Efficient and selective syntheses of S -Acyl and N -Acyl glutathiones

Article abstract of DOI:10.1055/s-0029-1219837

Selective syntheses of S-acyl glutathiones are achieved in 79-98% yields using 1-acyl-1H-benzotriazoles in the presence of potassium bicarbonate in aqueous methanol at 20C. N-Acylation of S-(p-nitrobenzoyl) glutathione with 1-acyl-1H-benzotriazoles follow

Full text of DOI:10.1055/s-0029-1219837

LETTER
1337
Efficient and Selective Syntheses of S-Acyl and N-Acyl Glutathiones
S
ynthese
s
of
S
-
Acy
l
l
and
N
a
-Acyl
G
lu
n
tathiones R. Katritzky,*a Nader E. Abo-Dya,a,b Srinivasa R. Tala,a Ebrahim H. Ghazvini-Zadeh,a Kiran Bajaj,a
Said A. El-Fekyb
a
Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
Fax +1(352)3929199; E-mail: katritzky@chem.ufl.edu
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
b
Received 8 February 2010
formate–Et3N9 (b) reactions of GSH with acyl chlorides or
anhydrides in trifluoroacetic acid,10 and (c) use of en-
zyme.11
Abstract: Selective syntheses of S-acyl glutathiones are achieved
in 79–98% yields using 1-acyl-1H-benzotriazoles in the presence of
potassium bicarbonate in aqueous methanol at 20 °C. N-Acylation
of S-(p-nitrobenzoyl) glutathione with 1-acyl-1H-benzotriazoles
followed by deprotection of the p-nitrobenzoyl groups under mild
conditions gave 63–78% yields of N-acyl glutathiones. These meth-
odologies should be useful for the S-acylation and N-acylation of
peptides and glycopeptides.
Published methods to synthesize N-acyl glutathiones have
utilized (a) potassium phosphate buffer, which provided a
mixture of N- and S-acylated products;12 (b) an activated
ester,13 but Bernardi et al. observed only S-acylation of
GSH using these conditions;2 (c) N-acylation of GSSG
with activated ester followed by reduction,2,8,14 but this
method fails when other susceptible groups like azide
present in the molecule during the reduction;8 (d) use of
trityl group to protect thiol group of GSH then N-acyla-
tion followed by trityl group deprotection,8 but overall
yields are very low. Some of these methods also suffer
from long reactions times (48 h).2,8,14
Key words: amino acids, acylation, glutathione, peptides, drugs
Glutathione (g-L-glutamyl-L-cysteinylglycine, GSH, 1), a
tripeptide derived from glycine, cysteine, and glutamic
acid, is critical to a variety of cellular functions.1 GSH,
which possesses a g-glutamyl linkage and a sulfhydryl
group as two characteristic structural features, plays an
important role in the human body antioxidant defense sys-
tem. In its reduced form, GSH (1) is the most abundant
nonprotein thiol in eukaryotic cells, and is involved in var-
ious physiological and metabolic functions of all mamma-
lian cells.2 The N- and S-blocked GSH derivatives inhibit
mammalian glyoxalase I from human erythrocytes.3
Recently, we have reported selective syntheses of S-acyl-
and N-acylcysteines (Scheme 1).15 However, our attempts
to synthesize S-acyl glutathiones according to the above
method for S-acylation of cysteine15 failed to proceed af-
ter several days. Furthermore, treatment of GSH with N-
acylbenzotriazoles in the presence of triethylamine did
not yield N-acyl glutathiones, according to the method
used for N-acylcysteines.15 Use of excess of triethylamine
gave low yields of S,N-diacylated product. Hence, the
methods successful for the selective syntheses of S-acyl-
and N-acylcysteines can not be used for selective synthe-
sis of S-acyl and N-acyl glutathiones.
S-Acetyl glutathione can act as selective apoptosis-induc-
ing agent in cancer therapy4 and as an antiviral agent
against herpes simplex virus-1 infection.5 S-Acetyl glu-
tathione is also a potent antioxidant, providing a rationale
for its combination with antiretroviral drugs.6
The hepatotoxic nonsteroidal anti-inflammatory drug
Zomepirac, containing a carboxylic acid group, under-
goes metabolic activation in vivo in rats, and in vitro in rat
hepatocytes, to form unstable acyl-linked metabolites that
react with GSH by transacylation forming the S-acyl glu-
tathione conjugate of the drug. Therefore, the detection of
S-acyl-linked GSH adducts of carboxylic acid drugs can
be used as an indirect marker of drug bioactivation to
chemically reactive transacylating metabolites.7
O
R
MeCN–H2O
r.t., 12 h
S
O
OH
N
SH
O
H2N
R
N
N
O
OH
+
H2N
SH
O
Et3N
OH
N-(4-Benzoylbenzoyl) glutathione is a photoaffinity label
of glutathione S-transferases.2 N-Acyl glutathiones bear-
ing an aryl azide moiety provide photoactivatable ana-
logues of GSH.8
MeCN–H2O
r.t., 2 h
R
N
H
O
Scheme 1 Selective synthesis of S-acyl and N-acylcysteines
Several literature reports of the selective S-acylation of
GSH utilize (a) coupling reagents such as ethyl chloro-
We now report facile, mild, and efficient syntheses of S-
acyl and N-acyl glutathiones, which should have general
applicability for S-acylation and N-acylation of biologi-
cally important larger peptides and glycopeptides. 1-
Acyl-1H-benzotriazoles are stable crystalline compounds
and advantageous for N-, O-, C-, and S-acylation.16a–j
SYNLETT 2010, No. 9, pp 1337–1340
x
x.
x
x.
2
0
1
0
Advanced online publication: 16.04.2010
DOI: 10.1055/s-0029-1219837; Art ID: S00510ST
© Georg Thieme Verlag Stuttgart · New York

