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Home , Disulfide, Dithiol, Hydroxy group, Selenol, Thiol

CHAPTER1 3

Thiol-disulfideinterchange

RAJEEVASINGH lmmunoGen,lnc., 748 Sidney St.,Cambrtdge, MA 02139 and

GEORGEM WHITESIDES

Departmentof ,Harvard university,12 oxford St.,Cambridge, MA02138

I. INTRODUCTION 634 II. METHODS USED IN FOLLOWING - INTERCHANGE 636 A. Spectroscopic(UV, NMR) Assays 636 B. Enzymatic Assays 631 C. AssaysBased on Chromatography 638 III. MECHANISM 638 A. Products 638 B. Dependenceon Solution pH, and on the pK" Valuesof 638 C. Kinetics 639 1. Rate law 639 2. Br/nsted relation 640 3. effects 641 a. Steric 641 b. Acidity 642 c. Charge 642 d. bonding 642 e. Reactionsinvolving cyclic 643 4. effects 643 5. Gas-phasestudies 645 6. 645 7. Comparison with selenolate-diselenideinterchange 646 D. Transition State Structure 646 E. TheoreticalCalculations on Thiol-Disulhde Interchanse 646 F. MechanisticUncertainties . . 641

SupplementS: The ,hr^irnrv of sulphur-containinllfunctional aroups Edited by S. Patai and Z. Rappoport O 1993John Wiley & Sons Ltd 633 634 R. Singh and G. M. Whitesides

IV. EQUILIBRIUMIN THIOL DISULFIDE INTERCHANGE - REACTIONS 6-l A. Equilibria Involving Monothiols 641 B. Equilibria Involving a,co- 648 V. APPLICATIONSOF THIOL-DISULFIDE INTERCHANGE IN 654 u. CONCLUDING REMARKS 655 VII. ACKNOWLEDGMENTS 656 VIII. REFERENCES 656

I. INTRODUCTION Thiol-disulfrde interchangeis the reaction of a thiol (RSH) with a disulfide(R'SSR'), with formation of a new disulfide (RSSR')and a thiol (R'SH) derived from the original disulfide(equation 1). The reactionis unique in organic chemistry:although it involves the cleavageand formation of a strong (the S-S bond; bond energy ca 60 kcal mol 1),it occurs reversiblyat room temperaturein water at physiological pH (ca 7)1-a.The reaction is moderately fast: a typical value of the observed rate constant is ca 10M-1min-r at pH 7 and room temperatures.The half-lifefor the reactionis ca 2h for mM concentrationsof thiol and disulfidein aqueoussolution at pH 7. and for alkanethiolswith normal valuesof pK^ (ca9-10). The yield of the reactionis quanti- tative if side-reactions-such as air oxidation of thiol to disulfide,and cleavageof disulfidebonds at high pH-are preventedr. RSH + R,SSR,= RSSR,+HSR, (I) - Thiol-disulfide interchangeis an Sr2 reaction.Thiolate anion (RS ) is the active if solution is made acidict.The reaction nucleophileand the reactioncan be stopped the -zo. is overall second-order:first-order each in thiolate and in disulfide(equations ) 4f In this chapter, we will distinguish between rhlol-disulfide interchange(the overall observedprocess, equation 1),and thiolate-disulfideinterchange (the reaction of thiolate anion with disulfide,equation 3). The two processesdiffer according to the extent to which thiol is dissociatedto thiolate anion under the reactionconditions. RSH = RS + H+ (pK^*t") (2) _SR, RS_+ R,SSR,= RSSR,+ (3) * H + SR' = HSR' (pK"*'tt) (4) 2s Thiolate-disulhde interchange is base catalyzed2l and involves the backside nucleophilicattack of thiolate anion along the S-S bond axis of the disulfide26.It showsless sensitivity to solventthan most S"2 reactionsinvolving oxygenand nitrogen nucleophilesls.The rates of thiolate-disulfideinterchange in polar aprotic (DMSO, DMF) are fasterby a factor of approximately 103than rates in polar protic solvents(water, ). For comparison,the rate enhancementfor oxyanions(which s aremore highlysolvated than thiolates)1on moving from protic to polar aproticsolvents is 106-107. Biologicallyimportant moleculescontaining the thiol or the disulfidegroup are widely distributedin naturel'2,and the unique position of thiols and disulfidesin biochemistry 3. hasbeen reviewed in severalexcellent books and articlest The thiol-containingamino -is presentin proteinsand ;examples include glutathione2- and trypanothione2s-30.Several cofactors coenzymeA, dihydrolipoamide, (-O3SCH2CH2SH)3r containthe essentialthiol functionalityl'2.The disulfidebonds 635

= 0 =f - .F -10 c 50 a) lTl q q) C LLJ 30

20

r0

0

-r0 0 30 60 90 120 150 180 Dihedralangle (HSSH or CSSC) ')of FIGURE 1. Relativeenergy lkJmol HSSH and MeSSMeas a function of the dihedralangle ({, RSSR):( ) MM2 (85);(--) 6-31G* [M. Aida and C. Nagata, Theor. Chint. Acta,70,73 11986)J,(- -) SCF and MP2 [C. J. Marsdenand B. J. Smith,J. Pht's. -) Chem.,92,347(1988)l; (- - OPLS [W L. Jorgensen,J. Ph1,s. Chem.,90, 6379 (1986)1. Reprinted with permission from Reference16. Copyright (1990)American Chemical Society 636 R. Singh and G. M. Whitesides

betweencysteine residues are important tertiary and quarternarystructural clcntcnrr in (especially extracellular proteins) such as immunoglobulins,, horntor.rc.. procollagenand albuminl-3. The cleavageof the disulfidebond(s) of many proteins1e .g. deoxyribonucleaseI) resultsin loss of activity32.Thiol-disulfide interchangemay plal a role in metabolic regulation of enzymatic activities3'::':+.76. activitiesof several chloroplastenzymes such as fructose-1,6-biphosphatase,NADP-, sedoheptulose-1,7-biphosphatase, NADP--3- dehydrogenase and phosphoribulokinaseare enhancedby reduction of their specificdisulfide bonds by photogeneratedreducing equivalents transferred via ferredoxinand thioredoxin3''rs-'17. The cleavageof disulfide bonds of B-adrenergicand other cell surfacereceptors by thiols activatesthe receptorin a manner similar to binding of agonist3s'3e. The thiol is essentialfor activity of many enzymesl'2such as thiol (, ficin, bromelain)a0,B-ketoacylthiolaseal-a3, enolaseaa, creatine kinase,glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and adenylate kinaser-3'11.These enzymes are inactivein mixed disulfideform, and can be reactivated using strongly reducing thiols (e.g. , lV,ly''-dimethyl-N,lV'-bis(mercapto- acetyl)hydrazine)l1'18.A thiolate anion may be involved in methanogenesisby 2elle couplingwith a Ni cofactorin methyl coenzymeM reductase3t.The reactivethiol functionalityis maskedas a trisulfidein the potent DNA-cleavageagents-calichemycin and esperimicin4s-48.The rate-determiningstep in the unmaskingof theseDNA-cleavage agentsis the cleavageof the trisulhdeby a thiol (e.g.)48. The thiolate anion formedundergoes intramolecular Michael-type attack on an enonesystem, which in turn facilitatesthe ring closureof the enediynemoiety to form the reactivearomatic diradical. Thiolate anion is a strong nucleophileand a good leavinggroup becauseof its high polarizability and low degreeof solvation.The thiol group is lessstrongly hydrogen- bonded,and the thiolate anion is lesssolvated, than the anion. The value of pKu of the SH group of butanethiol in DMSO (ca 17)is 7 units higher than in water (ca 10);the correspondingpKu value for the OH group of is 12 units higher in DMSO (ca28) than in water (cctI6)ae-s1. The optimum CSSC dihedral angle in the disulfide bond is ca90 (Figure l). The energy barrier to rotation around CSSC bond is c'u7.5 kcalmol-1 s2. Five-membered cyclic disulfidesare strainedand have a CSSC dihedral angle of ca 30. s3. shows concatenation:molecular sulfur S, exists as an eight-memberedring; organosulfur compoundscontaining tri- and polysulfidelinkages (RS"R) are found in natureand have beencharacterizedsa's s. This chapter focuseson the physical-organicaspects of the thiol-disulfide interchange reaction.The biochemicalaspects of the reaction are only lightly touched upon here, and we recommendReferences 1-3 to the readerfor a detailedreview of the biochemistrv.

