Thiol-disulfideinterchange - Whitesides Research Group

Thiol-disulfideinterchange - Whitesides Research Group

C H A P T E1R3
Thiol-disulfide interchange
RAJEEVA
SINGH
lmmunoGen,lnc., 748 Sidney St.,Cambrtdge,MA 02139
and
G E O R G EM W H I T E S I D E S
Departmentof Chemistry,Harvard university,12 oxford St.,Cambridge,MA02138
I . INTRODUCTION
II. M E T H O D S U S E D I N F O L L O W I N G T H I O L - D I S U L F I D E
INTERCHANGE
A. Spectroscopic(UV, NMR) Assays
B. Enzymatic Assays
C. AssaysBasedon Chromatography
III. MECHANISM
A. Products
B. Dependenceon Solution pH, and on the pK" Values of Thiols
C. Kinetics
1. Ratelaw
2. Br/nsted relation
3. Substituent effects
a. Steric
b. Acidity
c. Charge
d. Hydrogen bonding
e. Reactions involving cyclic disulfides
4. Solvent effects
5. Gas-phasestudies
6. Catalysis
7. Comparison with selenolate-diselenideinterchange
D. Transition State Structure
E. TheoreticalCalculationson Thiol-Disulhde Interchanse
F. MechanisticUncertainties
. .
SupplementS: The ,hr^irnrv of sulphur-containinll
functional aroups
Edited by S. Patai and Z. Rappoport O 1993 John Wiley & Sons Ltd
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R. Singh and G. M. Whitesides
IV . E Q UI LI B RI U M I N T H I O L D I S U L F I D E I N T E R C H A N G E
REACTIONS
A. Equilibria Involving Monothiols
B. Equilibria Involving a,co-Dithiols
V . A P P LI CA T I O N SO F T H IO L -D ISU L F ID E IN TE R C H A N GE
IN BIOCHEMISTRY
u. CO NCLUDI N G R EMA R K S
VII. ACKNOWLEDGMENTS
VIII . RE F E RE NC ES
6-l
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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 reaction is unique in organic chemistry:although it involves
the cleavageand formation of a strong covalent bond (the S-S bond; bond energy
ca 60 kcal mol 1),it occurs reversibly at room temperature in water at physiological pH
(ca 7)1-a.The reaction is moderately fast: a typical value of the observed rate constant
i s c a 1 0 M - 1 m i n - r a t p H 7 a n d r o o m t e m p e r a t u r e sT. h e h a l f - l i f ef o r t h e r e a c t i o ni s
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 quantitative if side-reactions-such as air oxidation of thiol to disulfide, and cleavageof
disulfidebonds at high pH-are preventedr.
(I)
Thiol-disulfide interchangeis an Sr2 reaction. Thiolate anion (RS ) is the active
nucleophileand the reactioncan be stoppedif the solution is made acidict. The reaction
-zo.
is overall second-order:first-ordereach 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 reaction conditions.
R S H + R , S S R , = R S S R , +H S R ,
(2)
RSH = RS + H+ (pK^*t")
_SR,
(3)
R S _+ R , S S R , = R S S R , +
*
(4)
H + SR'= HSR' (pK"*'tt)
2s
and involves the backside
Thiolate-disulhde interchange is base catalyzed2l
nucleophilic attack of thiolate anion along the S-S bond axis of the disulfide26.It
showslesssensitivityto solventthan most S"2 reactionsinvolving oxygen and nitrogen
nucleophilesls.The rates of thiolate-disulfide interchangein polar aprotic solvents
(DMSO, DMF) are faster by a factor of approximately 103 than rates in polar protic
solvents(water,methanol).For comparison,the rate enhancementfor oxyanions(which
s
are more highly solvatedthan thiolates)1 on moving from protic to polar aprotic solvents
i s 1 0 6 -1 0 7 .
Biologicallyimportant moleculescontainingthe thiol or the disulfidegroup are widely
distributedin naturel'2,and the unique position of thiols and disulfidesin biochemistry
3.
has beenreviewedin severalexcellentbooks and articlest The thiol-containingamino
acid cysteine-is presentin proteinsand peptides;examplesincludeglutathione2-and
trypanothione2s-30.Severalcofactors coenzymeA, dihydrolipoamide,coenzyme M
( - O 3 S C H 2 C H 2 S H ) 3 r c o n t a i n t h e e s s e n t i atlh i o l f u n c t i o n a l i t y l ' 2T. h e d i s u l f i d eb o n d s
635
=
f
=
.-F
c
0
-10
50
a)
lTl
q
q)
C
LLJ
30
20
r0
0
-r0
0
30
60
90
120
150
180
D i h e d r a al n g l e ( H S S Ho r C S S C )
F I G U R E 1 . R e l a t i v ee n e r g yl k J m o l ' ) o f H S S H a n d M e S S M e a s
a f u n c t i o n o f t h e d i h e d r a l a n g l e ( { , R S S R ) :( ) M M 2 ( 8 5 ) ;( - - )
6-31G* [M. Aida and C. Nagata, Theor. Chint.Acta,70,73
1 1 9 8 6 ) J( -, - ) S C F a n d M P 2 [ C . J . M a r s d e na n d B . J . S m i t h ,J . P h t ' s .
C h e m . , 9 2 , 3 4 7( 1 9 8 8 ) l ;( - - - ) O P L S [ W L . J o r g e n s e nJ,. P h 1 , s .
Chem.,90, 6379 (1986)1. Reprinted with permission from
Reference16. Copyright (1990) American Chemical Society
636
R. Singh and G. M. Whitesides
betweencysteineresiduesare important tertiary and quarternary structural clcntcnrrin
proteins(especially
extracellularproteins)suchas immunoglobulins,enzymes,horntor.rc..
procollagenand albuminl-3. The cleavageof the disulfidebond(s)of many proteins1e.g.
deoxyribonuclease
I) resultsin loss of activity32.Thiol-disulfide interchangemay plal
a role in metabolic regulation of enzymatic activities3'::':+.76. activities of several
chloroplastenzymessuch as fructose-1,6-biphosphatase,
NADP-malate dehydrogenase,
sedoheptulose1,7-biphosphatase,NADP-glyceraldehyde-3-phosphatedehydrogenase
and phosphoribulokinase are enhancedby reduction of their specificdisulfide bonds by
photogeneratedreducing equivalentstransferredvia ferredoxinand thioredoxin3''rs-'17.
The cleavageof disulfide bonds of B-adrenergicand other cell surfacereceptorsby thiols
activatesthe receptorin a manner similar to binding of agonist3s'3e.
The thiol functional group is essentialfor activity of many enzymesl'2such as thiol
proteases (papain, ficin, bromelain)a0,B-ketoacylthiolaseal-a3,enolaseaa,creatine
kinase,glyceraldehyde-3-phosphate
phosphofructokinase
dehydrogenase,
and adenylate
kinaser-3'11.Theseenzymesare inactivein mixed disulfideform, and can be reactivated
using strongly reducing thiols (e.g. dithiothreitol, lV,ly''-dimethyl-N,lV'-bis(mercaptoacetyl)hydrazine)l1'18.
A thiolate anion may be involved in methanogenesisby 2elle
redox coupling with a Ni cofactorin methyl coenzymeM reductase3t.The reactivethiol
functionalityis maskedas a trisulfidein the potent DNA-cleavageagents-calichemycin
and esperimicin4s-48.
The rate-determiningstepin the unmaskingof theseDNA-cleavage
agentsis the cleavageof the trisulhde by a thiol (e.g.glutathione)48.
