HCON(CH)2. rate constantsfor thiolate-disulfideinterchange are faster in it is an sp2 reactionin which thiolateanion attacks the disulfide bond along the S-S axis. The symmetry and structureof the (22) Freter.R: Pohl.E. R.; Hupe, D. J. J. Org. Chem.1979, 44. transitionstate are not known in detail,although the chargeis t71t*t771. concentratedon the terminalsulfur atoms. (23) Sandstrom,J. Dt'namic,\'MR Spectroscop)':Academic: London. r982. The influence of solventon the rate of reactionshas not been (24) Binsch.G.; Kessler,H. Angew,.Chem., Int. Ed. Engl. 1980. 19. exploredand wouldprovide mechanistically useful information 4t l-428. concerningcharge distributions and about differencesin solvation (25) Binsch,G. Top.Stereochem. 1968, J, 97-192. (26) of groundand transitionstates. This work reportscomparisons Binsch.G.ln Dvnamicl\'luclear Magnetic Resonance Spectroscopl:1 Jackman,L. M., Cotton,F. A., Eds.;Academic: of rate constants New York, 1982;pp 45-81. for representativethiolate-disulfide interchange (27) Selenol-diselenideinterchange in waterresults in exchange-averaged reactionsin water and in the polar aprotic solventsDMSo and lH NMR spectra:Pleasants, J. C.;Guo, W.; Rabenstein,D. L. J. Am. Chem. DMF. lt had threeobjectives: to clarify the roleof solvation; Soc.1989, ll1,6553-6558. Tan, K. S.;Arnold, A. P.;Rabenstein. D.L. Can. to indicatcwhethcr changes in the polarityof the reactionmedium J. Chem.1988, 66, 54-60. For the slowerthioltisulfide interchange,separate 'H NMR resonanceswere observed for thiol anddisulfide in aqueoussolution: would providea method of controllingthe rate of the reaction; Rabenstein,D. L.; Theriault,Y. Can. J. Chem. 1984,6'2, 1672-1680. and to evaluatethc possibilitythat a weaklysolvated thiorate Theriault.Y.; Cheesman,B. V.; Arnold,A. P.; Rabenstein,D. L. Can.J. nucleophilcmight be exceptionallyreactive. The observationof Chem.1984,62,1312-1319.Theriault, Y.; Rabenstein,D. L, Can.J. Chem. 1985,6J,2225-2231. a correlationbetween reactivity and solvationmight providethe (28) oNuno.written by Prof.C. H. Bushwelleret al. is availablefrom the basisfor strategiesfor the designof catalysts for thiolate-disulfide QuantumChemistry Program Exchange, Department of Chemistry,Indiana interchanee. U niversity. ll92 J. Am. Chem. Soc..Vol. I12, No. 3, 1990 Singh and Whitesides

-SR Table L Rate Constantsfor DegenerateThiolate-Disulfide Interchange: RS- + RSSR : RSSR + 10-3ka.b lGI (M-t s-t; (kcal/mol) ATI ASI RS- M' solvent (291K) (291 K) (kcal/mol) (call(deg mol)) -10 HOCH2CH25- Na' Dzo 0.0077 t6.2 IJ -l K+ Dro 0.0095 16.r IJ I -13 K+ DMF-d7 20 r 1.5 8 K* DMSO-d6 21 l.l t1 cH3cH2cH2cH2s- Na+ DMF-di +J l.l K+ DMSO-d6 54 1.0 cH3c(cH3)2cH2s- K+ DMF-d7 l5 t.t K+ DMSO-d6 l6 1.1 -10 HOC(CH3)2CH25- K+ DMSO-d6 t.l r3.2 l0 -16 HOCH2C(CHr)2CH25- K+ DMSO-d6 0.67 r 3.5 9 bRate oUncertaintiesare k, *10%: AGl, +0.1 kcal/mol;lHt, +1 kcal/mol;A^Sl, *2 cal/(degmol). constantswere inferred from visual com- parisonof thc simulatedrH NMR Iine shapeswith the experimentalline shapes;details are given in the ExperimentalSection.