II. METHODSUSED IN FOLLOWINGTHIOL-DISULFIDE INTERCHANGE

A. Spectroscopic(UV, NMR) Assays

The kinetics of thiol disulfide interchange reactions involving formation of a chromophoric thiolate are convenientlyfollowed by UV spectroscopy.The reactionof thiolates with excessEllman's reagent [EllS-SEll, 5,5'-Dithiobis(2-nitrobenzoicacid)] is usedfor quantitativeestimation of thiol by measuringthe absorptiondue to Ellman's thiolate(EllS ) at 412nm (equation5)so-su. Reactions of thiols with 4,4'-dipyridyldisulfide and 2,2'-dipyridyl disulfide generatechromophoric thiols: 4-thiopyridone (r3z+n-: t 19600M cm- 1)and 2-thiopyridone(e.o.,_ : 8080M- 1cm t) respectivelyso-t'u. The kinetics and equilibria of reactions involving cyclic five- and six-membered disulfidescan be followed at 330nm and 290 (or 310nm) respectively.Five-membered 13. Thiol-disulfide interchanse 637 RS_.,;b,_, el'o*o,

Ellman's Reagent EIIS-SEII (5)

---* RS-S + OrN

EIIS E4r2n^:13600 M tcm-t

cyclic disulfides absorb in the UV region at 330nm (e:147M tcm t) and six- membered disulfides absorb at 290nm (e: 290M tcm t) or 3l0nm (e:110M I a-- tys,s:,63-6s. 1H NMR spectroscopycan be used to determinethe position of equilibria in thiol disulfideinterchange reactionsro'13'18, and to follow the kineticsof the reactions(either in the reactingsystem or after quenchingwith acid)18'6668. This method is usefulwhere the methyleneprotons e and p to the sulfur in the reactantsand products differ in chemical shift and can be integratedaccurately. Dynamic lHNMR lineshapeanalysisls'1'and spin-transfermethodse'6e have beenused to determinethe rate constantsof degenerate intermolecularthiolate-disulfide interchangereactions: RS- + RSSR: RSSR+ RS Analysisof 1H NMR lineshapes,where the resonancesare exchange-broadened,is useful for determining the ratesof fast degenerateintermolecular interchangereactions between thiolatesand disulfides(ft. c:al0 107M t ,- tlts'tz. 16. spin-transfermethod between pairs of exchangingprotons and (a to sulfur) is applicablefor slower rates (ft. ca2-60M-1 s-r1o'oo.

B. Enzymatic Assays The kinetics of reduction of oxidized glutathione (GSSG) by thiols is conveniently followedsusing a fastenzymatic reaction involving glyoxalase-I.The glutathione(GSH) that is releasedis convertedto S-lactoylglutathione by reactionwith methylglyoxalin the presenceof glyoxalase-I(GX-I), and the appearanceof S-lactoyl glutathione is followed spectrophotometricallyat 240nm (e:3370M-r cm 1) (equations6 8)s.The ratesof reactionsinvolving aminothiols cannot be determinedby this method because they react rapidly with methylglyoxal and form speciesthat absorb strongly at 240nm and thus interfere with the spectroscopicmeasurements. This assayis subject to errors due to oxidation of thiol groups if air is not carefullyexcluded. RS_+ GS-SG = RS SG+ GS (6) GS- + CH3COCHO = GS-- CH(OH) COCH. (7) GS-CH(OH) COCH. + GX-I = GS-CO-CH(OH)CH1 + cX-I (8) The equilibria of thiol disulfide interchange reactions between ry,o-dithiolsand lipoamidecan be determinedconveniently by the addition of lipoamide dehydrogenase and NAD*. NADH is produced;this compound is covenientlymonitored at 340nms't0 F

638 R. Singh and G. M. Whitesides

.SH *CoNH2 R ,\/ \sn S-S (9) a? -1tlr ftcoNH2 \s HS SH

&coNH2 \ / + NAD' HS SH (l0) llpoamrde dehydrogenase ,^ -\---r- r *CONH2 / +NADH + H- S-S

(equations9 and 10).The equilibrium constantsfor e,to-dithioland lipoamide are then calculatedfrom the measuredequilibrium constant of the z,o- with respectto NAD* and from the standard value of the equilibrium constant for linoamide and NADH.

C. Assays Based on Chromatography

The kinetics and equilibrium constants of thiol-disulfide interchange reactions involving cysteinederivatives or peptidescontaining cysteinehave been determinedby HPLC71 and gel-filtrationchromatographyl2 on the reactionmixtures after quenching with acid.The equilibrium productsof the reactionof glutathioneand cystinehave been separatedby -exchangechromatographyT3 or by electrophoresisof the 3sS-labeled compoundsTa. of the equilibrium mixture of alkanethiols and disulfideshas been usedto estimateequilibrium constantsTs.

III. MECHANISM

A. Products

Thiol disulfideinterchange of a monothiol (RSH) with a disulfide(R'SSR') involves multiple equilibria(equations 1 and 11):the reactionproducts include all possiblethiols (RSH and R'SH), symmetricaldisulfides (RSSR, R'SSR') and mixed or unsymmetrical disulfides(RSSR').

RSH + RS-SR,= RS- SR+ R,SH (l l)

B. Dependence on Solution pH, and on the pK" Values of Thiols Becausethe thiol-disulfide interchangereaction requires thiolate anion, the observed rate of reaction(and, in systemsin which the participatingthiols have differentvalues of pKu, the observedposition of equilibrium)depends upon the pH of the solution and 13. Thiol-disulfide interchange 639 the extent of ionization of the various thiols. For a thiol of pK" 10,only 0.1?; of thiol is presentas thiolate at pH J, and ll" of thiol is in thiolate form at pH 8; the observed rate constantof the thiol-disulfideinterchange at pH 8 is thereforel0 times fasterthan that at pH 7.The thiolatecan be generatedby addition of base,e.g. potassium /-butoxide, in polar aprotic solvents(DMSO, DMF)1s. Thiol is a much weaker nucleophilethan thiolate, and direct reaction between thiol and disulfide has not been observed.The reaction of thiolate with disulfide is effectively quenched by addition of acid and conversionof RS- to RSH.

C. Kinetics

1. Ratelaw The thiol disulfideinterchange reaction is overall second-order:first-order in thiolate and in disulfide6'7.For a representativereaction of a thiolate(RS )with Ellman'sdisulfide (EllS-SEll,equation 5) the rate laws are given by equatiorrsl 2 and 13. The rate constant ft*, derivedfrom equation 12,based on the concentrationof the thiolate,is independent of pH; the observedrate constant koo.o, based on total concentrationof thiol (equation13), dependsupon pH. The value of the rate constant ft*, is usefulfor interpretationsof reactivity(such as Br/nsted correlationsof rates or equilibrium constantswith values of pK" of thiols).The calculationof the observedrate constantkou.a 3t the pH of reaction is straightforwardfrom equation 13 usingthe valueof the total thiol presentin solution, [RSH],o,u,: IRS ] + [RSH], and is usefulfor predictingrates at the samepH. The two rate constants koo.oand ft*, are related to each other by equation 14 and can be interconverteduding the valuesof the pK" of thiol and the pH of solutions'o.Table I liststhe valuesof rate constantsfor representativethiol-disulfide interchangereactions in water.

(dIEllS fldt): kRS[RS-] [EllS-SEll] (t2) (dIEllS ]/dr) : ft"b,,r[RSH],.,"r[EllS-SEll] (13) k*, : koo.u(l+ lo'(a-PH) (14\

TABLE 1. Representativerate constantsof thiol-disulfide interchangereactions in water

Reactants pKo k*, ftnb.,l" Temp I ') - I ') Thiol Disulfide (thiol) (M min (M min ( c) Ref.

