Thio-mediated synthesis of derivatized N-linked glycopeptides using isonitrile chemistry

Article abstract of DOI:10.1016/j.tetlet.2009.06.005

The reaction of oligosaccharide isonitriles with peptide thioacids in the presence of bulky thiophenol as activator to provide N-linked glycopeptides at room temperature is described.

Full text of DOI:10.1016/j.tetlet.2009.06.005

Tetrahedron Letters 50 (2009) 4666–4669
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Tetrahedron Letters
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Thio-mediated synthesis of derivatized N-linked glycopeptides using
isonitrile chemistry
Xiangyang Wu a, Yu Yuan a, Xuechen Li a, Samuel J. Danishefsky a,b,
*
a Laboratory for Bioorganic Chemistry, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue, New York, NY 10065, USA
b Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction of oligosaccharide isonitriles with peptide thioacids in the presence of bulky thiophenol as
activator to provide N-linked glycopeptides at room temperature is described.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 May 2009
Revised 19 May 2009
Accepted 1 June 2009
Available online 6 June 2009
A central focus of our laboratory is the development of en-
hanced methods for the synthesis of glycopeptides and glycopro-
teins.1 In this regard, we have taken note of the paucity of
techniques for the efficient merger of complex glycan and peptidyl
domains. Although recent years have seen breakthrough advances
in one’s capacity to synthesize large peptide and glycan units in
isolation, the efficient merger of these fragments remains a daunt-
ing challenge. Standard glycan–peptide coupling techniques,
which typically involve appendage of the carbohydrate to an aspar-
tate residue through Lansbury2 or other standard aspartylation
conditions to provide N-linked glycans, are complicated by the
poor nucleophilicity of the anomeric terminus of the glycan do-
main, as well as by the poor solubility of the protein or peptide
fragments. The efficiency of aspartylation may be compromised
by a competing side reaction where, in the presence of the requi-
site base, the peptide undergoes ring closure to provide an asparti-
mide side product.3 The development of more efficient
aspartylation methods thus remains an important goal.
Toward this end, we recently disclosed the development of a
novel isonitrile-based ‘two-component coupling’ (2CC) reaction.
As outlined in Scheme 1, we have been able to accomplish 2CC-
based aspartylation between a mono-saccharide isonitrile 1 and
an aspartic acid 2, through microwave heating at 150 °C, to provide
glycopeptide 3 in 82% yield.4
As shown in Scheme 1, the 2CC reaction originally required
high temperatures, which could well be problematic with mainte-
nance of sensitive polypeptidic glycan structures. We also sought
to develop a milder version of the 2CC reaction that could be ap-
plied to the research objective at hand.5 Chupp and co-workers
had reported on the reaction of a very simple thio acid with an
isonitrile at room temperature to afford N-thioformyl amide in
modest yields.6 Our group7 has already successfully synthesized
a series of dipeptides in good yields at room temperature, by
employing a similar strategy. Unfortunately, this method suffers
a significant limitation when extended to the coupling of dipep-
tide thioacids with dipeptide isonitriles, presumably due to the
competing formation of oxazolones and thioanhydrides. With
these issues in mind, we hoped to develop a modified 2CC proto-
col, which might allow for efficient glycan aspartylation at room
temperature.4,5
Our initial attempts to couple monosaccharide isonitrile 4 with
aspartic thioacid 57 provided the desired N-thioformyl glycopep-
tide 6 in poor yield, due to formation of high levels of thioforma-
mide 7 (Scheme 2). In light of earlier precedent, we explored the
introduction of thiophenol to assist in the overall addition rear-
rangement sequence. We were pleased to discover that the cou-
pling yield was increased to 36% in the presence of thiophenol as
activator. When 2,6-dimethyl thiophenol was utilized as the medi-
ating agent, the coupling efficiency was further improved (60%).
Nonetheless, significant amounts of 7 were still observed. We sur-
mised that perhaps the hydrogen atom of the –NHAc group was
associating with the lone pair of the adjacent nitrogen of the FCMA,
thus activating it for thioformamide formation through nucleo-
philic attack.
Accordingly, we prepared the –NPhth containing monosaccha-
ride isonitrile, 8.8 Indeed, coupling of 8 with aspartic thioacid 5,
in the presence of bulky 2,6-dimethyl thiophenol as activator, pro-
ceeded smoothly to provide the desired product 9 in 88% yield
(Scheme 3). Notably, little or no thioformamide side product was
observed.
Armed with these encouraging findings, we sought to prepare
more complex N-thioformyl glycopeptides (Scheme 4). The re-
quired dipeptide thioacid 10 and tripeptide thioacid 13 were syn-
thesized from previously known procedures.7 In the event,
monosaccharide isonitrile 8 was coupled with dipeptide thioacid
10 under the above-described conditions (DCE, 2,6-dimethyl thio-
phenol, rt). This reaction led to the formation of the desired
* Corresponding author. Tel.: +1 212 639 5501; fax: +1 212 772 8691.
E-mail address: s-danishefsky@ski.mskcc.org (S.J. Danishefsky).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.06.005

