Synthesis of S-glycosyl amino acids and S-glycopeptides via photoinduced click thiol-ene coupling

Article abstract of DOI:10.1016/j.tetlet.2010.11.097

We report on the photoinduced addition of glycosyl thiols to alkenyl glycines (thiol-ene coupling) to give S-glycosyl amino acids in high yields (53-90%). One of the amino acids thus prepared was used in the construction of a tripeptide via solution phase

Full text of DOI:10.1016/j.tetlet.2010.11.097

Tetrahedron Letters 52 (2011) 444–447
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of S-glycosyl amino acids and S-glycopeptides via photoinduced
click thiol–ene coupling
⇑
Michele Fiore, Mauro Lo Conte, Salvatore Pacifico, Alberto Marra, Alessandro Dondoni
Dipartimento di Chimica, Laboratorio di Chimica Organica, Università di Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on the photoinduced addition of glycosyl thiols to alkenyl glycines (thiol–ene coupling) to give
S-glycosyl amino acids in high yields (53–90%). One of the amino acids thus prepared was used in the
construction of a tripeptide via solution phase chemistry. Moreover, a one-pot two-step approach to
an S-glycopeptide was developed using glutathione (GSH) as model starting material. This approach
involved GSH S-alkenylation followed by photoinduced hydrothiolation by a glycosyl thiol.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 21 October 2010
Revised 11 November 2010
Accepted 17 November 2010
Available online 23 November 2010
Keywords:
Carbohydrates
Glycosides
Peptides
Radical reactions
Thiols
While native O- and N-glycosidic bonds of glycopeptides are
prone to hydrolytic cleavage by O- and N-glycosidases, synthetic
C- and S-analogs are expected to be stable toward such enzymatic
degradation. Therefore, much effort has been devoted in the last
decades to synthesizing C- and S-glycosyl amino acids and their
assembly in glycopeptides.1,2 Glycopeptides with these non-native
linkages can be used as probes for biochemical studies and leads in
drug discovery, such as, for example, vaccines. It has to be noticed,
however, that the replacement of the oxygen atom of an O-linked
glycopeptide with a sulfur atom is the most straightforward way
for isosteric mimicry because the differences existing between
the C–S and C–O bond lengths as well as the C–S–C and C–O–C
bond angles result in small variation between the positions of car-
bon atoms in C–O and C–S glycosidic linkages.3 Nevertheless, the
longer C–S bond and the weaker stereoelectronic effect in S-linked
derivatives allow greater flexibility. Among the various methods
for S-glycosyl amino acid and peptide synthesis,1c the coupling of
an activated sugar derivative with a protected cysteine residue is
most widely exploited.4 Cyclic S-glycopeptide, however, have been
preferentially prepared by peptide synthesis with S-glycosylated
amino acid building blocks.5 Substantial advancements have been
made in very recent years. A method reported by Davis and
coworkers involves coupling of a carbohydrate thiosulfonate with
a cysteine residue to give a disulfide glycosyl amino acid that in
turn is desulfurized by a phosphine.6 This approach was applied
to a protein containing a single cysteine residue to give the thioe-
ther-linked glycoprotein. Reported from Davis laboratory is also
another method that appeared in a very recent publication7 when
our work described below was in progress.8 It involves thermally
or photochemically induced free-radical addition of glycosyl thiols
to homoallylglycine in a protected and unprotected form to give al-
kyl-tethered S-glycosyl glycines. Also this work was propaedeutic
to the synthesis of S-linked protein glycoconjugates.
The recent disclosures from the Davis group prompted us to re-
port here on a similar approach to S-glycosyl amino acids, that is,
based on the photoinduced hydrothiolation of alkenyl glycines by
glycosyl thiols (Scheme 1). This photoreaction, currently referred
to as thiol–ene coupling (TEC),9 is known to proceed by a radical
mechanism and, therefore, allows to add thiols to non-activated al-
kenes in an anti-Markownikov fashion.10 Main features of photoin-
duced TEC include the occurrence by irradiation at close to visible
light under aerobic conditions at room temperature in aqueous sol-
vents and orthogonality to a wide range of functional groups. Ow-
ing to its efficiency and specificity, TEC has been recently exploited
by us for the glycosylation of natural cysteine containing peptide
such as glutathione (GSH) and the protein bovine serum albumin
(BSA).11
NH2
CO2H
NH2
CO2H
O
O
hν
SH
S
+
n
R
R
n
R = H or sugar
⇑
Corresponding author. Tel.: +39 0532 455176; fax: +39 0532 240709.
E-mail address: adn@unife.it (A. Dondoni).
Scheme 1. General equation for S-glycosyl amino acid synthesis via photoinduced
TEC.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.11.097

