Synthesis of sugar nucleotides by application of phosphoramidites

Article abstract of DOI:10.1021/jo802021t

(Chemical Equation Presented) A new method for the construction of pyrophosphates is reported based on the coupling of a sugar phosphate and a nucleoside phosphoramidite. The in situ formed phosphate-phosphite intermediate was subsequently oxidized with tBuOOH. Three UDP-N-acetylglucosamine derivatives were prepared using this one-pot procedure in good yields.

Full text of DOI:10.1021/jo802021t

Synthesis of Sugar Nucleotides by Application of
Phosphoramidites
Henrik Gold,‡,† Pieter van Delft,‡,† Nico Meeuwenoord,†
Jeroen D. C. Code´e,† Dmitri V. Filippov,† Gerrit Eggink,§
Herman S. Overkleeft,† and Gijs A. van der Marel*,†
FIGURE 1. UDP-GlcNAc and analogues.
Leiden Institute of Chemistry, Bioorganic Chemistry,
Gorlaeus Laboratories, P.O. Box 9502,
oligosaccharides.4 Glycosyltransferases that incorporate non-
natural monosaccharides or accept non-natural substrates for
glycosylation may become a powerful tool for the synthesis of
non-naturally occurring bioconjugates. Furthermore, synthesis
of labeled or orthogonally tagged sugar nucleotides will aid the
efforts in glycomics.5 Thus, suitable procedures are required
that make sufficient quantities of nucleoside diphosphate sugars
available, in which the sugar moiety may be a natural or a
modified monosaccharide.
2300 RA, Leiden, The Netherlands, and Agrotechnology and
Food Sciences Group, Wageningen UniVersity and Research
Centre, P.O. Box 17, 6700 AA Wageningen, The Netherlands
marel_g@chem.leidenuniV.nl
ReceiVed September 12, 2008
In this paper we present a new method for the preparation of
nucleoside diphosphate sugars by the synthesis of UDP-N-
acetylglucosamine (UDP-GlcNAc, 1) and the unnatural conge-
ners 2 and 3 (Figure 1). The modified sugar nucleotides 2 and
3 were selected by their virtue of being potential chain
terminators in enzyme mediated polymerization reactions. For
instance, hyaluronic acid (HA), a negatively charged and high
molecular weight polymer, can be assembled using hyaluronan
synthase (HAS), UDP-GlcNAc, (1) and UDP-glucuronic acid
(UDP-GlcA).6 Incorporation of an N-acetylglucosamine in which
the C3-OH function is capped with a methyl functionality (2)
or removed (3) would prohibit chain elongation at that position.
Thus, it is expected that the length of the HA oligomer can be
tuned by the use of 2 or 3 as additional substrates in the HAS
reaction. Recently the group of Fairbanks reported the potential
of 3-OH modified UDP-glucose derivatives as a new class of
inhibitors of the biosynthesis of pathogenic oligosaccharides.7
A key step in the synthesis of nucleoside diphosphate sugars
is the introduction of the pyrophosphate function between the
5′-OH position of a nucleoside and the anomeric hydroxyl of a
saccharide unit. A variety of procedures has been reported that
can roughly be classified in two main categories. The first one
comprises the reaction of a protected glycosyl donor such as a
halogenide with the tetrabutylammonium salt of a nucleoside
5′-diphosphate.8 This approach is often hampered by a low
anomeric diastereoselectivity, although there are some
exceptions.8a In the second category, the pyrophosphate moiety
can be introduced by coupling of a monosaccharide-1-phosphate
with an activated nucleoside 5′-monophosphate.9 The anomeric
configuration of the final product can be controlled by stereo-
A new method for the construction of pyrophosphates is
reported based on the coupling of a sugar phosphate and a
nucleoside phosphoramidite. The in situ formed phosphate-
phosphite intermediate was subsequently oxidized with
tBuOOH. Three UDP-N-acetylglucosamine derivatives were
prepared using this one-pot procedure in good yields.