The thiolate anion
formed undergoesintramolecularMichael-typeattack on an enonesystem,which in turn
facilitatesthe ring closureof the enediynemoiety to form the reactivearomaticdiradical.
Thiolate anion is a strong nucleophileand a good leaving group becauseof its high
polarizability and low degreeof solvation.The thiol group is less strongly hydrogenbonded, and the thiolate anion is lesssolvated,than the alkoxide 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 butanol 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'u7.5kcalmol-1 s2. Five-membered
cyclic disulfidesare strainedand have a CSSC dihedral angle of ca 30. s3. Sulfur shows
concatenation:molecular sulfur S, exists as an eight-memberedring; organosulfur
compoundscontainingtri- and polysulfidelinkages(RS"R)are found in nature and have
s.
been characterizedsa's
This chapter focuseson the physical-organicaspectsof the thiol-disulfide interchange
reaction.The biochemicalaspectsof the reaction are only lightly touched upon here,
and we recommend References1-3 to the readerfor a detailedreview of the biochemistrv.
I I . M E T H O D SU S E D I N F O L L O W I N GT H I O L - D I S U L F I D EI N T E R C H A N G E
A. Spectroscopic (UV, NMR) Assays
The kinetics of thiol disulfide interchange reactions involving formation of a
chromophoric thiolate are convenientlyfollowed by UV spectroscopy.The reaction of
thiolates with excessEllman's reagent [EllS-SEll, 5,5'-Dithiobis(2-nitrobenzoic
acid)]
is usedfor quantitativeestimationof thiol by measuringthe absorption due to Ellman's
thiolate(EllS ) at 412nm (equation5)so-su.
Reactionsof thiols with 4,4'-dipyridyldisulfide
and 2,2'-dipyridyl disulfide generate chromophoric thiols: 4-thiopyridone (r3z+n-:
1 9 6 0 0 M t c m - 1 ) a n d 2 - t h i o p y r i d o n e( e . o . , _ : 8 0 8 0 M - 1c m t ) r e s p e c t i v e l y s o - 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
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13. Thiol-disulfide interchanse
RS_.,;b,_, el'o*o,
Ellman's Reagent
EIIS-SEII
(5)
---*
RS-S
+ OrN
EIIS
E 4 r 2 n ^ :1 3 6 0 0M
tcm-t
cyclic disulfides absorb in the UV region a t 3 3 0 n m ( e : 1 4 7 M t c m t ) a n d s i x membered disulfides absorb at 290nm (e: 2 9 0 M t c m t ) o r 3 l 0 n m ( e: 1 1 0 M I
a-- tys,s:,63-6s.
1H NMR spectroscopycan be used to determinethe position of equilibria in thiol
disulfideinterchangereactionsro'13'18,
and to follow the kineticsof the reactions(either
in the reactingsystemor after quenchingwith acid)18'6668.This method is usefulwhere
the methyleneprotons e and p to the sulfur in the reactantsand products differ in chemical
s h i f t a n d c a n b e i n t e g r a t e da c c u r a t e l yD
. y n a m i c l H N M R l i n e s h a p ea n a l y s i s l s ' 1 ' a n d
spin-transfermethodse'6ehave been used to determinethe rate constantsof degenerate
intermolecular thiolate-disulfide interchangereactions:RS- + RSSR: RSSR + RS
Analysisof 1H NMR lineshapes,
wherethe resonances
is useful
are exchange-broadened,
for determining the rates of fast degenerateintermolecular interchangereactionsbetween
thiolatesand disulfides(ft. c:al0 107M t ,- tlts'tz. 16. spin-transfermethod between
pairs of exchangingprotons and carbons (a to sulfur) is applicable for 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 fast enzymaticreactioninvolving glyoxalase-I.The glutathione(GSH)
that is releasedis convertedto S-lactoylglutathione by reaction with 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
rates of 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 assay is subject to errors
due to oxidation of thiol groups if air is not carefullyexcluded.
R S _+ G S - S G = R S S G + G S
GS- + CH3COCHO = GS-- CH(OH)
GS-CH(OH)
(6)
(7)
COCH.
COCH. + GX-I = GS-CO-CH(OH)CH1
+ cX-I
(8)
The equilibria of thiol disulfide interchange reactions between ry,o-dithiols and
lipoamide can be determinedconvenientlyby 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
R
\sn
, \ / *CoNH2
S -S
(9)
a?
\s
-1tlr
&coNH2
\
HS
+
/
SH
ftcoNH2
SH
HS
NAD'
(l 0 )
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-dithiol 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 cysteinederivativesor peptidescontaining cysteinehave been determinedby
HPLC71 and gel-filtrationchromatographyl2on the reaction mixtures after quenching
with acid.The equilibrium products of the reactionof glutathioneand cystinehave been
separatedby ion-exchangechromatographyT3or by electrophoresisof the 3sS-labeled
compoundsTa.Gas chromatography of the equilibrium mixture of alkanethiols and
disulfideshas been used to estimateequilibrium constantsTs.
III. MECHANISM
A. Products
Thiol disulfideinterchangeof a monothiol (RSH) with a disulfide(R'SSR')involves
multiple equilibria (equations1 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 interchangereactionrequiresthiolate anion, the observed
rate of reaction (and, in systemsin which the participating thiols have different values
of pKu, the observedposition of equilibrium) dependsupon the pH of the solution and
639
13. Thiol-disulfide interchange
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-disulfide interchangeat 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
c o n v e r s i o no f R S - t o R S H .
C. Kinetics
1 . R a t el a w
The thiol disulfideinterchangereactionis overall second-order:first-orderin thiolate
reactionof a thiolate(RS )with Ellman'sdisulfide
For a representative
and in disulfide6'7.
(EllS-SEll,equation 5) the rate laws are given by equatiorrsl 2 and 13. The rate constant
ft*, derivedfrom equation 12,basedon the concentrationof the thiolate,is independent
basedon total concentrationof thiol (equation 13),
of pH; the observedrate constantkoo.o,
dependsupon pH. The value of the rate constant ft*, is useful for interpretationsof
reactivity (such as Br/nsted correlationsof rates or equilibrium constantswith values
of pK" of thiols).The calculationof the observedrate constantkou.a3t the pH of reaction
is straightforwardfrom equation 13 using the value of the total thiol presentin solution,
[RSH],o,u,: IRS ] + [RSH], and is usefulfor predictingratesat the samepH. The two
rate constants koo.oand ft*, are related to each other by equation 14 and can be
interconverteduding the values of the pK" of thiol and the pH of solutions'o.Table I
lists the valuesof rate constantsfor representativethiol-disulfide interchangereactions
in water.
(dIEllS fldt):
(t2)
(13)
k R S[ R S - ] [ E l l S - S E l l ]
(dIEllS ]/dr) : ft"b,,r[RSH],.,"r[EllS-SEll]
(14\
k * , : k o o . u (+l l o ' ( a - P H )
r a t e c o n s t a n t so f t h i o l - d i s u l f i d e i n t e r c h a n g er e a c t i o n si n w a t e r
TABLE 1. Representative
Reactants
Thiol
Mercaptoethanol
3-Mercaptopropanoic acid
Mercaptoethanol
Propanethiol
Thiophenol
Dithiothreitol
Dithiothreitol
Disulfide
pKo
(thiol)
(M
k*,
I
min
')
(M
ftnb.,l"
- I
')
min
Temp
( c)
Ref.
