Molecular transporter between polymer platforms: Highly efficient chemoenzymatic glycopeptide synthesis by the combined use of solid-phase and water-soluble polymer supports

Article abstract of DOI:10.1002/anie.200463065

(Figure Presented) The molecule standing at platform one ...: Rapid glycopeptide synthesis is achieved with a heterobifunctional "molecular transporter" (see scheme), which interfaces two types of polymer platforms for combined chemical and enzymatic synt

Full text of DOI:10.1002/anie.200463065

Communications
Glycopeptide Synthesis
regioselective protection and stereoselective glycosylation
reactions. Although progress in solid-phase chemical syn-
thesis meanwhile allowed the construction of a variety of
oligosaccharides,[3] these methods still entail limitation of the
target structures and technical difficulty for general biochem-
ists or medical scientists. Enzymatic synthesis is a potential
alternative to the chemical synthesis of complex oligosac-
charides because of the specificities of both the stereochem-
istry and regioselectivity in the glycosylation reactions.[4]
However, glycosyl acceptor substrates immobilized on solid
supports are not suited for these enzymatic reactions in terms
of efficiency and versatility in practical synthesis.
In the course of our studies of enzymatic synthesis, based
on the cluster effect[5] of sugar-attached water-soluble poly-
mers as multivalent acceptor substrates,[6] our interest has
been focused on the efficient synthesis of glycopeptides as
important signal molecules in cellular recognition.[7] To
achieve a concerted and efficient glycopeptide synthesis
based on a combined chemical and enzymatic strategy, we
thought that the advent of an appropriate methodology to
combine solid-phase peptide synthesis and liquid-phase
carbohydrate synthesis was highly desirable.[4] Herein, we
report a novel strategy of rapid and efficient synthesis of
glycopeptides by using a convenient “molecular transporter”
that interfaces two different polymer supports.
Our synthetic strategy is summarized in Figure 1 as
follows: a) solid-phase synthesis of the photosensitive O-
GlcNAc-peptides terminated by the molecular transporter 1,
b) deprotection and release of the transporter from the resin,
c) chemoselective blotting of the molecular transporter that
carries glycopeptide primers using a water-soluble polymer
with alkoxyamino functional groups,[8] d) one-pot sugar
elongation with glycosyltransferases, and e) release of full-
length glycopeptides from the transporter on the polymer
platform with a photoselective cleavage reaction.
The molecular transporter 1 was synthesized from l-
proline, which was to be used as the N-terminal residue of the
target peptide, by modification with a reactive ketone group
with a photolabile linker moiety, 4-[4-(1-hydroxyethyl)-2-
methoxy-5-nitrophenoxy]butyric acid[9] (see the Supporting
Information).[10] This heterobifunctional linker acts as a trans-
porter between the two different polymer platforms and allows
both chemoselective blotting and photoselective cleavage
(catch and release) of glycopeptides (Figure 1, steps c and e).
We demonstrated the feasibility of our method by
constructing a dodecapeptide with a sialyl LewisX tetrasac-
charide residue (7) as a model compound. Scheme 1 shows
the solid-phase synthesis of a transporter molecule carrying
an intermediate glycopeptide (3) on a Fmoc-Arg(Pbf)-
NovaSynTGA resin using Fmoc-protected amino acids
(Fmoc-AA) and Fmoc-Ser(Ac3GlcNAcb)-OH.[11] Next, the
intermediate on the transporter (3) was blotted onto the
water-soluble polyacrylamide derivative 4[8] by chemoselec-
tive ligation of the ketone group of 3 with the alkoxyamino
group of 4 without any purification process. We suggest that
this blotting step using polymer 4, which we proved is
completed in three hours at room temperature under mild
conditions (pH 5.0–5.5) by monitoring with reversed-phase
(RP) HPLC (Figure 2), is crucial. As anticipated, polymer 4
Molecular Transporter Between Polymer
Platforms: Highly Efficient Chemoenzymatic
Glycopeptide Synthesis by the Combined Use of
Solid-Phase and Water-Soluble Polymer
Supports**
Masataka Fumoto, Hiroshi Hinou,
Takahiko Matsushita, Masaki Kurogochi, Takashi Ohta,
Takaomi Ito, Kuriko Yamada, Akio Takimoto,
Hirosato Kondo, Toshiyuki Inazu, and Shin-
Ichiro Nishimura*
Polymer-supported synthesis is a practical and convenient
method because it simplifies purification of the final products
and makes combinatorial processes feasible. Chemical syn-
thesis on solid-phase polymers made the automated synthesis
of nucleotides (DNA/RNA) and peptides (proteins) possi-
ble,[1,2] and they are now indispensable devices for the
investigation of the functional roles of genomes and proteins
as well as the development of a variety of therapeutic
reagents. Chemical synthesis of glycoconjugates, however, is a
much more difficult task than the synthesis of nucleic acids or
polypeptides because complex structures of glycoconjugates
require extremely time-consuming and tedious procedures of
[*] Dr. H. Hinou, Dr. T. Ohta, Prof. S.-I. Nishimura
Glycochemosynthesis Team, Research Center for Glycoscience
National Institute of Advanced Industrial Science and Technology
(AIST)
Sapporo 062-8517 (Japan)
Fax: (+81)11-857-8441
E-mail: tiger.nishimura@aist.go.jp
T. Matsushita, Dr. M. Kurogochi, Prof. S.-I. Nishimura
Division of Biological Sciences, Graduate School of Science
Hokkaido University, Sapporo 001-0021, Japan
