Efficient microwave-assisted synthesis of unsymmetrical disulfides

Article abstract of DOI:10.1021/jo902695a

"Chemical equation presented" An efficient synthesis of unsymmetrical disulfides exemplified for cysteines and penicillamines is described. The use of dimethyl sulfoxide mediated oxidation accelerated by microwave irradiation afforded various unsymmetrical disulfides in one step and in high yields.

Full text of DOI:10.1021/jo902695a

pubs.acs.org/joc
Furthermore, unsymmetrical disulfides serve as protecting
Efficient Microwave-Assisted Synthesis of
Unsymmetrical Disulfides
groups, especially in peptide chemistry. For instance, the
tert-butyl disulfide is a widely applied, acid- and base-stable,
yet reduction-sensitive blocking group for the nucleophilic
and easy oxidizable cysteine thiol.8-12
€
Kristina Gormer, Herbert Waldmann, and Gemma Triola*
Although several methods are known,13 options for the
synthesis of unsymmetrical cysteine disulfides are fairly
limited,14-17 typically involving multistep sequences or
highly pH-dependent conditions.18
Department of Chemical Biology, Max Planck Institute of
Molecular Physiology, Otto Hahn Strasse 11,
44227 Dortmund, Germany, and Department of Chemistry,
TU Dortmund, Otto Hahn Strasse 6, 44227 Dortmund,
Germany
To overcome these limitations we investigated the synth-
esis of Fmoc-protected penicillamine, which can be regarded
as a sterically highly demanding derivative of cysteine and
therefore also a challenging candidate compound for method
development.
gemma.triola@mpi-dortmund.mpg.de
Received December 28, 2009
Initial investigation of Tesser’s method, which had
been successfully applied to the synthesis of cysteine
disulfides before,16 met with failure. Thus treatment
of Fmoc-penicillamine with methoxycarbonylsulfenyl
chloride and subsequent thiol-mediated heterolytic
fragmentation in the presence of base led to the forma-
tion of the activated thiocarbonate, but the subsequent
thiolysis failed, probably because of steric hindrance
(Scheme 1).
In the light of this failure, an alternative method for the
preparation of unsymmetrical disulfides that could be ap-
plied to sterically demanding thiols such as penicillamine was
sought.
An efficient synthesis of unsymmetrical disulfides exem-
plified for cysteines and penicillamines is described. The
use of dimethyl sulfoxide mediated oxidation accelerated
by microwave irradiation afforded various unsymmetri-
cal disulfides in one step and in high yields.
Oxidation of thiols by dimethyl sulfoxide (DMSO) as an
oxidizing agent has occasionally been used for the formation
of disulfide bridges in peptides, proteins,19 and small mole-
cules.13 This method is applicable over a wide pH range (pH
3-8) in contrast to air oxidation and is not prone to side
reactions with other nucleophilic amino acids such as Met,
Trp, or Tyr (common problems encountered with other
stronger oxidizing agents such as iodine).20 However, its
application to the synthesis of unsymmetrical disulfides has
proven difficult because of mixed product formation, long
reaction times, and reduced reactivity in particular with
tertiary thiols.13
Disulfide bonds are present in numerous proteins, where
they play an important role in stabilizing their tertiary
structure.1,2
Apart from this biological relevance, unsymmetrical dis-
ulfides have also emerged as invaluable tools in biochemistry
and medicinal and biological chemistry due to their selective
formation in the presence of other functional groups and
reversibility of their formation under reducing conditions.
Hence, disulfide bond formation has been used for facilitat-
ing protein refolding and for the synthesis of prodrugs with
increased hydrophobicity or cell permeability. Inside the cell
the disulfide bond is then conveniently reduced as a result of
the high glutathione levels, thus releasing the active
compound.3-7
(8) Beekman, N. J. C. M.; Schaaper, W. M. M.; Tesser, G. I.; Dalsgaard,
K.; Kamstrup, S.; Langeveld, J. P. M.; Boshuizen, R. S.; Meloen, R. H.
J. Pept. Res. 1997, 50, 357–364.
(9) Besret, S.; Ollivier, N.; Blanpain, A.; Melnyk, O. J. Pept. Sci. 2008, 14,
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(10) Gottlieb, D.; Grunwald, C.; Nowak, C.; Kuhlmann, J.; Waldmann,
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J. B.; Shinde, R.; Contag, C. H. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
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(14) Hunter, R.; Stellenboom, N.; Caira, M. R. Synlett 2008, 252–254.
