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.