Oligosaccharides and glycoconjugates play important roles
in a variety of biological processes. The biosynthesis of these
ubiquitous and structurally diverse biopolymers is catalyzed by
glycosyltransferases.1 These enzymes transfer a monosaccharide
residue in a regio- and stereoselective manner from a specific
sugar nucleotide donor to acceptors such as (oligo)saccharides,
lipids, or proteins. On the basis of sequence similarities, 91
families of glycosyltransferases can be discerned.2 While
eukaryotic glycosyltransferases employ a limited number of
sugar nucleotides as glycosyl donors (UDP-Glc, UDP-GlcNAc,
UDP-GlcUA, UDP-Gal, UDP-GalNAc, GDP-Fuc, GDP-Man,
and CMP-Neu5Ac), the structures of nucleoside diphosphate
sugars in prokaryotes are both numerous and diverse.3 Advances
made in engineering of glycosyltransferases show promise that
these enzymes may be used for the synthesis of relevant
(4) Hancock, S. M.; Vaughan, M. D.; Withers, S. G. Curr. Opin. Chem. Biol.
2006, 10, 509.
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S. C.; Jakeman, D. L. Org. Lett. 2007, 9, 1227. (c) Uchiyama, T.; Hindsgaul, O.
J. Carb. Chem. 1998, 17, 1181. (d) Ernst, C.; Klaffke, W. J. Org. Chem. 2003,
68, 5780. In a procedure reported by Hannessian et al. unprotected glycosyl
donors (MOP glycosides) were used in the reaction with free UD: (e) Hanessian,
S.; Lu, P.-P.; Ishida, H. J. Am. Chem. Soc. 1998, 120, 13296.
† Leiden Institute of Chemistry.
‡ H.G. and P.v.D contributed equally to this work.
§ Wageningen University and Research Centre.
(1) Watkins, W. Carb. Res. 1986, 149, 1.
(2) (a) Coutinho, P. M.; Deleury, E.; Davies, G. J.; Henrissat, B. J. Mol.
Biol. 2003, 328, 307. (b) The glycosyltransferase family server at: http://
www.cazy.org/fam/acc_GT (accessed Aug 25, 2008).
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9458 J. Org. Chem. 2008, 73, 9458–9460
10.1021/jo802021t CCC: $40.75 2008 American Chemical Society
Published on Web 11/08/2008

SCHEME 1. Synthesis of Sugar Phosphates 9a-9c
SCHEME 2. One-Pot Reaction for the Synthesis of Sugar
Nucleotides 1-3
2-acetamido-2-deoxy-R-D-glucoside (4)13 in 84% total yield over
5 steps. The C4-OH and C6-OH were masked using a di-
tert-butylsilylene group, which greatly enhances solubility of
GlcNAc derivatives. The remaining hydroxyl of 5 was acetylated
(6a) after which the anomeric benzyl group was removed with
Pd(OH)2 and hydrogen gas to produce hemiacetal 7a. The
anomeric phosphate was stereoselectively introduced by treat-
ment of 7a with LDA at -78 °C, followed by addition of
tetrabenzylpyrophosphate (TBPP) to give the R-dibenzyl-
phosphotriester 8a. Hydrogenolysis of the benzyl groups in 8a
and addition of one equivalent of tetrabutylammonium hydrox-
ide yielded GlcNAc-R-1-phosphate 9a.
selective synthesis of the sugar 1-phosphate. The original method
of Khorana and Moffat using nucleoside phosphoromorpholi-
dates as activated phosphates has been most extensively
pursued.9a,b Improvements of this method in terms of reaction
time and yield have been reported by the utilization of a catalyst,
such as tetrazole.10 Recently, cyclo-saligenyl nucleosyl phos-
photriesters were presented as active esters to attain nucleoside
diphosphate glucopyranoses.11
The extensive use of phosphoramidite chemistry in synthesis
of (modified) DNA/RNA fragments12 encouraged us to explore
the implementation of this chemistry in the formation of
pyrophosphate moieties. UDP-GlcNAc 1 was selected as a first
target to explore whether the reaction of an activated phos-
phoramidite with a phosphate and subsequent oxidation would
lead to the desired pyrophosphate functionality.
