Reprinted from The Journal of Organic Chemistry, 1991,Yol. 56. Copyright O 1991by the American Chemical Society and reprinted by permission of the copyright owner. SelenolsCatalyze the Interchange Reactionsof Scheme L Catalysis of Thiol-Disulfide Interchange by Dithiols and Disulfides in Water Selenol -H* RajeevaSingh and GeorgeM. Whitesides+ RSH+ ) H' Departmentof Chemistry,Haruard Uniuersity,Cambridge, Massachusetts02138 Iuncatatvzeo ReceiuedApril 2, 1991 I The mechanism of thiol-disulfide interchange reaction R'SSR' RSSR' involvesthe nucleophilicattack of thiolate along the S-S R"SeH rl bond axis of the disulfide.t The reaction is kinetically secondorder: first order in thiolate and in disulfide.2{ At \ '-";Jfil'-' ) pH 7, only 0.1-l% of typical thiol groups is present as VV ",,",r,"0 thiolate in water, and the apparent rate constantof the - R"seSR' -4t- | 1'. /tt-' thiol-disulfide interchange reaction is small (ftou'a= 0.1 R,sH ,,r- , s'J M-r s-1).2'5Protein disulfide isomerase(EC 5.3.4.1)has H* been suggestedto act as a catalyst for thiol-disulfide in- fg terchangein vivo, but its lack of specificity and the fact that it induces only moderate rate enhancementsmake its biological role uncertain.6-e b As part of a program examining thiolate-disulfide in- I terchange,we surveyeda number of types of compounds (aromatic thiols, nonthiol nucleophiles,and cations) as l: d y i potential catalystsfor this reaction. The only significant dl/ ," rate enhancement was obtained with phenylselenol.s {",_d,'LlJ'lt-'ilfir flil a*. A-- - Selenolateis a strong nucleophiletoward diselenidesand a good leavinggroup in selenolatediselenideinterchange.lo The pK" of selenolsis =7,11and at pH ? the attack of 1 I selenolon diselenideis rapid (ftoua= 1.65x 10?M-l s-1).10 I We expectedselenolate to be a strong nucleophiletoward I disulfide and to be a good leaving group in the attack of I thiolate on selenosulfide(RS-SeR). SchemeI showsthe steps involved in the catalysisof a thiol-disulfide inter- lilI llilt, illl changereaction by selenol. iill ilfl ilI ilil, (1) Rosenfield,R. tli E.; Parthasarathy,R.; Dunitz, J. D. J. Am. Chent. irll ililt Soc. 1977,99, 4860-4862. [ (2) Szajewski,R. P.;Whitesides, G. M. J. Am. Chem. Soc. 1980,102, *--,'[ .-rn-r,l.a- 20Lr-2026. B - (3) Whitesides,G. M.; Lilburn, J. E.; Szajewski,R. P. J. Org. Chem. t977.42.332-338. AA (4) Freter, R.; Pohl, E. R.; Wilson, J. M.; Hupe, D. J. J. Org. Chem. 3.0 PPM 1979,44, l77t-t774. Figure l. tH NMR spectra (500 MHz) of reaction mixtures (5) Singh, R.; Whitesides,G. M. J. Am. Chem. Soc. 1990, /12, initially containing dihydroasparagusicacid (DHA, 5 mM) and 1190-1197,6304-S309. bis(2-hydroxyethyl)disulfide (ME'"', 5 mM) in phosphate-buffered (6) Freedman, R. B. Nature 1987,329,196-19?. (50 pD (A) presence (7) Lang, K.; Schmid, F. X. Nature lgt8, 331,453-455. mM) D2O at 7 under argon: in the of 10 mol (8) Pain, R. Trends Biochem. Soc. 1987,12,309-312. To Z-aminoethaneselenol(0.5 mM), quenched with DCI after 1.5 (9) Wetzel, R. Trends Biochem. Soc. 1987, 12,4i8-482. min; (B) in the absenceof selenol,quenched with DCI after 2.2 (10) Pleasants,J. C.; Guo, W.; Rabenstein,D. L. J. Am. Chem. Soc. min. The peak assignmentsare a = CH2OH (MEo'), b = CHzOH 1989,l1l,6553-6558. Wu, Z. P.;Hilvert, D. J. Arn.Chem. Soc. 1989. //1. (ME), c = CH (DHAo'), d,€ = CH2 (DHAo'), f = CH2S(MEo'), 45i3-4514. Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1990, 1/2, g = CH,CHz (DHA), h = CH2S(ME), x = CHzNH3*,y = CH2Se. 5647-5648. (11)Arnold, A. P.;Tan, K. S.; Rabenstein,D. L.Inorg. Chem.1986, In this study we have surveyed in greater detail the 25,2433-2437. Some representative values of pK" for selenols are as follows: 2-hydroxyethaneselenol, 6.6; selenocysteine,5.4; selenocyste- ability of several alkaneselenols to caLalyze thiol-disulfide amine, 5.5; selenoaceticacid, 6.9; selenopropionicacid, 7.3. interchange reactions. We have examined thiol-disulfide 0022-3263l9t I 1956-6931$02.50/0@ 1991 American Chemical Societv 6932 J. Org. Chem.,Vol. 56, No. 24, 1991 Notes Table I. Catalysis of Thiol-Dieulfide Interchange by Selenols in Water at pH 7o kob6d(M-l min-l) ^mtlobrd e*r boM bobsd reactantsD catalyst equit' ''cet " uncet m ffi method DHA + ME"' *H3NCH2CHTSeH 0.05 70 2.3 30 150 LIV 0.10 110 2.3 48 L2O UV (+HsNCH2CH2Se)2 0.05d 35 2.6 13 65 UV 0.025d 18 2.6 7 70 LTV +HBNCH2CH2SeH 0.10 70 418 36 NMR DTT + MEO' (+H3NCH2CHrSe)2 0.05d 130 915 75 I_lV 0.025d 65 97 70 LIV +HaNCH2CH2SeH 0.10 420 11 38 76 NMR HOCH2CH2SeCN 0.05 170 11" 16 64 NMR L-Cys+ MEo' +HsNCH2CH2SeH 0.05 30 27 1 NMR N-AcCys+ MEo' +H3NCH2CH25eH 0.05 9 61 NMR HOCH2CH2SeCN + DTT 0.05 8 6"1 NMR 'Reactions were carried out in buffered deoxygenatedaqueous solution at room temperature (23-24 "C) under an atmosphereof argon. The etror in the obseNed ratc constantsis *10%. bThe initial concentrationsof thiols and disulfides were 4 mM each in the UV erpe ments, 10 mM eachin the NMR elperimenLsinvolving cysteineor N-acetylcysteinewith +H3NCH2CH"SeHas catalyst, and 5 mM eachin all other NMR expedments, 'The quantity of RSeH used is given in terms of equivalentsof the reactants-thiols and disulfides. dThe equivalentaof RSeH reported are two times the initial molar equivalentsof (+H3NCHrCHrSe)r.'The valuesof the uncatalyzedrate con- stant, &:#i, for the HOCH2CHTSeCNexpe ments are assumedto be the sameas det€rurinedby the NMR method for +HTNCHTCHTSeH etDeltments. interchangereactions involving both monothiolsor dithiols obseruedrate constantsand do not contain correctionsfor and usedselenols and precursorsof selenols(diselenide, the valuesof pK" of the sulfur and seleniumcompounds. selenocyanate)as catalysts. Assays were based on lH They cannot,therefore, be related directly to the reactions NMR and UV spectroscopicanalyses. in SchemeI. The valuesof concentrationsof selenolsrued for calculatingthe ratio of observedrate constantsadjust€d Results and Disc rabrer shows theapparent."Hl'o11,""* *.*r"r [fl:lli,f;i:i*T,Tm1:l::li.'i,"ti,tlt?"rlili#;#f thiol-disulfide interchangereactions in t}re presenceor cyanate)and are thereforeapprorimate measures ofthe absenceof selenols.The reactionsinvolving strongly re- cata.lwicconcentrations of theielenol. The valuesof rate ducingdithiols (eqsI ald 2; DHA is dihydroasparagusic consLntsobtained from UV exoerimentsare more accu- COO- rate than those from NMR experiments. Most of the work has used 2-aminoethaneselenol(AE- -----> + HOV,,\S.S:IOX SeH) as the selenium-containingcomponent. The reasons SH SH for this choice are that its diselenide,bis(2-aminoethyl) diselenide (AESeo', selenocystamine),is commercially available,and both AESeo'and AESeH are water soluble. DHA MEOI reducing dithiols (e.g.,dithiothreitol (DTT)) coo' Strongly I reduce diselenideto selenol. The value of the equilibrium O z Ho-'Asn (1) constant (0.t+ M, eqs 5 and 6) for the reduction of sele- S-S HO OH LT *HgN\.