A novel method for the formation of N-glycosides using hydroxamate

Article abstract of DOI:10.1016/S0040-4039(03)00473-8

Direct formation of Asn-linked carbohydrate by N-glycosylation has been difficult, because of the lack of nucleophilicity of carboxamide nitrogen. We report here the novel N-glycosylation using Asn hydroxamate as a glycosyl acceptor. Reaction with glycosyl fluoride or glycosyl trichloroacetimidate afforded N-glycoside and subsequent reduction with SmI₂ gave Asn-linked glucose. Carbamate derived hydroxamates proved to have even enhanced reactivity to give N-glycosides in high yields.

Full text of DOI:10.1016/S0040-4039(03)00473-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 2853–2856
A novel method for the formation of N-glycosides using
hydroxamate
Jun Nakano,a,b Tsuyoshi Ichiyanagi,a,† Hiromichi Ohtab and Yukishige Itoa,c,
*
aRIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bDepartment of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-0061, Japan
cCREST, JST, Kawaguchi, Saitama 332-0012, Japan
Received 6 January 2003; revised 10 February 2003; accepted 14 February 2003
Abstract—Direct formation of Asn-linked carbohydrate by N-glycosylation has been difficult, because of the lack of nucleophilic-
ity of carboxamide nitrogen. We report here the novel N-glycosylation using Asn hydroxamate as a glycosyl acceptor. Reaction
with glycosyl fluoride or glycosyl trichloroacetimidate afforded N-glycoside and subsequent reduction with SmI2 gave Asn-linked
glucose. Carbamate derived hydroxamates proved to have even enhanced reactivity to give N-glycosides in high yields. © 2003
Published by Elsevier Science Ltd.
N-Glycosylation is an important post-translational
modification of proteins. It is mediated by the multisub-
unit enzyme oligosaccharyltransferase (OST) that trans-
fers tetradecasaccharide (Glc3Man9GlcNAc2) from
dolichol diphosphate (Dol-PP) to the asparagine (Asn)
side chain of the nascent protein (Scheme 1, Eq. (1)).1
Besides the size of the oligosaccharide (M.W. 2370)
transferred, the remarkable feature of this transforma-
tion is that generally unreactive carboxamide nitrogen
reacts as a nucleophile. In fact, direct formation of the
N-glycoside linkage with Asn by chemical glycosylation
is yet to be realized.2 Conventionally, synthesis of Asn-
linked oligosaccharide has been achieved by the cou-
pling of glycosylamine derivatives, obtainable by the
treatment of reducing sugar with ammonium
bicarbonate3a or reduction of glycosyl azide,3b with
activated aspartic acid (Eq. (2)). We report here the
N-glycosylation using hydroxamate, which was used
successfully for the formation of Asn-carbohydrate
linkage.
In order to chemically activate the carboxamide group
of Asn, our attention was turned to the use of hydrox-
amate as the nucleophile. It has been known that the
acidity of hydroxamate is substantially higher than that
of caboxamide.7 In addition, the nucleophilicity of
nitrogen should be enhanced by the a-effect of oxygen.
Contemplating these, we surmised that the nitrogen of
1 may have enough nucleophilicity to serve as a glyco-
syl acceptor (Scheme 1). After coupling, removal of the
benzyloxy group should be possible under reducing
conditions (Eq. (3)).
In order to test this hypothesis, hydroxamate 1a was
prepared from Fmoc-Asp-OtBu as depicted in Scheme
2. Our initial attempt was directed to the Mitsunobu-
type reaction8 between 1a and tetra-O-benzyl glucose
2a. Although the coupling reaction proceeded cleanly
under Tsunoda’s conditions9 (TMAD, Bu3P/toluene),
the product proved to be the O-glycoside 3 (Table 1,
entry 1). On the other hand, glycosyl fluoride 2b gave
10
N-glycoside 4 when activated by Ag(I)–Cp2MCl2
Biosynthetic incorporation of N-linked oligosaccharide
requires Asn-X-Ser/Thr (X: any amino acid except Pro)
as the consensus sequence.4 To reconcile this require-
ment with the exceptional activity of the Asn side
chain, intramolecular hydrogen bonding between
hydroxy group and carboxamide has been proposed.5,6
(entries 2–8). As silver salts, AgOTf and AgSbF6 gave
comparable results (entries 2, 5, 7), while AgBF4 (entry
3) and AgPF6 (entry 4) were not suitable for this
purpose. Cp2HfCl2 (entry 6) was somewhat less effec-
tive than Cp2ZrCl2. Reaction with the trichloroacetimi-
date 2c proceeded in a similar efficiency (entry 9).
Subsequent removal of the benzyloxy group was
achieved cleanly by SmI2 mediated reduction (THF, rt,
10 min)11 in the presence of MeOH (25 equiv.) to afford
glycosyl asparagine 5 in 88% yield. Although the
product obtained here is uncommon aGlc1“Asn,12 this
Keywords: N-glycoside; asparagine; hydroxamate.
* Corresponding author. E-mail: yukito@postman.riken.go.jp
† Current address: Department of Biochemistry and Biotechnology,
Tottori University, Tottori-shi 680-8553, Japan.
0040-4039/03/$ - see front matter © 2003 Published by Elsevier Science Ltd.
doi:10.1016/S0040-4039(03)00473-8