Uridine phosphoramidite 1014 (Scheme 2) was easily syn-
thesized in four steps from commercially available uridine, in
93% total yield. The cyanoethyl group was selected as a
phosphite protection because of its easy removal under anhy-
drous, basic conditions.
With sugar phosphate 9a and phosphoramidite 10 in hand,
the pyrophosphate formation was examined. The reaction
progress was monitored by 31P NMR spectroscopy (CD3CN,
162 MHz). The activator dicyanoimidazole15 and phosphate
monoester 9a (δ ) -1.2 ppm) were simultaneously added to a
solution of phosphoramidite 10 (δ ) 149.5, 149.4 ppm) in
CD3CN. Within 30 min at room temperature, complete disap-
pearance of amidite 10 and formation of two diastereomeric
phosphate-phosphite intermediates 11a [δ ) 130.3 (d, J ) 5.8
Hz), 128.3 (d, J ) 4.5 Hz), -10.9 (d, J ) 4.5 Hz), -11.0 (d,
J ) 5.8 Hz) ppm] was observed. Subsequent oxidation with
anhydrous tert-butylperoxide16 gave the diastereomeric cyano-
ethyl protected pyrophosphate 12a [δ ) -10.7 (d, J ) 16.2
Hz), -11.4 (d, J ) 17.8 Hz), -13.5 (d, J ) 17.8 Hz), -13.6
The tetrabutylammonium salt of GlcNAc-R-1-phosphate 9a
(Scheme 1) was synthesized, starting from the known benzyl
(9) (a) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc. 1958, 80, 3756. (b)
Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc.
1961, 83, 659. (c) Freel Meyers, C. L.; Borch, R. F. Org. Lett. 2001, 23, 3765.
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979. (e) Marlow, A. L.; Kiessling, L. L. Org. Lett. 2001, 3, 2517. (f) Timmons,
S. C.; Jakeman, D. L. Carb. Res. 2008, 343, 865. (g) Yamazaki, T.; Warren,
C. D.; Herscovics, A.; Jeanloz, R. W. Can. J. Chem. 1981, 59, 2247. (h) Nunez,
H. A.; O’Connor, J. V.; Rosevear, P. R.; Barker, R. Can. J. Chem. 1981, 59,
2086. (i) Imperiali, B.; Zimmerman, J. W. Tetrahedron Lett. 1990, 31, 6485.
(10) (a) Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144. (b) Busca,
P.; Piller, V.; Piller, F.; Martin, O. R. Bioorg. Med. Chem. Lett. 2003, 13, 1853.
(c) Muller, T.; Danac, R.; Ball, L.; Gurr, S. J.; Fairbanks, A. J. Tetrahedron
Asymmetry 2007, 18, 1299. (d) Danac, R.; Ball, L.; Gurr, S. J.; Fairbanks, A. J.
Carb. Res. 2008, 343, 1012. (e) Chang, R.; Moquist, P.; Finney, N. S. Carb.
Res. 2004, 339, 1531. (f) Stolz, F; Blume, A.; Hinderlich, S.; Reutter, W.;
Schmidt, R. R. Eur. J. Org. Chem. 2004, 3304.
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(14) Filippov, D.; Kuyl-Yeheskiely, E.; van der Marel, G. A.; Tesser, G. I.;
van Boom, J. H. Tetrahedron Lett. 1998, 39, 3597.
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Peterson, K.; Pieken, W. Nucl. Acid. Res. 1998, 26, 1046.
(16) Anhydrous conditions were used to prevent possible hydrolysis of the
labile phosphite-phosphate anhydride 11.
(11) Wendicke, S.; Warnecke, S.; Meier, C. Angew. Chem., Int. Ed. 2008,
47, 1500.
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J. Org. Chem. Vol. 73, No. 23, 2008 9459

(d, J ) 16.2 Hz) ppm]. The protecting groups were immediately
removed by a three step procedure. First the cyanoethyl group
was removed by treatment with anhydrous DBU, followed by
addition of HF/Et3N to deblock the di-tert-butylsilylene group
and ammonium hydroxide to unmask the remaining alcohol
functions. Purification of the obtained crude product by ion
exchange chromatography gave pure UDP-GlcNAc 1 [δ )
-10.8 (d, J ) 21.1 Hz), -12.5 (d, J ) 21.1 Hz) ppm] in 76%
yield.