Oxidizedglutathione
Oxidized glutathione
9.6
10.6
3.4x 103
1 . 2x 1 0 4
It.7
3.2
30
30
5
5
Ellman's disulhde
Ellman's disulfide
E l l m a n ' sd i s u l f i d e
Papain-S-SCH-.,
Oxidized mercaptoethanol
9.6
10.5
6.6
9.2
9.2
1 . 5x
6.4x
1 . 3x
5 . 3x
3 . 7x
3 . 7x 1 0 4
2 . 0x 1 0 4
9 . 6x 1 0 s
3 . 3x 1 0 3
2.3n
30
25
30
30
25
6
7
6
11
18
107
101
106
10s
102
'Values of kon"oare at pH 7. hThe value of the rate constant is corrected statisticallyfor the presenceof two thiol
g r o u p so n d i t h i o t h r e i t o l .
Y
640
R. Singh and G. M. Whitesides
2. Brdnsted relatron
The log of the rate constants(/<*. ) of thiolate disulhdeinterchangereactionsfollows
a Br/nsted correlation with the values of 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.Alkyl and aryl thiolates show separatecorrelation lines with
Ellman'sdisulfideT,but show a similar slope,fnu" :0.5. An aromatic thiolate reactswith
Ellman's reagentTor the mixed disulfideEIIS-SCH2CH2CH2OH76at a rate faster by a
factor of 6 than an aliphatic thiolate of the same pKu. The higher reactivityof aromatic
thiolates in comparison to aliphatic thiolates is probably due to greater softness(and
weakersolvation)of the formerT'8.Br/nsted correlationshave also beenreportedfor 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 pKuRSH
for the
nucleophilic(nuc),central (c) and leaving group (lg) thiols on the rate of the reaction.
Equations 17 and 18 represent different types of fits to data (ft*, is in units of
M l m i n - 1 ) s . E q u a t i o n l T r e p r e s e n t tsh e b e s t f i t t o a l l t h e a v a i l a b l ed a t a , a l t h o u g h i t
is suspectbecausethe valuesof pn,. and Br"arenot obviouslycompatiblewith a transition
statewith chargesymmetricallydistributedbetweenthe terminal sulfur atoms.The data
included in the correlation are taken from a range of studiesand are not necessarily
directly comparable. Severaldifferent 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
beenestimatedas ca -03 to -0.4 from a limited set of data for reactionsof RS- with
E l l S - S E l l ,R S - S E l l ,a n d H O C H , C H , C H , S - S E l l 7 6 .
logk*, : C + f,,"pKun'" *f"pKu'* p,rpK"'*
r\
R'u"S + R'S-SRrg
R n u c s S R "+ S R r g
logk*r :7.0*
0 . 5 0 p K , " u-"0 . 2 7 p K u ' - 0 . 7 3 p K u r s
l o g f t * r - : 6 . 3* 0 . 5 9p K , ' u " - 0 . 4 0p K u ' - 0 . 5 9p K " r s
(15)
(16)
(17)
(18)
The data on which equations17 and l8 are based cover a range of different (and
perhapsnot directly comparable)thioland disulfidestructures.A study of the Brpnsted
coefficientsof the nucleophilic,central and leavinggroup thiols using a carefullylimited
and consistentset of thiols and disulfidesu'oLrldbe usefulmechanistically.In the absence
of unambiguouslyinterpretabledata, these correlations(equations17 and l8) should be
consideredas kinetic modelsfor thiolate-disulfideinterchangereactions.Although they
do not have an unimpeachablemechanisticfoundation, they are useful in predicting
rate constants (ft*r ) in water. The value of kp" can be converted to the observed rate
constant (ft.0.0)at the pH of reaction using 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 value of the pH of the reactionmixture;
at pH 7 a nucleophilic thiol of pKu: 7 will show the maximum observed rate of
thiol-disulfide interchange(Figure 2).This predictioncloselymatchesthe observedrates
of thiol-disulfide interchanges'6and is useful in designing reagentsthat 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 <<pH, thiol is presentas thiolate. but its
13. Thiol-disulhde interchanse
641
1.0
a
0. 8
E
+
I
a
tr
0.6
o. 4
I
.n
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 degree of 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
c o n c e n t r a t i o no f t h i o l a n d t h i o l a t e i n s o l u t i o n i s g i v e n b y k o u , o : 9 l r n s -
nucleophilicityis low. A thiol of pK": pH balancesthe proportion of thiol presentas
thiolate and adequate nucleophilicityof 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 carbon I to sulfur. It is significantly large even when
two of the three adjacent thiols 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
' s 1 1 i nb u t a n o l i s c a 1 0 6 - f o l ds l o w e rt h a n t h a t f o r 1 - b u t y lt h i o l a t ew i t h
(k. :10-7M
bis(1-butyl)disulfide(/c. : 0.26M I s 1)24.The observedrate constantfor the reaction
with the mixed disulfideof penicilof penicillamine(-OOC-CH(NHJ)-C(CH3)2SH)
- 1.;
is ca 10s-foldslower than
lamine and glutathioneat pH J .4 (l;"b,d: 0.00047M 1min
that of penicillaminewith glutathione disulfide which contains no substitution n to sulfur
( k o 6 . 6 : 3 7 M l m i n - 1 1 6 6 .T h e o b s e r v e dr a t e c o n s t a n tf o r t h e r e a c t i o n o f g l u t a t h i o n e
lmin t) is also ca2000-fold lower than for
with penicillamine disulfide (0.012M
The equiliglutathionewith mixed penicillamine-glutathione
disulfide (27 M 1min 1166.
brium constant for the formation of bis(r-butyl)disulfideis small in the reaction of r-butyl
thiol with mixed 1-butyl /-butyl disulfide,i.e. the formation of bis(r-butyl)disulfide is
7.
disfavoredTs'7
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 threefold lower 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 cystamine(* H3NCH2CH2SSCH2CH2NH3 )78.
\
642
R. Singh and G. M. Whitesides
The thiol groups in some proteins appear to be relativelyinaccessible,
possibly due to
a combination of stericeffectand other factorssuch as local hydrophobicitv or charse
charge repulsion.
b. Acidity. The rate constants of thiolate disulfide interchange reactions vary
significantlywith the aciditiesof the substratethiols: The reaction of mercaptoethanol
w i t h E l l m a n ' sd i s u l f i d e( k n s : 1 . 5 x 1 0 7M r m i n r ) 6 i s s i g n i f i c a n t l yf a s t e rt h a n t h a t o f
m e r c a p t o e t h a n owl i t h g l u t a t h i o n ed i s u l f i d e( / . * , : 3 . 4 x l 0 r M r m i n r ) s i n w a t e r :t h e
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 disulfide inteichangereactions.