E-mail: shin@glyco.sci.hokudai.ac.jp
M. Fumoto, Dr. K. Yamada
Research Association for Biotechnology
Minato-ku, Tokyo 105-0003 (Japan)
M. Fumoto, T. Ito, Dr. A. Takimoto, Dr. H. Kondo
Shionogi & Co. Ltd.
Chuo-ku, Osaka 541-0045 (Japan)
Dr. K. Yamada
Hitachi High-Technologies Co.
Minato-ku, Tokyo 105-8717 (Japan)
Dr. T. Inazu
Department of Applied Chemistry, School of Engineering,
and Institute of Glycotechnology
Tokai University, Kanagawa 259-1292 (Japan)
[**] This work was partly supported by a grant for “Development of
Methodologies and Databases for Structural Glycoproteomics”
from the New Energy and Industrial Technology Development
Organization (NEDO). We are grateful to Professor Y. Nakahara of
Tokai University for valuable suggestions and comments. We also
thank Ms. S. Oka, Ms. M. Kiuchi, and Mr. T. Hirose of the Center of
Instrumental Analysis, Hokkaido University, for mass spectrometric
measurements and amino acid analysis.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200463065
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Chemie
Figure 1. Glycopeptide synthesis using molecular transporter 1.
Scheme 1. a) 1. Piperidine, DMF; 2. Fmoc-AA or Fmoc-Ser(Ac3GlcNAcb)-OH or 1, HBTU, HOBt, ethyldiisopropylamine, N-methylpyrrolidone/
DMF; 3. Ac2O, HOBt, ethyldiisopropylamine, DMF; b) 90% trifluoroacetic acid in water; c) NaOH, MeOH; d) AcOH/AcONa buffer (pH 5.0–5.5).
DMF=dimethylformamide; HBTU=O-(benzotriazol-l-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; HOBt=1-hydroxy-lH-benzotriazole.
À
Pbf=2,2,5,7,8-pentamethyldihydrobenzofurane-5-sulfonyl; Fmoc=9-fluorenylmethoxycarbonyl; R’=(CH2)6 polyacrylamide.
captured only the full-length peptide 3, since free amino
groups of the incomplete peptides were capped by acetylation
prior to the removal of the Fmoc groups. After one-pot sugar
elongation reactions by recombinant glycosyltransferases in
the presence of sugar nucleotides as glycosyl donor substrates,
selective cleavage reaction by photoirradiation at 365 nm
proceeded smoothly and the target product 7 was obtained in
12% overall yield from the initial solid-phase synthesis
(Scheme 2). As shown in Figure 3, the purity of the crude
compound 7 released from the transporter was satisfactory
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Communications
enough for further biological assay, which suggests that
blotting and one-pot glycosylation reactions on the water-
soluble polymer mediated by molecular transporter 1 will
become an efficient and versatile strategy for the high-
throughput synthesis of a glycopeptide library.
Interestingly, it was also demonstrated that irradiation
with a MALDI-TOF MS N2 gas laser efficiently cleaved the
transporter at its photosensitive position in the presence of
2,5-dihydroxybenzoic acid as matrix. The product-ion peaks
can be used for monitoring and characterizing glycosylation
reactions on the polymer support by subsequent precise
structural analysis in the TOF/TOF mode.[12,13] As indicated in
Figure 4, precise structural characterization of compound 7
was carried out by matrix-dependent selective fragmentation
(MDSF)[12b] in the MALDI-TOF/TOF MS mode.
Figure 2. RP-HPLC analysis of the chemoselective ligation of the
ketone group of compound 3 with the alkoxyamino group of polymer
4. a) Chromatogram of the crude mixture of 3 before blotting;
b) chromatogram after blotting for 3 h.
In conclusion, we have established a simple and efficient
method for rapid glycopeptide synthesis by using a hetero-
Scheme 2. a) UDP-Gal (5 mm), b-1,4-galactosyltransferase (100 mUmLÀ1),
MnCl2 (10 mm), 0.1% BSA, HEPES buffer (50 mm, pH 7.0), 100%; b) CMP-
NANA (5 mm), a-2,3-sialyltransferase (37 mUmLÀ1), 0.1% BSA, HEPES buffer
(50 mm, pH 7.0), 100%; c) GDP-Fuc (5–7 mm), a-1,3-fucosyltransferase (20–
30 mUmLÀ1), MnCl2 (10 mm), 0.1% BSA, HEPES buffer (50 mm, pH 7.0),
100%; d) UV irradiation (365 nm). UDP=uracil diphosphate; BSA=bovine
serum albumin; HEPES=N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic
acid; CMP-NANA=cytidine-5’-monophospho-N-acetylneuraminic acid sodium
salt; GDP=guanosine diphosphate.
Figure 4. MALDI-TOF/TOF MS analysis of compound 7 by the MDSF
method in the presence of a) 2,5-dihydroxybenzoic acid (DHB) or
b) a-cyano-4-hydroxycinnamic acid as the matrix.
Figure 3. RP-HPLC analysis of glycopeptide 7. a) Crude product 7;
b) product 7 purified by preparative-scale RP-HPLC.
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Angewandte
Chemie
bifunctional molecular transporter, which interfaces two
types of polymer platforms for chemical and enzymatic
synthesis. The merit of our strategy is evident: chemical
blotting based on a terminal ketone group of the molecular
transporter permits selective and quantitative transportation
of a variety of products synthesized by general solid-phase
synthesis. Moreover, the photoselective cleavage reaction of
water-soluble polymers was suitable for an efficient and mild
procedure to yield complex and unstable target compounds
safely. The present two-dimensional synthetic scheme using a
versatile molecular transporter will greatly accelerate both
practical enzymatic synthesis with immobilized glycosyltrans-
ferases[14] and functional identification of biologically impor-
tant glycopeptides.
Received: December 27, 2004
Published online: March 22, 2005
Keywords: enzymes · glycopeptides · polymers ·
.
solid-phase synthesis · synthetic methods
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