Preparation of oligosaccharides by homogenous enzymatic synthesis and solid phase extraction

Article abstract of DOI:10.1039/c1cc13293h

This communication describes a method for enzymatic preparation of bioactive glycans, which integrated the high-efficiency of homogenous phase enzymatic reaction and fast separation of solid phase extraction.

Full text of DOI:10.1039/c1cc13293h

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COMMUNICATION
Preparation of oligosaccharides by homogenous enzymatic synthesis and
solid phase extractionwz
Wenjun Wang,a Chen Jin,a Lina Guo,a Yu Liu,a Yue Wan,a Xin Wang,*ab Lei Li,*a
Wei Zhao*a and Peng George Wangabc
Received 3rd June 2011, Accepted 16th August 2011
DOI: 10.1039/c1cc13293h
This communication describes a method for enzymatic preparation
of bioactive glycans, which integrated the high-efficiency of homo-
genous phase enzymatic reaction and fast separation of solid phase
extraction.
Preparation of bioactive glycans is of great significance for
probing their biological functions and therapeutic applications.
Although organic synthesis has been successfully applied in
preparing grams of biomacromolecules such as polypeptides
and nucleotides, when turning to glycans, it is much less efficient.
A milestone work in this field was conducted by Seeberger and
coworkers, utilizing a cross-linked polystyrene to synthesize
a pentamannoside automatically.1 To overcome the inherent
deficiencies in solid phase glycans synthesis, a fluorinated tag
Scheme 1 Preparation of oligosaccharides with a novel enzymatic
was introduced by Pohl and coworkers to homogenous
chemical glycans synthesis.2 The target molecules can be
efficiently purified by fluorous solid-phase extraction (FSPE)
via fluorous–fluorous interactions. Compared to the traditional
organic method, enzymatic synthesis works in a high stereo
and regio selective manner avoiding multistep protection/
deprotection and has been extensively used in glycan
preparation.3 By applying enzymatic synthesis on controlled-
pore glass (CPG), Wong and coworkers prepared SLex with a
relatively low yield of 35% due to low reactivity, non-linear
kinetics, stereochemical complexity and analytical difficulty.4
A water-soluble polymer was later used instead of a solid
phase to compensate the drawbacks,5 whereas complicated
chemical steps were required for ligating and releasing the
glycans.
synthetic strategy and solid phase extraction.
of sugar acceptors with
a cleavable hydrophobic tag;
(2) enzymatic synthesis of glycans by glycosyltransferases in
a homogeneous phase; (3) digestion of the sugar acceptors by
special exo-glycosidases; (4) fast separation and concentrating
of products via solid phase extraction (SPE); (5) releasing
the hydrophobic tag and removing it by centrifugation or
evaporation. Compared to the widely used size exclusive
chromatography approach, which suffers from the dilution of
desired compounds, SPE is able to enrich a tagged compound,
thus exhibiting more delightful application potentials in large
scale preparation of glycans. To ensure the purity, efficiency
and recovery, glycosidases were introduced to assist with SPE.
A fluorenylmethyloxycarbonyl group and a phenylmethyl-
oxycarbonyl group were chosen as hydrophobic tags for the
following concerns: (1) they can be easily removed under basic
conditions or by catalytic hydrogenation, respectively; (2) they
are inert in glycosylation reactions (pH = 7.5). To facilitate
the synthesis of the corresponding glyco-conjugates, an amine group
was chosen as a ‘‘plug-in’’ group. Fluorenylmethyloxycarbonyl-
(2-aminoethyl)-4-O-b-D-galactopyranosyl-b-D-glucopyranose
(LacFmoc, compound 2) and N0-phenylmethyloxycarbonyl-
(2-aminoethyl)-2-(acetylamino)-2-deoxy-b-D-glucopyranose
(GlcNAcCbz, compound 8) were synthesized following the
method as illustrated in Scheme 2. Only one step is required
to transfer amide-containing acceptors to the corresponding
hydrophobic-tag labeling ones.