(15) Szymelfejnik, M.; Demkowicz, S.; Rachon, J.; Witt, D. Synthesis
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Senter, P. D. Bioorg. Med. Chem. Lett. 2003, 13, 539–542.
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Bioorg. Med. Chem. Lett. 2002, 12, 3591–3594.
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Vasseur, J. J.; Vives, E. Bioorg. Med. Chem. Lett. 2005, 15, 5084–5087.
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(16) Rietman, B. H.; Peters, R. F. R.; Tesser, G. I. Synth. Commun. 1994,
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(17) Cros, E.; Planas, M.; Bardaji, E. Lett. Pept. Sci. 2002, 9, 1–4.
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(19) Tam, J. P.; Wu, C. R.; Liu, W.; Zhang, J. W. J. Am. Chem. Soc. 1991,
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DOI: 10.1021/jo902695a
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Published on Web 02/01/2010
J. Org. Chem. 2010, 75, 1811–1813 1811
2010 American Chemical Society

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Gormer et al.
JOCNote
SCHEME 1. Synthesis of Unsymmetrical Disulfide 1: Unsuc-
cessful Attempt via Activation as Thiocarbonate and Successful
Formation of 1 by Dimethyl Sulfoxide Mediated Oxidation
TABLE 1. Scope of Dimethyl Sulfoxide Mediated and Microwave-
Enhanced Synthesis of Unsymmetrical Disulfides of Cysteine and Peni-
cillamine
We have now found that in the formation of cysteine and
penicillamine disulfides these limitations can be overcome by
means of microwave-mediated rate acceleration. To explore
unsymmetrical disulfide formation of Fmoc-protected peni-
cillamine 1, S-trityl protected Fmoc penicillamine was S-
deprotected and treated with buffered ammonium acetate
containing acetonitrile, 20% DMSO and an excess of 2-
methylpropane-2-thiol at room temperature. Under these
conditions compound 1 was indeed formed, but conversion
did not reach more than 50% even after long reaction times
(10 days). Encouraged by these initial results, we tried to
accelerate the reaction and to enhance the product formation
by applying microwave irradiation. Therefore, an analogous
reaction mixture was irradiated for 5 min at 150 °C with 150
W using a single-mode microwave instrument. Under these
conditions the highly sterically hindered unsymmetrical
disulfide 1 was quantitatively formed and isolated in 81%
yield after column chromatography (Scheme 1) (Table 1,
entry 1).
To demonstrate the general applicability of this method, a
set of thiols was submitted to the described conditions to
afford the corresponding unsymmetrical disulfides of Fmoc-
cysteine and -penicillamine.
Microwave-assisted disulfide formation proceeded in
high yields with aromatic thiols such as phenylthiol and
Fmoc-penicillamine or Fmoc-cysteine (Table 1, entries 3
and 4, respectively) or even quantitative with benzylthiol
(Table 1, entry 5). Aliphatic thiols were also converted in
high yields (Table 1, entries 6-9). Unsymmetrical disulfides
of Fmoc-cysteine with aliphatic thiols containing addi-
tional unprotected functional groups such as amines
(Table 1, entry 7), alcohols (Table 1, entry 8) and esters
(Table 1, entry 9) were also obtained in high yield; thus
demonstrating the broad scope of the reaction. Although
initial experiments were performed with 7-10 equiv of
thiol, eventually 5 equiv proved sufficient without reducing
the yield.
Finally, an unsymmetrical disulfide composed of a cy-
steine and biotin derivative (10) was synthesized under the
described conditions and biotin-tagged Fmoc-cysteine 11
was isolated in 44% yield. Given the substantial effort
required to synthesize thiol 10, only 3 equiv was used, which
might have led to the reduced yield.
Although dimethyl sulfoxide oxidation mainly leads to
homodimer formation and subsequently leads to complex
product mixtures,13 no penicillamine homodimer formation
was observed.
In light of these promising results, we explored the scope of
this fast and high-yielding transformation for the synthesis
of unsymmetrical disulfides of Fmoc-protected cysteine and
penicillamine. The synthesis of protected Fmoc-Cys-(St-Bu)-
OH (2) also proceeded efficiently, giving the desired disulfide
2 in quantitative yield (Table 1, entry 2), proving the method
as an advantageous alternative for the synthesis of this costly
protected cysteine derivative. Since cysteines are prone to
racemization,21 we investigated if any epimerization oc-
curred during the reaction.