DMSO and in DMF than in watcr by a factor of approximately T ("C) 90705030 10 -10 -50 x 103. Thc log of the rate constantdepends linearly on the 3 5 solventcomposition in mixtures of D2O and DMSO-d6 @q 2, Figurc2). There is no evidencefor specificsolvation of thiolate 4 Jfogk=llGtGXD,o r3 Q) '\*\- anionby water(specific solvation would be reflectedin nonlinearitl I in thisplot. especially at smallvalues of 1p,6). This observation 1 \ suggeststhat hydrogenbonding between water and the thiolate is relativelyunimportant. anion 2.5 3.0 4.0 4.5 The rate of thiolate-disulfideinterchange reaction shows only modestsensitivity to stericeffects and to groupscapable of in- 1o3T'1( x-1) tramolecularhydrogen bonding. Introductionof methyl groups Figure3. Plotof log k vs I for the thiolate-disulfideinterchange of slowsthe reactiononly by factorsof 3-20. This lT 0 to the C-S bond sodium2-hydroxyethanethiolate (3.8M) andits disulfide (1.9 M) in D2O insensitivityis expectedfor an Sp2 reactioninvolving front-side (t); potassium2-hydroxyethanethiolate (66mM) andits disulfide (31 attackof RS- alongthe S-S axisof the disulfide.Intramolecular mM) in DMF-d7 (r); potassium3-hydroxy-2,2-dimethylpropanethiolate hydrogenbonding between hydroxyl and thiolate doesnot sig- (0.20M) andits disulfide(0.10 M) in DMSO-de(o); potassium2- nificantlymodify the rate of interchangeof 2-hydroxlethane- hydroxy-2-mcth1'lpropanethiolate(0.18M) and its disulfide (0.09 M) in thiolate: the rate constantfor thiolate-disulfideinterchange DMSO-d^(O) Therate constants have units of M-r s-r. reactionof potassiuml-butanethiolate is onll nrice that of po- tassium2-hydroxyethanethiolate in DMSO. Introductionof a rntcrprctcdsinrlarlr.r' Thc determinationof lS+ and JH+ by \ \1R involvcerrors. and theirvalues hydroxylgroup either li or 1 to the C-S bondin stericallihindered thc dr nanric nrcthodmar alkyl thiolatesslows the interchangereaction bi approximatell shouldbc trcatcdri ith caution.lr''rl a factorof l5 in polar aproticsolvents. A gem-dimethyleffectl0 The Thiolate-Disulfidelnterchange Reaction Is Not Easily and weakersolvation of the hydroxyl group in the sterically Catalyzed. Ratesof many thiolate-disulfidereactions in water hinderedcase may result in greater intramolecularhydrogen follow a Bronstedrelation in the valuesof pK" of the participating bondingthan in the stericallyunhindered 2-hydroxyethanethiolate. species(eq 3 is one expressiondescribing this relation).32 We On the basisof theseobservations, we suggestthat differences havesurveyed a number of aromatic thiols having valuesof pKu in rateswith changesin solventfor 2-hydroxyethanethiolateare centeredaround pH 7, in the hopethat thesespecies might, by representativeof differencesexpected for other alkane thiolates. virtue of chargedelocalization and possibleweak solvationin water, Arrheniusplots yielded the thermodynamicparameters sum- proveto be more reactivein thiolate-disulfideinterchange than marizedin Table I (Figure 3). For HOCH2CH2S-,A11' de- would be predictedon the basisof eq 3.33 Theseand a range going -7AS' is approximateli' creasedin from D2O to DMF; log k = 6.3 + 0.59 pKunu"- 0.40pK.' - 0.59p6"ts (3) constant. We rationalizethe valueof the entropyof activation in thesereactions as a compromisebetween two factors. The of othertypes of potentialcatalysts did not showuseful catalytic reactioninvolves two particlesgoing to one in the transitionstate activity. The detailedresults are given in the Experimental and is thus intrinsicallyentropically disfavored. The observation Section. that ,\S+ is - -10 cal/(deg mol) suggestsa partial compensation We screenedfor catalysisin water by incubatingthe potentially of the unfavorableloss in translationaland rotationalentropy by catalyticadditive with an equimolarmixture of 2-carboxy-1,3- someother factor,presumptively solvent release. Thermodynamic propanedithioland bis(2-hydroxyethyl)disulfide. We followed data for other thiolate-disulfideinterchange reactions have been