Mercaptoethanol Oxidized glutathione 9.6 3.4x 103 It.7 30 5 3-Mercaptopropa- Oxidized glutathione 10.6 1.2x 104 3.2 30 5 noic acid Mercaptoethanol Ellman's disulhde 9.6 1.5x 107 3.7x 104 30 6 Ellman's disulfide 10.5 6.4x 101 2.0x 104 25 7 Ellman'sdisulfide 6.6 1.3x 106 9.6x 10s 30 6 Dithiothreitol Papain-S-SCH-., 9.2 5.3x 10s 3.3x 103 30 11 Dithiothreitol Oxidized mercapto- 9.2 3.7x 102 2.3n 25 18

'Values of kon"oare at pH 7. hThe value of the rate constant is corrected statisticallyfor the presenceof two thiol groupson dithiothreitol. Y

640 R. Singh and G. M. Whitesides

2. Brdnsted relatron

The log of the rate constants(/<*. ) of thiolate disulhdeinterchange reactions follows a Br/nsted correlation with the valuesof pK" of the thiols. The Br/nsted plot for the reaction of thiolates with oxidized glutathiones has a Br/nsted coefficient(slope), f",.. of 0.5. The Br/nsted coefficientsare also ca 0.4-0.5 for the reaction of thiolates with Ellman's disulfide6'7. and thiolates show separatecorrelation lines with Ellman'sdisulfideT, but show a similar slope,fnu" :0.5. An aromaticthiolate reacts with Ellman's reagentTor the mixed disulfideEIIS-SCH2CH2CH2OH76 at a rate fasterby a factor of 6 than an aliphaticthiolate of the samepKu. The higher reactivityof aromatic thiolatesin comparisonto aliphatic thiolatesis probably due to greater softness(and weakersolvation)of the formerT'8.Br/nsted correlationshave also beenreported for the reaction of thiolates with 4,4'-dipyridyl disulfideseand 2,2'-dipyridyl disulhde6o. A Br/nsted correlation(equation l5) for thiol disulfideinterchange (equation l6) has been assembledempirically by systematicexamination of the influenceof pKuRSHfor the nucleophilic(nuc), central (c) and (lg) thiols on the rate of the reaction. Equations 17 and 18 represent different types of fits to data (ft*, is in units of lmin-1)s. M EquationlT representsthe bestfit to all the availabledata, although it is suspectbecause the valuesof pn,. andBr"are not obviouslycompatible with a transition statewith chargesymmetrically distributed between the terminal sulfur .The data included in the correlation are taken from a range of studiesand are not necessarily directly comparable.Several different setsof Br/nsted coefhcientsgive similar fits to the observed data (equations17 and 18); this observation suggeststhat the Br/nsted coefficientsare not sharply defined.We recommend equation l8 for general use based on the symmetry of the valuesof /inu"and p,r. The value of f" for the central thiol has beenestimated as ca -03 to -0.4 from a limited set of data for reactionsof RS- with EllS-SEll,RS-SEll, and HOCH,CH,CH,S-SEll76.

logk*, : C + f,,"pKun'" *f"pKu'* p,rpK"'* (15) r\ - R'u"S + R'S-SRrg Rnucs SR"+ SRrg (16)

logk*r :7.0* 0.50pK,"u"-0.27 pKu'-0.73pKurs (17) log ft*r- : 6.3* 0.59pK,'u" - 0.40pKu' - 0.59pK"rs (18)

The data on which equations17 and l8 are basedcover a range of different(and perhapsnot directly comparable)thioland disulfidestructures. A study of the Brpnsted coefficientsof the nucleophilic,central and leavinggroup thiols usinga carefullylimited and consistentset of thiols and disulfidesu'oLrld be usefulmechanistically. In the absence of unambiguouslyinterpretable data, these correlations(equations 17 and l8) should be consideredas kinetic modelsfor thiolate-disulfideinterchange reactions. Although they do not have an unimpeachablemechanistic foundation, they are useful in predicting rate constants (ft*r ) in water. The value of kp" can be converted to the observedrate constant(ft.0.0) at the pH of reactionusing equation 14. The optimum value of pK" of the nucleophilicthiol for the maximum observedrate (koo.o)of thiol-disulfide interchange at a given pH can be predicted by a Brdnsted correlation(equation l9)t'u.The optimum value of pKu of the nucleophilicthiol (based on the assumptionof pnu":0.50)is thereforethe valueof the pH of the reactionmixture; at pH 7 a nucleophilic thiol of pKu: 7 will show the maximum observed rate of thiol-disulfideinterchange (Figure 2). This predictionclosely matches the observedrates of thiol-disulfide interchanges'6and is usefulin designingreagents that reduce disulfide bonds rapidly at pH 718'20.For a thiol of pKu>>pH, only a small fraction of the total thiol is presentas thiolate;for a thiol of pKu <

- a 0.8 E + 0.6 aI tr o.4 .nI E

il o.2

G

0.0 56789 eKo FIGURE 2. Comparison of plots of the log ol rate constant of the thiolate-disulfide interchangereaction (1ogk*, , -) vs pK" of a nucleophilic thiol, and of the degreeof dissociation at pH I (), ----) vs pKu of thiol. The values of rate constant (k*, ) are calculated using equation 17, assuming the value of 0.u" as 0.5, and the value of pKu of both the central and leaving group thiols as 8.5. The observed rate constant (koo.o)in terms of the total concentrationof thiol and thiolate in solution is given by kou,o:9lrns- nucleophilicityis low. A thiol of pK": pH balancesthe proportion of thiol presentas thiolate and adequatenucleophilicityof the thiolate. pH: pK"+ log[(1- p)lp] (le)

3. Subslifuenteffects

a. Steric. The steric effectis most pronounced when all three thiols in the transition state are fully substituted at the I to sulfur. It is significantly large even when two of the three adjacentthiols in the transition state are fully substituted at the carbon r to sulfur. The rate constant for reaction of r-butyl thiolate with bis(r-butyl) disulfide (k. :10-7M 's 11inbutanol is ca 106-foldslower than that for 1-butylthiolate with - - bis(1-butyl) disulfide (/c. : 0.26M I s 1)24.The observedrate constantfor the reaction of (-OOC-CH(NHJ)-C(CH3)2SH) with the mixed disulfideof penicil- - - 1.; lamine and glutathioneat pH J.4 (l;"b,d: 0.00047M 1min is ca 10s-foldslower than that of penicillaminewith glutathione disulfidewhich contains no substitution n to sulfur (ko6.6:37M lmin-1166.The observedrate constantfor the reactionof glutathione lmin t) with penicillamine disulfide (0.012M is also ca2000-fold lower than for - 1166. glutathionewith mixed penicillamine-glutathionedisulfi de (27M 1min The equili- brium constant for the formation of bis(r-butyl)disulfide is small in the reaction of r-butyl thiol with mixed 1-butyl /-butyl disulfide,i.e. the formation of bis(r-butyl)disulfide is disfavoredTs'77. Steric effectsare small for alkyl substitution at carbon p to sulfur: the rate constant for degeneratethiolate disulfide interchange of neopentyl thiolate with its disulfide is only threefoldlower than that of 1-butyl thiolate with its disulfidels.The reaction of thiol group of the Bovine serum albumin (BSA) with Ellman's disulhdeis anomalously * slower,by a factor of 14,than that with (* H3NCH2CH2SSCH2CH2NH3 )78. \

642 R. Singh and G. M. Whitesides

The thiol groups in some proteinsappear to be relativelyinaccessible, possibly due to a combinationof stericeffect and other factorssuch as local hydrophobicitvor charse charge repulsion.

