Chemical synthesis and isolation of UDP-2-deoxy glucose and galactose

Article abstract of DOI:10.1080/00397911.2016.1227849

2-Deoxy sugars are attractive compounds in synthetic chemistry with regard to reactivity and stereoselectivity. Moreover, their ability to inhibit enzymes and metabolism is significant in biology. In this study, uridine-5′-diphosphate (UDP)-2-deoxy glucose (11) and galactose (12) were synthesized chemically. These sugar donors for glycosyltransferases were obtained α-selectively via phosphorylation using thioglycosides, coupling reaction with uridine-5′-monophosphate (UMP)-morpholidate, and moderate deacetylation. Isolation was carried out by sequential silica-gel chromatography using two kinds of developing solvents in a refrigerator. The structures were elucidated from the NMR results. Investigation of stability showed that the synthesized UDP-2-deoxy sugars were hydrolyzed much faster in buffer (pH 4) than the natural UDP sugars.

Full text of DOI:10.1080/00397911.2016.1227849

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Chemical Synthesis and Isolation of UDP-2-Deoxy
Glucose and Galactose
Atsushi Miyagawa, Shunya Takeuchi, Shinji Itoda, Sanami Toyama, Kenta
Kurimoto, Hatsuo Yamamura & Yukishige Ito
To cite this article: Atsushi Miyagawa, Shunya Takeuchi, Shinji Itoda, Sanami Toyama,
Kenta Kurimoto, Hatsuo Yamamura & Yukishige Ito (2016): Chemical Synthesis and
Isolation of UDP-2-Deoxy Glucose and Galactose, Synthetic Communications, DOI:
10.1080/00397911.2016.1227849
To link to this article: http://dx.doi.org/10.1080/00397911.2016.1227849
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Date: 13 September 2016, At: 08:46

Chemical Synthesis and Isolation of UDP-2-Deoxy Glucose and Galactose
Atsushi Miyagawa1, Shunya Takeuchi1, Shinji Itoda1, Sanami Toyama1, Kenta
Kurimoto1, Hatsuo Yamamura1, Yukishige Ito2
1Department of Materials Science and Engineering, Graduate School of Engineering,
Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi, Japan,
2Synthetic Cellular Chemistry Laboratory, RIKEN, Hirosawa, Wako, Saitama, Japan
Corresponding author to Atsushi Miyagawa, E-mail: miyagawa.atsushi@nitech.ac.jp
Abstract
2-Deoxy sugars are attractive compounds in synthetic chemistry with regard to reactivity
and stereoselectivity. Moreover, their ability to inhibit enzyme and metabolism is
significant in biology. In this study, UDP-2-deoxy glucose (11) and galactose (12) were
synthesized chemically. These sugar donors for glycosyltransferases were obtained α-
selectively via phosphorylation using thioglycosides, coupling reaction with UMP-
morpholidate and moderate deacetylation. Isolation was carried out by sequential silica
gel chromatography using two kinds of developing solvents in a refrigerator. The
structures were elucidated from the NMR results. Investigation of stability showed that
the synthesized UDP-2-deoxy sugars were hydrolyzed much faster in buffer (pH 4) than
the natural UDP-sugars.
1

GRAPHICAL ABSTRACT
KEYWORDS: UDP-sugar; 2-Deoxy sugar; Isolation; Stability
INTRODUCTION
In biosynthesis of carbohydrates, sugar nucleotides perform as glycosyl donors to
construct oligosaccharides and polysaccharides. Additionally, they are also utilized in
biosynthesis of glycosylated natural products.[1-4] Glycosyltransferases catalyze the
transfer of a monosaccharide from a sugar nucleotide either to an aglycon or to a growing
oligosaccharide to form a specific glycosidic bond. The inhibition of the reaction causes
a wide variety of biological effects.[5-7] Thus, sugar nucleotide analogs are potential leads
for therapeutic agents, development of which have been attempted by various
modifications. Indeed, deoxy sugars, which lack a selected hydroxy group, exist as a part
of antibiotic agents including vancomycin and erythromycin in nature.[8-13]
2