(3 I ) In the caseof potassium2-hydroxyethanethiolate and its disulfidein were to be 7.7 kcal/mol and -13 (29) From the dataof Creighton(Creighton, T. E. "/. Mol. Biol.1975,96, DMF-d7,the valuesof AIl+ and A,St found 767-776)we havecalculated the rateconstant Ik = l9 M-rs-r(25 "C)] for cal/(deg mol) with carefulcalibration of temperature.The valuesof AIll the reactionof dithiothreitolwith bis(2-hydroxyethyl)disulfide in waterIt and AS* calculatedwithout calibrationof temperaturewere 10.6kcal/mol = 16ob(l+ lgp(a-pH).kob = 4.5M-rs-r;pH = 8.7;pK"(dithiothreitol) = 9.21. and-2 cal/(degmol). The valueof AGr wasalmost unaffected by the error We have to divide Creighton'srate constantby two (to accountfor the in temperature. presenceof two symmetricalthiol groupsin dithiothreitol)in order to compare (32) Two other equationsgive similar Bronstedcorrelations. The 0 'C)] with our rate constant[9.5 M-' t-t 124 for HOCH2CH2S-K*/ coefficientsare thereforenot sharplydefined by the availabledata. These (HOCH2CH25)2interchange in D2O. The identityof thesetwo rateconstants Bronstedequations should be consideredprimarily as kinetic modelsfor -0.98) is accidentalbut doesindicate that the two independentmeasurements are thiol-disulfideinterchange in water.r5A Bronstedslope (B = hasbeen in closeagreement. Some representativevalues of rate constantsof thio- observedfor the reactionof symmetricalalkyl disulfideswith triphenyl- late*disuliideinterchange from the literature are k = 57 M-r s-r for the phosphinein 50Vodioxane-water: Overman, L. E.; O'Connor,E. M. J. Am. oCl5 reactionof, mercaptoethanol and oxidizedglutathione in water,pH 7, 30 Chem.Soc. 1916, 98,'77 1-7'l 5. and k = 2 x 105M-l s-l for the reactionof mercaptoethanolwith Ellman's (33) Catalysisof acylationof hydroxyl groups using N,N-(dimethyl- reagentin water,pH 7, 30 oC.16 amino)pyridine(DMAP) is an exampleof the strategyof enhancingnucleo- (30) Schleyer,P. v. R. J. Am. Chem.Soc. 1961,8J, 1368-1373. Ingold, philicityand chargedelocalization: Hofle, G.;Steglich, W.; Vorbruggen,H' C. K. "/. Chem.Soc. 1921. 119.305-329. Angew.Chem., Int. Ed. Engl. 1978,17, 569-583. Th io I at e- Di su lfi de I nt erc' hange J. Am. Chem. Soc.,Vol. I I2, No. 3, t990 ll93

rl Table IL Comparisonsof Ratesof Reactionsin Water and in polar lns....s.snl NonproticSolvents

rRj l0-3(ft,or/kH20) reactlon or l0-l(r(.or/KHro) ({=SorO) solvent X=S X=O ref iF\ * RX + RXXR RXXR * \:I li \\* RX- DMSO-d6 L.) ns-+ RSSR / 1\, nssn* DMF-d7 2.2 I ,r/ \\_ C2H5X-+ [{+: C2HsXH DMSO 700 1.6x lOe a I C6H.X * Mel ' DMF 32 1900 b p-O2NC6HaX-* * /tt Mel DMF 0.24 15 b \- Dp C6H.X * p-OrNCuHal- DMF 4.0 lxl04 b oArnett, E. M.; Small,L. E. J. Am. Chem.Soc. 19l1-.99. g0g-g16. tJupe,D. J.; Jencks, W. P.J. Am.Chem. Soc.1977.99,15t-464. ,Clare, B.W.: Cook, D.; Ko, E. C. F.;Mac, Y. C.;Parker, A. J.J. Ant.Chem. Soc. 1966.88,l9ll-1916. Parker. A. J.Chem. Rer,. 1969, 69.1-32. Figure4. Hypotheticalplot of freeenergy vs reactioncoordinate Therate for comparison-sarereported for DMF andmethanol at 0 oC thiolate-disulfideintcrchange reaction in watcrancl in DolaraDrotlc solvents(DMSO. DMF). the morestrongl\ dcstabilized (Figure .l). This inferencedoes not dependstrongl\ on the structurcof the transitionstate, pro- thc rateof reactionby monitoringthe increascin absorpriorrar ridcd that thc churgcis dclocalizedover the threesulfur centers, 330 nm due to the product4-carboxl'- I .J-dithiolanc trq +t. .rsis indccdinl'crrcd from the Bronstedrelation that describes (HSCH2)2CHCOO-+ (HOCH2CH2S)z * the reaction in $ iitcr. s ---cH" Thc proposalthat the increasein rate in going from water to DMSo or D\4 F correlaresprimarily with the dielectricconstant + 2HocH2cH2sH(4) IS--