1338
A. R. Katritzky et al.
LETTER
O
O
O
HO
HO
N
HN
O
N
N
O
HN
O
O
O
O
KHCO3
S
O
HO
N
H
SH
MeOH–H2O
(2:1)
HO
N
H
R
NH2
R
NH2
r.t., 0.5 h
2a–g
3a–g
1
Scheme 2 Synthesis of S-acyl glutathiones 3a–g
Table 1 Preparation of S-Acyl Glutathiones 3a–g
Table 2 Preparation of S,N-Diacyl Glutathiones 5a–e and N-Acyl
Glutathiones 6a–e
RCOBt 2
Product
3a
Yield (%) Mp (°C)
RCOBt 2
Yield (%) Mp
Yield of 6 Mp
Fmoc-Val-Bt 2a
naproxen-Bt 2b
4-MeOC6H4COBt 2c
4-MeC6H4COBt 2d
2-naphthylCOBt 2e
4-O2NC6H4COBt 2f
Cbz-Ala-Bt 2g
79
89
95
97
98
91
94
165–167
of 5
(°C)
(%)
(°C)
3b
204–205
222–224
228–230
233–235
203–205
184–185
Cbz-Ala-Bt 2g
Cbz-Phe-Bt 2h
Cbz-Leu-Bt 2i
5a, 84
5b, 82
5c, 89
5d, 80
5e, 84
132–134 6a, 65
162–163 6b, 65
150–152 6c, 78
140–141 6d, 80
183–184 6e, 63
182–183
203–204
198–200
210–212
174–175
3c
3d
3e
4-O2NC6H4COBt 2f
Fmoc-Val-Bt 2a
3f
3g
group. 4-Nitrobenzoyl is a known protecting group for
thiols.17 S-(4-Nitrobenzoyl) glutathione (3f) was synthe-
sized by S-acylation of GSH (1, Scheme 2), 3f was used
an intermediate to make N-acyl glutathiones 6a–e. S-(4-
Nitrobenzoyl) glutathione (3f) was acylated with one
equivalent of each of various 1-acyl-1H-benzotriazoles
2a,f–i in the presence of one equivalent of triethylamine
in aqueous acetonitrile for 1.5 hours to afford the corre-
sponding S,N doubly acylated GSH derivatives 5a–e, in
80–89% yields. Mild deprotection of the 4-nitrobenzoyl
groups from these double acylated glutathione derivatives
5a–e using pyrrolidine in dry THF–methanol for four
hours at room temperature, gave the corresponding N-acyl
glutathiones 6a–e in 63–80% yields (Scheme 4, Table 2).
Treatment of GSH (1) with one equivalent of 1-acyl-1H-
benzotriazoles 2a–g in the presence of one equivalent of
potassium bicarbonate at 20 °C in aqueous methanol for
15 minutes gave exclusively S-acyl glutathiones 3a–g in
79–98% yields as the only products isolated (Scheme 2,
Table 1). Of the two carboxylic acid groups present in the
GSH, one forms a zwitterion with the amino group, but
the other is free; we used potassium bicarbonate to neu-
tralize it and then selectively S-acylated GSH.
Reaction of GSH (1) with two equivalents of 1-acyl-1H-
benzotriazole 2c in the presence of triethylamine at 20 °C
in 1.5 hours gave the S,N-diacylation product 4 in 75%
yield (Scheme 3).
In conclusion, we have developed facile and efficient
methods for selective syntheses for both S-acyl
glutathiones18 and N-acyl glutathiones19,20 in good yields
under mild reaction conditions using 1-acyl-1H-benzotri-
azoles. In comparison with literature methods, our meth-
ods comprise: (1) mild and simple reaction and workup
conditions, (2) higher yields for the most of our products
(65–98%), and (3) reduced reaction times (15 min to 6 h).
These methodologies should have general applicability
for the S-acylation and N-acylation of biologically active
larger peptides and glycopeptides.
In GSH, an anionic thiol group is more nucleophilic than
an amino group, thus for selective N-acylation of GSH,
we first protected thiol group of 1 with a 4-nitrobenzolyl
O
O
HN
O
N
O
O
N
N
S
O
O
O
N
H
Et3N
NH
O
MeCN–H2O (3:1)
1 +
r.t., 1.5 h
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
OMe
OMe
OMe
2c
4, 75%
Acknowledgment
Scheme 3 Synthesis of bisacyl glutathione 4
We thank Dr. C. D. Hall for helpful discussions.
Synlett 2010, No. 9, 1337–1340 © Thieme Stuttgart · New York

