Kinetics and mechanism of the addition of nucleophiles to α,β-unsaturated thiol esters

Article abstract of DOI:10.1021/jo060191+

Compounds containing the UV-absorbing chromophores p-methoxycinnamate, p-methoxycinnamide, or anthranilate and an α,β- or α,β,γ,δ-unsaturated thiol ester (crotonyl or sorboyl) have been prepared. These compounds are subject to nucleophilic attack at the C

Full text of DOI:10.1021/jo060191+

Kinetics and Mechanism of the Addition of Nucleophiles to
r,â-Unsaturated Thiol Esters
Rosemarie F. Hartman and Seth D. Rose*
Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-1604
srose@asu.edu
ReceiVed January 28, 2006
Compounds containing the UV-absorbing chromophores p-methoxycinnamate, p-methoxycinnamide, or
anthranilate and an R,â- or R,â,γ,δ-unsaturated thiol ester (crotonyl or sorboyl) have been prepared.
These compounds are subject to nucleophilic attack at the CdC conjugated to the thiol ester carbonyl
group. The kinetics of the reactions of these thiol esters with N-acetyl-L-cysteine (NAC),
N-acetylcysteamine, and N2-acetyl-L-lysine (NAL) have been studied, and the thiol addition products
have been identified. The reaction rates increased at higher pH, and the reaction of NAC thiolate with a
crotonyl thiol ester in 1:1 (v/v) acetonitrile/aqueous HEPES exhibited buffer catalysis as a result of
protonation of the enolate intermediate. At the same concentration, NAC underwent ∼300-fold more
reaction than NAL with a crotonyl thiol ester at pH 9.8. Additionally, a crotonyl thiol ester was found
to be 7.9 times more reactive than a sorboyl thiol ester toward NAC addition. These unsaturated thiol
esters may serve as a means of covalently binding UVA and UVB sunscreens to the outer layer of skin
to provide long-lasting protection.
Introduction
characterized by increased pigmentation and stratum corneum
thickness, decreased hydration, and loss of elasticity.7 Addition-
ally, UV exposure results in immunosuppression.8
Numerous studies have demonstrated the effectiveness of
sunscreen use in preventing pyrimidine dimer formation,9 p53
mutations,10 precancerous lesions, such as actinic keratoses,11,12
skin cancer,13 photoaging of skin,14,15 and also photoimmuno-
One of the more serious daily assaults on skin is exposure to
solar ultraviolet (UV) radiation. Such exposure can have a
variety of detrimental effects,1 including induction of skin
cancer.2 Shorter wavelength radiation (UVB, 290-320 nm) is
generally responsible for formation of cyclobutane pyrimidine
dimers and other photoproducts, and it causes specific p53 tumor
suppressor gene mutations, which are thought to be an early,
essential event in skin cancer.3,4 The more abundant UVA
radiation (320-400 nm) can penetrate more deeply into the skin
than UVB, reaching the dermis,5 and it has been linked to skin
cancer, both nonmelanoma and malignant melanoma.6 Effects
of chronic exposure to UVA include photoaging of the skin,
(7) Labat-Robert, J.; Fourtanier, A.; Boyer-Lafargue, B.; Robert, L. J.
Photochem. Photobiol. B: Biol. 2000, 57, 113-118.
(8) Schaefer, H.; Chardon, A.; Moyal, D. In Methods in Enzymology;
Packer, L., Sies, H., Eds.; Academic Press: San Diego, CA, 2000; Vol.
319, pp 445-465.
(9) Ley, R. D.; Fourtanier, A. Photochem. Photobiol. 1997, 65, 1007-
1011.
(10) Ananthaswamy, H. N.; Ullrich, S. E.; Mascotto, R. E.; Fourtanier,
A.; Loughlin, S. M.; Khaskina, P.; Bucana, C. D.; Kripke, M. L. J. InVest.
Dermatol. 1999 112, 763-768.
(1) Pillai, S.; Oresajo, C.; Hayward, J. Int. J. Cosmet. Sci. 2005, 27,
17-34.
(2) Elmets, C. A. In Pharmacology of the Skin; Mukhtar, H., Ed.; CRC
Press: Boca Raton, FL, 1992; pp 389-416.
(11) Naylor, M. F.; Boyd, A.; Smith, D. W.; Cameron, G. S.; Hubbard,
D.; Neldner, K. H. Arch. Dermatol. 1995, 131, 170-175.
(12) Thompson, S. C.; Jolley, D.; Marks, R. New Engl. J. Med. 1993,
329, 1147-1151.
(3) Berg, R. J. W.; van Kranen, H. J.; Rebel, H. G.; de Vries, A.; van
Vloten, W. A.; van Kreijl, C. F.; van der Leun, J. C.; de Gruijl, F. R. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93, 274-278.
(4) Ananthaswamy, H. N.; Loughlin, S. M.; Cox, P.; Evans, R. L.; Ullrich,
S. E.; Kripke, M. L. Nat. Med. 1997, 3, 510-514.
(5) Chadwick, C. A.; Potten, C. S.; Nikaido, O.; Matsunaga, T.; Proby,
C.; Young, A. R. J. Photochem. Photobiol. B: Biol. 1995, 28, 163-170.
(6) Moan, J.; Dahlback, A.; Setlow, R. B. Photochem. Photobiol. 1999,
70, 243-247.
(13) Stern, R. S.; Weinstein, M. C.; Baker, S. G. Arch. Dermatol. 1986,
122, 537-545.
(14) Schaefer, H.; Moyal, D.; Fourtanier, A. J. Dermatol. Sci. 2000, 23
(Suppl. 1), S64-S74.
(15) Seite´, S.; Colige, A.; Piquemal-Vivenot, P.; Montastier, C.; Four-
tanier, A.; Lapie`re, C.; Nusgens, B. Photodermatol. Photoimmunol. Pho-
tomed. 2000, 16, 147-155.
10.1021/jo060191+ CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/18/2006
6342
J. Org. Chem. 2006, 71, 6342-6350

Conjugate Addition to R,â-Unsaturated Thiol Esters
suppression.4,14 In addition to dramatic tumor prevention by
sunscreens applied to mice prior to UVB exposure (100% vs
2% tumor development) and significant reduction in p53
mutations,16 sunscreen use in a human clinical study15 showed
that daily use of a broad-spectrum-absorbing sunscreen protected
against many of the changes associated with photoaging. An
Australian study comparing daily sunscreen use to discretionary
use only (control group) over a period of 4.5 years found a
significant decrease in the incidence of squamous but not basal
cell carcinomas.17
Users typically fail to apply and maintain the 2 mg/cm2
coverage required to achieve the specified SPF.18 A sunscreen
capable of covalently bonding to skin would potentially avert
loss, for example, due to rubbing off and exposure to water.
Such a strategy would entail linking a UV-absorbing moiety to
a skin-bonding functionality that would react with nucleophilic
groups (e.g., -NH2, -SH) in skin proteins. To avoid generation
of the byproducts inherently produced in nucleophilic substitu-
tion reactions, formation of adducts of nucleophiles via conju-
gate addition to R,â-unsaturated electrophiles seemed preferable.
Although numerous unsaturated systems are known to undergo
conjugate addition (e.g., R,â-unsaturated ketones, N-alkylma-
leimides, R,â-unsaturated nitriles), most appear too reactive for
topical, cutaneous use.
in conjugation with the C(dO)S functionality. Thiols also add
to R,â-unsaturated systems containing electron-withdrawing
groups, such as vinyl26 sulfones, sulfonates, and sulfonamides,
enones,27 and R,â-unsaturated esters28 and imides.29 R,â-
Unsaturated thiol esters undergo conjugate addition reactions
with thiols,22,30 but kinetic data are lacking.