Among them, 2-deoxy sugars which lack 2-hydroxy group near anomeric position are
intriguing in both chemical and biological points of view. Chemical synthesis of 2-deoxy
glycoside is nontrivial, which usually rely upon thermodynamic control, as neighbouring
group participation is not feasible without introducing streocontrolling auxiliary.[14-19]
While, biological activity of 2-deoxy glucose has been investigated in numerous
studies,[20-23] especially in terms of its ability to inhibit glycolysis and glycosylations,
which causes depletion of cellular ATP and aberrant glycosylation of proteins, it is
difficult to evaluate only the activity of deoxy sugars in total metabolism. By using 2-
deoxy sugar nucleotides, the activity for each enzyme can be analyzed in detail. Indeed,
Palcic group reported the pioneering work, which achieved synthesis of UDP-2-deoxy
galactose. However, the product contained UMP or β-isomer and the mixture was used
for galactosyltransferase catalyzed reactions.[24,25] In this paper, we wish to report our
own effort to chemically synthesis of UDP-2-deoxy glucose and galactose, which were
obtained in extensively purified forms.
RESULTS AND DISCUSSION
To begin with, 2-deoxy glucose was converted to peracetate, whose 1-O-acetyl group was
deprotected. Unexpectedly, the attempt to phosphorylate 1-OH group with phosphoryl
3

chloride and BuLi[26,27] was unsuccessful. As Oberthur et al. previously achieved
synthesis of 2-deoxy L-sugar phosphates using glycosyl halides[28] the similar approach
was examined. However, 2-deoxy glucosyl phosphate is known to be unstable under
acidic conditions,[29,30] and the use of Lewis acids for the activation should be avoided.
Therefore, an alternative approach based on thioglycoside activation by IDCP as a
promoter was selected, which proved to be suitable for the preparation of UDP-2-deoxy
sugars (Scheme 1).
Namely, peracetylated glycal (1 and 2) was treated with O,O'diethyl dithiophosphate to
afford α-glycosyl dithiophosphate 3 (96%) and 4 (91%), respectively. They were
phosphorylated with dibenzyl phosphte in the presence of IDCP as promotor.[31-33]
Analysis by TLC indicated that the phosphorylation was complete without noticeable
side reaction. Then, silica gel chromatography was conducted. Whereas hydrolysis of
the product (5 or 6) during chromatographic purification over silica gel was observed,
compound 5 was isolated in 32% yield (only α-form) along with the hydrolysis product
(30%), when the purification was conducted at 5 ºC. Similarly, the galactose congener 6
was obtained in 26% yield. In this case, formation of nearly equal amount of α- and β-
isomers was observed by TLC. However, through silica gel column the chromatography,
4

most of the latter isomer vanished, suggesting moisture sensitivity of the β-phosphate.
The dibenzyl phosphate (5 or 6) was hydrogenated with platinum oxide to afford the
deprotected phosphate (7 or 8). Then, the monophosphate was coupled with UMP-
morpholidate in presence of 1H-tetrazole. Synthesis of sugar nucleotides is often
complicated by difficulty of purification. Especially, UMP and UMP dimer produced as
by-products retards facile isolation of UDP-sugars from the reaction mixture. The
isolation required sequential silica gel column chromatography by using two kinds of
developing solvents. Namely, the reaction mixture including UDP-glucose, glucose 1-
phosphate, UMP and UMP dimer was concentrated and applied to first silica gel
chromatography which was developed by MeCN−H2O. As shown in Fig. 1(a), this first
chromatography was able to remove UMP and UMP dimer, whereas glucose 1-phosphate
remained (5.6 ppm in Fig. 1 (a)). Next, the resulting mixture was separated on second
silica gel chromatography with the developing solvent of EtOAc−MeOH−H2O. By the
second chromatography, UDP-glucose was isolated and confirmed the structure by 1H
NMR spectrum (Fig. 1 (c)). As expected, purification by only the second silica gel
chromatography gave the mixture of UDP-glucose, UMP and UMP dimer (7.8 ppm in
Fig. 1 (b)), and only glucose-1-phosphate was separated. Then, the isolated UDP-2-
5

deoxy acetyl glucose (9) was deprotected in MeOH−Et3N−H2O at −20 ºC to afford the
desired product (11). The same procedure was applied to 6 to give the UDP-2-deoxy
acetyl galactose (10) in 50% yield (two steps), which was converted to UDP-2-deoxy
galactose (12) quantitatively (Scheme 1).
Stability of synthesized 11 and 12 were compared with UDP-glucose and -galactose at 37
ºC in buffer solutions at pH 4 (acetate buffer), 7 (phosphate buffer) and 10 (carbonate
buffer), and periodically analyzed the decomposition of UDP-sugars by HPLC. Whereas
UDP-2-deoxy glucose had similar retention time to UDP, semi-quantitative estimation
revealed that UDP-2-deoxy glucose and galactose were decomposed to under ca. 10%
within 1 hour at pH 4 (Fig. S1 (c)), while natural UDP-glucose and galactose were stable
(Fig. S1 (a) and (c)). However, in pH 7 and 10 solutions, all UDP-sugars remained
unchanged with no apparent decomposition. Therefore, synthesized UDP-deoxy sugars
could be utilized for enzyme reaction under neutral condition.
CONCLUSION
In summary, UDP-2-deoxy glucose and galactose were synthesized chemically and
isolated by twice silica gel chromatography. These UDP-sugars are unstable in the
6

