Emollient, humectant, and fluorescent α,β-unsaturated thiol esters for long-acting skin applications

Article abstract of DOI:10.1016/j.bioorg.2008.06.004

We describe compounds in which an emollient or a humectant bears an α,β-unsaturated thiol ester capable of reacting with nucleophilic amino acids in stratum corneum proteins. These compounds should serve as long-lasting moisturizers for skin. The emollient derivatized was octadecyl propanoate, and the humectant was poly(ethylene glycol). These hydrophobic and hydrophilic compounds, as well as a fluorescent, dansyl-containing thiol ester, were found to react within minutes with the thiol N-acetylcysteamine upon addition of a catalytic amount of an organic base in chloroform. The structures of the products resulting from conjugate addition to the unsaturated thiol esters were determined by NMR spectroscopy. In the case of the α,β,γ,δ-unsaturated (sorboyl) thiol ester, both the 1,4-addition product and the β,γ-unsaturated-1,6-addition product formed, followed by diadduct. An in vivo test of the fluorescent α,β-unsaturated thiol ester showed that this compound persisted on skin for 3 weeks vs. 6 days for the non-bonding control compound.

Full text of DOI:10.1016/j.bioorg.2008.06.004

Bioorganic Chemistry 36 (2008) 265–270
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Emollient, humectant, and fluorescent
a,b-unsaturated thiol esters
for long-acting skin applications
1
*
Carmen Robinson , Rosemarie F. Hartman, Seth D. Rose
Department of Chemistry & Biochemistry, Arizona State University, P.O. Box 871604, Tempe Campus, Tempe, AZ 85287-1604, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2008
Available online 26 August 2008
We describe compounds in which an emollient or a humectant bears an a,b-unsaturated thiol ester capa-
ble of reacting with nucleophilic amino acids in stratum corneum proteins. These compounds should
serve as long-lasting moisturizers for skin. The emollient derivatized was octadecyl propanoate, and
the humectant was poly(ethylene glycol). These hydrophobic and hydrophilic compounds, as well as a
fluorescent, dansyl-containing thiol ester, were found to react within minutes with the thiol N-acetylcy-
steamine upon addition of a catalytic amount of an organic base in chloroform. The structures of the
products resulting from conjugate addition to the unsaturated thiol esters were determined by NMR
Keywords:
Moisturizers
Emollients
Humectants
Michael-type addition
Conjugate addition
spectroscopy. In the case of the
and the b, -unsaturated-1,6-addition product formed, followed by diadduct. An in vivo test of the fluo-
rescent ,b-unsaturated thiol ester showed that this compound persisted on skin for 3 weeks vs. 6 days
for the non-bonding control compound.
a,b,c,d-unsaturated (sorboyl) thiol ester, both the 1,4-addition product
c
a
a,b-Unsaturated thiol ester
Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction
persist on the time scale of the sloughing off and renewal of
skin cells. In addition to moisturizers, we have also synthesized
Moisturizers for skin are typically composed of emollients and
humectants. Emollients are hydrophobic compounds that provide
an occlusive barrier to prevent water loss, whereas humectants
are hydrophilic compounds that hydrate the stratum corneum.
Glycerin [1], which penetrates and hydrates skin, as well as main-
tains stratum corneum lipids in a non-crystalline state, is typically
added as well. Moisturizers are used to relieve symptoms of dry
skin (xerosis) and to restore normal barrier function to skin [2,3].
In addition, moisturizer use has been found to reduce irritant con-
tact dermatitis [4]. The effect of a single application of moisturizer
is typically short lived. Repeated applications of moisturizers, par-
ticularly ones with a high (>15%) glycerol content, have been found
to persist for as long as 4 days [5].
a,b-unsaturated thiol esters containing a fluorescent moiety that
can serve to visualize binding of the thiol-ester-containing agents
to skin.
The
a,b-unsaturated thiol ester moiety is subject to Michael-
type addition by nucleophiles [6,7]. This functional group is more
reactive toward Michael-type addition than the corresponding
oxygen esters [8]. It is, however, significantly less reactive than
typical sulfhydryl-reactive reagents, such as maleimides [9], which
would be expected to be too harsh for large-surface-area topical
use. In previous studies of
a,b-unsaturated thiol esters, we found
that reaction with the cysteine thiolate was approximately 300
times faster than that of the lysine amino group at pH 9.8. We
found that addition of cysteine and lysine occurred under basic
conditions, with the pH-rate profile reflecting the abundance of
the reactive species, i.e., the thiolate anion of cysteine and the neu-
tral amino group of lysine [7].
The skin is rich in nucleophiles such as sulfhydryl and amino
groups that could be used for attachment of beneficial agents such
as humectants and moisturizers to skin, thereby affording resis-
tance to loss by washing and mild abrasion. For reaction with skin
We describe herein the preparation and reactivity of com-
nucleophiles, we have selected the
a,b-unsaturated thiol ester
pounds in which an emollient or humectant bears an a,b-unsatu-
functional group. As a Michael acceptor, this group is capable of
covalent bonding to nucleophilic groups that add to the C@C con-
jugated to the thiol ester C@O group. The structures of the com-
pounds are shown in Fig. 1. We aim to provide moisturizers that
rated thiol ester group capable of reacting with proteins in the
stratum corneum, the outermost layer of the skin.
