Preparation of pseudo glycoamino acid and its application to glycopeptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2011.09.121

A pseudo glycoamino acid composed of a 1,3-diol structure and an amino acid was synthesized. This amino acid analog can act as an alternative acceptor to an amino acid containing GlcNAc for transglyco-sylation by Endo-M. A pseudo glycopeptide was synthesi

Full text of DOI:10.1016/j.tetlet.2011.09.121

Tetrahedron Letters 52 (2011) 6504–6507
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journal homepage: www.elsevier.com/locate/tetlet
Preparation of pseudo glycoamino acid and its application to
glycopeptide synthesis
⇑
Yusuke Tomabechi, Toshiyuki Inazu
Department of Applied Chemistry, School of Engineering, Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A pseudo glycoamino acid composed of a 1,3-diol structure and an amino acid was synthesized. This
amino acid analog can act as an alternative acceptor to an amino acid containing GlcNAc for transglyco-
sylation by Endo-M. A pseudo glycopeptide was synthesized using the pseudo glycoamino acid by a solid
phase procedure. We attempted transglycosylation of N-glycan to the peptide using Endo-M.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 8 August 2011
Revised 22 September 2011
Accepted 26 September 2011
Available online 1 October 2011
Keywords:
Endo-M
Transglycosylation reaction
Glycoconjugates
Pseudo glycoamino acid
Glycopeptides
Oligosaccharides on cell surfaces play important roles in biolog-
ical processes such as cell–cell interactions, cell adhesion, and
immunogenic recognition.1 These glycoconjugates are generated
by cells through a complicated synthesis. In addition, there is a
growing demand for chemo-enzymatic synthesized O- and
N-glycans2 because their peptide conjugates act as useful probes
for studying glycoprotein-mediated biological reactions. Moreover,
these glycopeptides may have therapeutic uses because it has been
reported that addition of carbohydrates could potentiate the effects
of drugs.3 Glycoamino acid is a key compound in glycopeptide and
glycoprotein syntheses. Unfortunately, glycoamino acid synthesis
is also a lengthy and complex process.
Endo-b-N-acetylglucosaminidase (ENGase, EC 3.2.1.96) is used
for catalyzing the hydrolysis of the b-(1-4)-glycosidic linkages
between N,N0-diacetylchitobiose moiety of N-glycans. This enzyme
is isolated from Mucor hiemalis (Endo-M) and is unique in that it
can act on all types of N-glycans including high-mannose, complex,
and hybrid-type oligosaccharides, catalyzing the hydrolysis and
transglycosylation of appropriate acceptors containing N-acetylglu-
cosamine (GlcNAc) residues.4 Consequently, Endo-M is an effective
tool for reconstructing and remodeling glycopeptides and glycopro-
teins.5 Further efforts are necessary for optimizing the transglycosy-
lation efficiency of the enzyme.6 Moreover, the transglycosylation
reaction was limited and could not be promoted by the enzyme in
the absence of GlcNAc (sugar) as an acceptor.
that the oxygen atom on the pyranose ring was not essential for its
activity; however, the narrow regions of the 1,3-diol structure from
4-OH to 6-OH of GlcNAc, primary, and secondary hydroxyl groups,
were essential for transglycosylation using Endo-M. Furthermore,
we determined that a functional group causing significant steric ef-
fects resided in the C-5 position of GlcNAc. This group interrupted
transglycosylation.
Based on these results, we designed the amino acid containing
1,3-diol structure that can be modified with a sugar chain using
Endo-M. To connect a 1,3-diol moiety to the amino groups of a sub-
strate by an amide linkage, we designed the 1,3-diol structure with
a carboxylic acid group and a ‘1,3-diol tag’ according to the previous
report (Fig. 1). This amino acid analog can act as an alternative
acceptor to an amino acid containing GlcNAc. Consequently, we
refer to the compound as a ‘pseudo glycoamino acid’.
The pseudo glycoaminoacid was prepared from a 1,3-diol tag and
an amino acid with an amino group side chain, such as Fmoc-
Lys(1,3-diol tag)-OH (1), Fmoc-Orn(1,3-diol tag)-OH (2), and
Fmoc-Dab(1,3-diol tag)-OH (3) (Fig. 2). Next, the pseudo glycoamino
acid was used in solid phase synthesis and a pseudo glycopeptide
was synthesized. Lastly, we attempted transglycosylation of
complex-type oligosaccharide blocks using Endo-M for obtaining
the pseudo glycopeptide.
First, the 1,3-diol tag was synthesized on the basis of proce-
dures in the literature (Scheme 1).8 The 1,3-diol tag derivative 4
We have studied the acceptor recognition site of Endo-M in de-
tail and previously reported its structure.7 Our work demonstrated
OH
HO
CO
⇑
Corresponding author.
E-mail address: inz@keyaki.cc.u-tokai.ac.jp (T. Inazu).
Figure 1. Structure of ‘1,3-diol tag’.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.09.121

