Chemoselective coupling of sugar oximes and α-ketoacids to glycosyl amides and N-glycopeptides

Article abstract of DOI:10.1016/j.tetlet.2014.02.056

The reaction of unprotected sugar hydroxylamines and oximes with α-ketoacids leads to the chemoselective formation of glycosyl amides following the decarboxylative condensation pathway of Bode's ketoacid hydroxylamine (KAHA) ligation. Sugar oximes with gl

Full text of DOI:10.1016/j.tetlet.2014.02.056

Tetrahedron Letters 55 (2014) 2197–2200
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Chemoselective coupling of sugar oximes and
amides and N-glycopeptides
a-ketoacids to glycosyl
Claudia Pöhner a, Vera Ullmann a, Ramona Hilpert a, Eric Samain b, Carlo Unverzagt a,
⇑
a Bioorganische Chemie, Gebäude NWI, Universität Bayreuth, 95440 Bayreuth, Germany
b CERMAV-CNRS, BP 53, 38041 Grenoble cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction of unprotected sugar hydroxylamines and oximes with
a
-ketoacids leads to the chemoselec-
Received 13 January 2014
Revised 11 February 2014
Accepted 14 February 2014
Available online 26 February 2014
tive formation of glycosyl amides following the decarboxylative condensation pathway of Bode’s ketoacid
hydroxylamine (KAHA) ligation. Sugar oximes with gluco configuration stereoselectively form b-linked
products. This method can be used for the convergent synthesis of N-acylated sugars and oligosaccha-
rides bearing small labels, spacers, or peptides in the acyl part.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
N-glycopeptides
Sugar oximes
Chemoselective coupling
Decarboxylative condensation
Synthetic N-linked glycopeptides1 have become valuable tools
for glycobiologists when studying the biological properties of com-
plex glycoconjugates including glycoproteins. Many methods have
been established over the years in order to provide the desired gly-
cosyl amides. The convergent assembly of glycosyl amino acids and
N-glycopeptides can be accomplished by coupling the appropriate
derivatives of aspartic acid with glycosyl amines,2,5 glycosyl iso-
thiocyanates,3 or pentenyl glycosides.4 Other amide forming reac-
tions comprise Staudinger ligations6,7 or the coupling of thioacids
with amines,8 isonitriles,9 or azides.10 Even though these methods
are valuable in many cases, additional methods for the stereoselec-
tive formation of glycosyl amides are desired. Recently, a chemose-
glucosyl hydroxylamine 1 in DMF at 60 °C. After 1 day the reaction
products were peracetylated and isolated by flash chromatography
(Scheme 1). We were pleased to find that the major product 4
(62%) contained an anomeric acetamide, indicating that also the
sugar hydroxylamine 1 can be employed as a substrate in a decarb-
oxylative condensation reaction. Notably, only the b-configured
acetamide was found. Due to the slow hydrolysis of 1 some per-
acetylated glucose (24%) was also found.
Since the stability of the labile glucosyl hydroxylamine 1 under
the reaction conditions was unclear we sought for more details of
the amidation process by in situ monitoring the reaction of 1 and 2
in DMSO-d6 in an NMR tube at 40 °C. When hydroxylamine 1 was
dissolved in DMSO-d6 rapid equilibration of the isomers 1, 1E, and
1Z occurred with only 6% of 1 still detectable after 1 h. After 48 h a
ratio of 1E/1Z/1 = 79.3:20.1:0.6 was found. However, upon addi-
tion of pyruvic acid 2 (1.2 equiv) to freshly dissolved 1 the signals
of the cyclic hydroxylamine 1 disappeared immediately and only
oximes 1E and 1Z could be detected (E/Z = 85:15, Scheme 2).
lective amidation by decarboxylative condensation of a-ketoacids
and hydroxylamines was developed and used for the chemoselec-
tive ligation of protected and unprotected peptide fragments
(KAHA ligation).11 We reasoned that this reaction might also be
applicable to anomeric hydroxylamines of carbohydrates. In this
Letter we demonstrate that decarboxylative condensation of
a-ketoacids and glycosyl oximes leads to glycosyl amides and
N-glycopeptides.