2. Materials and methods
2.1. General
* Corresponding author. Fax: +1 480 965 2747.
E-mail address: srose@asu.edu (S.D. Rose).
Present address: Isis Pharmaceuticals, Inc., 1896 Rutherford Road, Carlsbad, CA
92008, USA.
1
Octadecyl 3-mercaptopropionate (OMP; octadecyl 3-sulfanyl-
propanoate) was a gift from Evans Chemetics (Iselin, NJ) and was
0045-2068/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2008.06.004

266
C. Robinson et al. / Bioorganic Chemistry 36 (2008) 265–270
H3C(CH2)17
O
S
H3C(CH2)17
O
S
O
O
O
O
1
2
S
S
O
n
O
O
3
S
S
HN
HN
HN
O
S
O
O
O
S
O
O
O S O
N
N
N
6
5
4
Fig. 1. Compounds studied are shown: emollients 1 and 2, humectant 3, fluorescent thiol esters 4 and 5, and fluorescent control compound 6.
used without further purification. THF was dried by distillation un-
der N2 from Na/benzophenone. Pyridine was distilled from BaO
and stored over 3 Å molecular sieves. All glassware was dried with
a flame before use. NMR spectra were measured with a Varian
Gemini spectrometer at 300 MHz for 1H and 75 MHz for 13C, or
an Inova spectrometer at 400 or 500 MHz for 1H and 125 MHz
for 13C, with tetramethylsilane as internal standard. Thin layer
chromatography was carried out with Analtech silica gel GHLF
Uniplates.
The product 2 (Rf = 0.55) was obtained as a white solid, 7.5 g
(67%), mp 43.5–45.5 °C. Anal. Calcd for C27H48O3S: C, 71.63; H,
10.69. Found: C, 71.24; H, 10.66. 1H NMR (500 MHz, CDCl3) d
(ppm) 0.89 (t, J = 7.0 Hz, 3H, CH3(CH2)17), 1.2–1.35 (m, 30H,
CH3(CH2)15), 1.61 (m, 2H, OCH2CH2), 1.87 (d, J = 6.5, 3H, CH3CH),
2.63 (t, J = 7.0, 2H, SCH2CH2), 3.19 (t, J = 7.5, 2H, SCH2), 4.08 (t,
J = 7.0, 2H, CO2CH2), 6.02 (d, J = 15.0 Hz, 1H, CH3CH@CHACH@CH),
6.20 (m, 2H, CH3CH@CH), 7.20 (dd, J = 15.5, 15.0 Hz, 1H,
CH3CH@CHACH@CH).
2.2. Synthesis
2.2.3. Poly(ethylene glycol) dimesylate (7)
Poly(ethylene glycol) (PEG) with an average molecular weight
2.2.1. Octadecyl 3-crotonoylsulfanylpropanoate (1)
(Mn) of 400 was dried by heating at 70 °C under vacuum for 3 h.
To a solution of crotonoyl chloride (13.5 g, 0.129 mol) in 250 mL
THF was added dropwise a solution of OMP (35.2 g, 0.098 mol) and
pyridine (7.7 g, 0.097 mol) in 75 mL THF. A white solid formed
immediately and continued to form. After 1 h at room tempera-
ture, the mixture was filtered, and the filtrate was rotavapped to
yield 35.6 g of a slurry that solidified to a waxy white solid upon
chilling. The crude material was diluted with chloroform and
washed twice with dilute sodium bicarbonate, and the organic
layer was dried with MgSO4 and rotavapped to yield 28.0 g crude
solid (67% yield). This material was purified batchwise by chroma-
tography (silica, 3% hexanes in methylene chloride, Rf = 0.47) to
yield 1 as a white solid in 25% overall yield, mp 32–34 °C. Anal.
Calcd for C25H46O3S: C, 70.37; H, 10.87. Found: C, 70.15; H,
10.82. 1H NMR (500 MHz, CDCl3) d (ppm) 0.89 (t, J = 7.0 Hz, 3H,
CH3(CH2)17), 1.2–1.4 (m, 30H, CH3(CH2)15), 1.62 (m, 2H, OCH2CH2),
1.88 (d, J = 6.5 Hz, 3H, CH3CH), 2.64 (t, J = 7.0 Hz, 2H, SCH2CH2),
3.18 (t, J = 6.5 Hz, 2H, SCH2), 4.09 (t, J = 6.5 Hz, 2H, CO2CH2), 6.13
(d, J = 15.5 Hz, 1H, CH3CH@CH), 6.91 (dq, J = 15.0, 7.0 Hz, 1H,
CH3CH@CH). 13C NMR (125 MHz, CDCl3) d (ppm) 14.2, 21.7, 22.8,
23.3, 24.3, 26.0, 28.6, 29.3, 29.4, 29.6, 29.7, 29.8, 29.8, 30.7, 32.0,
34.4, 36.4, 38.9, 51.0, 65.1, 170.2, 171.5, 196.9.