Herein, we report an improved in-solution enzymatic synthesis
and purification strategy for the preparation of glycans. The
strategy (Scheme 1) includes 5 steps: (1) chemical preparation
a College of Pharmacy, Nankai University, Tianjin 300071, PR China.
E-mail: wzhao@nankai.edu.cn, lilei2108@gmail.com,
wangxinnk@nankai.edu.cn; Fax: +86-22-23507880;
Tel: +86-22-23507880
b State Key Laboratory of Elemento-organic Chemistry,
Nankai University, Tianjin 300071, PR China
c Department of Chemistry and Biochemistry, The Ohio State
University, Columbus, OH 43210, USA
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc13293h.
z This article is part of the ChemComm ‘Glycochemistry and glyco-
biology’ web themed issue.
c
11240 Chem. Commun., 2011, 47, 11240–11242
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Table 1 Enzymatic synthesis and purification of trisaccharides
Yielda
(%)
Entry Donor
Enzymes
Product
a
Lgt C, GalE
85
b
WbsJ
70
80
Scheme 2 The synthesis of sugar primers with a hydrophobic tag.
NmCSS,
Pd2, 6SiaT
c
In proof-of-concept experiments, Gb3 (compound 3), type V
human blood group H-antigen (compound 5) and 60-sialyllactose
(compound 7) were prepared using three glycosyltransferases
(a1,4-galactosyltransferase (Lgt C),6 a1,2-fucosyltransferase
(WbsJ)7 and a2,6-sialyltransferase (Pd2,6SiaT)8) respectively.
In the preparation of 3, UDP-Glc C4-epimerase (GalE) was
employed to convert uridine 50-diphosphoglucose (UDP-Glc,
compound 1) to precious uridine 50-diphosphogalactose
(UDP-Gal).9 And in the preparation of 7, cytidine 50-mono-
phosphate sialic acid synthase (NmCSS) was introduced to
generate cytidine 50-monophosphate sialic acid (CMP-Sia)
from sialic acid and cytidine triphosphate in situ.8
a
Isolated yield from SPE.
(detailed procedure and results are described in ESIw). The
usage of glycosidases is essential for the purity of the products.
The products obtained are 97% pure (Fig. 1B) when glycosi-
dase was included, but only 85% pure without glycosidase.
These results revealed the efficiency of enzymatic synthesis and
purification.
The conversions from compound 2 to compounds 5 and 7
were catalyzed by WbsJ or Pd2,6-SiaT, respectively. Following
the same procedure mentioned above, compound 5 was
obtained with a total yield of 70% (Table 1, entry b). As to
the highly hydrophilic compound, such as compound 7, NaCl
was added to the reaction mixture to a final concentration of
Gb3 (compound 3) was synthesized in a system containing
acceptor 2, UDP-Glc, GalE and LgtC. The reaction was
allowed to proceed at 37 1C (pH = 7.5) with brief agitation
and was monitored by TLC and 13C DEPT135. After quenched
by boiling for 5 min, b-galactosidase and b-glucosidase were
added to a digest intact acceptor. After brief centrifugation, the
supernatant was directly loaded on the SPE (C18 as a stationary
phase). The Fmoc tagged Gb3 was enriched on the column,
whereas the byproducts, including UDP, UDP-Glc, glucose,
galactose and buffer salts, flowed through the column. After
being eluted with 20% methanol, the Fmoc tag was released by
adding 10% piperidine in water. Released pure Gb3 was
collected from the supernatant after brief centrifugation. The
yield of this reaction is 85% (Table 1, entry a). Furthermore,
compound 3 can be coupled to BSA under the EDC/sulfo-
NHS system to form a hapten-carrier protein complex
Scheme 3 Enzymatic synthesis and purification of P1 trisaccharide in
tandem.th.
Fig. 1 (A) HPLC profile of GB3-Fmoc purified without glycosidases, peak 1 is Lactose-Fmoc, and peak 2 is Gb3-Fmoc. Two peaks were
confirmed by LC-MS; (B) HPLC profile of GB3-Fmoc purified with glycosidases.
c
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200 mM before loading to the SPE column. After deprotection,
compound 7 can be afforded with a yield of 80% (Table 1,
entry c).
financial support. L. Li is grateful to China Postdoctoral
Science Foundation (No. 20100480040) for support. X. Wang
acknowledges Natural Science Foundation of Tianjin (No.