LETTER
Syntheses of S-Acyl and N-Acyl Glutathiones
1339
O
O
O
O
N
HO
HO
R
N
N
HN
O
HN
O
O
O
O
S
O
S
O
HO
N
H
HO
N
H
HN
O
2a,f–i
NH2
Et3N, aq MeCN
1.5 h, r.t.
R
NO2
NO2
O
5a–e
3f
HO
HN
O
O
O
SH
pyrrolidine
HO
N
H
THF-MeOH (3:1)
4 h, r.t.
HN
O
R
6a–e
Scheme 4 Synthesis of N-acyl glutathiones 6a–e
2000, 65, 8210. (f) Katritzky, A. R.; Wang, M.; Yang, H.;
Zhang, S.; Akhmedov, N. G. ARKIVOC 2002, (viii), 134.
(g) Katritzky, A. R.; Shestopalov, A. A.; Suzuki, K.
ARKIVOC 2005, (vii), 36. (h) Katritzky, A. R.; Tala, S. R.;
Singh, S. K. Synthesis 2006, 3231. (i) Katritzky, A. R.;
Chen, Q.-Y.; Tala, S. R. Org. Biomol. Chem. 2008, 6, 2400.
(j) Katritzky, A. R.; Angrish, P.; Todadze, E. Synlett 2009,
2392.
References and Notes
(1) Pompella, A.; Visvikis, A.; Paolicchi, A.; Tata, V. D.;
Casini, A. F. Biochem. Pharmacol. 2003, 66, 1499.
(2) Bernardi, D.; Battaglia, E.; Krisch, G. Bioorg. Med. Chem.
Lett. 2006, 16, 1601.
(3) Al-Timari, A.; Douglas, K. T. Biochim. Biophys. Acta 1986,
870, 160.
(4) Donnerstag, B.; Ohlenschläger, G.; Cinatl, J.; Amrani, M.;
Hofmann, D.; Flindt, S.; Treusch, G.; Träger, L. Cancer Lett.
1996, 110, 63.
(5) Vogel, J.-U.; Cinatl, J.; Dauletbaev, N.; Buxbaum, S.;
Treusch, G.; Cinatl, J. Jr.; Gerein, V.; Doerr, H. W. Med.
Microbiol. Immunol. 2005, 194, 55.
(17) Ané, A.; Josse, S.; Naud, S.; Lacône, V.; Vidot, S.; Fournial,
A.; Kar, A.; Pipelier, M.; Dubreuil, D. Tetrahedron 2006,
62, 4784.
(18) General Procedure for the Preparation of Compounds
3a–e
To a solution of GSH (1 equiv) in H2O (3 mL), a solution of
1-acyl-1H-benzotriazole (1 equiv) in MeOH (8 mL) was
added, and the mixture was stirred for 5 min at r.t. An aq
KHCO3 solution (2 equiv) in H2O (1 mL) was added
dropwise to the reaction mixture at a rate of 0.2 mL/min. The
reaction progress was monitored by TLC (disappearance of
1-acyl-1H-benzotriazoles, which have Rf values in the range
from 0.6–0.7 using solvent mixture of hexanes–EtOAc (2:1)
as eluent; together with the appearance of benzotriazole at Rf
value 0.45), which indicated the completion of the reaction
within 10–15 min. MeOH was evaporated under reduced
pressure and dilute HCl (6 M)/Na2HPO4 (1 N) was added
until pH of the mixture was adjusted to 5. The precipitate
formed was collected on a Buchner funnel, was washed with
H2O, EtOAc, and hexanes to afford the desired compound.
(19) General Procedure for the Preparation of Compounds
5a–e
(6) Fraternale, A.; Paoletti, M. F.; Casabianca, A.; Orlandi, C.;
