Synthesis of α-S-linked glycopeptides in water containing solution

Article abstract of DOI:10.1016/S0040-4039(03)01465-5

α-Thiols of N-acetylglucosamine and of N-acetylgalactosamine react with β-bromoalanine containing peptides at pH 8.5 under phase transfer conditions or, alternatively, in aqueous DMF, cleanly to α-S-linked glycopeptides. Thus, mimetics of important O-glycopeptides can be readily prepared.

Full text of DOI:10.1016/S0040-4039(03)01465-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 6063–6067
Synthesis of a-S-linked glycopeptides in water
containing solution
Xiangming Zhu and Richard R. Schmidt*
Fachbereich Chemie, Universita¨t Konstanz, Fach M 725, D-78457 Konstanz, Germany
Received 23 May 2003; revised 10 June 2003; accepted 12 June 2003
Abstract—a-Thiols of N-acetylglucosamine and of N-acetylgalactosamine react with b-bromoalanine containing peptides at pH
8.5 under phase transfer conditions or, alternatively, in aqueous DMF, cleanly to a-S-linked glycopeptides. Thus, mimetics of
important O-glycopeptides can be readily prepared.
© 2003 Elsevier Ltd. All rights reserved.
It has been well established that glycopeptides and
glycoproteins play a central role in various biological
functions.1 However, glycopeptides and particularly gly-
coproteins are often difficult to obtain in homogeneous
form from biological sources because they appear as a
population of different glycoforms. The synthesis of pure
native glycopeptides and finally glycoproteins is therefore
necessary in order to further explore the function of these
compounds in biological systems.2 On the other hand, the
field of glycopeptide mimetics3 is driven by the urgency
of certain biological problems, which overrides the
preference for a native structure. For example, a native
O-linked glycopeptide may lack the stability or
bioavailability required for a specific therapeutic applica-
tion or a basic scientific investigation. As a result, more
and more glycopeptide mimetics have been designed and
synthesized as potential therapeutic agents, including
S-linked glycopeptides.4,5 Replacement of the anomeric
oxygen of O-glycopeptides by sulfur (Fig. 1), thus leading
to S-linked glycopeptides is a modification tolerated by
most biological systems which, in addition, increases the
stability of the peptide-sugar linkage against chemical
degradation as well as against enzymatic cleavage.6 Thus,
particularly valuable compounds for biological studies
are provided.
The synthesis of several S-linked glycopeptides has been
recently reviewed.2a Most of these compounds were
obtained by the co-translational approach, wherein the
S-linked glycosyl amino acid was pre-prepared and then
incorporated during the course of peptide assembly. Only
one paper was devoted to the synthesis of S-linked
glycopeptides in a post-translational strategy: 1-Thio
sugars were attached to dehydroalanine containing pep-
tides by Michael addition; however, no diastereoselectiv-
ity was observed in this addition.4b In view of the
advantage of the post-translational strategy7 in synthe-
sizing peptide conjugates, we described very recently a
convenient synthesis of S-linked glycopeptides from
glycosyl bromides and cysteine or homocysteine contain-
ing peptides in aqueous solution.5 This procedure is a
significant advance in the convergent assembly of S-
linked glycopeptides because it demonstrates that under
mild basic, aqueous conditions peptides can be S-glyco-
sylated effectively. By this procedure, as mimetics of
native b-O- and b-N-linked glycopeptides, b-S-linked
glycopeptides were obtained efficiently, as shown in
reaction 1 in Figure 2.
Figure 1. Comparison of a native O-linked glycopeptide (I) and
S-linked glycopeptide (II).
Amongst O-linked glycoproteins a-linkage between a
GlcNAc and a Ser or Thr residue, respectively, is very
common.8 In addition, the Tn antigen,9 featuring Gal-
NAc a-O-linkage to a Ser or a Thr residue, is a
* Corresponding author. E-mail: richard.schmidt@uni-konstanz.de
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01465-5

