Chemoenzymatic syntheses of carbasugar analogues of nucleoside diphosphate sugars: UDP-carba-Gal, UDP-carba-GlcNAc, UDP-carba-Glc, and GDP-carba-Man

Article abstract of DOI:10.1039/b821058f

Chemoenzymatic syntheses of several NDP-carba-sugars have been successfully carried out, and these essential cofactor analogues are expected to be selective inhibitors of glycosyltransferase enzymes. The Royal Society of Chemistry.

Full text of DOI:10.1039/b821058f

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Chemoenzymatic syntheses of carbasugar analogues of nucleoside
diphosphate sugars: UDP-carba-Gal, UDP-carba-GlcNAc,
UDP-carba-Glc, and GDP-carba-Manwz
Kyung-Chang Seo,a Young-Geol Kwon,a Dae-Hee Kim,b In-Sook Jang,c Jin-Won Choc
and Sung-Kee Chung*a
Received (in Cambridge, UK) 25th November 2008, Accepted 9th January 2009
First published as an Advance Article on the web 6th February 2009
DOI: 10.1039/b821058f
Chemoenzymatic syntheses of several NDP-carba-sugars have
been successfully carried out, and these essential cofactor
analogues are expected to be selective inhibitors of
glycosyltransferase enzymes.
Isosteric analogues of the NDP-sugars are of interest as
potential GT inhibitors due to their inherent stability towards
enzymatic cleavage. One such example of NDP-sugars
analogues is the isosteric C-glycosidic phosphonate, in
which the phosphoester oxygen atom is substituted by a
carbon atom.6 Another example involves replacement of the
glycosyl moiety with a carbasugar (pseudosugar). Chemical
syntheses and the inhibitory activities of the guanosine
50-(5a-carba-b-L-fucopyranosyl diphosphate) [GDP-carba-fucose]
and uridine 50-(5a-carba-a-D-galactopyranosyl diphosphate)
[UDP-carba-galactose] have been reported for fucosyl-
transferase (FucT) and galactosyltransferase (GalT),
respectively.7 Both carbasugar analogues showed good inhi-
bitory activity against the GT enzymes with Ki values of
about 60 mM in in vitro bioassays. Herein, we report successful
examples of chemoenzymatic syntheses of several NDP-
carba-sugars.
Carbohydrates, in addition to their well-known function as a
substance for energy storage, have versatile biological roles in
life processes. In eukaryotes, more than half of all proteins are
glycosylated, and the carbohydrate appendages in glycoproteins
have significant structural and physiological influences in
protein folding, stability, translocation, activity, and signal
transduction, thus mediating important cell–cell recognition
processes in immune response, fertilization, cell growth,
cell–cell adhesion, viral infection, inflammation, induction
and metastasis of cancer. Hence, understanding the
relationship between the glycosylation pattern and cellular
physiology, especially the onset of disease states such as cancer
and inflammation, is a major goal of the current glycobiology
research.1,2
In order to examine the feasibility of this approach, we
first examined the chemoenzymatic synthesis8 of known
UDP-carba-Gal from carba-galactose-1-phosphate, an
NDP-carba-sugar chemically prepared by Hindsgaul.7c
Carba-a-D-galactose-1-phosphate (10 mM), prepared from
(1S, 2R, 3S, 4S, 5R)-2,3,4-(trisbenzyloxy)-5-(benzyloxy-
methyl)-cyclohexan-1-ol,9 was incubated with UMP, ATP,
MgCl2 6H2O, acetyl phosphate, glucose-1-phosphate,
Tris-HCl in water in the presence of Escherichia coli enzyme
extracts containing UMP kinase (UMK),10 acetate kinase
(ACK),11 glucose-1-phosphate uridyltransferase (GlcU),12
and galactose-1-phosphate uridyltransferase (GalU)13 for
5 h at 37 1C (Scheme 1). HPLC analysis [Hypersil ODS,
4.6 ꢀ 250 mm, 5 mm particle size, eluted with potassium
phosphate buffer (100 mM, pH 7.0)–MeOH = 95 : 5 (v/v),
1.0 mL flow rate, at 270 nm] showed essentially a single
product which had a slightly longer retention time than that
of UDP-Gal. The product was separated and subjected to
mass spectral analysis, which showed m/z 563, consistent with
the UDP-carba-Gal structure.