Therefore, the same reaction was performed with Fmoc-D-
cysteine and both disulfides (2a, 2b) were separately carboxy-
methylated. Analysis of both enantiomers by normal phase
chiral HPLC showed that the reaction proceeds without
racemization (Supporting Information).
In conclusion, we have developed an efficient method for
the preparation of unsymmetrical cysteine and penicillamine
disulfides. Application of microwave irradiation reduces the
(21) Han, Y. X.; Albericio, F.; Barany, G. J. Org. Chem. 1997, 62, 4307–
4312.
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Gormer et al.
JOCNote
reaction time dramatically to 5 min, avoids racemization, is
widely applicable to aromatic as well as aliphatic thiols, and
tolerates the presence of different unprotected functionalities
such as amines, alcohols, and esters. The experimental
procedure is operationally easy and leads to high yields in
short reaction time without using toxic reagents. The method
allows the synthesis of several differently protected cysteines
and penicillamines and can also be used for selective attach-
ment of structurally more demanding biomolecules, such as
biotin, to cysteine. In particular, the methodology gives
access to sterically highly demanding disulfides such as the
Fmoc-penicillamine-disulfide 1.
Analytical data: 1H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J =
7.5 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.57 (d, J = 6.6 Hz, 1H),
7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 4.37-4.10 (m,
4H), 3.16 (dd, J = 13.4, 3.9 Hz, 1H), 2.94 (dd, J = 13.3, 9.9 Hz,
1H), 2.70 (q, J = 7.3 Hz, 2H), 1.21 (t, J = 7.3 Hz, 3H); 13C NMR
(101 MHz, DMSO-d6) δ 172.6, 155.9, 143.8, 140.7, 127.6, 127.1,
125.3, 120.1, 65.6, 53.8, 46.6, 40.1, 31.6, 14.3; IR ν~ = 3339, 3043,
3017, 2962, 2925, 2870, 1713, 1691; LC-MS (ESI) calcd for
C20H22NO4S2: 404.09848 [M þ H]þ, found 403.75 [M þ H]þ,
t
R = 10.16 min; HR-MS m/z calcd for C20H22NO4S2 404.09848
[M þ H]þ, found 404.09832 [M þ H]þ; [R]20D = -25.22 (CHCl3,
c 5).
(L)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-((2-amin-
oethyl)disulfanyl)propanoic Acid. (7). After deprotection of
Fmoc-Cys(Trt)-OH (280 mg, 0.48 mmol) under acidic condi-
tions, the reaction was performed under the described condi-
Experimental Section
Microwave irradiation was performed in a single-mode Dis-
covery system coupled to an Explorer system (CEM). Reactions
were carried out under stirring in a 10 mL closed reaction vessel
and temperature was controlled by an IR temperature sensor.
(L)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(tert-but-
yldisulfanyl)-3-methyl Butanoic Acid. (1). Fmoc-Pen(Trt)-OH
(435 mg, 0.71 mmol) was deprotected following reported con-
ditions.22 The residue was dissolved in 2.5 mL of buffer contain-
ing 10 mM NH4OAc in acetonitrile/water (3:2). 2-Methyl-2-
propanethiol (0.8 mL, 7.13 mmol) and 0.75 mL of dimethyl
sulfoxide were added, and the reaction mixture was irradiated
using a single-mode microwave instrument (P = 150 W, t = 2
min ramp, 5 min hold, Tmax = 150 °C). After TLC had indicated
complete consumption of the starting material, the reaction
mixture was concentrated and extracted with brine and CH2Cl2.
The organic layer was dried over MgSO4, filtered, and concen-
trated. The crude residue was purified by flash chromatography
using CH2Cl2/MeOH (97:3) as eluent to yield the desired
product (266 mg, 81%). Analytical data: 1H NMR (400 MHz,
DMSO-d6) δ 12.90 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.83 (d, J =
9.2 Hz, 1H), 7.77 (d, J = 5.4 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H),
7.32 (t, J = 7.3 Hz, 2H), 4.37-4.13 (m, 4H), 1.37 (s, 3H), 1.33 (s,
3H), 1.26 (s, 9H); 13C NMR (101 MHz, DMSO-d6) δ 171.4,
156.1, 143.7, 140.6, 127.6, 127.0, 125.5, 125.4, 120.1, 66.0, 61.3,
50.5, 46.6, 46.5, 30.2, 25.9, 24.2; IR ν~ = 2962, 2921, 2896, 2860,
1714; LC-MS (ESI) calcd for C24H30NO4S2 460.16108 [M þ
H]þ, found 459.67 [M þ H]þ, tR = 10.54 min; HR-MS m/z calcd
for C24H30NO4S2 460.16108 [M þ H]þ, found 460.16073 [M þ
H]þ; [R]20D = 0.28 (CHCl3, c 5).