As a model compound for glycosyl hydroxylamines we selected
glucosyl-b-hydroxylamine 112 since it appears to be a rare example
where a crystalline compound (1) readily precipitates from a
methanolic solution of the sugar and free hydroxylamine. Pyruvic
O
CH3
HO
Ac2O
pyridine
OH
OH
OAc
O
O
2
O
O
H
N
H
N
HO
HO
HO
HO
AcO
AcO
NHOH
CH3
CH3
acid 2 was chosen as a small
a-ketoacid and was incubated with
OH
OH
OAc
DMF, 60 °C
O
O
1
3
4 (62 %)
⇑
Corresponding author. Tel.: +49 921 552670; fax: +49 921 555365.
E-mail address: carlo.unverzagt@uni-bayreuth.de (C. Unverzagt).
Scheme 1. Chemoselective amidation of glucosyl hydroxylamine 1 with pyruvic
acid 2.
http://dx.doi.org/10.1016/j.tetlet.2014.02.056
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

2198
C. Pöhner et al. / Tetrahedron Letters 55 (2014) 2197–2200
monitored by 1H NMR in DMSO-d6 (SI, Figs. 1–3). When the N-acet-
OH
yl glucosamine oxime 613 (E/Z = 4:1 + traces of cyclic hydroxyl-
amine in DMSO-d6) was reacted with pyruvic acid 2 at 40 °C in
DMSO-d6 only the b-linked acetamide 7 was found. In contrast
OH
N
OH
OH
HO
HO
OH
OH
DMSO-d6
1Z
O
O
HO
HO
HO
the galactose oxime 814–16gave rise to a mixture of anomers (
a/
NHOH
NHOH
HO
OH
OH
OH
b = 1:0.85). Similarly, oxime 10 of mannose,12,14,15 which required
OH
OH
HO
HO
higher reaction temperatures, resulted in a mixture of anomers
1
(crystalline)
1
N
OH
(a/b = 1:1) and showed only low conversion (Scheme 4).
1E
Since the decarboxylative condensation of sugar oximes yields
anomerically modified glycans we sought to apply this method
to the synthesis of carbohydrates modified with a chemically sta-
ble spacer. By means of an incorporated azido group numerous
conjugation methods should be applicable to the spacered prod-
ucts. 6-Azido hexanoic acid was converted to the sulfur ylide 12
NMR ratios in DMSO-d6:
pH 7 after 2 d: 1 / 1E / 1Z = 0.6 : 79.3 : 20.1
pH ~ 2-3 after 1 min (1 + 1.2 eq. pyruvic acid): 1 / 1E / 1Z = 0 : 85 : 15
Scheme 2. Isomerization of glucosyl hydroxylamine 1 in DMSO-d6 under neutral
and acidic conditions.
and converted to the
a
-ketoacid 13 by a mild oxone treatment.17
The sugar oximes 1 and 6 were reacted with spacer 13 in a DMF/
The mixture of 1 and 2 in DMSO-d6 was warmed to 40 °C and
periodical monitoring of the reaction progress by 1H NMR showed
the slow formation of the desired amide 3 accompanied by some
free glucose (Scheme 3). The ratio of the signals corresponding to
the open chain sugar oximes 1E and 1Z remained constant over
the course of the reaction.
pyridine mixture, which was found to reduce the competing trans-
oximation of the a-ketoacid (SI, Figs. 7, 8). The spacered products
14 and 15 were obtained stereoselectively and were readily iso-
lated by flash chromatography in yields of 60% and 71% respec-
tively (Scheme 5).
Encouraged by these results, we investigated the use of unpro-
tected sugar oximes in the convergent assembly of N-glycopep-
tides (Scheme 6). To this end tripeptide 16 was assembled on the
Although hydroxylamine 1 rapidly isomerizes to open-chain
oximes 1E and 1Z in DMSO-d6 under acidic conditions the b-con-
figured amide 3 was selectively formed. Despite the unfavourable
equilibrium it can be assumed that the reaction proceeds via cyclic
b-configured hydroxylamine 1. Different solvents and mixtures
were tested to identify the best reaction conditions. DMF was
found to give the highest yields followed by DMSO, NMP, and pyr-
idine. When triethylamine was added the yields dropped dramat-
ically and aqueous mixtures gave no product due to competing
hydrolysis to glucose 5 (SI, Table 1). No amide product 3 was ob-
served when the glucose oximes 1E/Z were replaced by the corre-
sponding glucose methoxime in the condensation reaction.