The UVB-absorbing p-methoxycinnamoyl chromophore is
widely used in sunscreen formulations as the ethylhexyl ester
(FDA-allowed concentrations: 2-7.5%), and the UVA-absorb-
ing anthranilate chromophore is generally found as the menthyl
ester (FDA-allowed concentrations: 3.5-5%). For the purpose
of skin bonding and the mechanistic studies reported herein,
the alkyl groups normally attached to these UV-absorbing
moieties were replaced by unsaturated thiol ester functionalities,
as shown below:
Thiol esters are electrophilic and have utility in the native
chemical ligation of proteins via intramolecular rearrangement.
Incorporation of unsaturation in conjugation with the thiol ester
carbonyl group offers a CdC for conjugate addition by
nucleophiles.19-21 The work described herein explores the
reactivity of the CdC in R,â-unsaturated thiol esters. The
kinetics and mechanism of conjugate addition reactions of
nucleophiles have been studied, and a comparison to other
electrophiles that undergo conjugate addition reactions has been
made to assess the suitability of R,â-unsaturated thiol esters as
mildly reactive bonding agents for sunscreens to nucleophilic
groups in skin.
It is possible that differing solubility characteristics of the
amides 1 and 3 compared to those of the esters 2, 4, 5, and 6
will influence skin permeability and may thereby alter the site
of covalent attachment of the compound within the stratum
corneum.
Synthesis of the p-methoxycinnamoyl-containing UV absorb-
ers is outlined in Scheme 1. The acid chloride of p-methoxy-
cinnamic acid 7 was treated with cystamine to give disulfide 8
or with hydroxyethyl disulfide to give disulfide 10. Reduction
to the corresponding thiols 9 and 11 was accomplished by
treatment with zinc/HCl. Subsequent reaction with crotonyl
chloride or sorboyl chloride gave compounds 1 and 2 or 3 and
4, respectively.
Preparation of the anthranilate-containing UV absorbers is
outlined in Scheme 2. Reaction of isatoic anhydride or N-
methylisatoic anhydride in DMF with hydroxyethyl disulfide
in the presence of 10% DMAP gave the disulfides 12 or 13.
These disulfides were each reduced by zinc and acid to the
corresponding thiols 14 and 15. In the case of the unmethylated
anthranilate, coupling with crotonyl chloride/pyridine produced
the dicrotonyl compound 6. The UV spectrum of this compound
was shifted somewhat toward the UVB region (λmax ) 315,
Results and Discussion
In this study, p-methoxycinnamoyl and anthranilate moieties
have been modified to contain a pendant R,â- or R,â,γ,δ-
unsaturated thiol ester, as shown below (1-6). These UV-
absorbing compounds are designed for covalent attachment to
nucleophilic groups (e.g., -NH2, -SH) in skin proteins in the
uppermost layers of the epidermis (e.g., the stratum corneum).
The thiol ester functional group provides increased reactivity
of the CdC toward conjugate addition by nucleophiles, com-
pared to an ordinary oxygen ester,22 and it is 20-30 times more
stable toward acid hydrolysis, although it is of comparable
stability to oxygen esters toward alkaline hydrolysis.23 Nucleo-
philes, such as DMAP24,25 and Ph3P,25 readily add to the CdC
(16) Ananthaswamy, H. N.; Ullrich, S. E.; Kripke, M. L. Exp. Dermatol.
2002, 11 (Suppl. 1), 40-43.
(23) Janssen, M. J. In The Chemistry of Carboxylic Acids and Esters;
Patai, S., Ed.; Wiley Interscience: New York, 1969; pp 734-736.
(24) Keck, G. E.; Boden, E. P.; Mabury, S. A. J. Org. Chem. 1985, 50,
709-710.
(17) Green, A.; Williams, G.; Neale, R.; Hart, V.; Leslie, D.; Parsons,
P.; Marks, G. C.; Gaffney, P.; Battistutta, D.; Frost, C.; Lang, C.; Russell,
A. Lancet 1999, 354, 723-729.
(25) Keck, G. E.; Welch, D. S. Org. Lett. 2002, 4, 3687-3690.
(26) Reddick, J. J.; Cheng, J.; Roush, W. R. Org. Lett. 2003, 5, 1967-
1970.
(18) Diffey, B. J. Photochem. Photobiol. B: Biol. 2001, 64, 105-108.
(19) Liu, C.-F.; Rao, C.; Tam, J. P. J. Am. Chem. Soc. 1996, 118, 307-
312.
(20) Xu, R.; Ayers, B.; Cowburn, D.; Muir, T. W. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 388-393.
(21) Dawson, P. E.; Kent, S. B. H. Annu. ReV. Biochem. 2000, 69, 923-
960.
(27) Atsriku, C.; Scott, G. K.; Benz, C. C.; Baldwin, M. A. J. Am. Soc.
Mass Spectrom. 2005, 16, 2017-2026.
(28) Lutolf, M. P.; Tirelli, N.; Cerritelli, S.; Cavalli, L.; Hubbell, J. A.
Bioconjugate Chem. 2001, 12, 1051-1056.
(29) Bednar, R. A. Biochemistry 1990, 29, 3685-3690.
(30) Dunn, M. F.; Bernhard, S. A. J. Am. Chem. Soc. 1969, 91, 3274-
3280.
(22) Schleppnik, A. A.; Zienty, F. B. J. Org. Chem. 1964, 29, 1910-
1915.
J. Org. Chem, Vol. 71, No. 17, 2006 6343

Hartman and Rose
SCHEME 1
SCHEME 2
264 nm, in 95% ethanol). The N-methylanthranilate derivative
13, however, upon treatment with crotonic anhydride/NaH,
produced crotonate thiol ester 5, which had a UV absorption
addition of 1 equiv of N-acetylcysteamine to 2. No reaction is
evident at this time. Within 2 min of introduction of 0.1 equiv
of a base (1,5-diazabicyclo[4.3.0]non-5-ene, DBN), however,
addition of the cysteamine thiol to the crotonate double bond
had occurred. This is clearly evidenced by the disappearance
of the signal due to the vinyl protons (doublet at δ 6.15 ppm,
multiplet at δ 6.96 ppm) and the shift of the signal due to the
methyl group protons from δ 1.9 to 1.3 ppm (Figure 2, bottom).
Additionally, the triplet at δ 1.3 ppm due to the cysteamine SH
proton is no longer present after reaction with 2 has occurred.
COSY, HMBC, and HMQC spectra confirmed the structure of
the product, 16, and similar results were found with 1 (Sup-
porting Information).
spectrum characteristic of unsubstituted anthranilates (λmax
)
357, 255 nm, 95% ethanol), and it thus serves as a UVA
absorber.
It was found that conjugate addition of nucleophilic groups
to the R,â-unsaturated thiol ester readily occurred, giving a
covalent adduct. The reactions of compound 1 or 2 with
N-acetylcysteamine, N-acetyl-L-cysteine, or N2-acetyl-L-lysine,
as protein model compounds, to form adducts 16, 17, and 18
are shown in Figure 1.
Thiol esters are more reactive toward conjugate addition than
are ordinary oxygen esters,31 which can be attributed to the
reduced resonance stabilization in thiol esters.32,33 This is at least
in part a consequence of the poorer 2p-3p orbital overlap in
the former, although it is not insignificant.34
An NMR study of the reaction of sorboyl thiol ester 4 with
N-acetylcysteamine revealed that it reacted more slowly than
the analogous crotonate 2. Fifteen minutes after addition of 0.1
equiv of DBN, 64% of the sorboyl vinyl protons remained, while
36% appeared in a mixture of monoadducts, as evidenced by
the appearance of two multiplets at δ 5.25 and 5.5 ppm in the
NMR spectrum. The kinetics of a competitive reaction of
N-acetylcysteamine with 2 and 4 were followed by HPLC, and
the results are described below. Reaction of the sorboyl thiol
ester 3 with N-acetylcysteamine was also slower than that of
the corresponding crotonyl thiol ester 1. Five minutes after
addition of 0.2 equiv of DBN, 45% of the sorboyl vinyl protons
remained, while 55% appeared in a mixture of monoadducts.