PEG dimesylate, a-methylsulfonyl-x-methylsulfonyloxypoly(oxy-
ethylene), was prepared as follows. To a solution of dried PEG
(7.89 g, 39.4 mmol OH-ends) and triethylamine (7.94 g, 78 mmol)
in methylene chloride at 0 °C was added a fourfold excess of meth-
anesulfonyl chloride (17.9 g, 156 mmol) dissolved in methylene
chloride. After 3 h, the reaction mixture was rotavapped to a syrup,
taken up in water, and the unreacted methanesulfonyl chloride
was destroyed by addition of NaHCO3. The product was then ex-
tracted with chloroform, whereupon the solution was dried with
MgSO4, and rotavapped to give 7 as a light yellow oil (9.96 g,
95%). 1H NMR (300 MHz, CDCl3) d (ppm) 3.07 (s, 6H), 3.6 (m,
28H), 3.8 (m, 4H), 4.4 (m, 4H).
2.2.4. Poly(ethylene glycol) dithiol (8)
A solution was prepared containing 7 (5.0 g, 18 mmol), diethyl-
enetriaminepentaacetic acid (53.8 mg, 0.05% by wt of reaction mix-
ture), and thiourea (2.85 g, 37 mmol) in water (100 mL), and it was
adjusted to pH 6.7 with aqueous potassium hydroxide. The reaction
mixture was refluxed for 2.5 h. The reaction mixture was then
cooled, and the resulting isothiouronium salt was hydrolyzed by
addition of dilute aqueous NaHCO3 (2.35 g, 1.5 equiv) followed by
1.5 h of reflux. The solution was then neutralized with 1 M H2SO4,
2.2.2. Octadecyl 3-sorboylsulfanylpropanoate (2)
and the PEG dithiol, a-sulfanylethyl-x-sulfanylpoly(oxyethylene),
To a solution of OMP (11.1 g, 31.1 mmol) in 30 mL of cyclohex-
ane at 45 °C was slowly added a solution of sorboyl chloride
(4.05 g, 31.0 mmol) in 35 mL of cyclohexane. After 15-h reflux,
the solvent was evaporated in vacuo to leave a brown liquid, which
was subjected to chromatography (10% ethyl acetate in hexanes).
was extracted into chloroform, dried with MgSO4 and rotavapped
to yield 2.18 g of 8 as a light amber oil. The material was stored un-
der argon at ꢀ20 °C. 1H NMR (300 MHz, CDCl3) d (ppm) 1.59 (t,
J = 6.8 Hz, 2H), 2.69 ppm (dt, J = 5.2 Hz, 6.8 Hz, 4H), 2.88 ppm (t,
J = 5.2 Hz, <1H, trace disulfide), 3.63 (m, 32H), 3.74 (m, 4H).

C. Robinson et al. / Bioorganic Chemistry 36 (2008) 265–270
267
2.2.5.
a
-Crotonoylsulfanylethyl-
x
-crotonoylsulfanylpoly(oxyethylene) (3)
2.2.9. 5-(Dimethylamino)-N-[2-(sorboylsulfanyl)ethyl]naphthalene-1-
sulfonamide (5)
A solution of 8 (2.1 g, 9.0 mmol SH-ends) and pyridine (0.656 g,
8.3 mmol) in 10 mL THF was added dropwise to a stirred solution
of crotonoyl chloride (0.857 g, 8.5 mmol) in 20 mL THF. The solu-
tion instantly became cloudy. After 16 h, the reaction was rotavap-
ped to dryness and the residue was taken up in chloroform, which
was washed extensively with aqueous NaHCO3 and dried with
Na2SO4 to give
poly(oxyethylene)
(300 MHz, CDCl3) d (ppm) 1.88 (dd, J = 7.6, 1.7 Hz, 6H, 2CH3),
3.15 (t, J = 6.6 Hz, 4H, 2SCH2), 3.6–3.7 (m, (OCH2CH2)nO), 3.68 (t,
J = 7.2 Hz, 4H, 2CH2OCH2CH2S), 6.14 (dd, J = 15.3 Hz, 1.8 Hz, 2H,
2CH3CH@CH), 6.91 (dq, J = 15.3, 7.2 Hz, 2H, 2CH3CH@CH).
Compound 5 was prepared according to the procedure de-
scribed in Section 2.2.8 (synthesis of 4) by use of sorboyl chloride
instead of crotonoyl chloride. Purification of the crude material
by flash chromatography (40% acetone in hexanes, Rf = 0.55) pro-
duced 5 as a greenish film (42% yield). 1H NMR (500 MHz, CDCl3)