Y. Tomabechi, T. Inazu / Tetrahedron Letters 52 (2011) 6504–6507
6505
was prepared from
D
-malic acid. The acid was reacted with triflu-
OH
H
N
(
oroacetic anhydride, subsequently the cyclic anhydride was
opened by MeOH. Carboxylic acid 5 was reduced to a hydroxyl
group using BH3–THF, and thereby producing a dihydroxy deriva-
tive 6. Two hydroxyl groups of 6 were protected by a benzylidene
group using benzaldehyde dimethyl acetal and a catalytic amount
of p-toluenesulfonic acid, thereby producing 7 in 63% yield (4
steps). The methyl ester of 7 was hydrolyzed by Na2CO3 solution
to obtain the desired product 4. Product 4 was used without fur-
ther purification.
HO
)
n
O
Fmoc
OH
N
H
O
1 :n = 3 (AA = Lys)
2 :n = 2 (AA = Orn)
:n = 1 (AA = Dab)
3
Figure 2. Structure of Fmoc-AA(1,3-diol tag)-OH.
N-a-Benzyloxycarbonyl-lysine a-benzyl ester (Z-Lys-OBn) and
4 were condensed using 1H-benzotriazol-1-yl-oxy-tris(pyrrolidi-
no)phosphonium hexafluorophosphate (PyBOP), producing the
amide derivative 8 in 80% yield (Scheme 2). The benzyl, benzyloxy-
carbonyl, and benzylidene groups of 8 were removed by hydroge-
nation in the presence of 10% Pd/C. The corresponding compound
was reacted with N-(9-fluorenylmethoxycarbonyl) succinimide
(FmocOSu) for obtaining the desired product 1 in 62% yield (2
steps). Pseudo glycoamino acids 2 and 3 were synthesized in the
same manner as 1.
OH
O
OH
O
OH
c
a, b
OMe
OH
OMe
ONa
HO
HO
d
HO
e
O
O
O
6
5
Ph
Ph
O
O
O
O
OMe
Next, transglycosylation of the complex-type oligosaccharide
block to 1 was attempted using Endo-M. Using 1 as the glycosyl
acceptor, transglycosylation of a complex-type oligosaccharide
block [(NeuAc-Gal-GlcNAc-Man)2-Man-GlcNAc–] with sialylglyco-
peptide (SGP) as the glycosyl donor was examined in the presence
of Endo-M under the same reaction conditions as described previ-
ously.7 The reversed-phase high-performance liquid chromatogra-
phy (RP-HPLC) profile of transglycosylation of SGP to 1 after 2 h is
shown in Figure 3. Characterization of the product was performed
by matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS), and the transglycosylated product
was confirmed.
The time course of transglycosylation of 1, 2, and 3 is shown in
Figure 4. The highest yield using 1 as an acceptor was 26% after 2 h.
Glycosylated 2 and 3 gave maximum yields of 14% and 6%, respec-
tively. These data showed that differences in the amino acid side
chain length strongly influenced the transglycosylation yield.
Moreover, these results indicated that the transglycosylation rate
was so low that it probably shortened the length of the alkyl amino
acid side chain. We also plan to further study transglycosylation to
longer side chain derivatives. When the GlcNAc derivative was
used as an acceptor, the transglycosylated product reached 18%
O
O
7
4
Scheme 1. Preparation of the ‘1,3-diol tag’ derivative 4. Reagents and conditions:
(a) (CF3CO)2O, rt; (b) MeOH, rt; (c) BH3–THF, 0 °C to rt; (d) PhCH(OMe)2, p-TsOH,
DMF, 50 °C, 63% (4 steps); (e) Na2CO3, dioxane–water (1:1), rt.
Ph
O
O
Z
OH
H
N
H
N
H2N
HO
n
n
n
O
O
a
b, c
Z
OBn
OBn
Fmoc
OH
N
H
N
H
N
H
O
O
O
n = 3
n = 2
n = 1
8
9
1: n = 3 62%
: n = 2 89%
3: n = 1 68%
: n = 3 80%
2
: n = 2 74%
10: n = 1 81%
Scheme 2. Preparation of pseudo glycoamino acids 1–3. Reagents and conditions:
(a) 4, PyBOP, Et3N, DMF, rt; (b) H2 atmosphere, Pd/C, AcOH, rt; (c) FmocOSu, Na2CO3,
dioxane–water (1:1), rt.
Figure 3. RP-HPLC profile of the reaction mixture incubated with Fmoc-Lys(1,3-diol tag)-OH (1) and SGP in the presence of Endo-M for 2 h, as detected by absorbance at
280 nm. The large peak at 18–19 min corresponds to the residual acceptor 1.