M. Fiore et al. / Tetrahedron Letters 52 (2011) 444–447
445
OAc
OAc
our delight all reactions went to completion in 1 h as shown by the
total disappearance of the alkene proton signal at d = 5.7 ppm in
the 1H NMR spectrum of the crude reaction mixture. Yields of iso-
lated S-glycosyl amino acids 6–11 that are presented in Table 1
demonstrate the high efficiency of TEC in each reaction. Notewor-
thy is the fact that no epimerization occurred in all cases being the
sugar disulfide the only observed side-product that arised from the
thiyl radical homocoupling. Thus it appeared quite reasonable to
conclude that the configuration of all asymmetric carbons of both
reagents was preserved under the photochemical conditions and
consequently assign the stereochemistry of isolated products as
identical to that of the reactants.
OAc
AcO
OAc
O
O
O
AcO
AcO
AcO
O
SH
SH
O
AcO
SH
AcO
AcO
AcO
AcHN
AcO
AcO
1
2
3
NHFmoc
CO2t-Bu
4
NHCbz
CO2Me
5
Figure 1.
To perform the reaction shown in Scheme 1, we selected the
model glycosyl thiols 1–3 and allyl and vinyl glycines 4 and 5,
respectively, as substrates (Fig. 1). Carbohydrates 1–3 and amino
acids 4 and 5 were known compounds and, therefore, were pre-
pared by literature methods (see Supplementary data). O-Acetyl
protecting groups in the sugar thiols were used because of their
compatibility with the planned photoreaction.12 Moreover orthog-
onal protective groups in the glycinyl moiety were employed in or-
der to obtain target S-glycosyl amino acids suitable for peptide
synthesis.
Taking advantage of the optimized conditions established in our
recent works in which TEC was conveniently exploited for S-disac-
charide synthesis,12 peptide and protein glycosylation,11 and calix-
arene glycoclustering,13 we performed the photoreactions of a
slight excess of thiols 1–3 (1.2 equiv) with alkenes 4 and 5 by irra-
diation at kmax 365 nm in the presence of 2,2-dimethoxy-2-pheny-
lacetophenone (DPAP) as the photoinitiator (0.1 equiv).
In order to demonstrate the viability of the approach to S-glyco-
peptides by the use of the S-glycosyl amino acids thus prepared,
the synthesis of a tripeptide was performed by solution phase
chemistry (Scheme 2). To this end, the amino ester 6 was trans-
formed into the free carboxylic acid by selective removal of tert-
butyl group and the crude product was coupled with phenyl alan-
inate 12 under basic conditions (i-Pr2EtN) and in the presence of
(benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluoro-
phosphate (PyBOP) as a condensing agent. The N-Fmoc protected
dipeptide 13 thus obtained was liberated of the Fmoc protective
group by basic treatment and the free amine was condensed with
N-Boc alanine 14 under basic conditions in the presence of PyBOP.
The pure final tripeptide 15 featuring a pendant glucoside residue
linked through a robust thioether linkage was isolated by chroma-
tography in 75% yield. This compound turned out to be a single dia-
stereomer by NMR analysis, thus indicating that all stereocenters
of the reactants employed remained unaltered throughout the
whole synthetic sequence.
Reactions were carried out in dichloromethane as a solvent at
room temperature in glass vials using a hand-held UVA generating
apparatus. No care was taken to exclude air and moisture. Much to
In a second instance we set out to develop a TEC-based post-
synthetic approach to S-glycopeptides. To this end we envisaged
introducing a chemical handle bearing an ene tag on a cysteine free
sulfhydryl group and then performing the photoinduced TEC with
Table 1
Photoinduced reactionsa of sugar thiols 1–3 with alkenyl glycines 4 and 5
Thiol
Alkene
Product (yield)b
OAc
OAc
NH3 Cl
CO2Et
O
AcO
AcO
NHFmoc
CO2t-Bu
O
S
Ph
AcO
S
AcO
AcO
1
4
AcO
CF3CO2H
12
6
H
N
PyBOP, i-Pr2EtN
CH2Cl2, r.t., 21 h
CH2Cl2, r.t., 3 h
6
(73%)
CO2Et
Ph
FmocHN
OAc
O
NHFmoc
CO2t-Bu
O
82%
AcO
AcO
S
13
2
3
1
2
4
4
5
5
AcHN
OAc
7
(53%)
NHBoc
O
AcO
OAc
AcO
AcO
OAc
S
AcO
CO2H
NHFmoc
CO2t-Bu
O
O
AcO
O
piperidine
O
14
S
AcO
AcO
H
AcO
PyBOP, i-Pr2EtN
CH2Cl2, r.t., 20 h
75%
DMF, r.t., 2 h
BocHN
CO2Et
Ph
N
N
H
8
(80%)
O
OAc
O
15
AcO
AcO
S
CO2Me
NHCbz
AcO
Scheme 2.
9
(90%)
OAc
O
AcO
AcO
S
CO2Me
NHCbz
NHBoc
CO2Et
NHBoc
HS
Br
AcHN
S
CO2Et
10 (89%)
Et3N, r.t., 18 h
87%
17
16
OAc
AcO
AcO
OAc
O
O
O
CO2Me
NHCbz
OAc
NHBoc
CO2Et
S
3
5
AcO
AcO
O
S
AcO
AcO
AcO
1, DPAP
CH2Cl2, hν, 15 min
S
11 (89%)
AcO
a
93%
18
All reactions were carried out with 1.2 equiv of thiol and 0.1 equiv of DPAP in
CH2Cl2 under irradiation at 365 nm for 30 min.
b
The products were isolated by column chromatography on silica gel.
Scheme 3.