Having established the value of cyanoethyl phosphoramidite
10 in the formation of UDP-GlcNAc 1, the synthesis of the
non-natural UDP-GlcNAc derivatives 2 and 3 was undertaken.
The requisite methylated and deoxygenated GlcNAc-R-1-
phosphate derivatives 9b and 9c were obtained from 5 as
depicted in Scheme 1. The methyl group at the C3-OH of 5
was selectively introduced by treatment with TMS-diazomethane
and BF3 ·OEt2 to give fully protected GlcNAc 6b, without
unwanted N-methylation. Alternatively, compound 5 was sub-
jected to the Barton-McCombie procedure. Thus, after forma-
tion of the xanthate ester and radical reduction with AIBN and
trin-butyltin hydride, the deoxygenated compound 6c was
obtained in 93% yield. The modified GlcNAc derivatives 6b
and 6c were then transformed into the corresponding R-phos-
phates 9b and 9c by the same sequence of reactions as described
for the conversion of 6a into 9a (Scheme 1). Dicyanoimidazole
mediated reaction of 9b and 9c with uridine phosphoramidite
10, followed by oxidation and the outlined deprotection
sequence, smoothly provided the modified UDP-GlcNAc deri-
vates 2 and 3 in 63% and 71% yield respectively.
ology. Current research is directed at the extension of the
methodology to other nucleophiles and nucleobases.
Experimental Section
General Experimental Procedure for Reaction of a Sugar
Phosphate (9a-c) and Nucleoside Phosphoramidite (10). Sugar
phosphate 9a-c (0.12 mmol, 1.2 equiv) and dicyanoimidazole (0.2
mmol, 2 equiv) were dissolved in 2 mL anhydrous MeCN and added
to a solution of phosphoramidite 10 (0.1 mmol, 1 equiv) in
anhydrous MeCN (1.5 mL). The reaction mixture was stirred at
room temperature for 30 min after which t-BuOOH (0.4 mmol, 4
equiv) was added. After 30 min of reaction time DBU (0.5 mmol,
5 equiv) was added and the reaction was stirred for an additional
30 min. The reaction mixture was stirred with triethylamine (1.6
mmol, 16 equiv) and NEt3 ·(HF)3 (0.8 mmol, 8 equiv) for four hours,
followed by addition of NH4OH 25% (40 mmol, 40 equiv). The
reaction was stirred for 12 h and then diluted with 4 mL MilliQ.
The mixture was washed with 4 mL of DCM and the organic phase
was extracted with 4 mL MilliQ. The combined water layers were
concentrated in vacuo, redissolved in MilliQ and centrifuged. The
supernatant was applied to a strong anion exchange column and
eluted with a gradient of ammonium acetate (0.05-0.5 M). The
fractions containing the product were collected and concentrated
under reduced pressure. Repeated lyophilization and filtration over
Amberlite Na+-form produced the desired sugar nucleotides 1, 2,
and 3 in 63-76% yield based on phosphoramidite 10.
Acknowledgment. This project is financially supported by
The Netherlands Ministry of Economic Affairs and the B-Basic
partner organizations (www.b-basic.nl) through B-Basic, a
public-private NWO-ACTS programme (ACTS ) Advanced
Chemical Technologies for Sustainability).
In conclusion, we have developed an efficient, robust and
rapid procedure for the synthesis of sugar nucleotides, using
easily accessible nucleoside phosphoramidites and sugar phos-
phates. Phosphoramidites were shown to be powerful phospho-
rylating agents in the formation of the pyrophosphate moiety.
The procedure presents an attractive alternative to existing
methods and is a valuable asset for future research in glycobi-
Supporting Information Available: Detailed experimental
procedures, including full characterization of unknown com-
pounds and copies of 1H, 13C, and 31P spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO802021T
9460 J. Org. Chem. Vol. 73, No. 23, 2008