b. Acidity. The rate constants of thiolate disulfide interchange reactions vary significantlywith the aciditiesof the substratethiols: The reactionof mercaptoethanol rmin r)6 with Ellman'sdisulfide (kns :1.5 x 107M is significantlyfaster than that of rmin mercaptoethanolwith glutathionedisulfide (/.*, :3.4 x l0r M r)s in water:the relevant values of pK" are 4.5 for EIISH and 8.7 for GSH. Brdnsted correlations (equations15-18) describethe effectof acidities(pK^) of the nucleophilic,cenrral and leaving group thiols on the rate constantsof thiolate disulfideinteichange reactions. The rate constantsfor thiolate-disulfideinterchangc (kns ) arc larger for incrcasing valuesof pK" for nucleophilicthiols, and for decreasingpKu valuesfor centraland leaving group thiols. The rate constant should be affectedmore by a change in the pK" valuei of the nucleophilicand leavinggroup thiols than that for the central thiol, beciuse the Br/nsted coefficientsare larger for the nucleophilicand leaving group thiols than for the central thiols. A mixed disulfide (R'SSR")may have two constituent thiols of different acidities (pKu*'tt ) pK.n"sH;.The cleavageof the mixed disulhdeR'SSR" by a nucleophilicthiolate RS- occursfavourably with releaseof the more acidic thiol (R"SH),and the lessaci

c. Charqe. The rates of thiol-disulfide interchangereactions in aqueoussolutions with chargedsubstituents vary by as much as a factor of 2.5 from the predictedrate constantsbased on structure reactivitycorrelations with unchargedsubstituents'e. The deviationsfrom predicted valuesbased on unchargedsubstituents are the greatestwhen the chargeis on the centralgrolrp, and the deviationsdecrease with increasingdistance of thecharge from the reactivesitete;e.g., both the ratesof reactionsof OTCCHTCH2S with the mixed disulfides'O2CCH2CH2SSC6H*NOr-pand O2CCH2CH,CH,SSC6H4- NOz-P are lower than the predicted values based on the Br/nsted correlation with unchargedsubstituents, but the deviationis lower with O,CCIH2CH,CH,SSC6H4- NOr-ptn.A similar effectof the negativecharge on R groupsis sein in the raG constantsfor degenerateRS /RSSR interchangereactionse. Thiolates without chargedsubstituents, such as mercaptoethanol thiolate, react 25 times faster with a positively charged analog of Ellman'sdisulfide than with the negativelycharged Ellman's disulfide; this rzitiodecreases to ca I to 3.5for a thiolatewith a positivelycharged substituent three bonds from sulfur (cysteineethyl ),and increasesto 120 for -)to. a thiolate with a nesativelvcharsed substituent(- O2CCHTS The electrostaticinfluence of the local cysteineenvironments in peptideshas been observedin thiol-disulfide interchangereactionsrt'at.76. rate constantsin water for the reactionof the negativelycharged Ellman's disulfide and a peptidecontaining cysteine with two positive neighbors,one positive and one neutral neighbor, or two neutral neighborsare 130,000,3350 and 370M-1s-r respectivelyat pH 7 and 20mM ionic strength8l.Electrostatic contributions totaling a fictor oi ZOOb(AG:4.3kcalmol-r) have been estimatedfor the fastestand the slowestthiol-disulfide interchangereactions of small charged substratesin 50\ methanol-water mixture; these contributions to I the free energy comprise *3.0kcalmol from attraction and - 1.3kcalmol- 1 from repulsionTl.

d. Hydroglenbonding. The rates of thiolate-disulfide interchange in polar aprotic solvents are not significantly affected by groups capable of intramolecuiar hydiogen bondingls. The rate constantfor the degeneratethioiate disulfideinterchang. reaction 13. Thiol-disulfide interchange 643

TABLE 2. Comparison of rate constants for degeneratethiolate disullide interchange(RS + -SR) RSSR=RSSR + in polar protic and polar aprotic solvents

19-: ;,c,tt AGI (M 1s 1.1(kcalmol-t) LHI AS+ -lmol RS_ M- Solvent (297K) (297K) (kcalmol t) (calK t)

HOCH,CH.S Na' DrO 0.0077 16.2 _tJ -10 ta -11 K' DrO 0.0095 l6.r I --) K* CD3OD 0.0040 16.6 13 l') la K* DMF-d? 20 I 1.5 IJ l-) K' DMSO-d6 2l r r.5 cH3cH2cH2cH2s- Na* DMF-d? 43 11.1 K* DMSO-d6 54 I 1.0 cH3c(cH3)2cH2s- K- DMF-d7 l5 1t.1 K* DMSO-d6 t6 |.7 - HOC(CH3)2CH2S K- DMSO-d6 1.1 13.2 t0 -10 n o - HOCHTC(CH3)2CH2S K DMSO-/6 0.67 13.5 16

'ljncertaintiesare: t; 1.AS]. lmol I. ft, tl0'l;,'AGl. +0.lkcalmol AH;. f lkcal mol t2cal K 'H bRateconstants were inferred from visualcomparison of the simulated NMR line shapeswith the experimental line shapes.The valuesfor CD.OD are unpublishcdobservations of R. Singh nnd G. M. Whitesides:all other values are frogr reference I 5.

of 2-hydroxyethanethiolateis only twofold lower than that of 1-butanethiolatc.In stericallyhindered thiolates, introduction of a either P or "; to the C- S bond slows the interchangeby approximately a factor of 15 in DMSO (Table 2). A gem-dimethyleffect and weakersolvation of the hydroxyl group in the stericallyhindered substratemay result in greaterintramolecular hydrogen bonding than in the sterically s. unhindered2-hydroxyethanethiolater

e. Reactionsint'oltin(J c),clic disul/ides. The rate constantfor degenerateintermolecular thiolate-disulfideinterchange involving cyclic five-membereddisulfides (1,2-) is higher than that involvingcyclic six-membereddisulfides (1,2-dithiane) by a factor of t)tt. ca 650 (AACI ca 3.8kcal mol The rate constants for the cyclic six- and seven-membereddisulfides are similar to thosefor noncyclicdisulfides't. The ring strain of 1,2-dithiolane(estimated by calorimetry) is higher than that of 1,2-dithianeby - 1; 3.7kcalmol 182. The agreementof the valueof AAGI (3.8kcal mol from kineticsand the value of ring strain (3.7kcalmol ') from calorimetry suggeststhat the ring strain in the cyclic five-membereddisulf-rde is completelyreleased in the transition stater'. In the transition state,the S-S bond is expectedto be longer than in the ground stateof disulfide,and the CSS angle at the central carbon is energeticallymost favorable at ca90"s:'a+. 161r geometry expectedfor the transition state is better matched by the structureof the ground stateof the cyclic five-membereddisulfide than that of , basedon X-ray cryslallographicstructural parametersr'.

4. Solventeffects The ratesof thiolate disulfideinterchange reactions are larger in polar aprotic solvents (DMSO, DMF) than in polar protic solvents(water, methanol) by a factor of ca 103 (Table2)1s. The natureof the counterions (Na*, K*), or additionof 18-crown-6to the reactioninvolving potassiumalkanethiolate, has no effecton the rate of thiolate disulfide interchansein DMSOt5. 644 R. Singh and G. M. Whitesides

The transition state is expectedto have a more delocalizednegative charge and thereforeto be lessinfluenced by solvation than the ground state thiolate.The higher ratesof thiolate-disulfideinterchange in polar aprotic solvents(DMSO, DMF) than in polar protic solvents(water, methanol) may be explainedby a smaller destabilizarion of the transition statethan that of the ground statethiolate, in going from polar protic solventsto polar aprotic solvents(Figure 3)15.The log of the rate constant depends linearlyon the solventcomposition in mixturesof water and DMSO (Figure4lts'tr. + r R l-T LRS..s..r*J

DMSO,DMF

- - water,methanol RS + RSSR RS + RSSR

FIGURE 3. Hypotheticalplot of free energy vs reaction coordinate for thiolate-disulhdeinterchange reaction in polar protic solvents (water. methanol)and in polaraprotic solvents(DMSO, DMF)

* ctl o

020406080 100 moloh DrO (or CD3OD)

FIGURE 4. Effectof additionoi DrO (l) or CD3OD (O) on log of rate constants (k) ol thiolate-disulfide interchange of potassium 2-hydroxyethanethiolateand bis(2-hydroxyethyl)disulfide in DM SO- - du. The values of rate constants are for 297K and are in M I s t r 13. Thiol-disulfide interchange 645 The correspondingplot for methanol-DMSO mixture, although not linear,also shows a gradual decreasein the log of the rate constant with an increasingmole fraction of methanol (Figure418s. The absenceof a sharp drop in rate on addition of small mole fractionsof water or methanol to DMSO suggeststhe absenceof specificsolvation of thiolate by polar protic solvents.In going from polar protic to polar aprotic solvents. the increaseof approximately 103in rate of reaction involving thiolate anion (RS-) is lessthan that (106-107)involvingS,2 reactionsof alkoxideanion (RO-;ts. The alkoxide anions are more solvatedin water than are thiolate anionss6'87. The rate of thiolate disulfideinterchange of 1,3-propanedithiolateand 1,2-dithiolane 1s-r) (cyclicfive-membered disulfide) is extremelyfast in DMSO (tRS c'cll08M and only ca 102slower than the diffusion limittT (equation20). This large rate arisesfrom two factors:(i) the ground stateof 1.2-dithiolzrneis destabilizedrelative to the transition statebecause of ring strain,and (ii)the thiolate is relativelymore destabilizedin DMSO than is the transition statewith its more delocalizedcharse17.