An NMR experiment was carried out to demonstrate the
reactivity of the R,â-unsaturated thiol ester functional group and
to identify the structure of the product. The spectrum was
recorded before (Figure 2, top) and after (Figure 2, middle) the
(31) Khandelwal, G. D.; Wedzicha, B. L. Food Chem. 1990, 37, 159-
169.
(32) El-Aasar, A. M. M.; Nash, C. P.; Ingraham, L. L. Biochemistry 1982,
21, 1972-1976.
(33) Erben, M. F.; Boese, R.; Della Ve´dova, C. O.; Oberhammer, H.;
Willner, H. J. Org. Chem. 2006, 71, 616-622.
(34) Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 1992, 114, 10297-
10302.
Conjugate addition to form a covalent adduct also occurred
in the case of the anthranilate-containing thiol esters. An NMR
6344 J. Org. Chem., Vol. 71, No. 17, 2006

Conjugate Addition to R,â-Unsaturated Thiol Esters
FIGURE 1. Conjugate addition of N-acetylcysteamine, N-acetyl-L-cysteine, or N2-acetyl-L-lysine to 1 or 2 to form the adducts 16, 17, and 18.
FIGURE 3. Kinetics of the reaction of 20.0 mM N-acetyl-L-cysteine
and 0.77 mM 1 in 1:1 (v/v) acetonitrile/72 mM HEPES (aqueous buffer
at pH 7.95) are shown. Reaction progress was monitored by the
disappearance of reactant 1 by HPLC.
FIGURE 2. Reaction of 2 with N-acetylcysteamine and DBN. Top
spectrum: 2 in CDCl3. Middle spectrum: 2 with 1 equiv N-
acetylcysteamine. Bottom spectrum: 2 min after addition of 0.1 equiv
of DBN.
study of the addition of N-acetylcysteamine to 5 found that, 2
min after addition of 0.1 equiv of DBN, 50% of the thiol ester
had reacted to form an adduct analogous to that shown in Figure
1 (Supporting Information). With compound 6, 75% of the thiol
ester had reacted to form an adduct after 2 min. Only the
S-crotonyl group is reactive in this molecule; the N-crotonyl
group remained unchanged in the presence of DBN/cysteamine
(Supporting Information).
Kinetics of the addition of amino acid derivatives to the thiol
esters was followed by HPLC. Reactions were carried out in a
1:1 mixture (v/v) of acetonitrile and 72 mM HEPES buffer at
23 °C. All points are the average of at least two determinations.
Conjugate addition of N-acetyl-L-cysteine (NAC) to 1 was
carried out under pseudo-first-order conditions, with greater than
HEPES-buffered 1:1 (v/v) water/acetonitrile, final pH 7.79. The line
10-fold excess N-acetyl-L-cysteine over 1. The observed rate
FIGURE 4. Reaction of 1 with N-acetyl-L-cysteine (25 mM) in
is the least-squares fit to the data (r2 ) 0.99). [HEPES]T is neutral
HEPES zwitterion concentration plus HEPES anion concentration.
equation is
V ) kobs [1]
(1)
A study of the effect of varying the concentration of HEPES
buffer revealed buffer catalysis, as shown in Figure 4.
Data were plotted according to the pseudo-first-order rate
The observation of buffer catalysis suggests the mechanism
shown in Scheme 3, in which rapid attack of NAC thiolate is
followed by rate-limiting protonation of the intermediate enolate
ion by acids, such as neutral zwitterionic HEPES in solution.
Equilibrium favors protonation of the enolate, as enolates of
simple thiol esters are only weakly acidic (pKa ∼ 21).34
equation
ln(At/Ao) ) -kobs
t
(2)
A plot of the data from a typical reaction is shown in
Figure 3.
J. Org. Chem, Vol. 71, No. 17, 2006 6345

Hartman and Rose
SCHEME 3
The mechanism in Scheme 3 exhibits general base catalysis,
with the following rate law, in which [HEPES]z is neutral
HEPES zwitterion concentration and Ke is defined in
Scheme 3:
FIGURE 5. Reaction of 1 with varying concentrations of N-acetyl-
L-cysteine in 1:1 (v/v) water/acetonitrile containing 36 mM HEPES at
a final pH of 7.79. The line is a least-squares fit of eq 3 to the data
(r2 ) 0.98).
V
) kobs ) Ke [NAC-S-](kHEP[HEPES]z + k0) (3a)
[1]
The effect of varying the concentration of N-acetyl-L-cysteine
was also studied, and the data are shown in Figure 5. The
reaction rate was dependent on N-acetyl-L-cysteine concentration
as expected.
In terms of the total HEPES concentration (i.e., zwitterion
and anion), [HEPES]T, and the fraction of [HEPES]T present
in zwitterionic form, fz, eq 3a becomes
The slope of the line in Figure 5 equals the slope of eq 3b
with [NAC] as the variable, which gives Ke fthiolate(kHEP fz-
[HEPES]T + k0) ) 0.247 M-1 min-1. Calculation of this
quantity by use of the values determined from Figure 4 was in
V
) kobs ) Ke fthiolate [NAC]T (kHEP fz [HEPES]T + k0)
[1]
(3b)
good agreement, giving 0.240 M-1 min-1
.
Rate-limiting protonation of anionic addition intermediates
has been observed in the addition of thiolates to R,â-unsaturated
nitriles35 and to cyanamide.36 In the case of N-ethylmaleimide,
at least partial rate-limiting protonation of the intermediate imide
anion was detectable but was relatively unimportant.37 Carbon-
sulfur bond formation, however, is rate limiting in the base-
catalyzed addition of thiols to R,â-unsaturated ketones.38
The neutral form of HEPES (i.e., the zwitterionic form whose
nitrogen atom is protonated) would be required for proton
transfer to the enolate shown in Scheme 3. Given the pKa of
HEPES of 7.80 in 1:1 (v/v) H2O/acetonitrile and that of NAC
of 10.77, the fraction of HEPES present in zwitterionic form,
Nucleophilic attack by the thiolate anion of N-acetyl-L-
cysteine on 1 would be expected to be much faster than attack
by the neutral N-acetyl-L-cysteine sulfhydryl group, as the
neutral thiol is much less nucleophilic than the thiolate in
conjugate addition reactions.28,29,39,40 Conjugate addition of
thiolates to Michael acceptors has been studied theoretically,
as well.41 A pH-rate profile for the reaction of 1 and N-acetyl-
L-cysteine is shown in Figure 6. Indeed, the reaction was
accelerated with increasing pH of the solution.
To assess the relative reactivities of the R,â-unsaturated and
the R,â,γ,δ-unsaturated systems, a competitive experiment with
crotonyl and sorboyl thiol esters was carried out. A 1:1 mixture
of 1 and 3 was treated with N-acetyl-L-cysteine in 36 mM
HEPES in 1:1 acetonitrile/water, pH 8.23. The results, presented
in Figure 7, show that the crotonate is 7.9-fold more reactive
than the sorbate.
fz, is 0.51 and the fraction of total NAC in thiolate form, fthiolate
,
is 1.05 × 10-3 at pH 7.79. The slope of the line in Figure 4,
(7.14 ( 0.47) × 10-2 M-1 min-1, equals the slope of eq 3b
with HEPES as the variable (Ke fthiolate [NAC]T kHEP fz), which
gives KekHEP ) (5.4 ( 0.4) × 103 M-2 min-1. The y-intercept,
A comparison of the reactivity of the thiol esters studied
herein with the reactivity of the widely studied N-ethylmaleimide
(NEM) can be made. The reactions of alkyl and aryl thiolates
in water and 95% ethanol, respectively, with NEM have second-
order rate constants29,37 in the range of ∼1-20 × 106 M-1
min-1. As shown above, thiolate addition to thiol ester 1 occurs
with a second-order rate constant (in the absence of buffer) equal
(3.42 ( 0.16) × 10-3 min-1 (Figure 4), equals Ke fthiolate
-
.