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J. Nakano et al. / Tetrahedron Letters 44 (2003) 2853–2856
Scheme 1. Enzymatic and chemical formation of Asn-linked saccharide.
Scheme 2. Formation of N-glycoside from hydroxamate.
result provides the first example of the formation of the
Asn-linked carbohydrate by direct glycosylation.
(Troc)-protected13 glucosamine donor 9 was highly suc-
cessful (entries 10–13). It selectively provided the b-
linked N-glycosides 10a–c in high yield. By contrast,
reaction of 9 with 1a did not proceed at all. Since the
facile conversion of allyloxycarbonyl masked glycosyl-
amine to N-glycosylated asparagine was reported previ-
ously,14 this reaction might well provide a novel access
to Asn-linked glycans.
Carbamate-derived hydroxamate proved to have an
even higher reactivity toward glycosyl donor (Scheme
3). Thus, 6a was prepared from benzyloxyamine and
allyl chloroformate and subjected to glycosylation with
2b. This reaction proceeded smoothly at subzero tem-
perature to afford the product in high yield (Table 1,
entry 10). It was smoothly reduced to 8 under condi-
tions identical to the preparation of 5. More gratify-
ingly, the reaction of 6a–c with trichloroethoxycarbonyl
In summary, a novel method for the formation of
N-glycoside was developed, that was applied to the
direct glycosylation of Asn side chain.

J. Nakano et al. / Tetrahedron Letters 44 (2003) 2853–2856
Table 1. Results of glycosylation reactions
2855
Entrya
Donor
Hydroxamate
Reagentsd
Temp./Time (h)
Product
Yield (%)
a:b
1
2a
2b
2b
2b
2b
2b
2b
2b
2c
2b
9
1a
1a
1a
1a
1a
1a
1a
1a
1a
6a
6a
6b
6c
A
B
C
D
E
F
E
G
H
B
B
B
B
rt/3
rt/3
rt/24
rt/24
rt/24
rt/24
rt/24
−20°C–rt/10
−78°C–rt/16
−20°C–rt/5.5
−20°C/1
−20°C/1
−20°C/1
3
4
4
4
4
4
4
4
4
7
10a
10b
10c
95
53
18
Trace
53
39
55
48
43
90
83
84
87
b
2b
3c
5.5:1
1.7:1
ND
2:1
4c
5c
6c
2.1:1
4:1
1.8:1
3.3:1
3.5:1
b
7c
8c
9b
10b
11b
12b
13b
9
9
b
b
a All reactions were performed in CH2Cl2, except entry 1 (toluene) and entry 7 (toluene–CH2Cl2), 9:1).
b Performed in the presence of molecular sieves 4A.
c Performed in the presence of molecular sieves AW-300.
d A: TMAD (3 equiv.)–Bu3P (3 equiv.), B–F: AgX (1.0 equiv.)–Cp2MCl2 (0.5 equiv.) (B: X=OTf, M=Zr; C: X=BF4, M=Zr; D: X=PF6,
M=Zr; E: X=SbF6, M=Zr; F: X=SbF6, M=Hf), G: BF3·OEt2 (1.0 equiv.), H: TMSOTf (1.0 equiv.).
Scheme 3. Reaction with carbamate-derived hydroxamate.
Acknowledgements
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We thank Ms. Akemi Takahashi for technical assis-
tance. This work was partially supported by Ministry of
Education, Culture, Sports, Science and Technology
[Grant-in-Aid for Scientific Research (B) No.
13480191]. T.I. is
a recipient of Basic Science
Researcher Fellowship from RIKEN (2001-2002).
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