+O=-O+O (20) S_ S_ S-S S-S S_S_ A comparison of the strengthsof the RS-"HOR complexesand RO-"HOR complexesby pulsedhigh-pressure mass spectrometry shows that complexesincorporat- ing alkoxidesare more stableby 2 Tkcalmol-rthan thoseincorporating thiolatess8. The weak contributionsof ionic to solvation in RS-(HzO), complexes are effectivelydissipated within the first 2-3 solventmolecules (rt : 2 -3)tt.

5. Gas-phasesludies

The reactionof ethanethiolate(CrHrS ) with dimethyl disulfide(CH-rSSCH.,) in the gas phase occurs exclusivelywith thiolate disulfide interchange;this reaction yields methanethiolate(CH35 ) and mixed ethyl methyl disulfide(CH3SSC2HT)tn. A possible sidereaction, the carbon-centeredsubstitution to yieldCHlSS and CH.SCrHr.is not observed8e.The valueof the rateconstant for the reactionof ethanethiolatewith dimethyl disulfide in the gas phase is estimatedas 3 x 10eM I s 1. Comparison of this rate constant with the collisional rate constant susseststhat the reaction occurs with a probability of 0.003per collisionse.

6. Catalysis A number of species aromatic thiols,nonthiol nucleophilesand cations have been surveyedas potential catalystsfor thiol-disulfide interchangein waterls; catalysisis observedonly with 's'to,and even with thesespecies the magnitudesof the catalysisare not largc. Selenolsare only effectiveas catalysts for thiol disulfide interchange reactions involving strongly reducing dithiolsle. The observed rate of reduction of bis(2- hydroxyethyl)disulhde b-v-' dithiothreitol in water at pH 7 is enhancedby a factor of 15 in the presenceof 5molu,,2-aminoethaneselenol.This catalyticactivity of selenolsis probably due to a combination of the low pK^(ca 5.5to 7) (and hencesignificantly high concentrationof RSe at pH 7) for selenols,and weak solvationand high polarizability (and hence high nucleophilicity)of the selenolateanion. The precursorsof selenols. diselenides(RSeSeR) and selenocyanates(RSeCN) can also be convenientlyused to catalyzethe thiol-disulfide interchangereactions involving stronglyreducing dithiols'e. Thiol-disulfideinterchange reactions involving monothiolsare not catalyzedby selenols. becausethese disulfides oxidize the selenolsto diselenides.Strongly reducinsdithiols at 646 R. Singh and G. M. Whitesides

even moderate concentrationscan reducediselenides to selenols.and thereforein the thiol-disulfide interchangereactions involving strongly reducing dithiols, the remainsin the reduced(and catalytic)statele.

7. Compartsonwrth selenolate-drselenrdernterchange

The observed rate of selenolate-diselenideinterchange for selenocysteamineand t)un selenocystamine(koo.o: 1.65x 107M-1s in water at pH 7 is fasteiby a factor of 107 than the correspondingthiol

D. Transition State Structure

A study of crystal structuresof compounds containing divalent sulfur (Y S Z: Y,Z + H) showsthat nonbondedcontacts of nucleophilesare directed along the extension of one of the covalent bonds to sulfur26.According to the frontier-orbial model. the HOMO of the nucleophileinteracts preferentially with the LUMO (o*) orbital of S y or S-2. Attractive nonbonded interactions may representthe incipient stages of chemicalreactions26 The preferredattack of the thiolate nucleophileon the diiulfide (S-S) bond is thereforealong the extensionof the S S bond. In the transition state the negativecharge rnust be delocalizedover the three sulfur atoms. The transition state is qualitativelypictured as having greater negativecharge at the terminal sulfurs than at the central sulfur, based on the value of ihe BrdnstJd 8. coefficients:fn,.: lJrr:0.5 (by symmetry):fi,= -0.3 to -0.4s The absenceof curvature in the Br/nsted plots for attack of thiolate anions having a range of pK" valueson a singledisulfide suggests that the transition statestructure cloes not .hang. with changes in structure of the thiolate anions or the disulfide groups for theie 7. thiol-disulfideinterchange reactionss Superpositionof plots of log kl, (rateconstant) vs log K. (equilibrium constant)for zr sericsof thiol disulfideinterchanee reactions. varying in equilibrium constant by a factor of approximately102'. shows gradual c_urvatureof the type expectedon the basisof the Hammond postulatcs.Although these data indicate a change in transition state structures,factors other than Hammond postulatebehavior, such as solvation,can causecurvature in Brdnstedplots. Although thiolateanions are not as stronglysolvated as alkoxideanions, interpretaiions suggesting a change in structure of the transition state from a curved Brdnsted plot should Ue treatedwith cautionso'eo'. The valueof AS; for thiol disulfideinterchange in polar protic (water.methanol) and polaraprotic (DMSO, DMF) solventsis c'a-10 to -l6calK-1mol I (Table2)ts. This value is lessthan that expectedfor complete localizationof two particlesin the transition state,and suggeststhat the decreasein entropy in the transition staterelative to two particlesin the ground stateis partially compensatedeither by releaseof solvent s, moleculesattached to the thiolatein lhe ground statel or by a relativelyloose transition- statestructure (with two weak, partial S..S bonds)or both.

E. Theoretical Calculations on Thiol-Disulfide Interchange

An ab initio MO study on the thiolate-disulfideinterchange reaction indicatesthat the reactionis a typical S"2 reactionand proceedsvia a singlelransitionstate with little xt I* t 7 13. Thiol-disulfide interchanee 647

conformationaldistortione2. The chargedistribution in the transition stateis calculated to be higher on the two terminal sulfurs and lower on the central sulfur, in agreement with the experimental results based on Br/nsted coeflicientse2.The geometry of the transition state has been suggestedto be a trigonal bipyramidal configuration at the central sulfur with the nucleophilic and leaving sulfurs in apical positions83.The participation of d orbitals is noi essentialin stabilizationof the transition state83. 0.4- 0.2- 0.4- RS...S ...SR R

F. Mechanistic Uncertainties The geometryof the transition state is unclear:the relativedispositions of the alkyl groups on the three sulfur atoms in the transition stateare not known. The symmetry of the transition statewith respectto the nucleophilicand leaving group thiols is still ambiguous,although microscopicreversibility would indicate a symmetricalstructure if there is a single transition state. Unsymmetrical transition states connectedby a symmetricalintermediate are possible,but seemunlikely. A more completecharacter- ization of the Br/nsted coefficients,and appropriate calculations, will both be useful in understandingthis issue.The transitionstate seems to be lesssolvated than the ground statethiolate, but the degreeof solvationof the transitionstate is not known. Resolving the question of solvation may be useful in designing strategiesfor catalysis of thiol-disulfide interchange.Strategies for catalysisbased on desolvationand destabiliz- ation of the ground statethiolates seem unlikely to producelarge effects. A more plausible strategy (although one that representsa difficult problem in molecular design) will be to stabilizethe transition state of the catalyzedreaction, perhaps by appropriate charge- charseinteractions in the charse-delocalizedtransition state.