Despite the significance of the glycan expression in the
cellular function, understanding the molecular basis of the
glycan function is incomplete, in part because biosynthetic
processes of glycans are not clearly elucidated.3 Production of
glycans and glycoconjugates is catalyzed by the action of
glycosyltransferases (GTs) and glycosidases during their move
through the eukaryotic secretory pathways, but GTs are
generally not as well studied as glycosidases.4 GT enzymes
catalyze the transfer of the activated monosaccharide units
from either the nucleoside diphosphate sugars (NDP-sugars;
Leloir type) or glycosyl phosphates (non-Leloir type) to a
specific free hydroxyl group of the acceptor molecule.
Eight NDP-sugars are known to be commonly used as the
essential sugar donors in most biosynthetic glycosylations in
eukaryotes.3b,5 An ability to modulate or inhibit this transfer
reaction is expected to provide a good opportunity for
intervention of the glycan biosynthesis and also for a
better molecular level understanding of the structure–function
relationship of glycans and glycoconjugates.
Encouraged by this result, we investigated chemoenzymatic
syntheses of several NDP-carba-sugar analogues including
uridine 50-(5a-carba-a-D-N-acetylglucosaminopyranosyl di-
phosphate) (UDP-carba-GlcNAc) (1), uridine 50-(5a-carba-a-
D-glucopyranosyl diphosphate) (UDP-carba-Glc) (2) and
guanosine 50-(5a-carba-a-D-mannopyranosyl diphosphate)
(GDP-carba-Man) (3) (Fig. 1).
a Department of Chemistry, Pohang University of Sciecnce and
Technology, Pohang, 790-784, Korea. E-mail: skchung@postech.edu;
Fax: (+82) 54-279-3399
b GeneChem Inc., Daejeon, 305-343, Korea
c Department of Biology, Yonsei University, Seoul, 120-749, Korea
w Dedicated to Professor Robert M. Coates of University of Illinois at
Urbana on the occasion of his retirement.
For the preparation of UDP-carba-GlcNAc (1), we
synthesized carba-a-D-GlcNAc-1-phosphate from compound
z Electronic supplementary information (ESI) available: Experimental
details and characterization data. See DOI: 10.1039/b821058f
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Scheme 2 One-pot enzymatic synthesis of UDP-carba-GlcNAc. ACK
(acetate kinase), UMK (UMP kinase), GlmU (GlcNAc-1-phosphate
uridyltransferase).
Scheme 1 One-pot enzymatic synthesis of UDP-carba-Gal. ACK
(acetate kinase), UMK (UMP kinase), GlcU (glucose-1-phosphate
uridyltransferase), GalU (galactose-1-phosphate uridyltransferase).
various NDP-carba-sugar cofactors and analogues. One of the
potential uses of NDP-carba-sugars may be their potential
inhibitory activity for the corresponding glycosyltransferases
(GTs). A preliminary study with UDP-carba-GlcNAc (1) for
UDP-N-acetylglucosaminyl transferase (OGT) on casein
kinase (CKII), a known OGT substrate, showed an inhibitory
activity comparable to a nonselective inhibitor, alloxan.16
For the synthesis of UDP-carba-glucose 2, carba-sugar diol
617 was didehydroxylated with PPh3, imidazole and iodine,18
and then dihydroxylated with a catalytic amount of OsO4 and
an excess NMO to give the opposite stereochemistry in a
carba-a-D-glucose derivative 7. The dihydroxy compound
was selectively monobenzoylated by treatment with triethyl
orthobenzoate and TSA, followed by hydrolysis in 80%
aqueous AcOH. The remaining hydroxyl group was
benzylated with benzyl trichloroacetimidate and triflic acid,
and the product was debenzoylated with NaOMe to give the
desired compound with only one hydroxyl group at the C1
position. This alcohol was phosphorylated by successive
treatments with dibenzyl diisopropylphosphoramidite and
1H-tetrazole, and then mCPBA. Removal of the benzyl
protective groups by hydrogenolysis gave the desired
Fig. 1 Target NDP-carba-sugars for chemoenzymatic synthesis.
