Efficient stereoselective synthesis of γ-N-glycosyl asparagines by N-glycosylation of primary amide groups

Article abstract of DOI:10.1021/ja0450298

The efficient and elegant synthesis of N-glycosides by N-glycosylation of asparagine-containing peptides is described. Glycosylation of primary amides with glycosyl N-phenyltrifluoroimidates in the presence of a catalytic amount of TMSOTf in nitromethane

Full text of DOI:10.1021/ja0450298

Published on Web 01/22/2005
Efficient Stereoselective Synthesis of γ-N-Glycosyl Asparagines by
N-Glycosylation of Primary Amide Groups
Hiroshi Tanaka, Yuki Iwata, Daisuke Takahashi, Masaatsu Adachi, and Takashi Takahashi*
Department of Applied Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology
2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
Received August 18, 2004; E-mail: ttakashi@apc.titech.ac.jp
Scheme 1
Post-translation modification of proteins involves phosphorylation
and glycosylation, which is an essential biological process to mature
their functions. N-glycosides attached to asparagines at the γ-posi-
tion through a â-glycosidic bond are found in various membrane
proteins and play significant roles in biological processes on the
cell surface.1 To elucidate the biological role of these glycoproteins,
their partial fragments, such as N-glycopeptides, have served as
effective biochemical probes and are attractive synthetic targets.2
Therefore, an effective methodology for the synthesis of the various
N-glycosyl peptides is required.
Most of the established methodologies for linking asparagines
Table 1. N-Glycosylation of Acetamide 10 with the Galactosyl
Imidates 6a-9a
and saccharides through an N-glycosidic bond to provide N-
glycopeptides 1 involve amidation of the asparaginic acid 5 with
the glycosylamines 4 or its equivalent (Scheme 1, path B).3
However, the â-glycosylamines 4 are sufficiently unstable not only
to epimerize at the anomeric position but also to hydrolyze to the
lactol during the reaction. Furthermore, the asparaginic acid 5 in
peptides easily undergoes cyclization to afford the corresponding
succinimide by activation of the carbonyl group4 (path C). To
minimize succinimide formation, careful control of the reaction
conditions is required. On the other hand, biological synthetic
processes of N-glycoproteins involve N-glycosylation of the primary
amides 2 with donor 3 (path A). The biosynthetic pathway suggests
an efficient and alternative approach for the chemical synthesis of
various N-glycosyl amides 1 from the corresponding stable amides
2.5 However, it is worth noting that the nitrogen of the amide group
showed very poor nucleophilicity toward glycosylation. Addition-
ally, O-glycosylation of the amide could also lead to a considerable
side reaction. In 1989, Kahne et al. reported that the coupling of a
N-silyl acetamide with a perbenzyl galactosyl sulfoxide provided
R-N-glycosyl acetamide as a major product.7,8 The N-trimethylsilyl
group enhances the nucleophilicity of the amide nitrogen. However,
the preparation of N-silyl asparagines is a difficult task because of
their instability.
entry
donor
activator
solvent
yield of 11 (%)
1
2
3
4
5
6
7
8
6a
6a
6a
6a
7a
8a
9ac
6a
6a
6a
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TESOTf
TBSOTf
TMSOTf
CH3NO2
CH3CN
EtCN
CH2Cl2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
CH3NO2
98
70
85
46
66
75
42
53
87
71b
9
10a
a With 1.5 equiv of acceptor used as base on donor 6a. b The yield was
estimated based on donor 6a.