10JCYBJC04100) for financial support. W. Wang thanks
Dr. Thomas Norberg in Uppsala University for useful suggestions.
P1 trisaccharide (Gal-a(1,4)-Gal-b(1,4)-GlcNAc, compound 10)
was also prepared via a cascade enzymatic catalyzed reaction10
starting from GlcNAcCbz (compound 8) and UDP-Glc catalyzed
by a b-1,4-galactosyltransferase (Lgt B),11 GalE and Lgt C
(Scheme 3). b-Galactosidase was subsequently added to digest
the unreacted compound 9 and b-N-acetylglucosaminidase
(HexA) was added to remove unreacted compound 8. The
final product was purified following the same procedure for
preparing compound 3. The Cbz tagged P1 trisaccharide was
separated by SPE as compound 3, and P1 trisaccharide was
released by catalytic hydrogenation. The total yield of the
reaction was 45%.
Notes and references
1 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science, 2001,
291, 1523–1527.
2 G. Park, K.-S. Ko, A. Zakharova and N. L. Pohl, J. Fluorine
Chem., 2008, 129, 978–982; F. A. Jaipuri and N. L. Pohl, Org.
Biomol. Chem., 2008, 6, 2686–2691; F. A. Jaipuri, B. Y. M. Collet
and N. L. Pohl, Angew. Chem., Int. Ed., 2008, 47, 1707–1710.
3 C.-H. Wong, R. L. Halcomb, Y. Ichikawa and T. Kajimoto,
Angew. Chem., Int. Ed. Engl., 1995, 34, 521–546; C.-H. Wong,
R. L. Halcomb, Y. Ichikawa and T. Kajimoto, Angew. Chem., Int.
Ed. Engl., 1995, 34, 412–432.
In summary, we reported a facile strategy for glycan
preparation. With the aid of the combined glycosyltransferases-
glycosidases strategy, this method utilizes a simple hydro-
phobic tag and SPE separation to get purified glycans. In
this report, 8 enzymes with various functions cooperated to
synthesize 4 bioactive glycans with a total yield of up to 85%,
demonstrating that this method integrates the high-efficiency
of homogenous phase enzymatic reactions and fast separation
of solid phase technology. Further studies on the large scale
preparation of more complicated glycans via the strategy are
undergoing.
4 M. Schuster, P. Wang, J. C. Paulson and C.-H. Wong, J. Am.
Chem. Soc., 1994, 116, 1135–1136.
5 X. Huang, K. L. Witte, D. E. Bergbreiter and C.-H. Wong,
Adv. Synth. Catal., 2001, 343, 675–681.
6 J. Shao, J. Zhang, P. Kowal, Y. Lu and P. G. Wang, Chem.
Commun., 2003, 1422–1423.
7 M. Li, X.-W. Liu, J. Shao, J. Shen, Q. Jia, W. Yi, J. K. Song,
R. Woodward, C. S. Chow and P. G. Wang, Biochemistry, 2007,
47, 378–387.
8 H. Yu, H. Yu, R. Karpel and X. Chen, Bioorg. Med. Chem., 2004,
12, 6427–6435.
9 X. Chen, P. Kowal, S. Hamad, H. Fan and P. G. Wang, Biotechnol.
Lett., 1999, 21, 1131–1135.
10 Z. Liu, Y. Lu, J. Zhang, K. Pardee and P. G. Wang, Appl. Environ.
Microbiol., 2003, 69, 2110–2115.
11 O. Blixt, J. Brown, M. J. Schur, W. Wakarchuk and J. C. Paulson,
J. Org. Chem., 2001, 66, 2442–2448.
We thank the National 973 Basic Research Program of
China (No. 2007CB914803, 2010CB529106) and National
Natural Science Foundation of China (No. 20802037) for
c
11242 Chem. Commun., 2011, 47, 11240–11242
This journal is The Royal Society of Chemistry 2011