oxygen) nucleophiles. In goingfrom polar useful,but it doessuggest that selenolsand relatedcompounds protic solvents(water, methanol) to polar nonproticsolvents (DMSO, may haveuseful catalytic activity.2T we will describestudies in DMF), the ratesof reactionsinvolving thiolate and this area in a separatepaper. alkoxy anionsincrease; the increaseis, however,much more for An effort to acceleratethe rate of thiolate-disulfideinterchanse the reactionsinvolving alkoxy anions than thiolates.4oThe arkoxy usinga two-phasebenzonitrile-water system16 was not successfiil. anionsare solvatedin water to a higherdegree than thiorates. What are the implicationsof theseresults for catalysisof Discussion t hiolate-disulfidc interchange'lMost strategiesfor catalyzingthis The rate constantfor representativethiorate-disulfide rnrer- reactionwould rcquirc dcveloping a speciesN: that wasboth an changereactions increases by - 103on going from *'ater as solvent exceptionalnucleophile trvard sulfur(eq 6) and an exceptional to poiaraprotic solvents (DMSO, DMF). The changein .\Gr leavinggroup front sulfur (eq 7t. Our studiesindicate that it is_directlyproportional to the compositionof the (eq -SR solvent 2). N- + RSSR - NSR + (6) This proportionality-particularlythe absenceof a sharpdrop -SR'- in rate on addingsmall quantities of water to DMSo-suggesrs NSR + N- + RSSR, (7) that hydrogenbonding to thiolateanion is a rerativelyunimportant is more difiicult to develophypernucleophilic species based on contributionto the free energyof solvationof this species.STwe sulfur than on oxygen. It appearsthat it will be difficult to increase infer, then,that the solventinfluences the rate of thiolatedisulfide the nucleophilicityof thiolateions relative to rheir basicitvbv interchangeprimarily through its dielectricconstant: In the lower interferingwith hydrogenbonding to them,since hvdrogen bonding dielectricconstant solvents (DMSo, DMF) both thiolateanion doesnot seemto be very importantin an1,event. The parallel and the transitionstate are energeticallydestabilized relative to to the a effectthat is importantfor oxygennucleophiles iuch as water,but the thiolateanion, with its more localizedcharge, is HOO-, CIO-, and Me2NO-alis not known to existwith sulfur

(34) Butler,J. C.; Whitesides,G. M. Unpublished results. (38) A valueof k - 3 x l0e M-r s-r hasbeen estimated for reactionof (35) For a reviewof the reactionof disuliideswith trivalentphosphorus CHTCH2S-with CH3SS!H' in vapor phaseby using flowingafrerglow compo_unds,see: Mukaiyama,T.; Takei,H. In phosphirus^chem- Topicsin techniques.comparison of thisrate constant with thecollisional rite constant islrl;Griffith, E. J.,Grayson, M., Eds.; york, Wiley-lnteiscience:New 1976; ilggeststhat the reactionoccurs with a probabilityof - 3 x per Vol. 8, pp 587-645. l0-3 collision: Grabowski,J. J.: Zhang,L. J. Am. Chem..Soc.1989, l l t, l*-1203. (36) The rateconstant ior thiol-disulfideinterchange of 2-carboxy-1,3- (39) For comparison,the rate constantfor a representativeSp2 reactron propa-nedithiol and bis(2-hydroxy_ethyl disu Ifide in ) benzoiitrile f w atermixt ure at carbon^-cl- + cHrcl * clcH, * cl--increases by a factoi of approx- wasthe sameas that in water. our choice of benzonitrile/waiersystem was rmatelyl0'' on goingfrom waterto the vaporphase. Jorgensen and Buikner ba.sedon the rapid decarboxylationof 3-carboxybenzisoxazoles oLservedin summarizeleading references in this area: Jorgensen,W. L.; Buckner,J. K. et al. (Kemp,D. paul, llt:lltgr_ly-Keyp S.; K.-c. "/.Am. Chem.Soc.1975, J. Phys. Chem. 1986, 90. 4651-4654. 97,_7305-7312.Kemp, D.S.; paul, eox, D. D.; K. G.,/, Am. Chem.Soc. (40) For relatedcalculations concerning the relative 1975,97,7312-7318\. nucleophilicitiesof OH andSll-, see:Howard, A. E.; p. (37) Kollman, A. J. Am. Chem.Soc.l9gg. The weak contributionof ionic hydrogenbond to solvationin RS-. 110. 1t95-'/200. (Hzo), complexesis dissipatedeffectively with'in the first 2-3 solventmole- (4 | ) Edwards,J. O.; Pearson,R. G. Am. Chem. Soc.1962, g4, t6-24. cules(n = 2-3) as the dissociationenergy decreases "/. rapidlyupon successive Hoz. S.; Buncel,E, Isr. J. Chem.1985, 26,313-319. Buncel,E.; Um, I.,/. hydration:Sieck, t-. W.; Meot-Ner(Miutner), phy;. M. J. C'hem.l9g9,9J, Chem.Soc., Chem. Commun. 1986, 595. Hudson,R. F.; p.; I 586-l588. Hansell,D. Wolfe,S.; Mitchell, D. L J. C'hem.Soc., Chem. Commun.l98S. 1406-1407. ll94 J. Am. Chem. Soc..Vol. I12, No. 3, 1990 Singh and Whitesides