IV. EQUILIBRIUMIN THIOL-DISULFIDEINTERCHANGE REACTIONS

A. EquilibriaInvolving Monothiols In the equilibria involving a monothiol (RSH) and a disulfide(R'SSR') (equations 1 and l1), the distribution of speciesis nearly random or statisticalif the pKu valuesof the thiols (RSH and R'SH) are similar,i.e.

Kr : {(IRSSR',][R'SH])^[RSH]IR',SSR'])l t 2 and

Kz : \(IRSSR]IR',SH]X[RSH] IRSSR',]) I : 0.5 The experimentalvalues of equilibrium constantsfor the interchangeinvolving glutathione (RSH) and cystine(R'SSR') in water at pH 7 are similar to thoseexpected from random distributiofl,Kr :3.7 and Kz:0.7923'73'e3. The equilibrium constantsfor thiol disulfide interchangereactions for a seriesof monothiols and disulfides,at valuesof pH in which the equilibrium concentrationof thiolate anion is small, are relativelyinsensitive to changesin (except for sterically hindered structures with alkyl substituentsat carbon 1 to sulfur)68.The formation of bis(r-butyl)disulfides is disfavoredin equilibria involving t-butyl thiol and mixed r-butyl l-butyl disulfideTs'71'e4,and the formation of penicillaminedisulfide is disfavored in equilibria involving penicillamine and mixed penicillamine cysteine disulfide68. The equilibrium constantfor the interchangeof a monothiol (RSH) with a disulfide \

648 R. Sineh and G. M. Whitesides

(R'SSR')is pH dependentif the valuesof pK" of the thiols (RSH and R'SH) are different. In general,the equilibrium mixture favours the most stablethiolate: the equilibrium is, in effect,driven to one side by the free energy of ionization of the most acidic thiol. At a value of pH betweenthe valuesof pK" of RSH and R'SH, the formation of the thiolate correspondingto the thiol of lower pK, is preferreds.The equilibrium of the thiol-disulfideinterchange reaction involving mercapioethanol(pK" : 4.0)ano Ellman's disulfide (pK" of EIISH ca 4.5) in aqueous buffer at pH 7 8 is shifted entirely toward the formation of Ellman's thiolate and the disulfrdeof mercaptoethanol.The amount of Ellman'sthiolate is approximatelyquantitatively equal to thaf of initial mercaptoethanol, and hence the utility of Ellman's assay. The valuesof the equilibriumconstant (K'o'a, of thiol-disulfideinterchange in aqueous medium can be dissectedint.o KsH (defined for thiols) and Ks (defined for thioiates;s. The equilibrium constantKsH showsno obviouscorrelation with the valuesof pKu, but t1 i{199.1tcedby..The plot of the log of the equilibrium constanf Kb vs 2(PK,*tn-pKuR'sH)is linear (slopeia 1.2)s.xs is=thereforestrongly influencedby the aciditiesof the participatingthiolss. Electrostaticeffects on equilibria of thiol-disulfide interchangereactions are small in magnitude,but occur in the expecteddirection. The formation of mixed disulfidewith unlike chargeson the two component thiols is favored, and the formation of mixed disulfidewith like chargeson the two componentthiols is disfavoredT2.In the equilibrium involving N-(A, bearingone negativecharge on the cysteinecaiboxylate) and,the 85 114 fragment of Kunitz soybeantrypsin inhibitor (B, bearing one positivecharge on the N-terminal leucineresidue next to cysteine),the proportions of disulfidesA-B, A-A and B-B in water at pH 7 and,lowionic strengih(20mM) are 72%,10% and 181"respectively, and at high ionic strength(1 M) are 6lg;, 15,1;and 24.,,, respectively.The expectedstatistical distributions are 50.t.,25'; an

B. EquilibriaInvolving a,oDithiols

Thiol-disulfide interchangeof a,rr;-dithiols(HS R SH) with a disulhde(R'SSR') can generatea varietyof productsranging from cyclicmonomeric disulfide, cyclic dimeric

6S-SR' + 4S-SR' mixed Lrt L,- ,*' disulfides *t" dH al + R',s-sR cyclic [\sH monomenc (21) \{ disulfide

.S-S\ cyclic RR dimeric Ls-sJ bis (disulhde) -I-s-R-s+ oligomer (cyclicor linear) 7 13. Thiol-disulhde interchange 649

bis(disulfide)to oligomeric disulhde (equation 2l). The product distribution depends on the nature of R, and on the concentrationsof the dithiol and the disulfide. Cyclic monomeric disulfidesare the major products for the thiol'_disulfideinterchange reactionsof 1,3-dithiolsto 1,6-dithiolsin which the two thiol groups are separatedby three to six atoms (equations22 and 23).The formation of the cyclic monomeric disulfide occurs via the intramolecular thiol disulfide interchange reaction of the intermediate mixed disulfide (kr, equation 22): this reaction is significantly faster than the corres- ponding intermolecularreaction. High effectiveconcentration (EC, seebelow and also Table 3, for footnote c) favors the formation of the cyclic monomeric disulfrdelt.

S .SH k1 4S-SR' A:_;l R + R/S-SR/ R + R'SH -r. + R'SH k_1 i I Ltt lt" ' \_s (22)

K"q : ( ISRS] [R',SH]2)/([HS-R--SH] [R',SSR',]) (23) The stability of the cyclic disulfideis an important factor in the overall equilibrium. Cyclic six-membereddisulfides have a CSSC dihedral angle of ca 60. and are more stable than cyclic five-membereddisulfides, which have a CSSC dihedral angle of ca 30'ra. The ring strain in cyclic five-membereddisulfrdes has beenestimated as 3.Tkcalmol-1 higher than that for the cyclic six-membereddisulhdest'. It has been estimatedthat the ring cleavage(k ,, equation 22) of cyclic five-membereddisulfides is faster by a factor of ca 600than that of cyclicsix-membered disulfidesr 7. On the other hand,the formation of the cyclic five-membereddisulfide(kr, equation 22)is fasterby a factor of ca 20 than that of the cyclicsix-membered disulhde, based on valuesof kinetic effectiveconcentrations for analogousreactionslT. The overallresult is that K"o for formation of a six-membered disulfidefrom the correspondingdithiol is more favorable than that for a five-membered disulfidefrom its dithiol by a factor of ca 3013'17. The reducing ability of s,rr-r-dithiolsdepends on two factors: (i) the stability of the monomeric cyclic disulfide, and (ii) the kinetic effective concentration for the intra- molecular ring-closurestep (kr, equation22). 1,4-Alkanedithiolsthat form strain-free cyclic six-membereddisulfides are the most reducing (K", ca 10-103M, equation23); 1,3-and 1,5-alkanedithiolsthat form five- and seven-memberedrings respectivelyare ca 10-fold less reducing (Table 3). Rings smaller than six-membered are less favored primarily for enthalpicreasons (ring strain,including angle strain in the CSSC group). Rings larger than six-memberedare lessfavored becauseof conformational entropy (low kinetic effectiveconcentration for the intramolecularring-closure)t:'t0. In 1,8-dithiols, the effectiveconcentration for intramolecular ring-closure is sufficiently low that intermolecular oligomeric disulfide formation becomes competitive with cyclic monomeric disulfide formationl3. The reduction potentials of dithiols that form oligomeric products are similar to those for monothiolss'13.1,2-Dithiols form cyclic bis(disulfide)dimers in relatively dilute solution (r'a l mM), but polymerize aI higher concentrations13. Molecularmechanics calculations of equilibriaof thiol-disulfideinterchange reactions involving a,o-dithiols with 1,2-dithianecorrelate well with experimentalresults, but do not give the absolutevalues of energiestu.The empiricalrelationship between calculated differencesin strain energy (ASE) and the experimentalvalues of AG is: AG c'a0.4ASE. Why the molecular mechanicscalculations overestimate strain is not known. This correlation may be a useful guide for designing e,rr;-dithiolsof appropriate reduction potential. -

650 R. Singhand G. M. Whitesides

TABLE 3. Equilibrium constants for thiol disulfide interchange

Eq. Structure K(ME") €o(V)" againstD References

Dithiols that form cyclic monomers'