446
M. Fiore et al. / Tetrahedron Letters 52 (2011) 444–447
Figure 2. 1H NMR spectra of the reaction mixture between 20 and 21 (Scheme 4) monitored at different times.
quantitative yield. Then, this crude product was coupled with ex-
SH
cess of unprotected sugar thiol 21 (1.2 equiv) under the above pho-
toinduced conditions with the only change that a mixture of
MeOH–H2O was employed as the solvent.
O
H
Br
CO2H
N
HO2C
N
H
NH3, MeOH
r.t., 3 h
NH2
O
The NMR spectrum of the reaction mixture recorded after
20 min irradiation revealed the presence of unreacted alkene 20
while this was completely consumed after 45 min (Fig. 2).
Thus, to our satisfaction, the target S-glycopeptide 22 was iso-
lated by chromatography in 97% yield. It is noteworthy that the
coupling of 20 with 21 gave 22 in similar yield upon exposure
(8 h) to unfocused sunlight. This and our earlier finding12 on the
use of sunlight for performing TEC reactions, demonstrate the abil-
ity of this click process to proceed under very mild and benign con-
ditions with the need of minor amounts of energy.
19
OH
O
HO
HO
SH
S
OH
O
H
21
CO2H
N
HO2C
N
H
DPAP, MeOH-H2O
NH2
OH
O
hν, 45 min
20
97%
O
HO
HO
S
In summary, two routes have been paved to S-glycopeptides.
One route consists of S-glycosyl amino acid synthesis from photo-
induced addition of sugar thiols to alkenyl glycine followed by
incorporation of the amino acid into a peptide. The second route,
that is, specific for a cysteine containing peptide such as glutathi-
one, involves peptide S-homoallylation followed by TEC with sugar
thiol. Thus, extension of this process to larger peptides and pro-
teins now becomes of interest. Finally, the observation that TEC
can proceed under ambient processing conditions and upon sun-
light irradiation, opens up the way for future applications particu-
larly for bioconjugation.
OH
S
O
H
N
CO2H
HO2C
N
H
NH2
O
22
Scheme 4.
a glycosyl thiol. In a model study serving to validate this idea, we
introduced a homoallyl chain in -cysteine 16 by reaction with 4-
L
bromo-1-butene in the presence of Et3N. Then, the S-butenyl cys-
teine 17 thus formed was submitted to the photoinduced reaction
(kmax 365 nm) with glycosyl thiol 1 in the presence of DPAP
(Scheme 3). The TEC reaction worked beautifully as it afforded in
only 15 min irradiation the S-thioglycosylbutyl cysteine 18 in
excellent isolated yield (93%). It has to be noted that the choice
of the butenyl as a chemical handle was made after having carried
out some experimentation with the use of the allyl group. In fact
the photoinduced reaction of 1 with S-allyl cysteine gave the
expected thioether in very low yield together with by-products
derived from coupling of various thiyl radicals (see Fig. S1 in
Supplementary data).
Acknowledgments
We thank Dr. Stefano Gotta (MET Profiling Unit, Siena Biotech
SpA, Italy) for the HRMS analyses.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.11.097.
Encouraged by this result we extended the approach to the S-
glycosylation of glutathione 19 (Scheme 4). The selective S-homo-
allylation of 19 by butenyl bromide in NH3/MeOH proceeded read-
ily and specifically due to the superior nucleophilicity of the free
sulfhydryl group with respect to terminal amino group of glutamic
acid.14 Thus, the S-homoallyl glutathione 20 formed in almost
References and notes
1. (a) Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5, 1384–1390; (b)
Dondoni, A.; Marra, A. Chem. Rev. 2000, 100, 4395–4421; (c) Pachamuthu, K.;
Schmidt, R. R. Chem. Rev. 2006, 106, 160–187.
2. For a list of references on recent syntheses of C-glycosyl amino acids, see:
Barghash, R. F.; Massi, A.; Dondoni, A. Org. Biomol. Chem. 2009, 7, 3310–3319.