The rate constants for thiolate-disulfide interchangc(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 leaving group 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<iic
R'S group is retainedin the new mixed disulfide(RSSR')76.
c. Charqe. The rates of thiol-disulfide interchangereactionsin aqueous solutions
with charged substituentsvary by as much as a factor of 2.5 from the predicted rate
constantsbasedon structure reactivitycorrelationswith unchargedsubstituents'e.The
deviations from predicted values based on uncharged substituentsare the greatestwhen
the chargeis on the centralgrolrp,and the deviationsdecreasewith increasingdistanceof
the chargefrom the reactivesitete;e.g.,
both the ratesof reactionsof OTCCHTCH2S with
t h e m i x e d d i s u l f i d e s ' O 2 C C H 2 C H 2 S S C 6 H * N O r -apn d O 2 C C H 2 C H , C H , S S C 6 H 4 NOz-P are lower than the predicted values based on the Br/nsted correlation with
u n c h a r g e ds u b s t i t u e n t sb, u t t h e d e v i a t i o n i s l o w e r w i t h O , C C I H 2 C H , C H , S S C 6 H 4 NOr-ptn.A similar effectof the negativechargeon R groups is sein in the raG constantsfor
degenerateRS /RSSR interchangereactionse.Thiolateswithout chargedsubstituents,such
as mercaptoethanol thiolate, react 25 times faster with a positively charged analog of
Ellman'sdisulfidethan with the negativelychargedEllman'sdisulfide;this rzitiodecreases
to ca I to 3.5for a thiolate with a positivelychargedsubstituentthree bonds from sulfur
(cysteineethyl ester),and increasesto 120 for a thiolate with a nesativelv charsed
-)to.
s u b s t i t u e n (t - O 2 C C H T S
The electrostaticinfluenceof the local cysteineenvironmentsin peptideshas been
observedin thiol-disulfide interchangereactionsrt'at.76. rate constantsin water for
the reactionof the negativelychargedEllman'sdisulfideand a peptidecontainingcysteine
with two positive neighbors,one positive and one neutral neighbor, or two neutral
n e i g h b o r sa r e 1 3 0 , 0 0 03, 3 5 0 a n d 3 7 0 M - 1 s - r r e s p e c t i v e l a
y t pH 7 and 20mM ionic
s t r e n g t h 8 l .E l e c t r o s t a t i cc o n t r i b u t i o n st o t a l i n g a f i c t o r o i Z O O b( A G : 4 . 3 k c a l m o l - r )
have been estimated for the fastestand the slowest thiol-disulfide interchangereactions
of small charged substratesin 50\ methanol-water mixture; these contributions to
the free energy comprise *3.0kcalmol I from attraction and - 1.3kcalmol- 1 from
repulsionTl.
d. Hydroglen bonding. The rates of thiolate-disulfide interchange in polar aprotic
solvents are not significantly affected by groups capable of intramolecuiar hydiogen
bondingls. The rate constant for the degeneratethioiate disulfideinterchang. reaction
13. Thiol-disulfide
643
interchange
TABLE 2. Comparison of rate constants for degenerate thiolate disullide i n t e r c h a n g e( R S +
-SR)
in polar protic and polar aprotic solvents
RSSR=RSSR +
RS_
M-
Solvent
HOCH,CH.S
Na'
K'
K*
K*
K'
Na*
K*
KK*
KKn
DrO
DrO
CD3OD
DMF-d?
DMSO-d6
DMF-d?
DMSO-d6
DMF-d7
DMSO-d6
DMSO-d6
DMSO-/6
cH3cH2cH2cH2scH3c(cH3)2cH2sHOC(CH3)2CH2S
H O C H T C ( C 3H) 2 C H 2 S
AGI
19-: ;,c,tt
( M 1 s 1 . 1( k c a l m o l - t )
LHI
AS+
-lmol
( 2 9 7K )
( 2 9 7K )
( k c a lm o l t ) ( c a l K
0.0077
0.0095
0.0040
20
2l
43
54
l5
t6
1.1
0.67
16.2
l 6 .r
16.6
I 1.5
_tJ
t a
I --)
13
IJ
t)
-10
-11
l')
l a
l-)
r r.5
11.1
I 1.0
1t.1
|.7
13.2
13.5
t0
o
-10
- 16
lmol I.
1.AS].
t; AH;.
' l j n c e r t a i n t i e s a r fet:,
t2cal K
t l 0 ' l ; , ' A G l . + 0 . l k c a lm o l
f lkcalmol
b R a t ec o n s t a n t sw e r e i n f e r r e df r o m v i s u a lc o m p a r i s o no f t h e s i m u l a t e d' H N M R l i n e s h a p e sw i t h t h e e x p e r i m e n t a l
: ll other
line shapesT
. h e v a l u e sf o r C D . O D a r e u n p u b l i s h c do b s e r v a t i o n so f R . S i n g h n n d G . M . W h i t e s i d e s a
values are frogr reference I 5.
of 2-hydroxyethanethiolateis only twofold lower than that of 1-butanethiolatc.In
stericallyhinderedthiolates,introduction of a hydroxy group either P or "; to the C- S
bond slows the interchangeby approximately a factor of 15 in DMSO (Table 2). A
gem-dimethyleffectand weakersolvationof the hydroxyl group in the stericallyhindered
substratemay result in greaterintramolecularhydrogen bonding than in the sterically
s.
unhindered2-hydroxyethanethiolater
e. Reactionsint'oltin(Jc),clicdisul/ides. The rate constantfor degenerateintermolecular
thiolate-disulfideinterchangeinvolving cyclic five-membereddisulfides(1,2-dithiolane)
is higher than that involvingcyclic six-membereddisulfides(1,2-dithiane)by a factor of
t)tt. The rate constants for the cyclic six- and
c a 6 5 0 ( A A C I c a 3 . 8k c a l m o l
disulfidesare similar to thosefor noncyclicdisulfides't.The ring strain
seven-membered
of 1,2-dithiolane(estimated by calorimetry) is higher than that of 1,2-dithianeby
1; from kineticsand
3.7kcalmol 182.The agreementof the value of AAGI (3.8kcal mol
the value of ring strain (3.7kcalmol ') from calorimetry suggeststhat the ring strain
is completelyreleasedin the transition stater'. In
in the cyclic five-membereddisulf-rde
the transition state,the S-S bond is expectedto be longer than in the ground state of
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 state of the cyclic five-membereddisulfidethan that of cystine,
b a s e do n X - r a y c r y s l a l l o g r a p h i sc t r u c t u r a lp a r a m e t e r s r ' .
4. Solventeffects
The ratesof thiolate disulfide interchangereactionsare larger in polar aprotic solvents
(DMSO, DMF) than in polar protic solvents(water, methanol) by a factor of ca 103
( T a b l e2 ) 1 s .T h e n a t u r eo f t h e c o u n t e ri o n s ( N a * , K * ) , o r a d d i t i o n o f 1 8 - c r o w n - 6t o t h e
reaction involving potassiumalkanethiolate,has no effecton the rate of thiolate disulfide
i n t e r c h a n s ien D M S O t 5 .
644
R. Singh and G. M. Whitesides
The transition state is expected to have a more delocalizednegative charge and
thereforeto be lessinfluencedby solvation than the ground state thiolate. The higher
ratesof thiolate-disulfideinterchangein polar aprotic solvents(DMSO, DMF) than in
polar protic solvents(water, methanol) may be explained by a smaller destabilizarion
of the transition state than that of the ground state thiolate, in going from polar protic
solventsto polar aprotic solvents(Figure 3)15.The log of the rate constant depends
l i n e a r l y o n t h e s o l v e n tc o m p o s i t i o ni n m i x t u r e s o f w a t e r a n d D M S O ( F i g u r e 4 l t s ' t r .