nucleophiles.Because z'-bonding to sulfur is lessimportant than to oxygen,it will be more difficult to manipulatethe polarizability and nucleophilicityof sulfur than of oxygenthrough remote o substituenteffects. It may be possibleto developspecies highly tro fr10 HOCH2CH2S- nucleophilictoward the disulfidegroup using phosphorus, selenium, T a o or otherelements, but we do not havefirm experimentalsupport o..'o r- f-'l for this approach. U to €rEs IT Thus, we concludethat the best strategiesfor "catalyzing" (HOCH2CH2S)2 thiolate-disulfideinterchange, especially under conditions com- patiblewith biologicalreactions, probably involve transferring the -1 reactantsfrom water to an environmentof lowerdielectric constant 300 310 320 330 340 350 and preorganizingthem into a configurationresembling the T (K) transitionstate. A strategyinvolving reaction in a low dielectric medium is basedon destabilizationof the reactantthiolate (RS-) Figure5. Elfect of temperatureon the 300 MHz rH NMR chemical relativeto the moredelocalized transition state IR3S3-]t. Sla- shifts of the methyleneprotons of (a) sodium2-hydroxyethanethiolate bilizing the transitionstate electrostatically relative to the reactant (1.2 M) and (b) bis(2-hydroxyethyl)disulfide (2.0 M) in D2O. The thiolateanion will requireboth preorganizationand precisear- temperaturedependence of the chemicalshifts for the methyleneprotons (a) rangementof the electrostaticpotential around the [R3S3-]+unit. of the thiolate and the disulfide(b) are small in comparisonto the differencein chemicalshifts betweenthe methyleneprotons of the thio- ExperimentalSection late and disulfide(-100 Hz at 300 MHz). The standardsfor mea- GeneralMethods. rH NMR spectrawere recorded with Bruker surementsof the chemicalshifts were silicone grease (for a) and DSS (for AM300and AM-500 instruments. The diiierence between the rH NMR b). chemicalshifts ol thcmcthvlene and hydroryl peaks of ethyleneglycol wasused to calibrate the temperature recorded bv theNMR spectrom- (vl)lGlr)l is quitc accurate(u'ithin 5% of the valuedetermined by eterin therangc oi temperaturesfrom 295--170 K: methanolw'as used completclinc shapcanallsis). The temperaturedependence of the for calibrationfrom 230-2906.r2-as Argon wasdeorr,gcnared br chcmicaishil'ts ior the nrcthllcncprotons of the thiolateand disulfide passingover Ridox (Fisher Scientific) and molecular sieves before use \\crc lr)casurcdindcpcndcntll and uere foundto be smallcompared to Distilledwater from a CorningAG-lb stillwas used to uashall glass- the dificrcncc rn chemical shifts betweenthe methyleneprotons of the ruarc.46 thiolateand disuliide(Figure 5). Materials. CambridgeIsotope Laboratories and Aldrich suppliedDrO In eachrcactive collrsion of thiolateand disulfide,only half of each disulfidemolecule converts to thiolate,although each thiolate converts (99.9Eo),DMSO-d6 Q9.96Va),and DMF-dj 09ok). Mercaptoethanol. bis(2-hydroxyethyl)disulfide, methyl thioglycolate,2,2-dimethylpropanol, to one-halfo[ a disulfidemolecule. In the degeneratethiolate-disulfide p-toluenesulfonylchloride, sodium deuteroxide (40% solutionin D2O), interchange,if we denote the rate of exchangeseen by NNIR as -dIRSSR]idt -d[RS-]/dt potassiumdeuteroxide (40% solutionin D2O),sodium hydrogen sulfide, and for the disulfideand thiolate,respectiicly, -dlRSSRj = (l/2)klRS-ltRSSRl -dIRS-]/dt = potassiumrerl-butoxide, thiolacetic acid, diethyl malonate,and me- we obtain f dt and thylmagnesiumbromide (a 3 M solutionin diethylether) uerc all pur- i([RS-][RSSRl. lf rps and rprr* are lifetimeof the thiolatcand di- chasedfrom Aldrich. Sodium l-butanethiolatewas purchascdlrrtnt suifidc.then Fluka. Proteindisulfide isomerase (8.C, 5.3.1.1)wasa gifr from Gcn- LllR\ 1,1dt = r,l )[RS ] = t[RS-][RSSR] (8) zyme. /'s 0.995 for 7-9 was taken as 0.| 5 s (calculatedfrom the reciprocalof the productof z- f points (Figurc 3).48 The determinationof ,\F1*and .