d 1.88 (dd, J = 6.5 Hz, 1.5 Hz, 3H, CH3CH), 2.89 (s, 6H, N(CH3)2),
2.96 (t, J = 6.5 Hz, 2H, CH2S), 3.12 (dt, J = 6.5 Hz, 6.0 Hz, 2H,
NCH2), 5.07 (t, J = 5.5 Hz, 1H, NH), 5.93 (d, J = 15.5 Hz, 1H,
CH3CH@CHACH@CH), 6.0–6.2 (m, 2H, CH3CH@CH), 7.08 (dd,
J = 15, 11 Hz, 1H, CH3CH@CHACH@CH), 7.17 (d, J = 8.0 Hz, 1H,
arom), 7.53 (m, 2H, arom), 8.25 (m, 2H, arom), 8.54 (d, J = 8.5 Hz,
1H, arom). Anal. Calcd for C20H24N2O3S2: C, 59.38; H, 5.98; N,
6.92. Found: C, 59.56; H, 6.08; N, 6.71.
a
-crotonoylsulfanylethyl-
x-crotonoylsulfanyl-
3
as an amber oil (1.7 g, 70%). 1H NMR
2.2.6. N,N0-Bis(5-dimethylaminonaphthalene-1-sulfonyl)-cystamine (9)
Dansyl chloride (4.0 g, 14.8 mmol) was dissolved in a solution of
900 mL acetone and 30 mL water, and a solution of cystamine dihy-
drochloride (1.66 g, 7.4 mmol, 1 equiv) in 100 mL 0.1 M aqueous
NaHCO3 was added in portions. The solution was maintained at pH
7.5 by addition of dilute aqueous sodium hydroxide. After 2 h at
room temperature, the reaction mixture was diluted with 400 mL
chloroform, and the solution was washed four times with aqueous
sodium bicarbonate, followed by water, whereupon it was dried
with MgSO4. Solvent was removed to yield3.86 g of 9 as a fluffy, crisp
yellow solid (84%), Rf = 0.73, 5% methanol in chloroform; Rf = 0.20,
30% acetone in hexane, mp 71–72 °C. 1H NMR (500 MHz, CDCl3) d
(ppm) 2.50 (t, J = 6.5 Hz, 4H), 2.89 (s, 12H, 2N(CH3)2), 3.10 (d,
J = 6.5, 5.5 Hz, 4H, 2CH2N), 5.23 (br t, 2H, 2NH), 7.18 (d, J = 7.5 Hz,
2H), 7.53 (m, 4H), 8.25 (m, 4H), 8.55 (d, J = 8.5 Hz, 2H).
2.2.10. 5-(Dimethylamino)-N-propylnaphthalene-1-sulfonamide (6)
A solution of dansyl chloride (0.92 g, 3.4 mmol) and triethyl-
amine (0.47 mL, 3.4 mmol) in 60 mL distilled methylene chloride
was treated with propylamine (0.28 mL, 3.4 mmol). The reaction
mixture was stirred for 1 h at room temperature. The resulting
reaction mixture was washed extensively with water, dilute
aqueous NaHCO3, and brine, whereupon it was dried with Na2SO4
and rotavapped to a crude oil 6 (0.93 g, 98%). Crude 6 was puri-
fied by chromatography (40% acetone in hexanes, Rf = 0.62) to
yield
6 as a
greenish-yellow solid (mp 92–94 °C). 1H NMR
(500 MHz, CDCl3) d 0.77 (t, J = 6.5 Hz, 3H, CH3), 1.41 (m, 2H,
CH2CH2CH3), 2.86 (m, 2H, CH2CH2CH3), 2.89 (s, 6H, N(CH3)2),
4.67 (t, J = 6.0, 1H, NH), 7.18 (d, J = 7.5, 1H, arom), 7.53 (m, 2H,
arom), 8.25 (m, 2H, arom) 8.55 (d, J = 8.5 Hz, 1H, arom). Anal.
Calcd for C15H20N2O2S: C, 61.61; H 6.89; N, 9.58. Found: C,
61.45; H, 6.80; N, 9.53.
2.2.7. 5-(Dimethylamino)-N-(2-sulfanylethyl)naphthalene-1-
sulfonamide (10)
Reduction of the disulfide 9 was carried out as follows. Com-
pound 9 (1.96 g, 3.25 mmol) dissolved in 400 mL absolute ethanol
was treated with 11.2 g finely powdered zinc and 25 mL glacial
acetic acid, added alternately in portions. After 4 h, the reaction
mixture was filtered to remove zinc and zinc salts, and the filtrate
was rotavapped to a volume of 50 mL. This was then diluted with
chloroform, and the organic phase was washed three times with
water, once with dilute aqueous NaHCO3 followed by brine, where-
upon it was dried with MgSO4 and rotavapped to yield 1.5 g (76%)
of a greenish-yellow film (30% acetone in hexane, Rf = 0.44). 1H
NMR (300 MHz, CDCl3) d (ppm) 1.2 (t, J = 8.5 Hz, 1H), 2.50 (m,
2H, CH2S), 2.90 (s, 6H, N(CH3)2), 3.08 (dt, J = 5.5, 6.5 Hz, 2H,
CH2N), 5.15 (broad t, 1H, NH), 7.20 (d, J = 7.5 Hz, 2H), 7.56 (m,
2H), 8.27 (m, 2H), 8.55 (d, J = 8.5 Hz, 1H).
2.3. Formation and characterization of N-acetylcysteamine adducts
2.3.1. Reaction of 1 with N-acetylcysteamine
The reaction of the
NMR spectroscopy. To a solution of 1 (22 mg, 0.052 mmol) dis-
solved in 0.6 mL of CDCl3 was added N-acetylcysteamine (5.7 L,
a,b-unsaturated thiol esters was followed by
l
0.053 mmol). The NMR spectrum was recorded and showed that
no reaction had occurred. A catalytic amount (0.1 equiv) of the
base DBN was added, and the NMR spectrum was recorded after
5 min. More than 60% of 1 had reacted, as judged by the dimin-
ished integration of the resonances at d 6.1 ppm and 6.9 ppm due
to the vinyl protons of the crotonoyl moiety. An NMR spectrum re-
corded 27 min after DBN addition showed that 1 had completely
reacted.