[NAC]T k0, which gives Kek0 ) (1.3 ( 0.1) × 102 M-1 min-1
The ratio then gives kHEP/k0 ) 41 ( 3 M-1, which allows
calculation of the ratio of buffer-catalyzed product formation
to spontaneous protonation (e.g., by water) at different values
of HEPES zwitterion concentration as [HEPES zwitterion] ×
kHEP/k0 (e.g., equal contributions at 24 mM HEPES zwitterion).
to kobs/(fthiolate[NAC]T) ) Kek0 ∼ 1.3 × 102 M-1 min-1
,
Thus, protonation of the enolate intermediate by proteins in the
skin could have an accelerating effect on the skin-binding
reaction of sunscreen molecules, such as 1.
considerably slower than NEM additions, although more sensi-
tive to buffer catalysis.
The 6-amino group of lysine in skin proteins would also be
expected to serve as a nucleophile capable of adding to 1, so
(35) Fishbein, J. C.; Jencks, W. P. J. Am. Chem. Soc. 1988, 110, 5075-
5086.
(36) Dalby, K. N.; Jencks, W. P. J. Chem. Soc., Perkin Trans. 2 1997,
(39) Lutolf, M. P.; Tirelli, N.; Cerritelli, S.; Cavalli, L.; Hubbell, J. A.
Bioconjugate Chem. 2001, 12, 1051-1056 and references cited therein.
(40) Jans, U.; Miah, M. H. J. Agric. Food Chem. 2003, 51, 1956-1960.
(41) Kunakbaeva, Z.; Carrasco, R.; Rozas, I. J. Mol. Struct. (THEOCHEM)
2003, 626, 209-216.
1555-1563.
(37) Semenow-Garwood, D. J. Org. Chem. 1972, 37, 3797-3803.
(38) Hiemstra, H.; Wynberg, H. J. Am. Chem. Soc. 1981, 103, 417-
430.
6346 J. Org. Chem., Vol. 71, No. 17, 2006

Conjugate Addition to R,â-Unsaturated Thiol Esters
FIGURE 6. The pH-rate profile for the reaction of 1 with N-acetyl-L-cysteine. Left panel: relative kobs (relkobs) versus pH (kobs ≡ 1.0 at pH 7.12).
Right panel: log (relkobs) versus pH. The reactions were carried out in 1:1 water/acetonitrile containing 36 mM HEPES at the final pH specified. The
lines are the result of a nonlinear least-squares fit (see Experimental Methods) of eq 3b, with kHEP/k0 set equal to 41 M-1 (as determined from
Figures 4 and 5).
FIGURE 7. Comparison of the relative reactivities of the crotonyl
thiol ester 1 (circles) and the sorboyl thiol ester 3 (triangles). A solution
containing 0.77 mM 1 and 0.77 mM 3 in acetonitrile was mixed with
an equal volume of 50 mM N-acetyl-L-cysteine in 72 mM HEPES
(aqueous buffer at pH 7.95), and the disappearance of the thiol esters
was monitored by HPLC.
FIGURE 8. The pH-rate profile for the reaction of 1 (0.77 mM) with
N2-acetyl-L-lysine (14.3 mM). The reactions were carried out at the
final pH specified in 1:1 (v/v) water/acetonitrile containing 76 mM
sodium borate buffer.
model studies of the reaction of N2-acetyl-L-lysine (NAL) with
1 were carried out. A pH-rate profile for the reaction is shown
in Figure 8.
evaporation removed the last traces of thionyl chloride to give a
1
A comparison of relative reactivities of NAC and NAL can
be made. For example, at pH 7.8, kobs for NAC at 25 mM is
0.00342 min-1 (y-intercept of plot in Figure 4). Correction to
14.3 mM NAC (×14.3/25) and for the relative rate increase
for pH 9.8 (×231, from Figure 6) allows comparison to kobs for
14.3 mM NAL at pH 9.8 (Figure 8, if there is no significant
buffer catalysis by boric acid). The resulting factor of ∼300
times greater reaction of NAC reveals that thiols are consider-
ably more reactive in mildly alkaline solution, but the relative
abundances and availability for reaction of cysteinyl and lysyl
groups in skin will determine the actual major sites of reaction
of compounds like 1 with skin.
dark yellow solid (16.14 g, 97%). Mp: 67-69 °C. H NMR (300
MHz, CDCl3): δ (ppm) 3.87 (s, OCH3), 6.51 (d, J ) 15.0 Hz,
CHCO), 6.94 (dd, J ) 6.9 Hz, 1.8 Hz, aromatic C(3)H, C(5)H),
7.53 (dd, J ) 6.9 Hz, 1.8 Hz, aromatic C(2)H, C(6)H), 7.79 (d, J
) 15.3 Hz, COCHCH). 13C (75.46 MHz, CDCl3): δ (ppm) 55.5,
62.1, 114.7, 119.4, 125.7, 131.1, 150.6, 162.9, 166.0.
A.2. Bis(p-methoxycinnamate) ester of 2-hydroxyethyl disul-
fide (10). Preparation was according to the literature.42 To 60 mL
of benzene were added 4.25 g (0.0216 mol) of 7 and 1.66 mL
(0.0135 mol) of 2-hydroxyethyl disulfide. The mixture was refluxed
for 1.5 h and allowed to stir at room temperature overnight. Solvent
was removed by rotary evaporation, and the residue was taken up
in chloroform, and the organic phase was washed with water. The
aqueous layer was back-extracted with chloroform. The combined
organic layers were dried over anhydrous MgSO4 and evaporated
to give 5 g of crude solid. This material was recrystallized from
absolute ethanol to give 3.6 g (70%) of white solid. Mp: 80-81
°C (found), 81.5 °C (lit).42 1H NMR (CDCl3): δ (ppm) 3.03 (t, J
) 6.6 Hz, CH2S), 3.83 (s, OCH3), 4.47 (t, J ) 6.8 Hz, CH2N),
6.30 (d, J ) 15.9 Hz, CHCO), 6.89 (d, J ) 9.0 Hz, aromatic C(3)-
Experimental Methods
A. Syntheses of p-Methoxycinnamates and p-Methoxycinna-
mides. A.1. p-Methoxycinnamoyl chloride (7). To 15.02 g of
4-methoxycinnamic acid in 200 mL of dry benzene with one drop
of pyridine was added a 2.4-fold excess of thionyl chloride. Care
was taken to trap the HCl vapor formed. The mixture was refluxed
overnight, after which the solvent and excess thionyl chloride were
removed by rotary evaporation. Repeated addition of benzene and
(42) Jung, L.; Richard, A.; Navarro, R. European Patent No. FR2504530,
1982.
J. Org. Chem, Vol. 71, No. 17, 2006 6347

Hartman and Rose
H, C(5)H), 7.46 (d, J ) 8.7 Hz, aromatic C(2)H, C(6)H), 7.65 (d,
J ) 15.9 Hz, CHCHCO). 13C (75.46 MHz, CDCl3): δ (ppm) 37.4,
55.2, 62.1, 114.2, 114.8, 126.9, 129.7, 144.9, 161.3, 166.8.
A.3. 2-Mercaptoethyl p-methoxycinnamate (11). To 100 mL
of ethanol/acetonitrile, 1:1 by volume, was added 1.2 g of 10. This
was treated with 3.5 g of zinc dust and 1 mL of concentrated HCl,
and the reaction mixture was stirred for 2 h at room temperature.