(^Y-rt 1500M - 0.354 DTT d.e \-/---sH

ff," 670M -0.344 DTT d,e \-\-s" HoF.,-, 180M -0.327 Lip d.e "n'1"-;; a---su 77M - 0.316 DTT d.e \,,-sH

l /\ 2 65M - 0.314 DTT d,e \stLSH or\f=-- sH 'c 63M - 0.313 DTT oZ\-,-- sn

|\._jnr^r" 44M -0.309 DTT d.e H'c'6-1^su 19M - 0.298 DTT ".6,s-s" H,c1/P ,N-\ rt" r5M -0.295 DTT \ ,,SH NJ H.c--( o

"'tV- 14M -0.294 H,c- \---^..^.. DTT d.e :)H

(Yt'n')4co:H 8.6M - 0.288 ME.DTT d.e HS SH

/--\/-sH 8.0M -0.287 DTT d.e \/LqH

(r'ontinuedI 13. Thiol-disulfide interchanse 651

TABLE 3. (continuedl

Eq. Structure K(ME'-) eo(V)' againstb References

_ .sH /"-- 6.7M - 0.285 DTT d.e S.*s"

/'-v-sH o.*sn 6.1M -0.284 DTT d,e

r', ,z-SH (X -0.279 DTT d,e v \_sH 4.4M

3.6M -0.277 DTT (-/rY-'" l- 511 d.e

//---SH 3.6M -0.277 DTT .^SH d.e

HS----r z-SH 3.1M -0.275 DTT H.c\-sH d.e

H.CX,---SH 2.9M -0.274 DTT d.e H.C \-SH

o\-- H.C. ' 'N' tsH 2.5M -0.272 DTT ilsH A.1'U Y

2.-rM -0.2'71 ME.DTT tl,e at"\_sH

CONMe, I a\su -0.269 (-,-t" 1.8M DTT I CONMet

},1-tt 1.2M -0.263 DTT d,e r \_qFl

/-SH 0.67M * 0.255 DTT d,e \-SH

6,6'-sucrose 0.30M -0.245 ME i disulfide HS(CH2)6SH 0.21M -0.240 DTT d.e

(r'ontinuedI -

652 R. Singh and G. M. Whitesides

TABLE 3. kttntinuedl

Eq. Structure K(ME"-) so(V)' againstb References

Monothiolsthat form dimers

1i}--JsH 2.6 -0.272 ME \-,/ L1 cH3(cH2)6sH -0.261 1.1 ME d -0.260 HOt^\--sH 1.0 ME d

A-t" -0.245 Y/ 0.31 ME (l Dithiols that.fbrm cvclic dimers r--rlt" tl 0.40M -0.254 \---\SH ME d.e a^"'?tn tt 0.38M -0.254 ME d,e \,/^.sH zl-sH t()l 0.32M -0.253 ME H.cr'*n e.g

0.035M -0.239 "t-,Arn ME d,e Dithiolsthat Jbrm polymers

HS"*-sH 4.8 -0.280 ME HS,, ,sH 4.0 -0.278 ME

,,A, 3.4 -0.276 "rt-Vfr"

3.1 -0.27s ME "i-Q-r"

3.0 -0.275 ME (1

rtt t^l 2.8 -0.274 HS---A\2\ ME k SH (continuedI 7 13. Thiol-disulhde interchanse 653

TABLE 3. (continued)

Eq. Structure K(ME"-) co(V)" againstD References

-{t' 1.8 - 0.268 ME -1_qglt

.t HS(CHr)8SH 1.7 -0.267 ME u .t HS(CH,),SH 1.4 -0.265 ME u

HS, 1.3 -0.264 ME YO)-s"--l \ r^\ HS-< ( ) >-Srr 0.20 -0.240 ME \"/

"i:,,(V)values vs standard hydrogen electrodeat pH 7.0 and 25"C. All r;,,(V)values are calculatedusing the r;n(V) valuesfor [-0.288V. D. R. Sanadi,M. Langleyand R. L. Searls,J. Bbl. Chem..234.178 (1959) and C. V. Massey,Biochem- Biophys. At'tu,37.314 (1960)l and the K"o valuebetween lipoic acid and the compound of interest. bAbbreviations:DTT, dithiothreitot; Lip, lipoic acid; ME, 2-mercaptoethand. 'The value of K(ME") for this group olcompounds is sometimescalled the effectiveconcentration (EC). dEquilibrations were carried out at 25 C. in a l/1 mixture of do-methanoliphosphatebufier (50mM. pH 7.0) in DrO, seeReference 13. "The equilibrium constants(K) in the Houk and Whitesidespaper (13) were systematicallyin correct by a factor of 103 (originating in error in manipulation of units during the original calculations)and have been adjusted accordingly. The values of equilibrium constants,which were obtained from equilibrium with DTT. have also been readjustedby a factor of approximately 2 so as to obtain a similar value to that reported in this paper. /Equilibrations werecarried out at 25 C in a l/l mixture of r/.-methanoliphosphatebuffer (50mM. pH 7.0)in DrO, seeG. V. Lamoureux and G. M. Whitesides,J. Orq. ('hem.,58.633(1993). ,Equilibrations were carried out in oln-methanolwith 0.02mM sodium methylate added, see Reference13. hEquilibrationswere carned out in DrO (pD 7.0.50mM phosphate).see Reference 18. iEquilibrationswere carriedout in D,O (pD 7.0,50mM phosphate).see Reference 20. iEquilibrationswere carried out in DrO (pD 7.0, 50mM phosphate).see W.J. Lcss and G. M. Whitesides.J. Orq. Chem.,58, 642 (1993). ftEquilibrationswere carried out in r/o-benzenewith 0.02mM tetramethylguanidineadded, see Referencel3.

The equilibrium constantfor the thiol disulfideinterchange of an r,co-dithiolwith a 'effective disulfide (equations22 and 23) has also been termed as the concentration' (ECles'so.The equilibrium expressionfor effectiveconcentration is a measureof the propensityof thiols to form the cyclic disulfidee6.The EC has also been interpretedin terms of the proximitr of thesethiol groups in the ground state (that is, as a kind of localconcentration),and thus usedto infer informationabout conformation.Considering that the value of the equilibrium constant is very strongly influencedby strain in the CSSC group and by ring strain (for cyclic disulfides),its interpretation in terms of 'proximity' 'concentration' and must be evaluatedwith the possibilityof contributions from theseterms in mindro. If the EC is used (and interpreted)just as an equilibrium constant,but as one with an easilyremembered reference value (EC ctt I 10M for an r,o-dithiol forming a strain-freecyclic disulfide) it has the virtue of being easy to remember and to interpret. The equilibrium expressionfor effectiveconcentration (EC : K"o, equations 22 and 23) involvesa ring-closurestep (kr) in the forward reaction(k, is a measureof the kinetic effectiveconcentration), and a ring-cleavagereaction (k ,) in the reversedirection. The ring-cleavagereaction, k ,, is faster for a more strained cyclic disulfide than for an unstrainedone, and correspondinglythe value of the equilibrium expressionfor effective concentration(EC : K"u) is lower for the more strainedcyclic disulfide. \

654 R. Singh and G. M. Whitesides

In casesinvolving cyclic disulfideswith ring strain, the trends of the equilibrium expressionfor effectiveconcentration (equilibrium EC) may be differentthan the trends of the kinetic valuesfor effectiveconcentration (kinetic EC). In a comparison of the formation of cyclic five-membered disulfide and cyclic six-membered disulf-rde,the equilibrium EC (relatedto K.o, equations22 and 23) is favoredfor the formation of the six-membereddisulfide by a factor of 30 over the five-memberecldisulfide. whereas the kineticEC (relatedto k,.equation 22)ishigher for the ring-closurereaction for formation of the five-membereddisulfide than that of the six-membereddisulfide by a factor of 20. The value of the equilibrium EC is easierto determinethan that of the kinetic EC. As we have indicated,however, the equilibrium EC is a direct measureof proximity only when there is no strain in the disulfidesor dithiols, and it is perhapsmost usefulas i measureof proximity when theseother factors(e.g. ring strain reflectingan unfavorable CSSC dihedral angle;terms destabilizingthe dithiol relativeto disulfide)are absentor can be independentlyestimatedl6.