\S* by dynamic and the peakwidth (Hz) at half heightof the singletpeak corresponding NMR spectroscopyis known to not be very accurate.23We haveesti- to the methylgroups of l-BuOH). In our experimenrs.the NMR Iine- mated the errors in ,\H+ and lS* b"'-calculating the standarddeviations shapeswere not very sensitiveto T2* for two reasons:(i) -\r,(- 100 Hz at 907cconfidence for the slopeand intcrceptoi the plot of ln (/c/?') vs at 300 MHz) )) llTz* (-6 Hz), and (ii) we avoidedverv fastand verv I slow exchangeregimes in our study by manipulatingthe concentration lT." Preparationof SamplesContaining Thiolates and Disulfidesfor Dy- of thiolateand disulfideand the temperature.We resimulatedthe plots namic NMR Spectroscopy:General Procedure. The solventwas deox- for HOCHTCH2S-K+/(HOCH2CH2S)2interchange in D2Owith correct y'genatedby bubblingargon through it for )30 min if the samplewas valuesof T2* [0.15s (HOCH2CHzS-K*),0.26s ((HOCH2CH2S))] and to containthiolate at )0.1 M concentration.lior a samplecontaining found the plotsto be similar to thoseobtained previously with 12* = 0.15 <0. I M concentration of thiolate,the solventwas degassed with at least s. For variousvalues of rate constants,simulated NMR plots were four freeze-pump-thawcycles. The NMR tube was stoppereduith a generatedfor the frequency range 540-l 260 Hz. The rate constantswere rubberseptum, and the top of the NMR tube was sealedwith paraffin inferredby comparisonof the simulatedplots with the experimentalplots wax (or ApiezonW wax ior high-temperatureNMR measurements)to to the best fit by eye. The coalescenceapproximation lr = I k' = f preventoxidation of the sample.aeSealing by paraffin wax was espe- cially usefulin rate studiesin mixturesof DMSO-d6 and D2O;the wax (42) van Geet,A. L. Anal. Chem. 1968,40,2227-2229. sealcould be broken easily(to introducesmall amountsof deoxygenated (43) van Geet,A. L. Anal. Chent.1970,42,679-680. D2O) and replaced.Potassium /erl-butoxide was usedas baseto generate (44) Raiiord, D. S.; Fisk, C. L.; Becker,E. D. Anal. Chem.1979,5t, thiolatefrom thiol in DMSO-d6and DMF-d7. Sodium l-butanethiolate 2050-2051. was useddirectly for one experimentin DMF-d7. Potassiumdeuteroxide (45) Ammann,C.; Meier,P.; Merbach,A. E. J. Masn. Reson1982,46. 319-321, (46) Thiola_tesare oxidizedrapidly by oxygen;the oxidationis catalyzed (48) The plotsof (ln klT) vs (I lT) for the thiolate-disulfideinterchange by metal ions.7 reactionsof sodium l-butanethiolatein DMF-d? and potassium2,2-di- (47) We prepared2,2-dideuterio-2-hydroxyethanethiol by reduction of methylpropanethiolatein DMF-d7 are also linear. The valuesof A,St are methylthioglycolate with lithiumaluminum deuteride. The rateof the thi- negativein bothcases; the values of .\Hf and JS+, however,could be inac- olate/disulfideinterchange for sodium2,2-dideuterio-2-hydroxyethanethiolate curatebecause the chemicalshifts of methyleneprotons of the thiolateand and its disulfidewas the sameas that for sodium2-hydroxyethanethiolate and disuliidevary with temperaturein both cases.The simulationsof the NMR its disulfide. The peakcorresponding to the methyleneprotons in sodium spectrawere done by varyingboth the rate constantand Ay. 2,2-dideuterio-2-hydroxyethanethiolateappeared as a singletin the 300 MHz (a9) The resultswere the samewhether the top of the NMR was rH tube NMR spectrum(JcHrco, - 0.15,/cHrcsr). sealedwith wax or flame-sealed. Thiolate- Disu lfi de I nt erc hange J. Am. Chem. Soc., Vol. ll2, lio. 3, 1990 I 195 and sodiumdeuteroxide were usedas basesfor generationof thiolatein Table III. The lnfluenceof PotentiallyCatalytic Additiveson the Dro. Rate of Thiol-Disulfide Interchangein Water at pH 7 Preparation of a Sample in D2O: RepresentativeProcedure. All flasks L L cat/ cat/ and the NMR tube werestoppered with rubbersepta and were flushed ,\ a additive vuncat additive funcat with argon beforeuse. Mercaptoethanol(0.238 g, 3.04 mmol), bis(2- hydroxyethyl)disulfide (0.230 g. 1.49mmol) and D2O (0.30mL) were papain placedin a flask. The contentsof the flask were degassedwith four cF3cH2sH freeze-pump-thawcycles. In anotherflask, NaOD (40 wt % solution in D2O, 0.306 g, 2.98 mmol) was deoxygenatedby bubbling argon thiamine hydrochloride throughit for 30 min. The solutioncontaining mercaptoethanol and the correspondingdisulfide was transferredto the flask containingsodium r- (KI) I deuteroxidesolution by cannulaunder argon. The resultingsolution was (NarSOr) I transferredto an NMR tube by cannulaunder argon. The top of the SOrF NMR tube wassealed with ApiezonW wax. The total volumeof the CNS- (NaCNS) I solutionwas 0.79 mL. The solutionwas 3.78 M in thiolateand 1.89M POrS:- (Na3PO3S) I in disulfide. The