X. Wu et al. / Tetrahedron Letters 50 (2009) 4666–4669
4667
OAc
O
OAc
CHO
N
CHCl3
CO2t-Bu
HO
O
AcO
AcO
AcO
AcO
CO2t-Bu
NHFmoc
NC
+
150 °C
MV
O
NHFmoc
N3
N3
O
1
2
3
82%
Scheme 1.
Nu:
OAc
OAc
O
S
CO2Bn
NHBoc
O
AcO
AcO
CO2Bn
NHBoc
HS
N
H
AcO
AcO
+
NC
NHAc
O
H
N
O
O
4
5
Me
OAc
O
OAc
O
CHS
N
rt
H
AcO
AcO
AcO
AcO
+
CO2Bn
H
N
CH2ClCH2Cl
NHAc
NHAc
NHBoc
O
S
6
7
15%
65%
no thiol
SH
Me
36%
42%
24%
SH
Me
60%
Scheme 2.
SH
Me
Me
OAc
O
OAc
O
NC
NPhth
CHS
N
AcO
AcO
AcO
AcO
CO2Bn
NHBoc
+
5
rt, DCE
88%
NPhth
8
O
9
Scheme 3.
SH
O
OAc
O
NC
NPhth
rt
Me
Me
HS
OMe
AcO
AcO
+
+
N
H
DCE
O
O
NHFmoc
8
10
O
OAc
O
CHS
N
O
AcO
AcO
OMe
+
N
Leu–OMe
N
H
NPhth
FmocHN
O
O
NHFmoc
O
11 (61%)
12 (20%)
O
SH
Me
OAc
O
rt
HS
O
OMe
Me
AcO
AcO
+
+
N
NC
DCE
H
NPhth
O
HN
8
13
NHFmoc
O
OAc
O
CHS
N
O
O
AcO
AcO
OMe
N
N
Leu–OMe
NPhth
H
+
O
O
HN
Gly–HN
NHFmoc
O
14 (46%)
15 (30%)
O
Scheme 4.

4668
X. Wu et al. / Tetrahedron Letters 50 (2009) 4666–4669
SH
NPhth
OBn
O
rt
BnO
AcO
CO2Bn
HS
Me
Me
O
+
+
O
BnO
NC
NPhth
DCE
OBn
O
NHBoc
5
16
NPhth
NPhth
OBn
O
OBn
O
CHS
N
BnO
AcO
BnO
AcO
H
N
O
O
O
+
O
OBn
BnO
BnO
CO2Bn
NHBoc
H
OBn
NPhth
NPhth
O
S
17 (85%)
18 (0%)
SH
Me
O
O
NPhth
Me
OMe
OBn
O
rt
DCE
Me
Me
BnO
AcO
+
HS
+
O
N
O
BnO
NC
NPhth
H
OBn
O
NHFmoc
10
16
Me
O
OBn
O
CHS
N
NPhth
O
O
Me
OMe
BnO
AcO
+
N
Leu–OMe
BnO
O
N
FmocHN
NPhth
H
OBn
O
O
O
NHFmoc
19 (55%)
12 (20% )
Scheme 5.
product, 11, in 61% yield. The modest yield reflected the formation
of still substantial levels of undesired aspartimide side product 12.3
Coupling of 8 with tripeptide thioacid 13 provided N-linked glyco-
peptide 14 in 46% yield.
To evaluate the scope of this methodology for the assembly of
more complex glycopeptides, we prepared disaccharide isonitrile
16 from a known procedure,8 taking advantage of the feasibility
of reducing an anomeric azide to the corresponding amine under
basic conditions without affecting the acetate group. The coupling
of 16 with aspartic thioacid 5 was performed in the presence of
bulky 2,6-dimethyl thiophenol as a mediator, to afford the N-thi-
oformyl glycopeptide 17 in excellent yield without formation of
thioformamide (18). Similarly, aspartylation of 16 with dipeptide
10 provided 19 in 55% yield, along with the aspartimide side prod-
uct, 12 (Scheme 5).
We next sought to identify an appropriate peptide-protecting
group which would mitigate against aspartimide formation and
be compatible with other protecting groups as well as with the gly-
cosidic bond. It seemed that a 2,4-dimethoxy benzyl group would
indeed fulfill this demand. Happily, the reaction of 8 with pro-
tected dipeptide thioacid 20, in the presence of 2,6-dimethyl thio-
phenol, afforded the fully protected glycopeptide 21 in 77% yield
(Scheme 6). Notably, no aspartimide or thioformamide side prod-
ucts were observed.
O
OAc
HS
SH
rt
O
N
CO2Me
AcO
AcO
Me
Me
+
+
NC
NPhth
DCE
77%
O
BocHN
8
20
MeO
OMe
OAc
O
O
CHS
N
AcO
AcO
N
CO2Me
NPhth
O
BocHN
21
MeO
OMe
Scheme 6.
OAc
O
CHS
AcO
AcO
Raney-Nickel, EtOH
80 °C (MV)
N
CO2Bn
NPhth
O
NHBoc
9
OAc
OAc
O
Me
H
O
AcO
AcO
AcO
AcO
N
CO2Bn
NHBoc
N
CO2Bn
+
NPhth
23 (85%)
NPhth
O
NHBoc
O
22 (trace)
Scheme 7.