R
r
+
l-T
LRS..s..r*J
D M S O ,D M F
RS
-
+ R S SR
RS
-
water,methanol
+ RS S R
FIGURE 3. Hypotheticalplot of free energy vs reaction coordinate for
thiolate-disulhdeinterchanger e a c t i o n i n p o l a r p r o t i c s o l v e n t s ( w a t e r .
methanol)
and in polaraprotics o l v e n t s( D M S O , D M F )
*
ctl
o
020406080
moloh
100
DrO (or CD3OD)
F I G U R E 4 . E f f e c to f a d d i t i o n o i D r O ( l ) o r C D 3 O D ( O ) o n l o g
of rate constants (k) ol thiolate-disulfide interchange of potassium
2-hydroxyethanethiolateand bis(2-hydroxyethyl)disulfide in DM SOdu. The values of rate constants are for 297K and are in M I s t
r
645
13. Thiol-disulfide interchange
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 (Figure 418s.The absenceof a sharp drop in rate on addition of small mole
fractions of 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
g , 2 r e a c t i o n so f a l k o x i d ea n i o n ( R O - ; t s . T h e a l k o x i d e
l e s st h a n t h a t ( 1 0 6 - 1 0 7 ) i n v o l v i n S
anions are more solvatedin water than are thiolate anionss6'87.
and 1,2-dithiolane
The rate of thiolate disulfideinterchangeof 1,3-propanedithiolate
( c y c l i cf i v e - m e m b e r e d i s u l f i d e )i s e x t r e m e l yf a s t i n D M S O ( t R S c ' c l 0 8 M 1 s - r ) a n d
only ca 102 slower than the diffusion limittT (equation20). This large rate arisesfrom
is destabilizedrelativeto the transition
two factors:(i) the ground state of 1.2-dithiolzrne
statebecauseof ring strain, and (ii)the thiolate is relativelymore destabilizedin DMSO
than is the transition state with its more delocalizedcharse17.
+O=-O+O
S_ S_
S-S
S-S
(20)
S_S_
A c o m p a r i s o n o f t h e s t r e n g t h so f t h e R S - " H O R c o m p l e x e sa n d R O - " H O R
massspectrometryshowsthat complexesincorporatcomplexesby pulsedhigh-pressure
i n g a l k o x i d e sa r e m o r e s t a b l eb y 2 T k c a l m o l - r t h a n t h o s e i n c o r p o r a t i n gt h i o l a t e s s 8 .
The weak contributions of ionic hydrogen bond to solvation in RS-(HzO), complexes
are effectivelydissipatedwithin the first 2-3 solvent molecules(rt: 2 -3)tt.
5 . G a s - p h a s es l u d i e s
The reaction of 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
s i d er e a c t i o n t, h e c a r b o n - c e n t e r esdu b s t i t u t i o nt o y i e l d C H l S S a n d C H . S C r H r . i s n o t
The valueof the rate constantfor the reactionof ethanethiolatewith dimethyl
observed8e.
disulfide in the gas phase is estimated as 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
o b s e r v e do n l y w i t h s e l e n o l s ' s ' t oa, n d e v e n w i t h t h e s es p e c i e st h e m a g n i t u d e so f t h e
catalysisare not largc.
Selenols are only effective as catalysts for thiol disulfide interchange reactions
involving strongly reducing dithiolsle. The observed rate of reduction of bis(2hydroxyethyl)disulhde b-v-'
dithiothreitol in water at pH 7 is enhancedby a factor of 15
i n t h e p r e s e n c eo f 5 m o l u , , 2 - a m i n o e t h a n e s e l e n T
o lh. i s c a t a l y t i ca c t i v i t y o f s e l e n o l si s
probably due to a combination of the low pK^(ca 5.5 to 7) (and hencesignificantlyhigh
concentrationof RSe at pH 7) for selenols,and weak solvationand high polarizability
(and hence high nucleophilicity)of the selenolateanion. The precursorsof selenols.
(RSeCN) can also be conveniently used to
diselenides(RSeSeR)and selenocyanates
catalyzethe thiol-disulfide interchangereactionsinvolving strongly reducingdithiols'e.
Thiol-disulfide interchangereactionsinvolving monothiols are not catalyzedby selenols.
Strongly reducins dithiols at
becausethesedisulfidesoxidize the selenolsto diselenides.
646
R. Singh and G. M. Whitesides
even moderate concentrationscan reduce diselenidesto selenols.and thereforein the
thiol-disulfide interchangereactions involving strongly reducing dithiols, the selenol
remainsin the reduced(and catalytic)statele.
7. Compartson wrth selenolate-drselenrde
rnterchange
The observed rate of selenolate-diselenide
interchange for selenocysteamine
and
s e l e n o c y s t a m i n( e
k o o . o : 1 . 6 5x 1 0 7M - 1 s t ) u n i n w a t e r a t p H 7 i s f a s t e i b y a f a c t o r o f
107 than the correspondingthiol <lisulfideinterchange reaction of cysteamineand
c y s t a m i n e( f t o t . a : 1 . 4 M - t s 1 ) , p o s s i b l y d u e t o ( i ) b e t t e r n u c l e o p h i l i c t t ya n d b e t t e r
leavinggroup ability of selenolatethan for thiolate, and (ii) low pK, of selenols(c,a5.5
to 7) and thereforehigh concentrationof the nucleophilicselenolatcanion at pH 76e.
In this system,the absoluterate constant(k*s" ) for the selenolatediselenideinteichange
is higher than that of the structurallyanalogousthiolate-disulfideinterchange(ft*s ) by
2.4x l}s.
D. Transition State Structure
A study of crystal structuresof compounds containing divalent sulfur (Y S Z:
Y,Z + H) showsthat nonbondedcontactsof nucleophilesare directedalong the extension
of one of the covalent bonds to sulfur26.According to the frontier-orbial model. the
HOMO of the nucleophileinteractspreferentiallywith the LUMO (o*) orbital of S y
or S-2. Attractive nonbonded interactions may represent the incipient stages of
chemical reactions26The 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 qualitatively pictured as having greater negativecharge
at the terminal sulfurs than at the central sulfur, based on the value of ihe BrdnstJd
c o e f f i c i e n t sf:n , . : l J r r : 0 . 5 ( b y s y m m e t r y ) :f i , = - 0 . 3 t o - 0 . 4 s 8 . T h e a b s e n c eo f
curvature in the Br/nsted plots for attack of thiolate anions having a range of pK"
valueson a singledisulfidesuggeststhat the transition state structurecloesnot .hang.
with changes in structure of the thiolate anions or the disulfide groups for theie
thiol-disulfideinterchangereactionss 7.Superpositionof plots of log kl, (rateconstant)
vs log K. (equilibrium constant) for zr sericsof thiol disulfide interchaneereactions.
varying in equilibrium constantby a factor of approximately102'. shows gradual
c_urvature
of 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 Brdnsted plots. Although
thiolateanionsare not as strongly solvatedas alkoxide anions,interpretaiionssuggesting
a change in structure of the transition state from a curved Brdnsted plot should Ue
t r e a t e dw i t h c a u t i o n s o ' e o1' e.
T h e v a l u eo f A S ; f o r t h i o l d i s u l f i d ei n t e r c h a n g ei n p o l a r p r o t i c ( w a t e r .m e t h a n o l )a n d
I (Table2)ts.
p o l a r a p r o t i c( D M S O , D M F ) s o l v e n t si s c ' a - 1 0 t o - l 6 c a l K - 1 m o l
This value is less than that expectedfor complete localizationof two particlesin the
transition state,and suggeststhat the decreasein entropy in the transition state relative
to two particlesin the ground stateis partially compensatedeither by releaseof solvent
moleculesattachedto the thiolatein lhe ground statels,or by a relativelyloosetransitionstate structure(with two weak, partial S..S bonds) or both.
E. Theoretical Calculations on Thiol-Disulfide Interchange
An ab initio MO study on the thiolate-disulfideinterchangereaction indicatesthat
the reactionis a typical S"2 reactionand proceedsvia a singlelransitionstatewith 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.
0.2- 0.4.