2.2.8. N-[2-(Crotonoylsulfanyl)ethyl]-5-(dimethylamino)naphthalene-
1-sulfonamide (4)
The reaction of 1 was also carried out on a larger scale. To 1
(171 mg, 0.40 mmol) in 10 mL of N2-purged CHCl3 was added N-
Thiol 10 (1.5 g, 5.0 mmol) obtained from 2.2.7 was dissolved in
100 mL of freshly distilled THF containing pyridine (0.930 g,
11.8 mmol), and the solution was added dropwise to a stirred solu-
tion of crotonoyl chloride (3.92 g, 37.5 mmol) in 30 mL THF. After
4 h, the solvent was removed in vacuo, and the residue was taken
up in chloroform. The resulting solution was washed with dilute
NaHCO3 followed by brine, and dried with Na2SO4. The crude prod-
uct was purified by flash chromatography on silica (30% acetone in
hexane, Rf = 0.41) to yield pure compound 4 as a sticky greenish
acetylcysteamine (30
lL, 0.28 mmol), followed by triethylamine
(39 L, 0.28 mmol). The reaction mixture was stirred for 2 h under
l
N2, after which it was subjected to chromatography (3% methanol
in chloroform, Rf = 0.54) to give 38.5 mg adduct as a white waxy so-
lid (26% yield after purification). Anal. Calcd for C29H55NO4S2: C,
63.81; H 10.16; N, 2.57. Found: C, 63.36; H, 10.07; N, 2.62.
1H NMR (400 MHz, CDCl3) d (ppm) 0.88 (t, 3H, J = 6.8 Hz,
CH3(CH2)17), 1.22–1.26 (m, 30H, (CH2)15), 1.32 (d, 3H, J = 6.4,
CH3CHCH2), 1.62 (m, 2H, CO2CH2CH2), 2.02 (s, 3H, NHCOCH3),
2.63 (t, J = 6.8 Hz, 2H, SCH2CH2CO), 2.70 (t, J = 6.4 Hz, 2H,
SCH2CH2NH), 2.71 (dd, J = 14.5, 7.8 Hz, 1H, CH3CHSCHaHb), 2.82
(dd, J = 15.4, 7.4 Hz, 1H, CH3CHSCHaHb), 3.15 (t, J = 7.2 Hz, 2H,
SCH2CH2CO), 3.27 (dq, J = 14.0, 6.8 Hz, 1H, CH3CHSCH2), 3.45 (dt,
J = 6.4, 6.0 Hz, 2H, SCH2CH2NH), 4.09 (t, J = 6.8 Hz, 2H, CO2CH2
(CH2)16CH3), 6.2 (br s, 1H, NH). COSY crosspeaks: d 0.88/1.2–1.3;
1.2–1.3/1.62; 1.32/3.27; 1.62/4.09; 2.63/3.15; 2.70/3.45; 2.71/
2.82; 2.71/3.27; 2.82/3.27; 3.45/6.2. Conjugate addition to the
film (1.47 g, 80%). 1H NMR (500 MHz, CDCl3)
d 1.87 (dd,
J = 6.5 Hz, 1.5 Hz, 3H, CH3CH), 2.90 (s, 6H, N(CH3)2), 2.95 (t,
J = 6.5 Hz, 2H, CH2S), 3.13 (dt, J = 6.5, 6.0 Hz, 2H, NCH2), 5.04 (t,
J = 6.0 Hz, 1H, NH), 6.02 (dd, J = 15.5, 1.5 Hz, 1H, CH3CH@CH),
6.83 (dq, J = 15.0, 7.0 Hz, 1H, CH3CH@CH), 7.18 (d, J = 7.5 Hz, 1H
arom), 7.55 (m, 2H, arom) 8.25 (m, 2H, arom), 8.55 (d, J = 8.5 Hz,
1H, arom). Anal. Calcd for C18H22N2O3S2: C, 57.12; H, 5.86; N,
7.40. Found: C, 57.02; H, 5.94; N, 7.33.

268
C. Robinson et al. / Bioorganic Chemistry 36 (2008) 265–270
a
,b-unsaturated thiol ester to produce the adduct (R = octadecyl-
0.7 equiv) was used in place of the thiol N-acetylcysteamine, no
addition products were detected. This is not surprising, as we
found that amines were ꢁ102-fold less reactive than thiols in a
similar reaction [7]. When the reaction was carried out in the ab-
sence of the N2 atmosphere, minor byproducts were found that re-
sulted from oxidation of OMP to the symmetrical disulfide, and of
OMP with N-acetylcysteamine to the mixed disulfide, as well as a
product resulting from N-acetylcysteamine attack at the sorboyl
carbonyl carbon.
oxycarbonylethyl) shown in Fig. 2a was evidenced by the COSY
crosspeaks observed between the methyl protons at d 1.32 ppm
and the methine proton at 3.27 ppm, and between this methine
proton and the adjacent diastereotopic methylene protons at d
2.71 and 2.82 ppm.