Complete reduction to the thiol was verified by TLC (silica, 20%
acetone in hexanes; disulfide Rf ) 0.24, thiol Rf ) 0.45). The
reaction mixture was filtered to remove zinc and zinc salts, and
the solvents were removed by rotary evaporation. The residue was
dissolved in chloroform, the resulting solution was washed once
each with water and dilute aqueous sodium bicarbonate, and the
organic layer was dried over anhydrous MgSO4. Removal of the
solvent by rotary evaporation gave 11 as an oil that solidified in
rotary evaporation to give a tan solid (6.75 g, 80%). Recrystalli-
zation from ethanol gave a white solid. Mp: 263-265 °C. 1H NMR
(DMSO-d6): δ (ppm) 2.84 (t, J ) 6.6 Hz, 2H, CH2S), 3.46 (dt, J
) 6.6, 6.3 Hz, 2H, NCH2), 3.75 (s, 3H, OCH3), 6.47 (d, J ) 15.9
Hz, CHCO), 6.93 (d, J ) 9.0 Hz, 2H, aromatic C(3)H, C(5)H),
7.37 (d, J ) 15.9 Hz, 1H, COCHCH), 7.48 (d, J ) 8.7 Hz, 2H,
aromatic C(2)H, C(6)H), 8.22 (t, J ) 5.9 Hz, 1H, NH).13C NMR
(DMSO-d6): δ (ppm) 37.4, 38.1, 55.2, 114.4, 119.4, 127.4, 129.1,
138.6, 160.3, 165.4.
A.7. 2-Mercaptoethyl p-methoxycinnamide (9). Preparation of
9 was generally carried out as described for 11, except the solvent
was 10% acetone in ethanol. Disulfide 8 (1.65 g, 0.0035 mol) in
150 mL of ethanol with 20 mL of acetone was treated with 3.5 g
of zinc powder, 15 mL of glacial acetic acid, and 8 mL of
concentrated HCl. After 1 h, the reaction was worked up as
described for 11 to yield 1.25 g (76%). 1H NMR (CDCl3): δ (ppm)
1.39 (t, J ) 8.1 Hz, SH), 2.75 (dt, J ) 6.6, 8.1 Hz, CH2SH), 3.57
(dt, J ) 6.6, 6.0 Hz, CH2NH), 3.82 (s, OCH), 6.28 (d, J ) 15.6
Hz, CHCO), 5.95 (br s, NH), 6.89 (d, J ) 9.0 Hz, aromatic C(3)H,
C(5)H), 7.46 (d, J ) 8.7 Hz, aromatic C(2)H, C(6)H), 7.60 (d, J )
15.3 Hz, CHCHCO).
1
the cold to a white waxy material. H NMR (CDCl3): δ (ppm)
1.54 (t, J ) 8.4 Hz, SH), 2.82 (dt, J ) 6.6, 9 Hz, CH2SH), 3.84 (s,
OCH), 4.32, (t, J ) 6.6 Hz, CH2O), 6.31 (d, J ) 15.9 Hz, CHCO),
6.9 (d, J ) 6.3 Hz, aromatic C(3)H, C(5)H), 7.48 (d, J ) 6.9 Hz,
aromatic C(2)H, C(6)H), 7.66 (d, J ) 15.9 Hz, CHCHCO).
A.4. S-Crotonyl-2-mercaptoethyl 4-methoxycinnamate (2).
Thiol 11 (0.6 g, 2.5 mmol) and 1.6 equiv (0.31 g) of pyridine were
dissolved in 40 mL of freshly distilled THF in a flame-dried, N2-
flushed addition funnel. The solution was added dropwise over 30
min to a solution of crotonyl chloride (3.8 mmol, 1.5 equiv) in 25
mL of THF. The reaction mixture became cloudy, and a precipitate
formed on the walls. The reaction mixture was stirred under a N2
atmosphere overnight. The THF was removed by rotary evaporation,
and the residue was dissolved in chloroform. The resulting solution
was washed with water (3 × 25 mL), dilute aqueous sodium
bicarbonate (3 × 25 mL), and brine, whereupon it was dried over
anhydrous Na2SO4, and the solvent was removed by rotary
evaporation to yield an oil. Crude product (0.75 g) was purified by
flash chromatography (silica gel, 15 vol % ethyl acetate in hexanes;
2 Rf ) 0.29; 11 Rf ) 0.35) to yield 0.29 g (38%) of 2 as a white
solid. A portion was recrystallized from methylene chloride/hexane
to give fine white needles. Mp: 80 °C. 1H NMR (CDCl3): δ (ppm)
1.89, (dd, J ) 6.6, 1.5 Hz, CH3), 3.27 (t, J ) 6.3 Hz, CH2S), 3.83
(s, OCH3), 4.32 (t, J ) 6.6 Hz, CH2O), 6.15 (dd, J ) 15.6, 1.5 Hz,
CHCHCH3), 6.29 (d, J ) 15.9 Hz, CHCHCO), 6.90 (dd, J ) 6.6,
2.1 Hz, aromatic C(3)H, C(5)H), 6.9-7.0 (m, CHCHCH3), 7.47
(dd, J ) 7.0, 2.1 Hz, aromatic C(2)H, C(6)H), 7.65 (d, J ) 15.9
Hz, CHCHCOO). Anal. Calcd for C16H18O4S: C, 62.73; H, 5.92.
Found: C, 62.71; H, 5.96. UV λmax 310 nm (ꢀ ) 2.23 × 104 M-1
cm-1).
A.8. S-Crotonyl-2-mercaptoethyl 4-methoxycinnamide (1).
Compound 1 was prepared from thiol 9 following the procedure
described above for compound 2. Crude product was purified by
flash chromatography (silica gel, 30 vol % acetone in hexanes; 1
Rf ) 0.23) to give 1 as a white solid in 24% yield. Recrystallization
from methylene chloride/hexane gave a white solid. Mp: 107-
1
109 °C. H NMR (CDCl3): δ (ppm) 1.89 (dd, J ) 7.1, 1.7 Hz,
CH3), 3.16 (t, J ) 6.6 Hz, CH2S), 3.60 (dt, J ) 6.6, 5 Hz, NHCH2),
3.83 (s, OCH3), 6.04 (br s, NH), 6.16 (dd, J ) 14, 1.8 Hz,
CHCHCH3), 6.23 (d, J ) 15.3 Hz, CHCHCONH), 6.88 (dd, J )
6.9, 21.8 Hz, aromatic C(3)H, C(5)H), 6.9-7.0 (m, CHCHCH3),
7.45 (d, J ) 8.4 Hz, aromatic C(2)H, C(6)H), 7.56 (d, J ) 16.2
Hz, CHCHCOO). 13C NMR (CDCl3): δ (ppm) 18.0, 28.3, 40.0,
47.6, 55.3, 114.2, 118.0, 127.5, 129.4, 129.9, 140.9, 141.8, 160.9,
166.5. Mp: 107-109 °C. Anal. Calcd for C16H19NO3S: C, 62.93;
H, 6.27; N, 4.59. Found: C, 62.74; H, 6.31; N, 4.51. UV λmax 224
nm (ꢀ ) 3.00 × 104 M-1 cm-1), λmax 292 nm (ꢀ ) 2.87 × 104
M-1 cm-1).
A.9. S-Sorboyl-2-mercaptoethyl 4-methoxycinnamide (3).
Compound 3 was prepared from thiol 9 following the procedure
described above for compound 2. After 4 h, the reaction was judged
complete by TLC, and the reaction mixture was worked up as
described for compound 2 to yield a brown oil. Crude product was
purified by flash chromatography (silica gel, 30 vol % acetone in
hexanes; 3 Rf ) 0.34). The product was obtained as a white solid
in 40% yield and was then twice recrystallized from methylene
A.5. S-Sorboyl-2-mercaptoethyl 4-methoxycinnamate (4).