X. Wu et al. / Tetrahedron Letters 50 (2009) 4666–4669
4669
N-Methylated natural peptides could well be valuable in the
Supplementary data
development of peptide-based drugs, due to their remarkable bio-
logical and pharmacological profiles, such as long in vivo half-life
and bioavailability.9,10 The direct adduct of our 2CC aspartylation
protocol is an N-thioformyl amide (cf. 9). We hoped to expand
the general utility of this protocol. It could be valuable to convert
the thioformyl function to an N–Me group. Toward this end, we
followed a precedent by Chupp6 and co-workers, using Raney-
nickel as catalyst. Unfortunately, our initial attempts to employ
those conditions were unsuccessful. Careful monitoring of the
reaction progress indicated that the starting material was very sen-
sitive to base. When the Raney-nickel catalyst was washed to neu-
trality with water directly prior to the reaction, the reduction of 9
proceeded at 80 °C (microwave, EtOH) to afford N-linked glycopep-
tide 23 as the major product (85% yield), along with trace amounts
of N-methyl glycopeptide 22 (Scheme 7).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.06.005.
References and notes
1. Herzner, H.; Reipen, T.; Schulz, M.; Kunz, H. Chem. Rev. 2000, 100, 4495–
4537.
2. Cohen-Anisfeld, S. T.; Lansbury, P. T. J. Am. Chem. Soc. 1993, 115, 10531–
10537.
3. (a) Bodanszky, M.; Natarajan, S. J. Org. Chem. 1975, 40, 2495–2499; (b)
Bodanszky, M.; Kwei, J. Z. Int. J. Pept. Protein Res. 1978, 12, 69–74.
4. (a) Li, X.; Danishefsky, S. J. J. Am. Chem. Soc. 2008, 130, 5446–5448; (b) Li, X.;
Yuan, Y.; Berkowitz, w. F.; Todaro, L. J.; Danishefsky, S. J. J. Am. Chem. Soc. 2008,
130, 13222–13224; (c) Li, X.; Yuan, Y.; Kan, C.; Danishefsky, S. J. J. Am. Chem.
Soc. 2008, 130, 13225–13227. We are also now pursuing the possibility that
this 2CC methodology could provide an opportunity for the development of
alternative aspartylation methods which might minimize formation of
aspartimide side product.
5. Wu, X.; Li, X.; Danishefsky, S. J. Tetrahedron Lett. 2009, 50, 1523–1525.
6. Chupp, J. P.; Leschinsky, K. L. J. Org. Chem. 1975, 40, 66–71.
7. Yuan, Y.; Zhu, J. L.; Li, X.; Wu, X.; Danishefsky, S. J. Tetrahedron Lett. 2009, 50,
2329–2333.
8. Chiesa, M. V.; Schmidt, R. R. Eur. J. Org. Chem. 2000, 3541–3554.
9. Gilon, C.; Dechantsreiter, M. A.; Burkhart, F.; Friedler, A.; Kessler, H. Synthesis
of peptides and peptidomimetics. In Houben-Weyl Methods of Organic
Chemistry; Goodman, M., Felix, A., Moroder, L., Tonolio, C., Eds.;
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1331.
In summary, we have developed a modified 2CC aspartylation
protocol which allows for the synthesis of N-thioformyl glycopep-
tides in good efficiency. These thioformyl groups can be converted
to N-formyl groups. The latter can be converted to NH functions as
previously described. The results of further investigations in glyco-
peptide synthesis will be disclosed in due course.
Acknowledgment
This work was supported by the NIH (CA103823 to S.J.D.).