solution with pH 4, while they are stable in the solutions with pH 7 and 10. These UDP-
sugar analogs will clarify the specificity of various glucosyltransferases and
galactosyltransferases, and might provide oligosaccharide analogs by enzyme reaction.
EXPERIMENTAL
1H, 13C and 31P NMR spectra were recorded at 400, 101 and 162 MHz using a Bruker
AVANCE 400 Plus Nanobay in chloroform-d or deuterium oxide. Unless otherwise
stated, all commercially available solvents and reagents were used without further
purification. Dry solvents were prepared by storage over molecular sieves which were
activated in vacuum at 200 °C. HPLC analysis was performed on a Shimadzu HPLC
system equipped with a TSKgel Amide-80 column (Tosoh Bioscience, 4.6 mm × 250
mm, 5 μm).
General Procedure For UDP-2-Deoxy Sugars
S-(3,4,6-Tri-O-Acetyl-2-Deoxy-Α-D-Glycosyl) O,O'Diethyldithiophosphate
Tri-O-acetyl glycal and O,O'diethyldithiophosphate were stirred in toluene at 0 °C. The
solution was concentrated and the residue was purified by silica gel chromatography to
give the corresponding diethyldithiophosphate in good yield.
7

3,4,6-Tri-O-Acetyl-2-Deoxy-Α-D-Glycosyl 1-O,O'Dibenzyl Phosphate
The diethyldithiophosphate, which was dissolved in dichloromethane, was added
molecular sieves 4Å, dibenzyl phosphate and IDCP and the mixture was stirred. The
mixture was filtered, washed and evaporated. The residue was purified by silica gel
chromatography in the chromatochamber at 5°C to give the corresponding dibenzyl
phosphate.
3,4,6-Tri-O-Acetyl-2-Deoxy-Α-D-Glycosyl Phosphate Bis(Triethylammonium) Salt
To the dibenzyl phosphate in ethyl acetate, platinum oxide (IV) was added and the
mixture was stirred in hydrogen atmosphere. The mixture was filtered and the filtrate
was added triethylamine. The solution was evaporated and the residue was purified by
silica gel chromatography to give a mixture including the corresponding phosphate.
Uridine 5'Diphospho-3,4,6-Tri-O-Acetyl-2-Deoxy-Α-D-Sugar
To a solution of the phosphate and UMP-morpholidate in pyridine, 1H-tetrazole was
added and the mixture was stirred. The mixture was evaporated and the residue was
purified by silica gel chromatography to give the corresponding UDP-acetyl-2-deoxy
sugar.
8

Uridine 5'Diphospho-2-Deoxy-Α-D-Sugar Triethylammonium Salt
The UDP-acetyl-2-deoxy sugar was stirred in triethylamine:methanol:water = 1:2:2 at
−20 °C. Then, the solution was evaporated and lyophilised to give the desired UDP-2-
deoxy sugar in good yield.
SUPPORTING INFORMATION
Supporting information for this article including full experimental procedures and
spectral data for compounds. This material can be found via the “Supplementary
Content” section of this article’s webpage.
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Scheme 1. Synthesis of UDP-2-deoxy Glc (11) and Gal (12). (a) HSP(S)(OEt)2, Tol, rt,
24 h for 3; HSP(S)(OEt)2, Tol, 0 ºC, 18 h for 4; (b) HOP(O)(OBn)2, IDCP, CH2Cl2, rt, 2
h for 5; HOP(O)(OBn)2, IDCP, CH2Cl2, rt, 1.5 h for 6; (c) PtO2, H2, rt, 3 h; (d) UMP-
morpholidate, 1H-tetrazole, rt, 48 h; (e) MeOH−Et3N−H2O, −20 ºC, 41 h for 11;
MeOH−Et3N−H2O, −20 ºC, 17 h for 12.
13

Figure 1. Purification of UDP-2-deoxy AcGlc (9) by silica gel chromatography with
developing solvent (a) EtOAc:MeOH:H2O = 7:3:1; (b) MeCN:H2O = 7:1; (c) MeCN:H2O
= 7:1 (first chromatography) and then EtOAc:MeOH:H2O = 7:3:1 (second
chromatography).
14