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X. Zhu, R. R. Schmidt / Tetrahedron Letters 44 (2003) 6063–6067
Figure 2. Strategies for synthesizing -S-linked glycopeptides and a-S-linked glycopeptides.
common human tumor cell surface motif, which is
typical for all O-linkages in mucins. Therefore, the
corresponding a-S-linked glycopeptides are important
targets for biological studies also because of the
expected enhanced chemical stability and enzymatic
resistance. However, attempts to use our earlier proce-
dure for the synthesis of a-S-linked glycopeptides were
met by difficulties in the preparation of the required
b-glycosyl bromides.
which was simply prepared by bromination of commer-
cially available Z-Ser-OBzl with CBr4/Ph3P12 in 84%
yield, as shown in Scheme 1. a-GlcNAc thiol 1 was
prepared as a white foam from glucosamine according
to Knapp’s procedure.10c Based on our earlier work,5
the subsequent coupling between 1 and 2 was per-
formed in the presence of tetra-n-butylammonium
hydrogen sulfate (TBAHS) in ethyl acetate and an
aqueous solution of NaHCO3 at pH 8.5, i.e. phase
transfer conditions (PTC); expectedly, the Z-Cys(a-Glc-
NAc)-OBzl 3 was smoothly produced in 87% yield
without observable epimerization of the a-carbon of
cysteine, which has been detected under different exper-
imental conditions.13 In this coupling, the thiolate
anion was generated in situ by the action of NaHCO3,
and the a-thioglycoside 3 was then readily formed via
nucleophilic displacement of the b-bromo atom of 2 in
one pot. The corresponding iodoalanine10c turned out
to be much less stable than 2 and exhibited some
tendency to elimination;14,15 however, generation of
bromoalanine 2 or of peptide containing 2 led to stable
and pure compounds which underwent clean reactions
with glycosyl thiols. The same reaction could also be
performed with 1 and 2 in a mixture of DMF/H2O in
the presence of NaHCO3 at pH 8.5; also de-O-acetyl-
ated 1 reacted under these conditions.15
In order to overcome this problem, we planned to
exchange the electrophile and nucleophile, i.e. elec-
trophilic bromide containing peptides and nucleophilic
1-thio sugars were devised to construct the desired
a-linkage to sulfur (reaction 2 in Fig. 2). We present
here this new and effective strategy for the synthesis of
a-S-linked glycopeptides in aqueous solution.
To the best of our knowledge, the synthesis of S-linked
glycopeptides with exclusively a-glycosidic linkage par-
ticularly under aqueous conditions has not been
reported, though a-S-glycosyl cysteines have previously
been prepared in different laboratories.10 Among them,
Knapp and his co-workers firstly used a-GlcNAc10c and
a-GalNAc10d thiols to prepare the corresponding Boc-
Cys(a-GlcNAc)-OMe and Boc-Cys(a-GalNAc)-OMe
by coupling with b-iodoalanine with a strong base in
dry DMF.
Encouraged by this result, the generality of the present
procedure for the synthesis of a-S-linked glycopeptides
was examined. The required b-bromoalanine containing
peptides were prepared from the corresponding serine
Our initial efforts to test the feasibility of the new route
focused at first on amino acid b-bromoalanine 211
Scheme 1. Synthesis of N,O-protected (
L)-bromoalanine and its reaction with glycosylthiol 1. Reagents and conditions: (a) CBr4,
PPh3; (b) NaHCO3, TBAHS, EtOAc/H2O.