R S . . S. . . S R
R
0.4-
F. Mechanistic Uncertainties
The geometry of the transition state is unclear:the relative dispositionsof the alkyl
groups on the three sulfur atoms in the transition state are not known. The symmetry
of the transition state with respectto the nucleophilicand leaving group thiols is still
ambiguous,although microscopicreversibilitywould indicate a symmetricalstructure
if there is a single transition state. Unsymmetrical transition states connected by a
symmetricalintermediateare possible,but seem unlikely. A more complete characterization of the Br/nsted coefficients,and appropriate calculations, will both be useful
in understandingthis issue.The transitionstateseemsto be lesssolvatedthan the ground
statethiolate,but the degreeof solvation of the transition state is not known. Resolving
the question of solvation may be useful in designing strategies for catalysis of
thiol-disulfide interchange.Strategiesfor catalysisbasedon desolvationand destabilization of the ground statethiolatesseemunlikely to producelargeeffects.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 chargetransition state.
charse interactionsin the charse-delocalized
I V . E Q U I L I B R I U MI N T H I O L - D I S U L F I D EI N T E R C H A N G ER E A C T I O N S
A . E q u i l i b r i a I n v o l v i n gM o n o t h i o l s
In the equilibria involving a monothiol (RSH) and a disulfide(R'SSR')(equations1
and l1), the distribution of speciesis nearly random or statisticalif the pKu values of
the thiols (RSH and R'SH) are similar, i.e.
K r : { ( I R S S R ' , ] [ R ' S H ] ) ^ [ R S H ] I R ' , S S R) l' ]t 2
and
K z : \ ( I R S S R ]I R ' , S H ] X [ R S H ] I R S S R ' , ]I) : 0 . 5
The experimentalvaluesof equilibrium constantsfor the interchangeinvolving glutathione
(RSH) and cystine(R'SSR')in water at pH 7 are similar to those expectedfrom random
d i s t r i b u t i o f l ,K r : 3 . 7 a n d K z : 0 . 7 9 2 3 ' 7 3 ' e 3 .
The equilibrium constants for thiol disulfide interchangereactions for a seriesof
monothiols and disulfides,at values of pH in which the equilibrium concentrationof
thiolate anion is small, are relatively insensitiveto changesin substituents(exceptfor
sterically hindered structures with alkyl substituentsat carbon 1 to sulfur)68.The
formation of bis(r-butyl)disulfidesis disfavoredin equilibria involving t-butyl thiol and
m i x e d r - b u t y l l - b u t y l d i s u l f i d e T s ' 7 1 ' ea4n,d t h e f o r m a t i o n o f p e n i c i l l a m i n ed i s u l f i d ei s
disfavored in equilibria involving penicillamine and mixed penicillamine cysteine
disulfide68.
The equilibrium constant for 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 stable thiolate: 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-disulfide interchangereactioninvolving 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'sthiolateis approximatelyquantitativelyequalto thaf of initial mercaptoethanol,
and hence the utility of Ellman's assay.
The valuesof the equilibrium constant(K'o'a, of thiol-disulfideinterchangein aqueous
medium can be dissectedint.o KsH (defined for thiols) and Ks (defined for thioiates;s.
The equilibrium constant KsH shows no obvious correlationwith the valuesof pKu, but
t1 i{199.1tcedby.steric effects.The plot of the log of the equilibrium constanf Kb vs
2(PK,*tn-pKuR'sH)
is linear (slope ia 1.2)s.xs is=thereforestrongly influencedby the
aciditiesof the participating thiolss.
Electrostaticeffectson equilibria of thiol-disulfide interchangereactionsare 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-acetylcysteine
(A, bearing one negativecharge on the cysteinecaiboxylate)
and,the 85 114 peptide fragment of Kunitz soybeantrypsin inhibitor (B, bearing one
positive charge on the N-terminal leucine residuenext 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 expectedstatisticaldistributions are 50.t.,25'; an<i25".,,reipectively.
The electrostaticeffectat low ionic strength(20mM) favors the formation of A B. and
disfavorsthe formation of A A more than that of B - B. becausethe two negative
chargeson A-A
are closer to each other than are the positive chargeson B- B. At
high ionic strengthsthe electrostaticeffectsare shieldecland the obseived distribution
is similar to that statisticallyexpectedT2.
B . E q u i l i b r i a I n v o l v i n ga , o D i t h i o l s
Thiol-disulfide interchangeof a,rr;-dithiols(HS R SH) with a disulhde (R'SSR')
can generatea varietyof productsrangingfrom cyclicmonomericdisulfide,cyclicdimeric
6S-SR'
Lrt
dH
[\sH + R',s-sR
*t"
+
al
\{
.S -S\
RR
Ls-sJ
-I-s-R-s+
4S-SR'
L,- ,*'
mixed
disulfides
cyclic
monomenc
disulfide
cyclic
dimeric
bis (disulhde)
oligomer
(cyclic or linear)
(21)
7
649
13. Thiol-disulhde interchange
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 corresponding intermolecularreaction.High effectiveconcentration(EC, see below and also
Table 3, for footnote c) favors the formation of the cyclic monomeric disulfrdelt.
.SH
R
Ltt
k1
+ R/ S - S R/
k_1
4 S -SR '
R
+ R'SH
lt"
A:_;l
-r.
'
S
I
i\_s
+ R'SH
(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-membereddisulfideshave 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-membereddisulfrdeshas been estimatedas 3.Tkcalmol-1
higher than that for the cyclic six-membereddisulhdest'. It has been estimated that the
ring cleavage(k ,, equation 22) of cyclic five-membereddisulfides is faster by a factor
of ca 600 than that of cyclic six-membereddisulfidesr7. 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 cyclic six-membereddisulhde,basedon valuesof kinetic effectiveconcentrations
for analogousreactionslT.The overall result is that K"o for formation of a six-membered
disulfide from 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 intramolecular ring-closurestep (kr, equation22). 1,4-Alkanedithiolsthat form strain-free
cyclic six-membereddisulfidesare the most reducing (K", ca 10-103M, equation 23);
rings respectivelyare
1,3- and 1,5-alkanedithiolsthat form five- and seven-membered
ca 10-fold less reducing (Table 3). Rings smaller than six-membered are less favored
primarily for enthalpic reasons(ring strain, including angle strain in the CSSC group).
Rings larger than six-memberedare lessfavored becauseof conformational entropy (low
kinetic effectiveconcentrationfor the intramolecular ring-closure)t:'t0.In 1,8-dithiols,
the effective concentration 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-Dithiolsform cyclic
bis(disulfide)dimers in relatively dilute solution (r'a l mM), but polymerize aI higher
13.
concentrations
Molecular mechanicscalculationsof equilibriaof thiol-disulfideinterchangereactions
involving a,o-dithiols with 1,2-dithianecorrelatewell with experimentalresults,but do
not give the absolutevaluesof energiestu.The empirical relationshipbetweencalculated
differencesin strain energy (ASE) and the experimental values of AG is: AG c'a0.4ASE.
Why the molecular mechanicscalculations overestimatestrain 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
K(ME")
Structure
€o(V)"
Eq.
againstD
References
Dithiols that form
cyclic monomers'
(^Y-rt
1500M
- 0.354
DTT
d.e
670M
-0.344
DTT
d,e
1 8 0M
-0.327
Lip
d.e
77M
- 0.316
DTT
d.e
65M
- 0.314
DTT
d,e
63M
- 0.313
DTT
44M
-0.309
DTT
19M
- 0.298
DTT
r5M
-0.295
DTT
14M
-0.294
DTT
d.e
8 . 6M
- 0.288
ME. DTT
d.e
8.0M
-0.287
DTT
d.e
\-/---sH
ff,"
\-\-s"
HoF.,-,
"n'1"-;;
a---su
\,,-sH
l
/\
2 \st
LSH
sH
or\f=-'c
sn
oZ\-,--
r^r"
|\._jn
d.e
H'c'6-1^su
".6,s-s"
H,c 1
/P
,N-\
rt"
\
,,SH
NJ
H.c--(
o
"'tV-
H,c- \---^..^..