2.3.2. Reaction of 2 with N-acetylcysteamine
In a 50-mL round-bottom flask, 181 mg (0.40 mmol) of com-
pound 2 was dissolved in 10 mL of chloroform, and the solution
was purged with nitrogen for 15 min prior to use. After addition
2.3.3. Reaction of 3 with N-acetylcysteamine
of 30
l
L (0.28 mmol) of N-acetylcysteamine and 39
l
L (0.28 mmol)
The reaction of 3 with N-acetylcysteamine was also followed by
NMR spectroscopy: 35.6 mg of 3 was dissolved in 0.7 mL CDCl3,
and the spectrum was recorded before and after addition of N-acet-
ylcysteamine (1 equiv per crotonoyl moiety), and again after
0.1 equiv of DBN was added. The reaction was complete after
5 min and produced the adduct shown in Fig. 2a, as evidenced by
the disappearance of the resonances at d 6.14 and 6.91 ppm, which
correspond to the crotonoyl vinyl protons, and by the upfield shift
of the CH3 doublet from d 1.88 to 1.28 ppm. 1H NMR (300 MHz,
CDCl3) d (ppm) 1.28 (d, J = 6.1 Hz, 6H, 2CH3CH), 1.98 (s, 6H,
2NHCOCH3), 2.64 (t, J = 6.6 Hz, 4H, 2SCH2CH2N), 2.65 (m, 2H,
2CH3CHCHaHb), 2.80 (m, 2H, 2CH3CHCHaHb), 3.1 (m, 4H,
2OCH2CH2S), 3.2– 3.4 (m, 6H, 2CH3CH and 2CH2NH), 3.6 (br s,
CH2CH2O)n, 6.3 (br s, 2H, 2NH). COSY crosspeaks: 1.28/3.4; 2.64/
3.2; 2.64/3.4; 2.66/2.8; 2.66/3.4; 2.8/3.4; 3.1/3.6; 3.4/6.3. The COSY
spectrum showed the coupling of the terminal methyl group (d
1.28 ppm) with the adjacent methine proton (d 3.4 ppm) and from
this to the methylene protons (d 2.65 and 2.80 ppm).
of triethylamine, the solution was stirred under reflux in a nitrogen
atmosphere for 2 h. The solvent was evaporated, and an approxi-
mately 1:1 mixture of the 1,4- and 1,6-monoadducts was isolated
by flash chromatography (silica, 3% methanol in chloroform)
88 mg, 38% yield. Anal. Calcd for C30H57NO4S2: C, 65.10; H 10.05;
N, 2.45. Found: C, 64.84; H, 10.07; N, 2.50. Alternatively, the mono-
adducts were isolated by preparative TLC (Rf = 0.66, 3% methanol in
chloroform). The isolated mixture of monoadducts was applied to
another preparative TLC plate, and the monoadducts were sepa-
rated by repetitive development with 3% methanol in chloroform.
The 1,6- and 1,4-addition products are shown in Fig. 2b. 1,6-Addi-
tion product: 1H NMR (300 MHz, CDCl3) d (ppm) 0.89 t, J = 7 Hz, 3H,
(CH2)15CH3), 1.30 (br m, 30H, (CH2)15), 1.31 (d, J = 7 Hz, 3H,
CH3CHSCH), 1.61 (m, 2H, OCH2CH2), 2.00 (s, 3H, acetyl), 2.55 (m,
1H, NHCH2CHH0), 2.62 (t, J = 7 Hz, 2H, OCOCH2), 2.65 (m, 1H,
NHCH2CHH0), 3.12 (t, J = 7 Hz, 2H, COSCH2), 3.29 (d, J = 7 Hz, 2H,
SCOCH2), 3.32 (m, 3H, CH3CH(SCH2CH2)CH), 4.10 (t, J = 7 Hz, 2H,
OCH2(CH2)16), 5.42–5.62 (m, 2H, olefinic), 6.00 (br s, 1H, NH). COSY
crosspeaks: d 0.89/1.30; 1.30/1.61; 1.31/3.44; 1.61/4.10; 2.55/2.65;
2.62/3.12; 3.29/5.52; 3.32/5.35; 5.35/5.52. 1,4-Addition product:
1H NMR (300 MHz, CDCl3) d (ppm) 0.89 (t, 3H, (CH2)15CH3), 1.30
(br m, 30H, (CH2)15), 1.61 (m, 2H, OCH2CH2), 1.71 (d, J = 7 Hz, 3H,
CH3CH@), 2.00 (s, 3H, acetyl), 2.55 (m, 1H, SCHH0CH2NH), 2.65
(m, 1H, SCHH0CH2NH), 2.61 (m, 2H, OCOCH2), 2.79 (d, J = 7 Hz,
2H, SCOCH2), 3.18 (t, J = 7 Hz, 2H, COSCH2), 3.40 (m, 1H, NHCHH0),
3.42 (m, 1H, NHCHH0), 3.63 (m, 1H, CH3CH=CHCH), 4.08 (t, J = 7 Hz,
2H, OCH2 (CH2)16), 5.28 (m, 1H, olefinic), 5.55 (m, 1H, olefinic), 6.00
(br s, 1H, NH). COSY crosspeaks: d 0.89/1.30; 1.30/1.61; 1.61/4.10;
1.71/5.55; 2.55/2.65; 2.61/3.18; 2.55–2.65/[3.40–3.42]; 2.79/3.63;
3.40/3.42; 3.63/5.28; 5.28/5.55. When the reaction was carried
out in the presence of a fourfold excess of each N-acetylcysteamine
and triethylamine, the ratio of monoadducts was only slightly
changed to 57% 1,6- and 43% 1,4-addition product, and the yield
was not significantly changed (31%). When an amine (morpholine,
2.3.4. Reaction of 4 with N-acetylcysteamine
The reaction of 4 with N-acetylcysteamine was also followed by
NMR spectroscopy and showed complete reaction within 4 min of
addition of 0.3 equiv DBN. 1H NMR (300 MHz, CDCl3) d (ppm) 1.28
(d, J = 7 Hz, 3H, CH3CH), 1.95 (s, 6H, N(CH3)2), 1.98 (s, 3H,