In the case of other monosaccharides (N-acetylglucosamine,
galactose, mannose) only the readily available oximes were used
in decarboxylative condensations. In all cases the rapid equilibra-
tion of open chain oximes appeared to provide sufficient amounts
of cyclic hydroxylamines required for the progress of the
amidation. The reaction of the different sugar oximes with 2 was
solid phase and oxidized to the corresponding
a-ketoacid follow-
ing the Wasserman method.18 However, subsequent incubation
with glucosyl hydroxylamine 1 did not yield the desired glycopep-
tide. Recent work by Giese19 showed that peptides bearing an
aspartate derived
a-ketoester side chain readily undergo backbone
cyclization under basic and acidic conditions suggesting that the
a-
ketoacid intermediate cyclized to an unreactive lactam (17). Thus,
BocAspNHMe20 was converted to model compound 18, which was
oxidized and purified. Instead of ketoacid 19 a mixture of the cyclic
hemiaminals 20 a,b was obtained (Scheme 6).
In peptide synthesis cyclization of aspartates leading to asparti-
mides is a general problem. In contrast glutarimides do not readily
form.21 Thus we assumed that the backbone cyclization might also
be reduced for glutamate over aspartate derived ketoacids. This ap-
proach would give rise to analogs of natural N-glycopeptides
where the sugar moiety is attached to the side chain of a glutamic
acid. In order to also improve the purity of the
a-ketoacids, we
O
O
HO
2
OH
OH
O
HO
2
OH
OH
OH
O
O
H
N
O
HO
HO
HO
HO
NHOH
O
H
N
HO
HO
HO
HO
OH
OH
DMSO-d6, 40 °C
N
OH
O
1
3
NHAc
NHAc
DMSO-d6, 40 °C
O
7
6
OH
OH
HO
HO
HO
HO
2
O
OH
OH
H
N
N
OH
OH
OH
DMSO-d6, 40 °C
O
O
9
8
OH
OH
OH
OH
OH
2
O
HO
HO
HO
HO
H
N
N
DMSO-d6, 60 °C
10
11
Scheme 4. Decarboxylative condensation of various sugar oximes with pyruvic
acid 2.
Scheme 3. Time course of product formation (3) in DMSO-d6 at 40 °C.

C. Pöhner et al. / Tetrahedron Letters 55 (2014) 2197–2200
2199
THF/H2O 2:1, rt.
oxone
With the sulfur ylide building block 21, tetrapeptide 22 was
synthesized by SPPS (84%). After mild oxidation with oxone the
O
S
HO
HO
N3
a
-ketoacid containing peptide 23 was reacted with GlcNAc oxime
NC
HO
N3
O
613 in DMF. The stereoselectively N-glycosylated tetrapeptide 24
was obtained in 7% yield. HPLC-MS revealed that ketoacid 23
was converted to the oxime side product 23o by liberated hydrox-
O
12
13
OH
OH
ylamine. Since the
a-ketoacid leads to acidic reaction conditions,
13
O
H
N
O
the hydrolysis of sugar oximes by water liberated by the
HO
NHOH
HO
N3
DMF/pyridine 3:1
60 °C
OH
OH
O
1
14 (60 %)
OH
OH
HO
OH
OH
OH
OH
OH
OH
O
OH
OH
O
O
HO
O
O
O
13
O
O
HO
OH
OH
25
N
OH
O
O
HO
O
OH
H
N
HO
HO
OH
NHAc
HO
OH
HO
HO
NHAc
OH
N
OH
N3
DMF/pyridine 3:1
60 °C
NHAc
15 (71 %)
6-azidohexanoyl spacer by decarboxylative
NHAc
O
a or b
23
6
O
O
H
N
H
N
Scheme 5. Introduction of
a
BocHN
N
H
OH
condensation.