Preparation was analogous to that described above for 2, with
sorboyl chloride substituted for crotonyl chloride. Flash chroma-
tography on silica gel with 15 vol % of ethyl acetate in hexanes
(Rf ) 0.18) gave a white solid (32% after chromatography). A
portion of this was recrystallized from methylene chloride/hexane
1
chloride/hexane. Mp: 128-129 °C. H NMR (CDCl3): δ (ppm)
1.87 (d, J ) 6.0 Hz, CHCH3), 3.17 (t, J ) 6.0 Hz, CH2S), 3.61 (t,
J ) 5.4 Hz, NHCH2), 3.82 (s, OCH3), 6.0-6.25 (m, 3H, sorboyl
CHs), 6.24 (d, J ) 15.6 Hz, CHCHCONH), 6.87 (d, J ) 9.0 Hz,
aromatic C(3)H, C(5)H), 7.16-7.28 (m, 1H, sorboyl CH), 7.43 (d,
J ) 9.0 Hz, aromatic C(2)H, C(6)H), 7.56 (d, J ) 15.3 Hz,
CHCHCONH). Anal. Calcd for C18H21O3NS: C, 65.23; H, 6.39;
N, 4.23. Found: C, 65.11; H, 6.38; N, 4.18. UV λmax 224 nm (ꢀ )
1.80 × 104 M-1 cm-1), λmax 292 nm (ꢀ ) 4.52 × 104 M-1 cm-1).
A.10. Adduct of 2 and N-acetylcysteamine (16). Synthesis of
compound 16 was carried out on a 40-mg scale. Compound 2 (40
mg) in CHCl3 was treated with 1 equiv of NAC and 0.1 equiv of
DBN. After 30 min, the product was purified by chromatography
(silica, 40% ethyl acetate/hexane to 100% ethyl acetate) to give 16
1
to give a fine white solid. Mp: 74-76 °C. H NMR (CDCl3): δ
(ppm) 1.86 (d, J ) 5.1 Hz, CH3), 3.27 (t, J ) 4.8 Hz, CH2S), 3.82
(s, OCH3), 4.31 (t, J ) 4.8 Hz, CH2O), 6.0-6.3 (m, 3H, sorboyl),
6.28 (d, J ) 11.7 Hz, CHCHCO), 6.88 (d, J ) 6.6 Hz, 2H, aromatic
C(3)H, C(5)H), 7.15-7.24 (m, 1H, sorboyl), 7.47 (d, 2H, aromatic
C(2)H, C(6)H), 7.6 (d, J ) 12.3 Hz, 1H, CHCHCO). Anal. Calcd
for C18H20O4S: C, 65.04; H, 6.06. Found: C, 64.95; H, 5.99. UV
λmax 228 nm (ꢀ ) 1.64 × 104 M-1 cm-1), λmax 294 nm (ꢀ ) 4.23
× 104 M-1 cm-1).
A.6. N,N′-(Dithiodiethylene)bis(p-methoxycinnamide) (8). Cys-
tamine dihydrochloride (8.0 g, 0.0356 mol) was converted to the
free base by treatment with 2 equiv of aqueous sodium hydroxide
followed by extraction with chloroform. The chloroform solution
was then dried with Na2SO4. To this chloroform solution were added
7 (7.0 g, 0.0356 mol) and triethylamine (7.12 g, 0.0712 mol), and
the solution was refluxed for 2.5 h. The solution was washed twice
with water, twice with dilute HCl, once with dilute NaHCO3, once
with brine, and dried (Na2SO4), and the solvent was removed by
1
in 53% yield. H NMR (CDCl3): δ (ppm) 1.30 (d, J ) 6.6 Hz,
SCHCH3), 1.98 (s, OdCCH3), 2.66 (t, J ) 6.3 Hz, NHCH2CH2S),
2.7-2.8 (m, OdCCH2), 3.2-3.3 (m, SCHCH3 and CH2CH2SCO),
3.41 (m, NHCH2CH2S), 3.81 (s, OCH3), 4.29 (t, J ) 6.6 Hz, OCH2-
CH2S), 6.1 (br s, NH), 6.27 (d, J ) 15.9 Hz, CHdCHCO), 6.88
(d, J ) 8.1 Hz, aromatic C(3)H, C(5)H), 7.45 (d, J ) 8.1 Hz,
aromatic C(2)H, C(6)H), 7.63 (d, J ) 15.9 Hz, CHdCHCO). 13C
NMR (CDCl3): δ (ppm) 21.6 (OdCCH3), 23.2 (SCHCH3), 28.0
6348 J. Org. Chem., Vol. 71, No. 17, 2006

Conjugate Addition to R,â-Unsaturated Thiol Esters
(OCH2CH2S), 30.6 (SCH2CH2NH), 36.3 (SCHCH3), 38.8 (CH2-
NH), 51.0 (OdCCH2), 55.4 (OCH3), 62.5 (OCH2CH2S), 114.4
(aromatic C(3), C(5)), 114.9 (CHCHCOO), 127.0 (aromatic C(1)),
129.8 (aromatic C(2), C(6)), 145.1 (CHCHCOO), 161.5 (aromatic
C(4)), 166.9 (CHCHCOO), 170.2 (NHCO), 196.5 (SCO). COSY,
HMBC, and HMQC spectra are shown in the Supporting Informa-
tion.
in methylene chloride/hexane and then sublimation to yield fluffy,
light yellow florettes. Mp: 33-35 °C. 1H NMR (CDCl3): δ (ppm)
1.56 (t, J ) 8.7 Hz, SH), 2.86 (t, J ) 7.4 Hz, 2H, CH2S), 2.92 (s,
N-CH3), 4.38 (t, J ) 6.6 Hz, 2H, CH2O), 6.66 (dd, J ) 15, 1.2
Hz, 1H, C(5)H), 6.72 (d, J ) 8.4 Hz, C(3)H), 7.43 (dd, J ) 8.0,
1.5 Hz, 1H, C(4)H), 7.92 (dd, J ) 8.4, 1.5 Hz, 1H, C(6)H).
B.5. S-Crotonyl-2-mercaptoethyl N-methylanthranilate (5).
The reaction was carried out under an argon atmosphere. Thiol 15
(3.52 g, 16.7 mmol) was dissolved in 80 mL of THF, and crotonic
anhydride (2.57 g, 16.7 mmol) was added, followed by 0.40 g (16.7
mmol) of NaH, added in portions. Vigorous H2 evolution was
observed. Additional portions of crotonic anhydride and NaH were
added until TLC showed that thiol was totally consumed. After
1.5 h, the solvent was removed by rotary evaporation not quite to
dryness, and the residue was diluted with water and extracted twice
with ether. The organic phase was washed with dilute aqueous
NaHCO3 followed by brine, and then it was dried with Na2SO4.
Removal of solvent in vacuo gave 4.92 g of crude product.
Purification by flash chromatography (silica, 10 vol % ethyl acetate
in hexanes) yielded 2.26 g of 5 (48%) as a white solid. Mp: 42-
B. Syntheses of Anthranilates. B.1. Bis(anthranilate ester) of
hydroxyethyl disulfide (12). A solution of isatoic anhydride (15.0
g, 0.092 mol), hydroxyethyl disulfide (7.08 g, 0.0459 mol), and
DMAP (1.11 g, 0.0091 mol) in DMF was heated under N2 at 95
°C overnight. The DMF was removed by rotary evaporation at 60
°C, and the brown liquid obtained was diluted with chloroform.
The resulting solution was washed twice with water, once with
brine, and dried with Na2SO4. The solvent was removed by rotary
evaporation to give a brown oil. Crystallization occurred either
spontaneously or upon dilution with 100 mL of methanol followed
by trituration with water (50 mL). The precipitate was collected
by filtration, washed with methanol, and air-dried to give an off-
white solid, 17.0 g (94%). Mp: 90-92 °C. TLC Rf ) 0.75 (silica,
hexane/acetone, 1:1, v/v). 1H NMR (CDCl3): δ (ppm) 3.07 (t, J )
6.6 Hz, 2H, CH2O), 4.54 (t, J ) 6.6 Hz, CH2S), 5.5 (br s, NH2),
6.61-6.66 (m, 2H, C(3)H, C(5)H), 7.26 (ddd, J ) 8.3, 6.9, 1 Hz,
1H, C(4)H), 7.86 (d, J ) 8.1 Hz, 1H, C(6)H). 13C NMR (CDCl3):
δ (ppm) 37.0, 61.6, 110.0, 115.8, 116.2, 130.8, 133.8, 150.1, 167.3.