4, which was in turn derived from (1R,2S,5S)-5-(tert-butyl-
diphenylsilanyloxymethy)-cyclohex-3-ene-1,2-diol.14 (Scheme
S1 of ESIz) The b-hydroxyl group of 4 was mesylated and
displaced with CsOAc to give the corresponding a-acetate
intermediate. The azido functionality of the acetate intermediate
was reduced and N-acetylated, and then the acetate was
hydrolyzed to the protected carba-GlcNAc. At this stage the
hydroxyl group was phosphorylated by successive treatments
with dibenzyl diisopropylphosphoramidite and 1H-tetrazole,
and mCPBA, and then all benzyl protecting groups were
removed to provide the desired carba-a-GlcNAc-1-phosphate
5. The one-pot enzymatic synthesis of UDP-carba-GlcNAc 1
was carried out from compound 5 in the presence of
GlcNAc-1-phosphate uridyltransferase (GlmU)15 and UTP,
which was continually regenerated from UMP by the three
enzyme system (Scheme 2).13 The incubation was performed
for 5 h at 37 1C, and quenched by heating the reaction mixture
at 100 1C, and the product (30 mg, 23% based on UMP) was
purified on a Shimadzu SPD-10Avp. The target compound (1)
was fully characterized by spectroscopy including 1H-, 13C-,
31P-NMR and mass spectrometry. Although the conversion
yield may be judged somewhat low, this result indicates that
the ring oxygen of GlcNAc may not be critical for the
recognition of GlcNAc-1-phosphate by GlmU in the
UDP-GlcNAc biosynthesis, and the chemoenzymatic
conversion could be generally applicable to the syntheses of
carba-a-D-glucose-1-phosphate
8
(Scheme S2 of ESIz).
Compound 8 was enzymatically converted to UDP-carba-Glc
2 (219 mg, 84% based on UMP) with glucose-1-phosphate
uridyltransferase11 from E. coli K-12 and the regenerated UTP
(Scheme 3).
Next, the carbasugar diol 6 was converted to 1,2-b-epoxide
via the orthoacetate and bromoacetoxy intermediates.19 Ring
Scheme 3 One-pot enzymatic synthesis of UDP-carba-Glc. ACK
(acetate kinase), UMK (UMP kinase), GlcU (glucose-1-phosphate
uridyltransferase).
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Notes and references
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Scheme 4 One-pot enzymatic synthesis of GDP-carba-Man. ACK
(acetate kinase), GMK (GMP kinase), ManC (mannose-1-phosphate
guanylyltransferase).
opening of the 1,2-b-epoxide with allyl alcohol and BF3
etherate in CH2Cl2 exclusively gave the carba-a-D-mannose
derivative 9. Benzyl protection of 9 followed by removal of the
allyl group with PdCl2 and NaOAc in aqueous AcOH
provided the 1-hydroxyl intermediate, which was phosphorylated
and the protecting groups were removed as previously
described to give desired carba-a-D-mannose-1-phosphate 10.
(Scheme S3 of ESIz). The enzymatic transformation of 10 to
GDP-carba-Man 3 proceeded uneventfully as shown in
Scheme 4. For the enzymatic NDP coupling reaction to obtain
GDP-carba-Man (3: 192 mg, 83% from GMP), we again
employed a GTP regenerating system based on two enzymes
(acetate kinase and GMP kinase20), and mannose-1-phosphate
guanylyltransferase.21
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In summary, we have successfully demonstrated chemoenzy-
matic syntheses of several NDP-carba-sugar analogues, which
are based on readily available sugar-1-phosphate nucleotide
transferase enzymes coupled with nucleotide trisphosphate
regenerating systems. To the best of our knowledge, this
represents the first chemoenzymatic synthesis of the
NDP-carba-sugar analogues including UDP-carba-Gal,
UDP-carba-GlcNAc, UDP-carba-Glc, and GDP-carba-Man.
The chemoenzymatic strategy should be readily applicable to
the preparation of other NDP-carbasugars, provided that the
carbasugar analogues are available.8,14,17 Furthermore, we
envisage that these NDP-carba-sugar analogues may poten-
tially be selective inhibitors of various glycosyltransferases
utilizing these NDP-sugars as requisite cofactors. However,
these NDP-carba-sugars are highly water soluble and not
readily taken up by cells, thus limiting their in vivo utilities.
It will be useful to develop membrane permeable versions of
the NDP-carba-sugar analogues for wider applications, and
we are currently working toward this goal by utilizing novel
molecular transporters recently developed in our laboratory.22
This work was supported by KISTEP/KOSEF
(BT-Glycobiology Program 200402087) and the Korea
Research Foundation (MOEHRD KRF-2005-070-C00078).
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T. Takeuchi, S. Futaki and S. K. Chung, Angew. Chem., Int. Ed.,
2006, 45, 2907; (b) K. K. Maiti, W. S. Lee, T. Takeuchi,
C. Watkins, M. Fretz, D. C. Kim, S. Futaki, A. Jones,
K. T. Kim and S. K. Chung, Angew. Chem., Int. Ed., 2007, 46,
5880; (c) G. Biswas, O. Y. Jeon, W. S. Lee, D. C. Kim, K. T. Kim,
S. Lee, S. Chang and S. K. Chung, Chem.–Eur. J., 2008, 14, 9161.
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Chem. Commun., 2009, 1733–1735 | 1735