NHCOCH3), 2.5–2.8 (m, 4H, CH2S and CH3CHaHb), 3.2 (m, 1H,
CH3CH), 3.1 (m, 2H, CH2NH), 3.4 (m, 2H, CH2NH), 4.5 (br s, 1H,
NH), 6.2 (br s, 1H, NH), 7.17 (d, J = 7.6 Hz, 1H, arom), 7.52 (m, 2H,
arom), 8.24 (m, 2H, arom), 8.52 (m, 1H, arom). Conjugate addition
to the a,b-unsaturated thiol ester gave the adduct shown in Fig. 2a,
as evidenced by the disappearance of the crotonoyl vinyl protons,
the upfield shift of the terminal methyl proton doublet from d
1.87 ppm to 1.28 ppm, the coupling of this to the methine proton
at d 3.2 ppm, and from the methine to the adjacent methylene
protons at d 2.6 ppm. COSY crosspeaks: 1.28/3.2; 3.2/2.7; 2.7/3.4;
3.1/2.6; 7.17/7.52; 7.15/8.2; 7.15/8.52.
O
a
HS
N
S
S
H
R
R
O
O
O
S
N
H
O
b
HS
N
H
S
S
S
R
O
R
O
R
+
O
S
O
S
O
N
N
H
H
1,4-addition product
1,6-addition product
(β,γ-unsaturated)
Fig. 2. (a) The crotonoyl thiol esters 1, 3, and 4 reacted with N-acetylcysteamine in DBN/chloroform to form the adduct shown; R = octadecyloxycarbonylethyl in 1,
-crotonoylsulfanylpoly(ethyleneoxy)ethyl in 3, and dansyl in 4. (b) Sorboyl thiol ester 2 reacted to form the 1,4- and the b, -unsaturated-1,6-addition products with
N-acetylcysteamine in DBN/chloroform; R = octadecyloxycarbonylethyl.
x
c

C. Robinson et al. / Bioorganic Chemistry 36 (2008) 265–270
269
2.4. In vivo test of skin binding of fluorescent compounds on human
skin
typical thiol odor of thiol hydrocarbons. Two emollients were pre-
pared: 1 with an ,b-unsaturated crotonoyl thiol ester and 2 with
a
the corresponding sorboyl ester. Syntheses of compounds 1 and 2
were accomplished in one step by treatment of the commercially
available thiol octadecyl 3-mercaptopropionate with the respec-
tive acid chloride, as outlined in Scheme 1.
Poly(ethylene glycol) was chosen as the humectant, as it is
hydrophilic, yet non-toxic and non-immunogenic. Additionally, be-
cause the poly(ethylene glycol) molecules can be modified with
Compounds 4, 5, and 6 were dissolved in ethanol, and 1 mg each
was applied separately to 1-cm2 patches of skin on the inner arm,
previously delineated with Scotch tape. After air drying for ꢁ1 h,
the application area was washed with soap and water. The material
was not visible on the skin in room light. For the first day only,
however, the green color of the compounds was faintly visible in
very bright sunlight. The compounds emitted bright fluorescence
when illuminated with long-wavelength UV light. The fluorescence
was photographed under long-UV irradiation with Kodak Ultra
Max 800 speed film. For the duration of the experimental period,
the material was subjected to 1–2 soap washings/day, and no other
skin products were applied to the test area. No signs of irritation or
redness were observed.
skin-reactive
a,b-unsaturated thiol ester moieties at each end,
crosslinking of skin proteins may occur and may serve to enhance
the persistence on skin and possibly strengthen skin. The dicroto-
noyl thiol ester 3 was prepared. Preparation of 3 made use of PEG
dithiol, as outlined in Scheme 2.
For eventual visualization of binding of the thiol esters on skin,
compounds containing the fluorescent dansyl group with an
a,b-
unsaturated crotonoyl thiol ester 4 and sorboyl thiol ester 5 were
synthesized. As a potential control in such studies, compound 6
that has a propyl group in place of the a,b-unsaturated thiol ester
3. Results and discussion
and lacks covalent binding capability was prepared, as shown in
Scheme 3.
3.1. Chemistry
We followed the reaction of crotonoyl thiol esters (1, 3, and 4)
with the simple thiol, N-acetylcysteamine, by NMR spectroscopy
in CDCl3. In each case, reaction occurred within minutes of addition
of 0.1–0.3 equivalents of an organic base, e.g., DBN or triethyl-
Structures of the compounds studied are shown in Fig. 1. The
emollient we chose to derivatize was octadecyl 3-mercaptopropio-
nate, which contains a hydrophobic C18 chain and an ester group.