Schiavano, G. F.; Chiarantini, L.; Clayette, P.; Oiry, J.;
Vogel, J.-U.; Cinatl, J. Jr.; Magnani, M. Antiviral Res. 2008,
77, 120.
(7) Grillo, M. P.; Hua, F. Drug Metab. Dispos. 2003, 31, 1429.
(8) Bernardi, D.; Ba, L. A.; Kirsch, G. Synthesis 2007, 140.
(9) Stadtman, E. R. Methods Enzymol. 1957, 3, 931.
(10) Galzigna, L. WO 9200320, 1992.
(11) Clelland, J. D.; Thornalley, P. J. J. Chem. Soc., Perkin Trans.
1 1991, 3009.
(12) Chen, H.-J. C.; Hsieh, C.-J.; Shen, L.-C.; Chang, C.-M.
Biochemistry 2007, 46, 3952.
(13) Karwatsky, J.; Daoud, R.; Cai, J.; Gros, P.; Georges, E.
Biochemistry 2003, 42, 3286.
(14) Kiwada, H.; Akimoto, M.; Araki, M.; Tsuji, M.; Kato, Y. JP
63002922, 1988.
(15) Katritzky, A. R.; Tala, S. R.; Abo-Dya, N.; Gyanda, K.; El-
Gendy, B. E.; Abdel-Sammi, Z. K.; Steel, P. J. J. Org. Chem.
2009, 74, 7165.
(16) (a) Katritzky, A. R.; Suzuki, K.; Wang, Z. Synlett 2005,
1656. (b) Katritzky, A. R.; Angrish, P.; Suzuki, K. Synthesis
2006, 411. (c) Katritzky, A. R.; Angrish, P.; Hür, D.; Suzuki,
K. Synthesis 2005, 397. (d) Katritzky, A. R.; Suzuki, K.;
Singh, S. K.; He, H.-Y. J. Org. Chem. 2003, 68, 5720.
(e) Katritzky, A. R.; He, H.-Y.; Suzuki, K. J. Org. Chem.
A solution of 1-acyl-1H-benzotriazole (1 equiv) in MeCN
(5 mL) and Et3N (2 equiv) was added to a solution of 3f
(1 equiv) in MeCN–H2O (1 mL – 2 drops). The reaction
mixture was stirred at r.t. for 1.5 h. After completion of the
reaction (by TLC as described above for compounds 3a–e),
the pH was adjusted to 3 using HCl (6 M). MeCN was
evaporated under reduced pressure, and the resulting crude
was triturated with Et2O (5 mL) until a friable solid was
formed. The solid was filtered and washed with additional
Synlett 2010, No. 9, 1337–1340 © Thieme Stuttgart · New York

1340
A. R. Katritzky et al.
LETTER
was stirred at 20 °C for 4 h. The solvents were then
evaporated, and the resulting crude solid was dissolved in
H2O (2 mL) and MeOH (0.2 mL). The resulting solution
was acidified to pH 1 using HCl (6 M) and stirred for 30 min.
The precipitate was filtered and washed with Et2O to afford
the desired product.
amount of Et2O (2 mL) and dried to give the desired
compound.
(20) General Procedure for the Preparation of Compounds
6a–e
Pyrrolidine (3 equiv) was added to a stirred solution of 5a–e
(1 equiv) in anhyd THF and MeOH (3:1, 4 mL). The mixture
Synlett 2010, No. 9, 1337–1340 © Thieme Stuttgart · New York

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