rH NMR spectrawere recorded at varioustemperatures Zn2+ I at 300 MHz (pulsewidth = 4 ps, pulsedelay = 4 s, acquisitiontime = [Zn(OAc)2] 2,1 s). The time delaybetween pulses was greater than the valueof f1. Cd2+[Cd(OAc)r] b The valuesof I, weredetermined by the inversion-recoverymethod as 1.9s (CHTOH) and 1.3s (CH2S-)for sodium2-hydroxyethanethiolate Fe2* (FeSoa) I in DrO and2.2 s (CH2OH) and 2.1 s (CI12S)ior bis(2-hldrorrethrl) Ag+ (AgNO3) b disuliidein D20. Preparationof a Samplein DMSO-du: RepresentatireProcedure. '\ll Co2+ b flasksand the NMR tube werestoppered with rubberseptrr and ucrc [Co(N03)2] I flushedwith argonbefore use. Mercaptoethanol(0.0019 g. 0.06-1mmol). alumina bis(2-hydroxyethyl)disulfide (0.0042 g. 0.027mmol). and D\1SO-r/n (0.50mL) wereplaced in a flask. The solutionuas degasscd*ith iour freeze-pumpthaw cycles.The degassedsolution uas transferrcdto thc Z" .rctivatcdcarbon I NMR tube by cannulaunder argon. In anotherflask. potassium lerl- PrC H.Ctl.COO- K+) j I butoxidc(0.0089 g.0.079 mmol)rras rreighed in the glovebor.Frorn r (,4." solutionof DMSO-rCuthat had beendeoxy'genated previousll b1 bubbling (\{eo)3P I argonthrough it for 45 min.72 iiL wastransferred by a gas-tightsy'ringe /-\ ,I to the flask containingpotassium terl-butoxide. From the resulting \",)-sn potassiumterl-butoxide solution (1.10 M), 50 pL wastransferred by a gas-tightsyringe to the NMR tube. The top oi the NMR tube was Proteindisulfide isomerase f sealedwith paraffinwax. The solutionwas 49 mM in disulfideand 100 [\ 1 mM in thiolate. TherH NMR sDectrawere recordedat 500 MHz. \..AsH After the tH NMR spectrahad beentaken, the solutionwas quenched ' interchangeof 2-carboxy-1,3-propanedithiol and by additionoi DCI (10 rrL oia 37 wt % solutionin D2O).50'5rTherH The thiol-disulfide N M R spectrumof the resultingsolution showed that no oxidation(<5To) bis(2-hydroxyethyl)disulfide (4 mM each) in 50 mM aqueousphos- of thiolateto disulfidehad occurred. phate buffer at pH 7 was followed by monitoring the formation of 4- = Screening for Cataly-sisin Water Using UV Spectroscopy. The carboxy-1.2-dithiolaneat 330 nm; t'"",/uuncar apparentrate of cata- thiol-disulfideinterchange of 2-carborl-I .3-propanedithiols2and bis(2- lyzedreaction/apparent rate of uncatalyzedreaction. In eachcase the = hydroxyethyl)disuliide uas monitoredat 330 nm: the product ,1- additive was present at a molar concentration l}Vo that of 2- carboxy-1.2-dithiolane(r = l-1-1V I cm r) isthe onlr speciesthat absorbs carborl-1.3-propancdithiol.Papain and proteindisulfide isomerase = at 330nm. Separatestock solutions of 2-carborr-l.-j-propanedrthiol and \\crc prescntirt ntolar conccntrations l% and 0.06Vnthat of 2- - b 'lH bis(2-hydroxyethyl)disuliide (each 8 mM)*ere preparedrn dcorrgcn- srf br)\\ I ..1-pfr)firncdrthiol Prccipitateuas observedin solution. = - (ref dReference atedaqueous phosphate buffer solution (50 m!1. pH 7l .\ quartzcu- \\1 R.i.rlrrrhrrricdr,.r r"n.rr I -i-1), 54. vettewith the materialto be testedas catalyst (1.2 pmol) uas stoppered plI p- with a rubber septum and flushed with argon. The solution of l- .{minothiophen<,rlin \queous Solution at 7. \ ilask containing (tl.(ttt59 \\ils with carboxy-1,3-propanedithiol(1.5 mL) wasadded by a gas-tightsyringe arninothiophcntrl g. 0 0-1rrrtrriol) stoppcred a rubber \lethrlcnc (0.9a and the cuvetteequilibrated at 25 oC. The bis(2-hydroxyethyl)disulfide septumand tlushcdrrrth argon. chloride mL) that had solution(1.5 mL) wasadded to the cuvettewith a gas-tightsyringe, and beendeoxygenatcd previousll bi bubblingargon through it for 30 min the increasein absorbanceat 330 nm was monitored. The initial con- was addedto generatea stocksolution of p-aminothiophenol.The so- lutionwas estimated by Ellman'sanalysis to be 5l mM in thiol.56 By centrationsin the assaymixture were [2-carboxy-1,3-propanedithiol]= using a gas-tightsyringe, 20 pL of this solutionwas added to a flask 4 mM, Ibis(2-hydroxyethyl)disulfide] = 4 mM, and [catalyst]= 9.4 mM. The uncatalyzedapparent rate constantwas 2.3 M-r min-r (see stopperedwith a rubber septum,and the methylenechloride was removed TableIII). by evaporationunder