B.2. Bis(N-methylanthranilate ester) of hydroxyethyl disulfide
(13). Preparation of 13 from N-methylisatoic anhydride was carried
out as described for 12. Crude product was obtained as a brown
liquid, which solidified somewhat upon standing. The crude product
was triturated with methanol, collected by filtration, and washed
with methanol to give a tan solid. Mp: 67-69 °C. A second crop
was obtained by dropwise addition of water to the methanol filtrate.
1H NMR (CDCl3): δ (ppm) 2.91 (s, 3H, NCH3), 3.06 (t, J ) 6.6
Hz, 2H, SCH2), 4.53 (t, J ) 6.6 Hz, 2H, CH2O), 6.60 (t, J ) 7.4
Hz, 1H, C(5)H), 6.69 (d, J ) 9.0 Hz, 1H, C(3)H), 7.39 (ddd, J )
7.8, 7.5, 1.8 Hz, C(4)H), 7.8 (br s, 1H, NH), 7.91 (dd, J ) 7.8, 1.5
Hz, C(6)H).
1
43 °C. H NMR (CDCl3): δ (ppm) 1.89 (dd, J ) 7.2, 1.8 Hz,
COCHCHCH3), 2.91 (s, NCH3), 3.32 (t, J ) 6.6 Hz, CH2S), 4.39
(t, J ) 6.6 Hz, CH2O), 6.15 (dd, J ) 15.6, 1.7 Hz, CHCHCH3),
6.63 (dd, J ) 7.8, 6.6 Hz, aromatic C(5)H), 6.73 (d, J ) 8.1 Hz,
C(3)H), 6.94 (dq, J ) 15.3, 8.1 Hz, CHCHCH3), 7.40 (ddd, J )
8.1, 7.8, 1.5 Hz, C(4)H), 7.90 (dd, J ) 8.2, 1.5 Hz, C(6)H). Anal.
Calcd for C14H17NO3S: C, 60.19; H, 6.13; N, 5.01. Found: C,
60.28; H, 6.20; N, 5.02. UV λmax 255 nm (ꢀ ) 1.55 × 103 M-1
cm-1), λmax 357 nm (ꢀ ) 6.96 × 103 M-1 cm-1).
B.6. N,S-Di(crotonyl)-2-mercaptoethyl anthranilate (6). The
reaction was carried out in N2-flushed glassware. A solution of 14
and 2.9 equiv of pyridine in dry THF was added via addition funnel
at room temperature to a stirred solution of 2.9 equiv of crotonyl
chloride in THF. After 3 h, the reaction mixture had a copious
solid and was reddish-brown in color. The reaction was complete
as judged by the absence of thiol 14 by TLC. The reaction mixture
was worked up as described for compound 2 to give a reddish oil
in 89% yield. Flash chromatography (silica gel, 20% ethyl acetate
B.3. 2-Mercaptoethyl anthranilate (14). Disulfide 12 (8.20 g,
0.021 mol) was slowly added to 500 mL of warm absolute ethanol,
and it dissolved with stirring. The solution was then cooled to 20
°C and was treated with 2 mL of glacial acetic acid, 25.4 g of zinc
powder, and 8 mL of concentrated HCl (added in portions). The
solution was stirred for 3 h, at which time the reaction was judged
to be complete by TLC (silica, 20% acetone in hexanes; disulfide
Rf ) 0.18, thiol Rf ) 0.45). The reaction mixture was then filtered,
and the ethanol was largely removed in vacuo. The remaining syrup
was dissolved in chloroform, and the organic phase was washed
with water, dilute aqueous bicarbonate, saturated aqueous bicarbon-
ate, and finally with brine. The organic fraction was dried (Na2-
SO4), and the solvent was removed in vacuo to give a syrup (2.37
1
in hexanes) gave a white solid (36% yield). Mp: 66-68 °C. H
NMR (CDCl3): δ (ppm) 1.89 (dd, J ) 7.1, 1.8 Hz, SCOCH-
CHCH3), 1.92 (dd, J ) 7.2, 1.8 Hz, NHCOCHCHCH3), 3.34 (t, J
) 6.6 Hz, CH2S), 4.45 (t, J ) 6.3 Hz, CH2O), 6.02 (dd, J ) 15.3,
1.5 Hz, NHCOCHCHCH3), 6.16 (dd, J ) 15.5, 1.8 Hz, SOCH-
CHCH3), 6.90-7.10 (m, 3H, aromatic C(5)H, NHCOCHCHCH3
and SOCHCHCH3), 7.54 (ddd, J ) 7.8, 8.1, 1.2 Hz, C(4)H), 8.02
(dd, J ) 8.7, 1.7 Hz, C(6)H), 8.79 (d, J ) 8.7 Hz, aromatic C(3)-
H), 11.1 (br s, NH). 13C NMR (CDCl3): δ (ppm) 17.8, 17.9, 27.1,
63.5, 114.5, 120.3, 122.2, 126.6, 129.6, 130.8, 134.7, 141.1, 141.8,
141.9, 164.3, 167.8, 188.5. Recrystallization was from methylene
chloride/hexanes. Anal. Calcd for C17H19NO4S: C, 61.24; H, 5.74;
N, 4.20. Found: C, 61.19; H, 5.75; N, 4.26. UV λmax 264 nm (ꢀ )
1.75 × 104 M-1 cm-1), λmax 315 nm (ꢀ ) 9.65 × 103 M-1 cm-1).
C. Time-Course NMR Spectral Study of Reaction of 2 and 4
with N-Acetylcysteamine. A solution of 17 mg of 2 (0.055 mmol)
1
g, 69%). H NMR (CDCl3): δ (ppm) 1.6 (br s, SH), 2.85 (t, J )
6.6 Hz, CH2S), 4.38 (t, J ) 6.6 Hz, CH2O), 5.64 (br s, NH2), 6.6-
6.7 (m, 2H, aromatic C(3)H, C(5)H), 7.26 (ddd, J ) 6.9, 6.9, 1.8
Hz, C(4)H), 7.87 (dd, J ) 8.1, 1.1 Hz, C(6)H). 13C NMR (CDCl3):
δ (ppm) 23.3, 65.4, 110.2, 116.1, 116.6, 131.0, 134.1, 150.4, 167.5.
B.4. 2-Mercaptoethyl N-methylanthranilate (15). Disulfide 13
(4.1 g, 0.0097 mol) was dissolved in 100 mL of acetonitrile, and
the solution was chilled in an ice bath. To the solution were added
in portions 18 g of zinc powder, 4 mL of glacial acetic acid, and
a total of 7 mL of concentrated HCl. After 2 h, reduction was judged
to be complete by TLC (silica, 20% acetone in hexanes; disulfide
Rf ) 0.41, thiol Rf ) 0.56). After filtration to remove solids, the
solvent was removed in vacuo, and the residue was dissolved in
chloroform. The resulting solution was washed with water, very
dilute aqueous bicarbonate, very dilute sodium hydroxide (gently
to avoid emulsion), and finally brine, whereupon it was dried with
anhydrous Na2SO4. The chloroform was removed in vacuo, and
the thiol was obtained as an oil (2.91 g, 71%), which was stored
under N2 at 4 °C. A small portion was purified by crystallization
1
was dissolved in 1 mL of CDCl3, and the H NMR spectrum was
recorded. One equivalent of N-acetylcysteamine was added, and
the spectrum was recorded. It showed that no reaction had occurred.
One-tenth equivalent of the base DBN (1,5-diazabicyclo[4.3.0]non-
5-ene) was added, and the NMR spectrum was recorded within 2
min. A COSY spectrum confirmed the structure of the adduct that
had formed. An analogous NMR study with 4 was carried out as
described above, except NMR spectra were recorded at times up
to 15 min after DBN addition.
D. HPLC Kinetics of Conjugate Addition. HPLC was carried
out with a reverse phase C18 column (300 × 3.9 mm) with isocratic
elution with acetonitrile/water (1:1, v/v) as the mobile phase. UV
detection was used (310 nm). Data reported are the average of at
least two determinations. The retention times of 1 and 3 were 4.6
and 7.2 min, respectively.