:)H
(Yt'n')4co:H
HS
SH
/--\/-sH
\ / L q H
( r ' o n t i n u e Id
651
13. Thiol-disulfide interchanse
TABLE 3. (continuedl
K(ME'-)
Structure
/"-- _
.sH
eo(V)'
Eq.
againstb
References
6 . 7M
- 0.285
DTT
d.e
o.*sn
6 . 1M
-0.284
DTT
d,e
(r',vX
4.4M
-0.279
DTT
d,e
rY-'"
3 . 6M
-0.277
DTT
//---SH
.^SH
3 . 6M
-0.277
3.1M
-0.275
DTT
2 . 9M
-0.274
DTT
2 . 5M
-0.272
DTT
2.-rM
-0.2'71
ME. DTT
1 . 8M
-0.269
DTT
1 . 2M
-0.263
DTT
d,e
0 . 6 7M
* 0 .2 5 5
DTT
d,e
0 . 3 0M
-0.245
ME
i
0 . 2 1M
-0.240
DTT
d.e
S.*s"
/'-v-sH
(-/
,z-SH
\_sH
l-
HS----r
511
z-SH
DTT
d.e
d.e
H.c\-sH
H.CX,---SH
H.C
\-SH
o\-H.C.
' 'N'
tsH
ilsH
d.e
d.e
A.1
'U Y
at"
\_sH
tl,e
CONMe,
I
a\su
(-,-t"
I
CONMet
},1-tt
r
\_qFl
/-SH
\-SH
6,6'-sucrose
disulfide
HS(CH2)6SH
( r ' o n t i n u e Id
-
652
R. Singh and G. M. Whitesides
TABLE 3. kttntinuedl
Structure
K(ME"-)
so(V)'
Eq.
againstb
References
Monothiolsthat
form dimers
1i}--JsH
\-,/
2.6
-0.272
ME
L1
cH3(cH2)6sH
1.1
-0.261
ME
d
HOt^\--sH
1.0
-0.260
ME
d
A-t"
Y/
0.31
-0.245
ME
(l
r--rlt"
tl
0.40M
-0.254
ME
d.e
a^"'?tn
tt
0.38M
-0.254
ME
d,e
0 . 3 2M
-0.253
ME
e.g
0.035M
-0.239
ME
d,e
4.8
-0.280
ME
4.0
-0.278
ME
3.4
-0.276
3.1
-0.27s
ME
3.0
-0.275
ME
(1
2.8
-0.274
ME
k
Dithiols that.fbrm
cvclic dimers
\---\SH
\,/^.sH
zl-sH
t()l
H.cr'*n
"t-,Arn
DithiolsthatJbrm
polymers
HS"*-sH
HS,,
,sH
,,A,
"rt-Vfr"
"i-Q-r"
rtt
t^l
HS---A\2\
SH
(continued
I
7
653
13. Thiol-disulhde interchanse
TABLE 3. (continued)
Eq.
againstD
K(ME"-)
co(V)"
-{t'
l-1_qg
t
1.8
- 0.268
ME
HS(CHr)8SH
HS(CH,),SH
1.7
1.4
-0.267
-0.265
ME
ME
HS,
1.3
-0.264
ME
0.20
-0.240
ME
Structure
Y O\ )--l- s "
r^\
HS-< ( ) >-Srr
\"/
References
.t
u
.t
u
"i:,,(V)values vs standard hydrogen electrodeat pH 7.0 and 25"C. All r;,,(V)values are calculated using the r;n(V)
v a l u e sf o r l i p o i c a c i d [ - 0 . 2 8 8 V . D . R . S a n a d i ,M . L a n g l e y a n d R . L . S e a r l s ,J . B b l . C h e m . . 2 3 4 .1 7 8 ( 1 9 5 9 )a n d
- i o p h y s .A t ' t u , 3 7 . 3 1 4( 1 9 6 0 ) la n d t h e K " o v a l u eb e t w e e nl i p o i c a c i d a n d t h e c o m p o u n d o f
C . V . M a s s e y ,B i o c h e m B
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, see Reference13.
"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 readjusted by a factor of approximately 2 so as to obtain a similar value to that reported in this paper.
/ E q u i l i b r a t i o n s w e r e c a r r i e d o u t a t 2 5 C i n a l / l m i x t u r e o f r / . - m e t h a n o l i p h o s p h a t be u f f e r ( 5 0 m M . p H 7 . 0 )i n
D r O , s e eG . V . L a m o u r e u x a n d G . M . W h i t e s i d e sJ, . O r q . ( ' h e m . , 5 8 . 6 3 3( 1 9 9 3 ) .
,Equilibrations were carried out in oln-methanolwith 0.02mM sodium methylate added, see Reference13.
h E q u i l i b r a t i o n sw e r e c a r n e d o u t i n D r O ( p D 7 . 0 . 5 0 m M p h o s p h a t e )s. e e R e f e r e n c e1 8 .
i E q u i l i b r a t i o n sw e r e c a r r i e d o u t i n D , O ( p D 7 . 0 , 5 0 m M p h o s p h a t e )s. e e R e f e r e n c e
20.
i E q u i l i b r a t i o n sw e r e c a r r i e d o u t i n D r O ( p D 7 . 0 , 5 0 m M p h o s p h a t e ) s. e e W . J . L c s s a n d G . M . W h i t e s i d e s J. .
Orq. Chem., 58, 642 (1993).
ftEquilibrations were carried out in r/o-benzenewith 0.02mM tetramethylguanidineadded, see Referencel3.
The equilibrium constant for the thiol disulfideinterchangeof an r,co-dithiolwith a
'effective
concentration'
disulfide (equations22 and 23) has also been termed as the
(ECles'so.The equilibrium expressionfor effectiveconcentration is a measure of 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 information about 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'
must be evaluatedwith the possibility of contributions
and
from theseterms in mindro. If the EC is used (and interpreted)just as an equilibrium
constant,but as one with an easily rememberedreferencevalue (EC ctt I 10 M 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
unstrained one, 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 values for 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 favored for the formation of the
six-membereddisulfideby a factor of 30 over the five-memberecl
disulfide.whereasthe
kinetic EC (relatedto k,.equation 22)is higherfor the ring-closurereactionfor formation
of the five-membereddisulfidethan that of the six-membereddisulfideby 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 perhaps most useful as i
measureof proximity when theseother factors(e.g.ring strain reflectingan unfavorable
CSSC dihedral angle;terms destabilizingthe dithiol relative to disulfide)are absent or
can be independentlyestimatedl6.
V . A P P L I C A T I O N SO F T H I O L - D I S U L F I D EI N T E R C H A N G EI N B I O C H E M I S T R Y
The subject of the thiol-disulfide interchangereaction is an important one in biochemistry,and has been discussedextensivelyelsewhere' 3. Here we will only outline
s o m eo f t h e i s s u e s .