This ester group may be responsible for the minimization of the
amine. Addition of the thiol to the a,b-unsaturated crotonoyl thiol
ester produced the Michael-type adduct, as shown in Fig. 2a. For
each compound, NMR spectroscopy showed disappearance of the
two vinyl resonances of the thiol ester and an upfield shift of the
signal for the crotonoyl methyl protons.
The sorboyl thiol esters were found to react more slowly than
the crotonoyl esters, as reported previously in the case of p-meth-
oxycinnamoyl-containing thiol esters [7]. For example, in the
crotonoyl chloride
1
H3C(CH2)17
O
SH
THF/pyr, 1 h, RT
sorboyl chloride
O
2
cyclohexane/pyr, 15 h, reflux
Scheme 1.
MsO
OMs
HO
OH
methanesulfonyl chloride/Et N
3
O
O
n
n
CH Cl , 3 h, 0º C
2
2
7
1. thiourea/ H O, pH 6.7
2
2.5 h, reflux
crotonoyl chloride
THF/pyr, 16 h, RT
HS
SH
3
O
2. NaHCO , 1.5 h, reflux
3
n
8
Scheme 2.
S
S
HN
SO2
NH
SO2Cl
SO2
cystamine di-HCl
propylamine
CH Cl /Et N, 1 h, RT
6
EtOH/H O, pH 7.5, 2 h, RT
2
2
2
3
N
N
N
9
SH
HN
SO2
crotonoyl chloride
4
5
Zn/acetic acid
EtOH, 4 h, RT
THF/pyr, 4 h,RT
9
sorboyl chloride
THF/pyr, 4 h, RT
N
10
Scheme 3.

270
C. Robinson et al. / Bioorganic Chemistry 36 (2008) 265–270
forearm skin gave similar results; thiols were most abundant in the
control
control
lower stratum corneum, and disulfide groups were evenly distrib-
uted throughout the stratum corneum [15]. Broekaert and col-
leagues (1982) also reported free thiols in the inner stratum
corneum, which remained throughout the entire horny layer [16].
6
4
6
5
Day 3
Day 5
3.2. Biology
The compounds dissolved in ethanol were applied to skin
(1 mg/cm2). No redness or irritation was observed, either immedi-
ately or during the entire course of the experiment. The com-
pounds were virtually invisible under normal room light but
fluoresced brightly under long-wavelength UV, although the fluo-
rescence of 5 was noticeably less intense than 4 or 6.
Day 12
We found that fluorescence of the
bearing compound (4) was visible on the skin for at least 21 days,
and that of the ,b, ,d-unsaturated (sorboyl) thiol ester (5) was vis-
a,b-unsaturated thiol ester-
Day 21
a
c
ible on the skin for 12 days (although it was very light). Fluores-
cence of the control compound (6) faded more quickly and was
visible for only 6 days. Photographs of the skin fluorescence are
shown in Fig. 3.
Day 4, room light
Fig. 3. Photographs of the applied fluorescent compounds 4, 5, and 6, taken over a
3-week period, show the persistence on the skin of the unsaturated thiol esters
relative to the non-bonding control compound. A photograph of the application
area taken under room light is included.
4. Conclusion
Aliphatic emollients, poly(ethylene glycol)-derived humectants,
and a dansyl chromophore bearing an
a,b- or a,b,c,d-unsaturated
presence of 2 equivalents of N-acetylcysteamine and 1 equivalent
of base (DBN), compound 2 reacted after 1.5 h to yield the 1,4-
and b,c-unsaturated-1,6-addition monoadducts, as shown in
Fig. 2b. Apparently, nucleophilic attack at the end of the conjugated
system is followed by protonation of the intermediate enolate at
(sorboyl) thiol ester have been synthesized and found to be reac-
tive toward an amino acid model compound, N-acetylcysteamine,
under mildly basic conditions. The crotonoyl thiol esters under-
went conjugate addition within minutes of treatment with a cata-
lytic amount of base. The corresponding sorboyl thiol esters
reacted more slowly and formed the 1,4- and 1,6-monoaddition
products, which under prolonged conditions further reacted to give
the diadduct. An in vivo test showed that the unsaturated thiol es-
ters persisted on the skin for up to 3 weeks, compared to 6 days for
the non-bonding control compound.
the carbon
a
to the carbonyl group. The absence of protonation
c
to the carbonyl group, which would produce the
a
,b-unsatu-
rated-1,6-addition product, implies that there is little if any stabil-
ization due to conjugation of the carbon–carbon double bond to
the thiol ester carbonyl group, similar to the case with oxygen es-
ters [6,10]. NMR spectroscopy showed that after 39 h, the monoad-
ducts had further reacted to form a diadduct. To determine the
structures of the monoadducts, the reaction was carried out on a
180-mg scale in refluxing chloroform, with 0.7 equivalents of tri-
ethylamine, under N2 atmosphere. The resulting monoadducts
were separated by chromatography and identified by two-dimen-
sional NMR. As a comparison, the oxygen ester octadecyl sorbate
was prepared [11] and allowed to react with N-acetylcysteamine
and 0.5 equivalent of DBN in refluxing cyclohexane–chloroform
(1:1, v/v). We found that this compound also produced the 1,6-
addition product under these vigorous conditions.
Acknowledgments
We thank Dr. Ronald Nieman for assistance with the NMR spec-
troscopy, and Daniel Rose M.D. and Cathryn Rose for helpful
discussions.
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