X. Zhu, R. R. Schmidt / Tetrahedron Letters 44 (2003) 6063–6067
6065
containing peptides using the same condition as for 2 or
by ligating the peptides by using N-Boc-protected ( )-
conditions to give the desired a-thioglycoside 11 in 79%
yield, as shown in Scheme 3. Similarly, glycodipeptide
12 was produced from the corresponding a-GalNAc
thiol 9, which was prepared following literature proce-
dure,10d and tyrosine (Tyr) containing dipeptide 10 in
63% yield. It is noteworthy that the indole ring of Trp
and the hydroxyl group of Tyr did not compete as
nucleophiles because of the presence of the highly
nucleophilic thiol group.
L
bromoalanine. Dipeptide bromide 4 was also prepared
in a two-step procedure,16 i.e. tosylation and then
bromination, but the yield was not better compared
with the other procedures. As expected, reaction of
thiol 1 with dipeptide bromides 4 and 5 under the
above PTC conditions provided the corresponding a-S-
linked glycodipeptides 6 and 7 in 78 and 83% yields,
respectively (Scheme 2).
In order to further exploit the above procedure, tripep-
tide bromides 13, 14 and 15 were prepared and sub-
jected to the thioglycosylation with 1 and 9, as shown
Tryptophan (Trp) containing dipeptide 8 was also thio-
glycosylated effectively with 1 under the same reaction
Scheme 2. Reaction of bromoalanine containing dipeptides with glycosyl thiol 1. Reagents and conditions: (a) solution of NaHCO3
(pH 8.5), TBAHS, EtOAc.
Scheme 3. Reaction of glycosyl thiols 1 and 9 with dipeptides 8 and 10. Reagents and conditions: (a) solution of NaHCO3 (pH
8.5), TBAHS, EtOAc.

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X. Zhu, R. R. Schmidt / Tetrahedron Letters 44 (2003) 6063–6067
Scheme 4. Reaction of glycosyl thiols 1 and 9 with tripeptides 13–15. Reagents and conditions: (a) solution of NaHCO3 (pH 8.5),
TBAHS, EtOAc.
in Scheme 4. Interestingly, all the couplings worked
well and afforded the corresponding a-S-linked gly-
cotripeptides 16, 17 and 18 in good yields (Scheme
4).17,18 18 was also fully deprotected.19
gemeinschaft and the Fonds der Chemischen Indus-
trie.
References
In summary, we have developed a convenient and
efficient synthesis of a-S-linked glycopeptides using a-
1. Grogan, M. J.; Pratt, M. R.; Marcaurelle, L. A.;
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2002, 102, 579–601; (c) Wittmann, V. In Glycoscience:
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3. Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5,
1384–1390. For more recent work on glycopeptide and
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Lett. 2003, 5, 243–246; Liu, H.; Wang, L.; Brock, A.;
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125, 1702–1703 and references cited therein.
glycosyl thiols and peptides containing ( )-b-bromoala-
L
nine as building blocks. An advantage of this procedure
is that peptide derivatives were not exposed to strong
basic conditions because of the use of a two-phase
system; the risk of elimination and subsequent epimer-
ization of the amino acid a-carbon were therefore
greatly reduced. In addition, it is noteworthy that the
a-linked coupling products with GalNAc, such as 12,
17 and 18 mimic the important Tn antigen structure.
We believe that the current study, together with our
earlier report,5 is of considerable potential for the syn-
thesis of S-linked glycopeptides. Studies on the applica-
tion of these procedures to specific, biorelevant
glycopeptide targets are in progress.
Acknowledgements
This work was supported by the Deutsche Forschungs-