O
OH
O
OH
OtBu
OH
HO
OH
OH
OH
OH
OH
O
O
O
HO
O
O
O
O
O
HO
O
O
O
HO
OH
O
N
a) O3, - 35 °C
DCM/HFIP 98:2
b) H3CCN/H2O
NHAc
OH
HO
NHAc
OH
H
O
OH
CN
26
HO
O
COOH
PPh3
O
O
Scheme 8. Coupling of tetrapeptide ketoacid 23 and oxime 25: (a) DMF, 40 °C (26:
4%); (b) DMF/pyridine 3:1, 60 °C (26: 30%).
H
FmocHN
N
COOH
FmocHN
N
COOH
N
H
N
H
O
16
17
HO
BocHN
NHBoc
O
O
NH
a) O3, - 35 °C
DCM/HFIP 98:2
b) H3CCN/H2O
O
tBuO
O
O
O
O
BocHN
Me
BocHN
Me
H
N
N
N
H
N
BocHN
Me
H
O
N
FmocHN
N
H
O
S
O
O
OH
HOOC
PPh3
NC
O
HO
27
19
20 a,b
18
NC
O
Scheme 6. Cyclization of aspartic acid derived
a
-ketoacids.
a
Fmoc-SPPS
HO
HO
BocHN
NHBoc
BocHN
THF/H2O 2:1, rt.
oxone
O
O
O
O
NHBoc
NH
NH
O
O
O
H
O
H
N
H
N
H
N
O
O
N
N
H
tBuO
tBuO
O
BocHN
N
H
OH BocHN
OtBu
OH
OtBu
O
H
N
H
O
O
O
O
N
N
N
FmocHN
N
H
FmocHN
N
S
O
O
O
O
O
O
O
O
22
23
OH
OH
CN
OH
COOH
HO
HO
HO
a or b
N
NHAc
OH
28
28c
HO
b
6
O
O
O
O
O
+
H
H
H
N
H
HO
N
N
N
BocHN
NHBoc
BocHN
N
OH
OtBu
HO
BocHN
OH
N
H
OH
O
O
H
O
O
O
O
OtBu
NH
O
O
NOH
23o
O
24
NH
NHAc
tBuO
O
O
HO
OH
H
N
N
FmocHN
N
H
Scheme 7. Glycopeptide formation with glutamic acid based tetrapeptide ketoacid
23: (a) DMF, 40 °C (24: 7%); (b) DMF/pyridine 3:1, 60 °C (24: 23 %).
O
O
HO
HO
H
N
O
HO
OH
O
selected sulfur ylide derivative 21 of glutamic acid,17 which can be
oxidized readily with oxone.22 This generally led to much higher
overall yields of ketoacids compared to the ozonolysis of phospho-
rous ylides (Scheme 7).
29
Scheme 9. Oxime coupling with reduced cyclization using pseudoproline building
block 28: (a) THF/H2O 2:1, rt, oxone; (b) N-glucosyl hydroxylamine 1, DMF/pyridine
3:1, 60 °C (29: 56%).

2200
C. Pöhner et al. / Tetrahedron Letters 55 (2014) 2197–2200
decarboxylative condensation is facilitated. Different solvent addi-
tives were tested revealing that oxime formation could be reduced
significantly by adding 25% of pyridine to the solvent. These condi-
tions also tolerated higher reaction temperatures (60 °C) giving
GlcNAc containing tetrapeptide 24 in 23% yield. A small amount
of nitrile side product was found, presumably via decarboxylative
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.02.
056.
dehydration of the a
-ketoacid oxime.23
References and notes
The decarboxylative ketoacid-oxime coupling was carried out
with the oxime of lacto-N-neohexaose 2524 (NMR ratio in D2O: E/
Z/cyclic form = 7:1.5:1.5) and peptide 23 (Scheme 8). Using DMF
as a solvent, only 4% of 26 were obtained, whereas in DMF/pyridine
3:1 the yield increased to 30%.