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Y. Tomabechi, T. Inazu / Tetrahedron Letters 52 (2011) 6504–6507
Figure 6. RP-HPLC profile of the reaction mixture incubated with [Lys(1,3-diol
tag)5]-vapreotide (11) and SGP in the presence of Endo-M after a reaction time of
8 h at an absorbance of 254 nm. The large peak at 18–19 min corresponds to
residual amounts of acceptor 11.
Figure 4. Time course of the transglycosylation reaction using Endo-M. d: Fmoc-
Lys(1,3-diol tag)-OH (1), N: Fmoc-Orn(1,3-diol tag)-OH (2), j: Fmoc-Dab(1,3-diol
tag)-OH (3), ꢀ:Fmoc-Asn(GlcNAc)-OH.
1,3-OH tag
H-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2
Figure 5. Structure of [Lys(1,3-diol tag)5]-vapreotide (11).
Fmoc-NH-Resin
Resin: Rink amide resin
1) 20% Piperidine/ NMP
2) Fmoc-AA-OMpt (4 equiv.) X2
-Phe-Cys(Acm)-Tyr(tBu)- -Trp(Boc)-Lys("1,3-diol tag")-Val-
H-
D
D
Cys(Acm)-Trp(Boc)-NH-Resin 12
1) TFA : Phenol : H2O : EDT : TIS = 81.5 : 5 : 5 : 2.5 : 1
2) HPLC
13
D-Trp-Lys("1,3-diol tag")-Val-Cys(Acm)-Trp-NH2( )
H-D
-Phe-Cys(Acm)-Tyr-
Yield 17% MALDI-TOF MS Calcd for C67H88N14O14S2Na
[M+Na]+ 1399.6.
Found: m/z 1399.6.
AgNO3, DIEA, H2O, DMSO
H-D-Phe-Cys(Ag)-Tyr-D-Trp-Lys("1,3-diol tag")-Val-Cys(Ag)-Trp-NH2
1) 1M HCl-DMSO
2) HPLC
5
Figure 7. Time course of transglycosylation using Endo-M. j: [Lys(1,3-diol tag)5]-
vapreotide (11), d: Fmoc-Lys(1,3-diol tag)-OH (1).
11
[Lys("1,3-diol tag") ]-Vapreotide(
)
Yield 40% MALDI-TOF MS Calcd for C61H77N12O12S2
[M+H]+ 1233.5.
Found: m/z 1233.2.
Peptide 13 was then dissolved in DMSO, and a solution of AgNO3
and DIEA in H2O was added.11 Next, a mixture of 1 M HCl and DMSO
(1:1, v/v) was added to the reaction mixture, and [Lys(1,3-diol
tag)5]-vapreotide (11) was obtained in 40% yield from 13.
Next, transglycosylation was performed using pseudo glycopep-
tide 11 as the acceptor, SGP as the glycosyl donor, and Endo-M (
Fig. 6). Surprisingly, synthesis from 11 gave 28% yield of the trans-
glycosylated product after 4 h (Fig. 7). The bioactivities of vapreo-
tide and the transglycosylated product were similar.12
Scheme 3. Preparation of [Lys(1,3-diol tag)5]-vapreotide (11).
yield after 1 h. These results suggested that an efficient acceptor
was required for flexibility. This demonstrates that transglycosyla-
tion activity of 1 is superior to that of the GlcNAc derivative.
The best performing pseudo glycoamino acid 1 was used in the
solid-phase synthesis of the glycopeptide analog 11 of vapreotide
(Fig. 5).9
Peptide resin 12 was prepared from Rink Amide Resin™ using an
Fmoc strategy by the dimethylphosphinothioic mixed anhydride
(Mpt–MA) method (Scheme 3).10 Resin 12 was treated with a
mixture of trifluoroacetic acid (TFA)/phenol/water/ethanedithiol
(EDT)/triisopropylsilane (TIS) = 81.5:5:5:2.5:1 for cleaving the resin
peptide and removing the side chain protecting groups. Following
this, crude peptide 13 was obtained. Purification of the peptide by
RP-HPLC gave 13 in 17% yield (based on the amino-acid analysis).
Studies of further application of the pseudo glycopeptide meth-
od are now in progress.
Acknowledgments
The authors thank Dr. Keiko Kuwata (Kyoto University) for
HRMS analysis. We also thank Professor Hironobu Hojo for amino
acid analysis and optical rotation measurement and Dr. Yasuhiro

Y. Tomabechi, T. Inazu / Tetrahedron Letters 52 (2011) 6504–6507
6507
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Kuroda, Professor Naoya Kojima, Associate Professor Kazuya
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Chemical Industry Co., Ltd, for providing Endo-M and SGP. Part of
this work was supported by grants from the Sasakawa Scientific
Research Grant from The Japan Science Society, Hi-Tech Research
from Tokai University, and Research and Study Project Funding
from the Tokai University Educational System General Research
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