^t's"-^NH3*---.> ( ) * DHAOI SH SH HO OH \,r DTT AESeot * HO:.AS.S:iOH ---;> \/ HO OH SH SH \'o /-f. *HgN\^seH +2 (s) ( ) DTT MEOI s-s HO OH \ ..s DTTO' AESEH + ' no-^r, (2) ( ) s-s lAESeHl2[DTTb'] K"q = = 0.14M (6) IAESe"']tDTTl DTTOI ME acid) are significantly catalyzedby the presenceof alkyl nocystamine ((+H3NCH2CHzSe)2,AESeo') by DTT is, selenols. Figure 1 illustrates the rate enhancementsob- however,significantly lower than that (9.4 x 104M) for servedin these thiol-disulfide interchangereactions in the the analogousreduction of bis(2-hydroxyethyl)disulfide presenceof catalytic amounts of selenol. (ME*) by DTT.I2 Monothiols are more weakly reducing Table I reports observedrate enhancements(khTd/kflHXt = u."t/u*""J and ratios of observed rate constants adjusted (&:}|o/tRSeHlh$H$,) (12)Houk, J.; Whitesides,G. M. J. Am. Chem.Soc. 1987,109, for the initial concentrationof selenol 6825-6836. estimated from eqs 3 and 4. We emphasizethat these are (13)Klayman, D. L. J. Org.Chem.l965, 30, 2454-2456. = - (14)Miyashita, M.; Yoshikoshi,A. Synthesis1980, 664-666. uuncat %dlME) / dt e8*gttME-llR(sH)2] (3) (15)Gunther, W. H. H. J. Ore.Chem.1967,32,3931-3933. = - (16)Dickson, R. C.;Tappel, A.L. Arch.Biochem. Biophys.1969, /30, ucat %dtMEl/dt &3PtdtME"'l[R(sH)zJ(4) 547-550;1969, /31, 100-110. 6933 than dithiols such as DTT and cannot reduce diselenide UV Assay for Catalysis of Thiol-Disulfide Interchange significantly (eq 7). The rate of thioi-disulfide interchange Involving DHA and MEo' by 2-Aminoethaneselenol '- (*H.,NCH2CHzSeH). Sl.ucksolutions of DHA and MEo' iboth 2ME * AESeo" \{Por + 2AESeH (7t 8 rrrM)were prepared frorn DHA (0.0131g) in 10.8mL of deox- ygenatedphosphate (0.1 phosphate,pH reaction of cysteine (or l/-acetylcysteine) with bis(2- l:'.rffer M in 7.0, 2 mM in EIITA), and from M[.]o'(0.0129 g) in 10.5mL of deoxygenated hydroxyethyl) disulfide (MBo') is not in presence enhanced phosphatebuf fer In a quartz cuvettecontaining i.5 mL of DHA of 2-aminoethaneselenol(+H3NCHTCH2SeH) 2- or stock soiution and 30 str,of 2-arninoethaneselenolstock soiution hydroxyethaneselenol(HOCH2CH2SeH) (Table I). The (40 mM) was added 1.5 mL of MEo" stock solution, and the thiol-disulficle reactions involving rnonothiols and di- increaseirr absorbai:ceat 330 nm was recorded. The initial sulfides are not catalyzed by selenol becauseselenoi i** ct-rncentrationsin the cuvetle were IDHA1 = lMEo'J = ci'itiat= oxidizedto diselenideby reactionwith disutfide. 4.0 mM, ["ii'rNCHzCH2SeHl = 0.4 mM. The apparent rate In the reactionsinvolving strongly reducing dithiols and eonstant.(&obsa, was calculatedusing the integrated form of the disulfides, the catalytic selenoi can be generated from rateequation diDHA*]lcir = koH[DHA][ME"']: &oM = (tlcy,^ 'fhe - lf cinl;6)ft.