argon to leavep-aminothiophenol as a solid residue. (0.0032g, When Cd2+,Ag+, and Co2+were tried as catalysts,precipitation oc- Into two flaskswere weighedbis(2-hydroxyethyl) disulfide (0.0031 g, curred. When aromaticthiols were used as catalysts, an initial increase 0.02 mmol) and 2-carboxy-1,3-propanedithiol 0.02 mmol) in absorbancewas observeddue to the formation of mixed disulfide separately.The two flaskswere stopperedwith rubber septaand flushed gas-tight betweenthe aromaticthiol and mercaptoethanol.5sThis interferencewas with argon. To eachflask wasadded by usinga syringeI mL avoidedby incubatingthe aromaticthiol (catalyst)with bis(2-hydroxy- of DrO (50 mM phosphatebuffer, pD 7) that had beendeoxygenated ethyl) disulfidein the cuvettefor l0 min beforeaddition of 2-carboxy- previouslyb1' bubbling iirgon through it for 2 h. The contentsof the two 1,3-propanedithiol. flaskswere mixed b) transfer throughcannula under argon; 4 min after rH NMR Assay of Catalysis of Thiol-Disulfide Interchangeby p- mixing, I mL ol the solutionwas transferredto the flask containing p-aminothiophenolunder argon. The initial concentrationswere = [(HOCH2CHzS)z],ni,i"r= l0 mM, IHSCH2CH(COOH)CHzSH]ini,r"r (50) Lowe,O. G. /. Org. Chem.1975,40,2096-2098. l0 mM, and IHSC6HaNHz(p)lint,1"1= 1 mM in the catalyzedsolution. (51) The additionof excessDCI wasavoided during quenching because The catalyzedreaction was quenched after l3 min by adding l5 pL of thiolsare oxidized to disulfidesin DMSO in the Dresenceof excessHCI:50 we DCI solution (37 wt Vo in D2O), and the uncatalyzedsolution was preparedbis(2.2-dimethylpropyl) disulfide by oxidationof 2.2-dimethyl- quenchedafter 14 min by addition of 15 pL of DCI solution. The propanethiolin DMSO in the presenceof HCI (see ExperimentalSection). acid-quenchedsolutions were then transferredunder argon to NMR (52) 2-Carboxy-1,3-propanedithiolis a useful substrate lor theassay be- tubes stopperedwith rubber septa. The top of the NMR tubeswere causeit is an easilyprepared crystalline solid, and it doesnot smellstrongly. (53) The observedrate enhancement of 1.7times comDares well with the sealedwith paraifin wax. The ratesof the reactionswere estimatedfrom theoreticalvalue of 1.6calculated from eq 3. the peak areasfor the methylenepeaks of mercaptoethanoland its di- (54) Proteindisulfide isomerase does not catalyzereactions of dithiothreitol sulfidein the rH NMR spectrum. with oxidizedglutathione: Creighton, T. E.; Hillson,D. A.; Freedman,R. B. J. Mol. Biol. 1980,I42, 43-62. (55) Campaigne,E.;Tsurugi, J.; Meyer,W. W. J. Org.Chem.1961,26, (56) Ellman,G. L. Arch.Biochem. Biophys. 1959, 82,70-77. Habeeb, 2486-2491. n. F. S. A. MethodsEnzvmol.1972. 25.45'7-464. ll96 J. Am. Chem.Soc.. Vol. II2. No. 3. 1990 Singh and Whitesides

SchemeI solutionwas stirred at room temperaturefor 20 h. The reactionmixture testednegative with Ellman'sreagent, thereby indicating the complete Eto?c- co,Et CO?H COrH \./ I oxidationof thiol. The reactionmixture was poured into 200 mL of aq HBr I 1 Acsrr ac Na,CQ. 5 'C | | ------\- ^\ .,,"...... ,...-....-...... "..."...... */-'\ ice-coldwater. The resultingsuspension was warmedto room temper- tl I I oC,2 OH OH ature and extractedwith petroleumether (30-60 x 75 mL). The petroleumcthcr layer wasdried (MgSOa) and concentratedat reduced pressure yield (0.29g,827a): 'H NMR (CDCl3)6 corH co?H to a colorlessliquid t- I- 2.76(s, 2 H). 0.99(s. 9 H).5e -AcSFI -^.- ac

-91-l 20605-01-0;2-carboxy-1,3-propanedithiol,20605-01-0;3-(acetylthio)-2-methylpropylthiolate,124351 ; potassium3-hydroxy-2,2-dimethyl- - methenyl-I -propanoicacid, 56I 40-22-8;sodium 2-hydroxyethanethiolate. propylt hiola te, | 24357 -9 2-2: bis(2-hvdroxyethyl ) d isulfide, 189 2-29 | ; -02- 31482-l I -41potassium 2-hydroxyethanethiolate, 7450-3 1 -9: sodiumbu- 4-carbox,v- l, 2-di t hiol a n e. 2224 4: sodium 2'2-d ideuterio- 2- hydroxy- tylthiolate,4779-86-6: potassium butylthiolate, 26385-25- I ; potassium cthanethiolare.124357-93-3: bis(2,2-dideuterio-2-hydroxyethyl) disulfide, 2,2-dimethylpropylthiolate,124357 -90'0; potassium 2-hydroxy-2- | 24357-94-4: butyl disulfide,629-45-8; selenylbenzene' 645-96-5.