Disulfrde-reducingreagents are used in biochemistry for a number of purposes,
especiallyin reduction of cystinegroups in proteins and in maintaining essentialthiol
groups in their reduced stateeT. e,rr-r-Dithiols,such as dithiothreitol (DTT)70,
N,l{'-dimethyl-N,N'-bis(mercaptoacetyl)hy
drazine(DMH)t 8 and meso-2.5-d,imercaptoA/,.4y',4/',N'-tetramethyladipamide
(DTA)20,have higher reduction potential than cystine
groups in proteins, and are useful disulfide-reducingreagents because they react
specificallywith the cystinedisulfideto be reducedwithout any unwanted sicle-reaction
with the protein. 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) reduce small organic
disulfidesand disulfidebonds in proteins c'a7 times faster than does DTT in water at
. e r c a p t o e t h a n o(lM E ,
p H l t 8 ' 2 0M
p K " c a 9 . 6 )i s i n e x p e n s i vaen d i s u s e di n l a r g ea m o u n t s
(0.1-0.7M) in bioc_hemicalmanipulations, for example in conjunction with SDS
gel-electrophoresiseT.
Mercaptoethanolis weakly reducingand it often generates
complex
reaction mixtures containinq mixed disulfidess.
ntt,
HO
HO
\
(
SH SH
DTT
N-N
/CH.
MerN
SH
ME
';-61-
^/
\"
DMH
bH (r-r
NMe,
DTA
The cyclic five-membered disulfide lipoamide is a cofactor of the pvruvate
dehydrogenasecomplexes'ee.The rate of ring opening of this cyclic five-membered
disulfide by thiolate*disulfide interchange 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 maintain a high flux through the pyruvatedehydrogenase
complexr7.
/
13. Thiol-disulfide interchanse
655
The valttesof pK, of thiol groups in proteins have been measuredkinetically from
t h e B r o n s t c d c o r r e l a t i o n o f t h i o l d i s u l f i d ei n t e r c h a n g er e a c t i o n s r r .T h e p K , o f t h e
actirc'-site
t h i o l i n p a p a i n i s e s t i m a t e da s c ' r r4 a t p H 6 , a n d t ' u 8 . 4 a t p H 9 1 r . A t l o w
P H { r , i 6 t t h e p r o x i m a t ep o s i t i v e l yc h a r g e dg r o u p i n c r e a s e tsh e a c i d i t y o f t h e a c t i v e - s i t e
t h r o l i n p a p a i n .T h e p K " o f t h e t h i o l g r o u p o f r e d u c e dl y s o z y m ei s c , r z1 1 .T h e s ev a l u e s
rrf pN.,. although semiquantitative,are useful for comparison with the values of pK"
t i c ' t c ' r r n i n ebdy o t h e r m e t h o d s rr .
Thc-redox equilibria betweenthe cystine-bridgedcyclic disulfidestructuresin proteins
lnd thcir correiponding reducedopen-chainz,r,,r-ditiriol
forms have been measuredfor
: i \ er i t l p r o t e i n s 3T. h e v a l u e o f t h e e q u i l i b r i u mc o n s t a n t( o r e q u i l i b r i u me x p r e s s i o nf o r
ciicctire concentration,equilibrium EC) for the thiol disulfide interchangereaction of
It protein z,a-r-dithiol
can be a useful measureof 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
s t r a i n1 6 ' e s ' e 6 .
T h e d i s u l f i d eb o n d s i n p r o t e i n sa r e f o r m e d a f t e r t r a n s l a t i o n r o o ' 1 0T1h. e p a t h w a y o f
sequentialdisulfide bond formation has been studied for bovine pancreitic rrypsin
i n h i b i t o r ( B P T I ) 1 0 2 ' ' 0 'ar n d f o r r i b o n u c l e a s A
e 1 0 4 .I n t h e c a s eo f B P T I . i n t e r p r e t a t i o n s
o f d i f f e r e n ts e t s o f d a t a h a v e l e d t o d i f f e r e n tc o n c l u s i o n s r 0 2 , l 0 T
3h
. e conilusion of
Weissman and Kim
that all well-populated folding intermediatesin the oxidative
foldingof BPTI contain only nativedisulfidebonds -,is still beingactivelydebateclI05.r 06.
The inclusionbodies,clbtainedfrom the expressionof eukaryoticproteinsin genetically
engineeredE. coli. may contain protein with unformed and mismatched disulfide
' 0 8 .T h e c o n v e r s i o n
b o n d sI 0 7 1
o f t h e ' w r o n g l y ' d i s u l f i d e - c o n n e c t epdr o t e i n t o t h e
'correctly'disulfide-connected
protein is a major problem in biotechnology.The general
approach is to reduce the 'wrongly' 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
m a k e i t s b i o l o g i c a lr o l e u n c e r t a i n r l r ' r 1 2T. h i o r e d o x i nh a s a c y s t e i n eo f l o w p K . , a n c ii t
reactswith disulfidesrapidl-vat pH 7. Thioredoxin is redox-coupledto NADPH via the
enzymethioredoxin reductase,and may be of metabolic significancein thiol-disulfide
i n t e r c h a n g er e a c t i o n s1r 3 I 1 s .
V I . C O N C L U D I N GR E M A R K S
Thiol disulfide interchange is a reversibleSr2 reaction that involves cleavageand
formation of a covalent S S bond. The active nucleophileis 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-disulfideinterchangereaction (k*, , based on the
concentrationof thiolate anion) is influencedby factorssuch as pK" of thiol and CSSC
dihedral angle in the disulfide.The rate constant(k*, ) increaseswith increasingvalues
of pK" of the thiols becauseof the increasingnucleophiticityof the thiolate anions.The
observedrate constant of reaction (/coo.o),
however,is optimum for the value of pK" of
the thiol equal to the pH of the soluiion. The most stable CSSC dihedral angle in the
disulfideis ca 90". Cyclic five-membereddisulfides,with CSSC dihedral angle of r,a 30 ,
are strained,and are cleavedc'a103timesfasterthan the less-strained
cyclicsix-membered
656
R. Singh and G. M. Whitesides
disulfide.An improved theoreticalconformationalanalysisof the ground state of cyclic
disulfides-in terms of the bond angles,bond lengths,and the CSSI and CCSS dihedral
angles-would be useful to predict the ring strains and rates of thiol disulfide
interchangereactions involving cyclic disulfides.
Thiolate-disulfideinterchangereactionsare 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"
c a 1 0 8M t s - t ) i n p o l a r a p r o t i c s o l v e n t s .
Disulfide bonds are presentin proteins and are formed from the cysteinethiols after
translation.The physical-organicstudy of severalbiochemicalissuesrelatedto the thiol
disulfideinterchange-the mode of formation of the'correct'disulfidebonds.the degree
of stability imparted to the protein by the disulfide bond. the strain in the large-iing
protein disulfides,the role of thiol-disulfide interchangein regulationof prorein activity
and the design of reagents that 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.ProfessorH. 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 Process EngineeringCenter
(Cooperative
A g r e e m e nC
t D R - 8 8 - 0 3 0 1 4 ) a n bd y t h e N a t i o n a l I n s t i t u t e so f H e a l t h( G r a n t
GM3e589).
VIII. REFERENCES
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Biochemistry
of'thesH Group.Acadcmicpress,London.1912.
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of Sulfur.PlenumPress.
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and G. M. Whitesidcs.
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1 5 . R . S i n g ha n d G . M . W h i t e s i d eJs.,A m .C h e mS. o r ' .l.1 2 . l l 9 0 ( 1 9 9 0 ) .
16.J. A. Burnsand G. M. Whitesides.
J. Am. Chent.
Sor'.,I12.6296(1990).
17. R. Singhand G. M. Whitesides,
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