V. APPLICATIONSOF THIOL-DISULFIDEINTERCHANGE IN BIOCHEMISTRY The subject of the thiol-disulfide interchangereaction is an important one in bio- chemistry,and has been discussedextensively elsewhere' 3. Here we will only outline someof the issues. Disulfrde-reducingreagents are used in biochemistry for a number of purposes, especiallyin reduction of cystinegroups in proteinsand in maintaining essentialthiol groups in their reduced stateeT. e,rr-r-Dithiols,such as dithiothreitol (DTT)70, N,l{'-dimethyl-N,N'-bis(mercaptoacetyl)hydrazine (DMH)t 8 and meso-2.5-d,imercapto- A/,.4y',4/',N'-tetramethyladipamide(DTA)20, have higher reductionpotential than cystine groups in proteins, and are useful disulfide-reducingreagents becausethey react specificallywith the cystinedisulfide to be reducedwithout any unwantedsicle-reaction with the . The value of the first pK" of DTT is 9.2s,and it is thereforcrelatively s]ow as a reducing reagentat pH 7. DMH and DTA (pK^ r'a 8) reducesmall organic disulfidesand disulfidebonds in proteins c'a7 times fasterthan does DTT in water at 8'20. pH lt Mercaptoethanol(ME, pK" ca 9.6)is inexpensiveand is usedin largeamounts (0.1-0.7M) in bioc_hemicalmanipulations, for example in conjunction with SDS gel-electrophoresiseT.Mercaptoethanol is weaklyreducing and it oftengenerates complex reactionmixtures containinq mixed disulfidess.

HO HO ntt, /CH. \ N-N ';-61- ( MerN (r-r NMe, SH SH SH bH DTT ME ^/ \" DTA DMH

The cyclic five-membereddisulfide lipoamide is a of the pvruvate dehydrogenasecomplexes'ee. The rate of ring opening of this cyclic five-membered disulfide by thiolate*disulfideinterchange is faster by a factor of ca 103 than that involving cyclic six- or seven-membereddisulfidesrT. The evolutionary sclection of lipoamide as a cofactor in pyruvate dehydrogenasecomplex may reflect the fast rate of ring opening of the cyclic five-memberedring by nucleophilesand the resultingability of the lipoamidesto maintaina high flux through the pyruvatedehydrogenase complexr 7. / 13. Thiol-disulfide interchanse 655

The valttesof pK, of thiol groups in proteins have been measuredkinetically from the Bronstcdcorrelation of thiol disulfideinterchange reactionsrr. The pK, of the actirc'-sitethiol in papain is estimatedas c'rr4 at pH 6, and t'u8.4 at pH 91r. At low PH {r,i 6tthe proximatepositively charged group increasesthe acidityof the active-site throl in papain.The pK" of the thiol group of reducedlysozyme is c,rz11. These values rrf pN.,. although semiquantitative,are useful for comparison with the values of pK" tic'tc'rrninedby other methodsrr. Thc-redox equilibria between the cystine-bridgedcyclic disulfide structures in proteins lnd thcir correiponding reducedopen-chain z,r,,r-ditiriol forms have been measuredfor :i\ eritl proteins3.The valueof the equilibriumconstant (or equilibriumexpression for ciicctire concentration,equilibrium EC) for the thiol disulfideinterchange reaction of It protein z,a-r-dithiolcan be a usefulmeasure of proximity of the two thiol groups in the protein if there is no ring strain associatedwith the correspondingcyclic disulfide. .{ high value of the equilibrium EC suggeststhat the two thiol groups are nearby spatially,are limited in mobility and can form a CSSC group with little or no angle strain16'es'e6. The disulfidebonds in proteinsare formed after translationroo'101.The pathway of sequentialdisulfide bond formation has been studied for bovine pancreitic rrypsin inhibitor (BPTI)102''0'rand for ribonucleaseA104. In the caseof BPTI. interpretations of differentsets of data have led to differentconclusionsr02,l03. The conilusion of Weissmanand Kim that all well-populatedfolding intermediatesin the oxidative foldingof BPTI containonly nativedisulfide bonds -,is still beingactively debatecl I05. r 06. The inclusionbodies, clbtained from the expressionof eukaryoticproteins in genetically engineeredE. coli. may contain protein with unformed and mismatched disulfide I 07'108. 'wrongly' bonds The conversionof the disulfide-connected 'correctly'disulfide-connected protein to the protein is a major problem in biotechnology.The general 'wrongly' approach is to reduce the disulfide-connectedprotein completelv and to oxidize it gradually with a redox buffer containing a mixtuic of thiol anA alsutnderou. Protein-disulfide isomcrase has been proposed as catalyst for the thiol-disulfide interchangeinvolving proteinsttn, Its low catalytic activity and absenceof specificity make its biologicalrole uncertainrlr'r12. has a cysteineof low pK., anciit reactswith disulfidesrapidl-v at pH 7. Thioredoxin is redox-coupledto NADPH via the enzymethioredoxin reductase,and may be of metabolic significancein thiol-disulfide interchangereactionsr 13 I 1s.

VI. CONCLUDINGREMARKS Thiol disulfide interchangeis a reversibleSr2 reaction that involves cleavageand formation of a covalentS S bond. The activenucleophile is the thiolate anion (RS ); the thiol (RSH) is not active.The rates of reaction of thiolate anions with disulfides show a Br/nsted correlationwith the valuesof pK" of thiols.The value of the Brdnsted coefficientfor the nucleophilicthiol (t'a0.5)is well studied,but a more completeanalysis of the Br/nsted coefficientsfor the central and leaving group thiols would be a useful step toward a better understandingof the structureof the transition state. The rate constant of the thiolate-disulfideinterchange reaction (k*, , basedon the concentrationof thiolate anion) is influencedby factorssuch as pK" of thiol and CSSC dihedralangle in the disulfide.The rate constant(k*, ) increaseswith increasingvalues of pK" of the thiols becauseof the increasingnucleophiticity of the thiolate anions.The observedrate constantof reaction(/coo.o), however, is optimum for the value of pK" of the thiol equal to the pH of the soluiion. The most stableCSSC dihedral angle in the disulfideis ca 90". Cyclic five-membereddisulfides, with CSSC dihedral angle of r,a 30 , arestrained, and arecleaved c'a 103 times faster than the less-strainedcyclic six-membered 656 R. Singh and G. M. Whitesides disulfide.An improvedtheoretical conformational analysis of the ground stateof cyclic disulfides-in termsof the bond angles,bond lengths,and the CSSI and CCSSdihedral angles-would be useful to predict the ring strains and rates of thiol disulfide interchangereactions involving cyclic disulfides. Thiolate-disulfideinterchange reactions are faster in polar aprotic solventssuch as DMSO and DMF than in water. The rate enhancementin going from water to polar aprotic solventsis lower than for reactionsof alkoxide anions.The thiolate-disulfide interchangeinvolving strained cyclic five-membereddisulfide is extremelv fast (kp" t ca 108M s-t) in polar aproticsolvents. Disulfide bonds are presentin proteinsand are formed from the cysteinethiols after translation.The physical-organicstudy of severalbiochemical issues related to the thiol disulfideinterchange-the mode of formation of the'correct'disulfidebonds. the degree of stability imparted to the protein by the disulfidebond. the strain in the large-iing protein disulfides,the role of thiol-disulfideinterchange in regulationof prorein activity and the design of reagentsthat can efficiently reduce disulfide bonds-would be important and useful.

VII. ACKNOWLEDGMENTS

We thank Watson Lees for important contributions to the analysesof equilibrium constants,and Dr Guy Lamoureux for helpful discussions.Professor H. Schera_ea(Cornell) directed our attention to the error in Reference13; we thank him for this help. Our researchwas sponsoredby the National ScienceFoundation under the Engineering ResearchCenter Initiative to the M.I.T. Biotechnology ProcessEngineering Center (CooperativeAgreement CDR-88-03014)and by the NationalInstitutes of Health(Grant GM3e589).

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