tions for compound
hydrochloride (463 mg, 4.08 mmol). After purification of the
1 in the presence of cystamine
reaction mixture by preparative HPLC, compound 7 was iso-
lated after lyophilization (194 mg, 80%). Analytical data: H
1
NMR (400 MHz, DMSO-d6) δ 8.01 (br, 2H), 7.89 (d, J = 7.5
Hz, 2H), 7.82 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.42
(t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 4.39-4.17 (m, 4H),
3.23-3.04 (m, 3H), 2.96-2.92 (m, 3H); 13C NMR (101 MHz,
DMSO-d6) δ 172.1, 156.1, 143.8, 140.7, 127.7, 127.1, 125.2,
120.1, 65.8, 53.0, 46.6, 39.3, 37.7, 33.9; IR ν~ = 3320, 3042, 2947,
1686, 1530; LC-MS (ESI) calcd for C20H23N2O4S2 419.10938 [M
þ H]þ, found 419.10 [M þ H]þ, tR = 7.67 min; HR-MS m/z
calcd for C20H23N2O4S2 419.10938 [M þ H]þ, found 419.10891
[M þ H]þ; [R]20D = -1.209 (MeOH, c 2).
(L)-1-(9H-Fluoren-9-yl)-3,11-dioxo-2,12-dioxa-7,8-dithia-4-aza-
tridecane-5-carboxylic Acid. (9). After deprotection of Fmoc-
Cys(Trt)-OH (510 mg, 0.87 mmol) under acidic conditions, the
reaction was performed under the described conditions for
compound 1, in the presence of methyl-3-mercaptopropionate
(0.66 mL, 6.12 mmol). After purification of the reaction mixture
by column chromatography using CH2Cl2/MeOH (97:3) as
eluent, compound 9 was isolated (346 mg, 86%). Analytical
1
data: H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 7.5 Hz,
2H), 7.72 (d, J = 7.4 Hz, 2H), 7.53 (br, 1H), 7.41 (t, J = 7.4 Hz,
2H), 7.32 (t, J = 7.4 Hz, 2H), 4.40-4.09 (m, 4H), 3.59 (s, 3H),
3.18 (m, 1H), 3.01-2.84 (m, 3H), 2.68 (t, J = 6.8 Hz, 2H);
13C NMR (101 MHz, DMSO-d6) δ 172.5, 171.6, 155.9, 143.8,
140.7, 127.6, 127.1, 125.3, 120.1, 65.7, 53.9, 51.5, 46.6, 40.7,
33.3, 32.6; IR ν~ = 3323, 2950, 2923, 2853, 1717; LC-MS (ESI)
calcd for C22H24NO6S2 462.10396 [M þ H]þ, found 461.86
[M þ H]þ, tR = 9.86 min; HR-MS m/z calcd for C22H24NO6S2
462.10396 [M þ H]þ, found 462.10362 [M þ H]þ;[R]20D =-17.28
(CHCl3, c 5).
(L)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(ethyldis-
ulfanyl)propanoic Acid. (6). After deprotection of Fmoc-Cys-
(Trt)-OH (510 mg, 0.87 mmol) under acidic conditions, the
reaction was performed under the described conditions for
compound 1 in the presence ethyl mercaptan (0.45 mL, 6.12
mmol). After purification by column chromatography using
CH2Cl2/MeOH (97:3), compound 6 was isolated (325 mg, 92%).
Supporting Information Available: Detailed experimental
and NMR data of all products and analytic chiral HPLC data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
€
(22) Lumbierres, M.; Palomo, J.; Kragol, G.; Roehrs, S.; Muller, O.;
Waldmann, H. Chem.;Eur. J. 2005, 11, 7405–7415.
J. Org. Chem. Vol. 75, No. 5, 2010 1813