J. Org. Chem, Vol. 71, No. 17, 2006 6349

Hartman and Rose
The structures of the adducts formed by the reaction of 1 with
N-acetyl-L-cysteine and N2-acetyl-L-lysine in the HPLC kinetics
experiments were confirmed by mass spectrometry (MS) and NMR
spectroscopy (1H and COSY; Supporting Information). By MS
(MALDI), the reaction mixture of 1 and N-acetyl-L-cysteine showed
m/z 469.135 (calcd for [M + H]+ 469.146) and 491.124 (calcd for
[M + Na]+ 491.128). For NMR spectroscopy, the N-acetyl-L-
cysteine reaction was carried out in a 1:1 (v/v) mixture of 50 mM
NaH2PO4 in D2O (pD 8.4) and acetonitrile-d3. Addition of the thiol
group of N-acetyl-L-cysteine to the crotonyl CdC was evidenced
by the disappearance of the vinyl resonances of 1 and the upfield
shift of the crotonyl methyl group from δ 1.75 to 1.17 ppm. The
COSY spectrum revealed that the new CH3 signal was coupled to
the multiplet at δ 3.2 ppm, which is due to the proton on the
crotonyl C-3 that is attacked by the thiol. In the case of N2-acetyl-
L-lysine, addition was carried out in a 1:1 (v/v) mixture of 50 mM
sodium borate in D2O (pD 10.2) and acetonitrile-d3. Spectral
changes analogous to those seen with N-acetyl-L-cysteine were
observed. The spectra of both adducts reflect the complexity
resulting from the formation of diastereomers (E and Z rotamers at
the amide linkage, R and S configurations at crotonyl C-3, and either
R configuration at C-2 of the reactant N-acetyl-L-cysteine or S
configuration at C-2 of the reactant N2-acetyl-L-lysine). Contributing
to the complexity is the presence of diastereotopic hydrogens in
the methylene groups. Coupling of the crotonyl C-2 hydrogens to
the C-3 hydrogen was not observed for either adduct, as the former
protons exchange with deuterium from solvent.
As shown in Scheme 3, the preequilibrium involves attack of a
dianion (carboxylate and thiolate) on a neutral species (thiol ester),
and the rate-limiting step involves protonation of the resulting
dianion (carboxylate and enolate) by either neutral (zwitterionic)
HEPES or water. To a first approximation, according to the theory
of salt effects on reaction rates, neither step should experience a
salt effect (i.e., dependence of reaction rate on zAzBΓ1/2, where zA
and zB are the charges on the reactants and Γ is the ionic strength)
because zA ) 0. This, and the use of HPLC for analysis, justified
the omission of inert salt to maintain constant ionic strength.
It was found to be convenient to determine pH values in reaction
mixtures from a pH calibration curve, created by measurement of
pH in the aqueous solution (pHAq) containing either 72 mM sodium
HEPES and 50 mM NAC or 154 mM sodium borate and 28.6 mM
NAL, followed by addition of an equal volume of acetonitrile and
then measurement of the pH of the water/acetonitrile solution
(pHmeas). Addition of 0.18 to that value produced pHAqACN. A plot
of pHAqACN (ordinate) versus pHAq (abscissa) was fitted with an
arbitrary function to allow intermediate values of pHAq used for
kinetic runs to be converted to pHAqACN by calculation, without
the need for delaying acquisition of kinetics data by a pH
measurement or exposure of the kinetics solution to the liquid-
junction salt bridge of the pH electrode. The deviation of calculated
values of pHAqACN did not in any case deviate from the actual values
of pHAqACN used for creation of the calibration curve by more than
0.07 units and typically by only 0.02 units or less. The possible
influence of varying HEPES (Figure 4) from 72 mM or NAC
(Figure 5) from 50 mM was neglected.
derived from values of slope and intercept were calculated on the
basis of propagation of errors by differential error analysis. The
line in Figure 5 was constrained to have a zero y-intercept, in accord
with eq 3. If an unconstrained fit is carried out, the slope is 2.79 ×
10-1 M-1 min-1 and the y-intercept is (the physically meaningless)
-5.89 × 10-4 min-1, with r2 ) 0.995. For the curves in Figure 6,
an equation of the form of eq 3b was fitted to the data by
minimization of the sum of the squared deviations of calculated
log kobs from experimental values of log kobs, with the ratio of rate
constants kHEP/k0 set equal to 41 M-1, as determined from Figures
4 and 5. The curve in Figure 8 is line-of-sight.
For determination of pKa values, the method of half-neutralization
was employed. The apparent pKa of HEPES under our reaction
conditions of 1:1 (v/v) H2O/acetonitrile was determined by treatment
of 5.0 mL of 72 mM HEPES in water, containing a 1:1 mixture of
zwitterion and anion by adjustment to pH 7.54,43 with 5.0 mL of
acetonitrile, and measurement of the pH.44 Values of pH meter
readings from duplicate runs were 7.61 and 7.63. These values, as
well as the other pH meter readings in 1:1 (v/v) water/acetonitrile,
were converted to actual pH values in 1:1 water/acetonitrile by
addition of 0.18 to the meter reading,45 giving pKa ) 7.80 for
HEPES in 1:1 (v/v) water/acetonitrile, which is reasonable, relative
to the value of 7.91 found in 50% aqueous acetone.46 The apparent
pKa of N-acetyl-L-cysteine in 1:1 (v/v) water/acetonitrile was
determined by treatment of 4.0 mL of 50 mM N-acetyl-L-cysteine
in water, containing a 1:1 mixture of thiol and thiolate by adjustment
to pH 9.52,47 with 4.0 mL of acetonitrile, and measurement of the
pH. After addition of 0.18 to the pH meter reading, the pKa value
of 10.77 resulted. By comparison, the pKa of mercaptoethanol
similarly shifts from 9.5 in water48 to 10.8 in 1:1 acetonitrile/water,49
and the pKa of methyl mercaptopropanoate shifts from 9.3 in water48
to 10.7 in 1:1 acetonitrile/water.49
Acknowledgment. We thank Amanda Gallegos for technical
assistance with the syntheses. We thank Drs. Ronald Nieman
and Doug Klewer for assistance with NMR spectroscopy, and
John Lopez with MS (MALDI). We gratefully acknowledge
financial support through the Office of Technology Collabora-
tions and Licensing grant program in the Office of the Vice
Provost for Research at Arizona State University.
Supporting Information Available: General experimental
methods and NMR spectra of synthetic intermediates, thiol esters,
and adducts with N-acetylcysteamine, N-acetyl-L-cysteine, and N2-
acetyl-L-lysine are presented, as well as UV absorbance spectra of
compounds 1-6. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO060191+
(43) Grace, S.; Dunaway-Mariano, D. Biochemistry 1983, 22, 4238-
4247.
(44) Bernasconi, C. F.; Sun, W. J. Am. Chem. Soc. 1993, 115, 12526-
12532.
(45) Allen, D.; Tidwell, T. T. J. Am. Chem. Soc. 1987, 109, 2774-2780.
(46) Azab, H. A. J. Chem. Eng. Data 1993, 38, 453-457.
(47) Friedman, M.; Cavins, J. F.; Wall, J. S. J. Am. Chem. Soc. 1965,
87, 3672-3682.
(48) Hengge, A. C.; Hess, R. A. J. Am. Chem. Soc. 1994, 116, 11256-
11263.
E. Curve Fitting of Kinetics Data. Curve fitting of the data in
Figures 3-5 and 7 was by the equally weighted, linear least-squares
method. Slope and y-intercept of the line in Figure 4 are reported
( their standard errors, and uncertainties in kinetic parameters
(49) Bernasconi, C. F.; Ali, M. J. Am. Chem. Soc. 1999, 121, 11384-
11394.
6350 J. Org. Chem., Vol. 71, No. 17, 2006