1. (a) Herzner, H.; Reipen, T.; Schultz, M.; Kunz, H. Chem. Rev. 2000, 100, 4495–
4537; (b) Buskas, T.; Ingale, S.; Boons, G.-J. Glycobiology 2006, 16, 113R–136R.
2. (a) Anisfeld, S. T.; Lansbury, P. T., Jr. J. Org. Chem. 1990, 55, 5560–5562; (b)
Cohen-Anisfeld, S. T.; Lansbury, P. T., Jr. J. Am. Chem. Soc. 1993, 115, 10531–
10537.
3. Günther, W.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1990, 29, 1050–1051.
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5. Kaneshiro, C. M.; Michael, K. Angew. Chem., Int. Ed. 2006, 45, 1077–1081.
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Soc. 2011, 133, 1597–1602.
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We then reevaluated the synthesis of glycopeptides with native
linkages by looking for a means to reduce the tendency of the
a-
ketoacid side chain to form cyclic lactams (as in Scheme 6). Start-
ing from sulfur ylide Asp peptide 27 we tested if a pseudoproline25
protection of the consensus sequence Thr moiety might be affect-
ing the cyclization behavior of the resulting ketoacid 28 (Scheme 9).
Although the cyclization was not completely inhibited (HPLC ratio:
ketoacid 28/cyclic lactam diastereomers 28c 57:43), it was possible
to reduce the cyclic product significantly. The
a-ketoacid peptide
28 was coupled with hydroxylamine 1 in DMF/pyridine 3:1. The
corresponding glycopeptide 29 was obtained in a yield of 56% after
purification. The protective conformational effect of pseudopro-
lines at consensus sequence Ser/Thr moieties was further exploited
for the synthesis of N-glycopeptides via Lansbury aspartylation.25
14. Beer, D.; Bieri, J. H.; Macher, I.; Prewo, R.; Vasella, A. Helv. Chim. Acta 1986, 69,
1172–1190.
15. Jacobi, H. Chem. Ber. 1891, 24, 696–699.
16. Rischbieth, P. Chem. Ber. 1887, 20, 2673–2674.
17. Ju, L.; Lippert, A. R.; Bode, J. W. J. Am. Chem. Soc. 2008, 130, 4253–4255.
18. Wasserman, H. H.; Ho, W.-B. J. Org. Chem. 1994, 59, 4364–4366.
19. Seufert, W.; Fleury, A.; Giese, B. Synlett 2006, 1774–1776.
20. Hernández-Gay, J. J.; Ardá, A.; Eller, S.; Mezzato, S.; Leeflang, B. R.; Unverzagt,
C.; Cañada, F. J.; Jiménez-Barbero, J. Chem. Eur. J. 2010, 16, 10715–10726.
21. Kates, S. A.; Albericio, F. Lett. Pept. Sci. 1994, 1, 213–220.
22. Kennedy, R. J.; Stock, A. M. J. Org. Chem. 1960, 25, 1901–1906.
23. Ahmad, A.; Spenser, I. D. Can. J. Chem. 1961, 39, 1340–1359.
24. Priem, B.; Gilbert, M.; Wakarchuk, W. W.; Heyraud, A.; Samain, E. Glycobiology
2002, 12, 235–240.
Conclusions
In summary we established a new method for the synthesis of
N-glycosyl amides and N-glycopeptides by decarboxylative con-
densation. In this approach unprotected sugar oximes are reacted
with a-ketoacids in dry organic solvents. Activation of the compo-
25. Ullmann, V.; Rädisch, M.; Boos, I.; Freund, J.; Pöhner, C.; Schwarzinger, S.;
Unverzagt, C. Angew. Chem., Int. Ed. 2012, 51, 11566–11570.
nents is not required, and a b-N-glycosidic linkage is selectively ob-
tained from gluco-configured sugars. The use of sugar oximes may
provide a useful alternative to glycosylamines in the synthesis of
N-glycopeptides and other N-acylated sugars and oligosaccharides.
Acknowledgments
We are grateful for the support by the Deutsche Forschungs-
gemeinschaft, the Fonds der Deutschen Chemischen Industrie
and the European Union.