In this communication, we have demonstrated the stereoselective
synthesis of glycosyl amino acids and peptides by N-glycosylation
of primary amides without any amide activating groups. As
illustrated in Table 1, treatment of acetamide 10 with 1.5 equiv of
the glycosyl â-N-phenyltrifluoroacetoimidate 6a9 in the presence
of a catalytic amount of TMSOTf at room temperature in ni-
tromethane10 provided the glycosyl acetamide 11 in excellent yield
with complete â-selectivity (entry 1). Dichloromethane did not work
well in the N-glycosylation as a solvent (entry 4). Both electron-
withdrawing and -donating groups on the leaving group reduced
the yield of 1111 (entries 5 and 6). Use of R-trichloroacetoimidate
9a12 as a donor provided the glycosyl acetamide 11 in dramatically
reduced yield (42%) along with a significant amount of the glycosyl
trichloroacetamide 12 (entry 7). The yield of 12 was 80% based
on donor 9a. N-substitution of the trifluoroimidate prevented the
released trifluoroacetamide from being glycosylated with the donor
due to its steric hindrance. Both TESOTf and TBSOTf were found
not to be effective for the N-glycosylation as a promoter in
comparison with TMSOTf (entries 8 and 9). Use of excess acceptor
10 provided glycosyl amide in good yield based on the donor 6a
(entry 10).
We next investigated N-glycosylation of the protected asparagine
13A with â-glycosyl imidates 6a-e12 (Table 2). The galactosyl,
glucosyl, and mannosyl imidates 6a-c attached with an acyl
protecting group at the C2 position and underwent N-glycosylation
to form the 1,2-trans glycosidic bond, providing the corresponding
â-N-glucosyl, â-N-galactosyl, and R-N-mannosyl13 asparagines
14aA-14cA in excellent yields (91-98%) (entries 1-3). The
N-Troc glucosamine 6d14 was stereoselectively converted to the
corresponding N-glycosyl asparagines 14dA in moderate yields
(68%) with complete â-selectivity (entry 4). On the other hand,
glycosidation of the perbenzyl-protected galactoside 6e provided
9
1630
J. AM. CHEM. SOC. 2005, 127, 1630-1631
10.1021/ja0450298 CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Table 2. N-Glycosylation of Asparagin 13A and Peptides 13B and
13C with Glycosyl Donors 6a-e
tripeptides in nitromethane. To our knowledge, this is the first
example of the synthesis of N-glycosyl peptides by N-glycosylation
of nonactivated primary amides.
In conclusion, we have demonstrated the efficient and elegant
synthesis of N-glycosides by N-glycosylation of asparagine-
containing peptides with glycosyl N-phenyltrifluoroimidates utiliz-
ing a catalytic amount of TMSOTf in nitromethane. This coupling
method allows for the synthesis of the various N-glycosyl amides
from the primary amide derivatives, which are effective biochemical
probes for elucidation of the role of glycopeptides.
Supporting Information Available: Experimental procedures for
the N-glycosylation and full characterization for compounds. This
material is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Kunz, H.; Schultz, M. In Carbohydrates in Chemistry and Biology; Ernst,
B., Hart, G. W., Sinay, P., Eds.; Wiley-VCH: Weinheim, Germany, 2000;
Vol. 1, Chapter 11, pp 267-304. (b) Varki, A. Glycobiology 1993, 3,
97-130. (c) Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357-
2364.
(2) Arsequell, G.; Valencia, G. Tetrahedron: Asymmetry 1999, 10, 3045-
3094. (b) Herzner, H.; Reipen, T.; Schultz, M.; Kunz, H. Chem ReV. 2000,
100, 4495-4537. (c) Allen, J.; Harris, C. R.; Danishefsky, S. J. J. Am.
Chem. Soc. 2001, 123, 1890-1897. (d) Sears, P.; Wong, C. H. Science
2001, 291, 2344-2350.
entry
donor
acceptor
product
yield (%)
R:
âa
(3) Marks, G. S.; Neuberger, A. J. Chem. Soc. 1961, 4872-4879. (b) Kholrlin,
A. Y.; Zurabyan, S. E.; Macharadze, R. G. Carbohydr. Res. 1980, 85,
201-208. (c) Handlon, A. L.; Fraser-Reid, B. J. Am. Chem. Soc. 1993,
115, 3796-3797. (d) Inazu, T.; Kobayashi, K. Synlett 1993, 869-870.
(e) Matsuo, I.; Nakahara, Y.; Ito, Y.; Nukada, T.; Nakahara, Y.; Ogawa,
T. Bioorg. Med. Chem. 1995, 3, 1455-1463. (f) Meinjohanns, E.; Meldal,
M.; Paulsen, H.; Dwek, R. A.; Bock, K. J. Chem. Soc., Perkin Trans. 1
1998, 549-560. (g) Ishikawa, A.; Takatani, M.; Nakahara, Y.; Ito, Y.