X. Zhu, R. R. Schmidt / Tetrahedron Letters 44 (2003) 6063–6067
6067
4. For the synthesis of S-linked glycopeptides or S-linked
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9. Winterfeld, G. A.; Ito, Y.; Ogawa, T.; Schmidt, R. R.
Eur. J. Org. Chem. 1999, 5, 1167–1171.
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sugar thiol (0.34 mmol) in EtOAc (3 mL) was added pH
8.5 solution of NaHCO3 (3 mL) followed by addition of
TBAHS (272 mg, 0.8 mmol). The mixture was vigorously
stirred at room temperature for 5 h, then diluted with
EtOAc and washed successively with saturated aqueous
NaHCO3 and brine. The organic layer was dried over
MgSO4, concentrated in vacuo to give a residue which
was purified by flash column chromatography to afford
the corresponding thioglycoside.
18. All synthesized compounds gave satisfactory elemental
1
analyses and were identified by optical rotations, H, 13C
NMR, MALDI-MS spectroscopy. Selected data com-
1
pound 17: [h]D=+63 (c 1.0 CHCl3); H NMR (CDCl3) l
9.01 (br s, 1H), 7.70 (d, J=7.6 Hz, 1H), 7.37 (d, J=7.9
Hz, 1H), 7.16 (m, 3H), 7.04 (d, J=6.5 Hz, 1H), 6.78 (d,
J=8.0 Hz, 1H), 5.68 (d, J=6.6 Hz, 1H), 5.19 (m, 2H),
4.86 (dd, J=11.3, 2.9 Hz, 1H), 4.65 (m, 4H), 4.52 (dd,
J=8.5, 4.9 Hz, 1H), 4.05 (m, 1H), 3.90 (m, 1H), 3.73 (s,
3H), 3.71 (m, 1H), 3.50 (dd, J=14.5, 3.6 Hz, 1H), 3.11
(dd, J=14.6, 6.6 Hz, 1H), 3.04 (m, 1H), 2.45 (m, 1H),
2.12, 2.04, 2.03, 2.02 (4s, 12H), 1.88 (m, 1H), 1.47 (s, 9H),
1.30 (m, 2H), 0.90 (m, 6H); 13C NMR (CDCl3) l 172.1,
171.9, 171.1, 170.5, 170.3, 170.1, 168.8, 155.4, 136.2,
127.7, 123.1, 122.3, 119.7, 119.1, 111.4, 110.0, 86.4, 80.5,
68.3, 67.9, 67.0, 61.7, 56.6, 55.3, 53.3, 52.2, 48.1, 37.7,
34.0, 28.2, 25.1, 23.4, 20.7, 20.6, 15.4, 11.5; MALDI-MS
m/z 886.3 [M+Na+], 902.2 [M+K+]. Anal. calcd for
C40H57N5O14S (864.0): C, 55.61; H, 6.65; N, 8.11. Found:
C, 55.45; H, 6.82; N, 7.83.
Compound 18: [h]D=+50 (c 0.5 CHCl3); 1H NMR
(CDCl3) l 6.98 (d, J=8.5 Hz, 1H), 6.09 (br s, 1H), 5.66
(d, J=8.4 Hz, 1H), 5.38 (m, 2H), 4.97 (dd, J=11.8, 2.9
Hz, 1H), 4.88 (dd, J=8.7, 4.9 Hz, 1H), 4.64 (dd, J=8.7,
5.6 Hz, 1H), 4.52 (t, J=6.8 Hz, 1H), 4.39 (m, 2H), 4.14
(m, 2H), 3.70 (m, 2H), 3.39 (dd, J=14.5, 4.4 Hz, 1H),
2.84 (dd, J=14.5, 5.9 Hz, 1H), 2.17, 2.11 (2s, 6H), 2.00
(s, 6H), 2.30–1.90 (m, 5H), 1.44 (s, 18H), 1.05 (d, J=6.8
Hz, 3H), 0.94 (d, J=6.7 Hz, 3H); 13C NMR (CDCl3) l
171.1, 170.7, 170.5, 170.4, 170.3, 169.8, 169.7, 155.3, 87.3,
81.3, 80.6, 68.4, 67.9, 67.3, 62.0, 59.8, 55.5, 54.0, 48.0,
47.3, 35.2, 31.4, 29.1, 28.2, 27.9, 24.9, 23.2, 20.7, 19.6,
17.3; MALDI-MS m/z 822.9 [M+Na+], 839.3 [M+K+].
Anal. calcd for C36H58N4O14S (802.9): C, 53.85; H, 7.28;
N, 6.98. Found: C, 53.64; H, 7.49; N, 6.51.
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17. General procedure for the synthesis of a-S-linked gly-
copeptides 3, 6, 7, 11, 12, 16, 17 and 18. To a solution of
appropriate bromide (0.2 mmol) and the corresponding
19. 18 was smoothly fully deprotected to give the correspond-
ing glycotripeptide by treatment with 6 mM NaOMe/
MeOH solution and 40% trifluoroacetic acid in CH2Cl2
successively: [h]D=+93 (c 1.0 MeOH); MALDI-MS m/z
521.4 (M+H+), 543.7 (M+Na+), 559.7 (M+K+).