M. Fiore et al. / Tetrahedron Letters 52 (2011) 444–447
447
3. Rich, J. R.; Szpacenko, A.; Palcic, M. M.; Bundle, D. R. Angew. Chem., Int. Ed. 2004,
43, 613–615.
8. For a preliminary report of these results see: Dondoni, A. Polym. Prepr. 2010, 51,
709–710.
4. (a) Käsbeck, L.; Kessler, H. Liebigs Ann. 1997, 165–167; (b) Elofsson, M.; Walse,
B.; Kihlberg, J. Tetrahedron Lett. 1991, 32, 7613–7616.
5. Gerz, M.; Matter, H.; Kessler, H. Angew. Chem., Int. Ed. Engl. 1993, 32,
269–271.
6. Bernardes, G. J. L.; Grayson, E. J.; Thompson, S.; Chalker, J. M.; Errey, J. C.; El
Oualid, F.; Claridge, T. D. W.; Davis, B. G. Angew. Chem., Int. Ed. 2008, 47, 2244–
2247.
9. For a brief commentary on the potential of this reaction in synthesis, see:
Dondoni, A. Angew. Chem., Int. Ed. 2008, 47, 8995–8997.
10. Griesbaum, K. Angew. Chem., Int. Ed. Engl. 1970, 9, 273–287.
11. Dondoni, A.; Massi, A.; Nanni, P.; Roda, A. Chem. Eur. J. 2009, 15, 11444–11449.
12. Fiore, M.; Marra, A.; Dondoni, A. J. Org. Chem. 2009, 74, 4422–4425.
13. Fiore, M.; Chambery, A.; Marra, A.; Dondoni, A. Org. Biomol. Chem. 2009, 7,
3910–3913.
7. Floyd, N.; Vijayakrishnan, B.; Koeppe, J. R.; Davis, B. G. Angew. Chem., Int. Ed.
2009, 48, 7798–7802.
14. Lo Conte, M.; Pacifico, S.; Chambery, A.; Marra, A.; Dondoni, A. J. Org. Chem.
2010, 75, 4644–4647.