Synlett 2002, 634-636.
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6a
6b
6c
6d
6a
6b
6c
6d
13A
13A
13A
13A
13A
13B
13B
13B
13B
13C
13C
13C
13C
14aA
14bA
14cA
14dA
14eA
14aB
14bB
14cB
14dB
14aC
14bC
14cC
14dC
98
99
91
68
68
94
78
88
31
39
10
19
0
â only
â only
R only
â only
77:23a
â only
â only
R only
â only
â only
â only
R only
(4) Bodanszky, M.; Natarajan, S. J. Org. Chem. 1975, 40, 2495-2499. (b)
Cohen-Anisfeld, S. T.; Lansbury, P. T. J. Am. Chem. Soc. 1993, 115,
10531-10537. (c) Miller, J. S.; Dudkin, V. Y.; Lyon, G. J.; Muir, T. W.;
Danishefsky, S. J. Angew. Chem., Int. Ed. 2003, 42, 431-434.
(5) Imperiali, B.; Shannon, K. L.; Unno, M.; Rickert, K. W. J. Am. Chem.
Soc. 1992, 114, 7944-7945. (b) Helenius, A.; Aebi, M. Science 2001,
291, 2364-2369.
9
10b
11b
12b
13b
(6) Nakano, J.; Ichiyanagi, T.; Ohta, H.; Ito, Y. Tetrahedron Lett. 2003, 44,
2853-2856.
a Ratio was estimated by HPLC analysis based on refractive index
(7) Kahne, D.; Walker, S.; Cheng, Y.; Engen, D. V. J. Am. Chem. Soc. 1989,
111, 6881-6882.
detection. b Two equivalents of donors 6 was used.
(8) Garcia, B. A.; Gin, D. Y. J. Am. Chem. Soc. 2000, 122, 4269-4279.
(9) Yu, B.; Tao, H. Tetrahedron Lett. 2001, 42, 2405-2407. (b) Yu, B.; Tao,
H. J. Org. Chem. 2002, 67, 9099-9102.
the R-linked N-glycosyl amide 14eA in 68% yield with moderate
selectivity (entry 5). To demonstrate the feasibility of N-glycosyl-
ation, the synthesis of glycopeptides 14B and 14C by N-glycosyl-
ation of peptides 13B and 13C with 6a-d was investigated (entries
6-13). The glycosyl dipeptides, 14aB-, 14bB-, and 14cB-modified
glucose, galactose, and mannose, were prepared in good yields with
excellent selectivity from dipeptide 13B. The N-Troc glucosamine
6d was converted to the corresponding glycosyl amide 14dB in
moderate yield. However, glycosylation of tripeptides resulted in
the reduced yields of the corresponding glycopeptides 14aC, 14bC,
and 14cC. Unfortunately, the glucosaminyl tripeptide 14dC was
not obtained under these reaction conditions. The low efficiency
of the reaction might have resulted from the low solubility of the
(10) Dziadek, S.; Kunz H. Synlett 2003, 1623-1626.
(11) Cai, S.; Yu, B. Org. Lett. 2003, 5, 3827-3830.
(12) 1H NMR signals of the anomeric position are broad at room temperature
due to the syn-anti isomerization around the carbon-nitorgen double
bond. We determined the anomeric configuration of glycosyl imidates
6a-e by low-temperature NMR experiments to be â. In the case of
glucose, galactose, and glucosamine derivatives, the H-H coupling
constants at the anomeric position are 8.2 and 8.7 Hz. In the case of
mannosyl imidate 6c, NOE was observed between H1 and H3, and H1
1
and H5. Additionally, the JC-HC-H coupling constant of C1 was 164
Hz: Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293-
297.
(13) Structure determination of R-linked mannosides 14cA-C was achieved
by NOE experiments. Details are shown in Supporting Information.
(14) Ellervik, U.; Magnusson, G. Carbohydr. Res. 1996, 280, 251-260.
JA0450298
9
J. AM. CHEM. SOC. VOL. 127, NO. 6, 2005 1631