One-Step Synthesis of Sugar Nucleotides

Article abstract of DOI:10.1021/acs.joc.0c01943

The chemical synthesis of sugar nucleotides requires a multistep procedure to ensure a selective reaction. Herein, sugar nucleotides were synthesized in one step using 2-chloro-1,3-dimethylimidazolinium chloride as the condensation reagent. The products were obtained in yields of 12-30%, and the yields were increased to 35-47% by the addition of a tuning reagent. NMR identification of the sugar nucleotides showed that mainly 1,2-trans-glycosides were present. The reported method represents a one-step route to sugar nucleotides from commercially available materials.

Full text of DOI:10.1021/acs.joc.0c01943

pubs.acs.org/joc
Note
One-Step Synthesis of Sugar Nucleotides
Atsushi Miyagawa,* Sanami Toyama, Ippei Ohmura, Shun Miyazaki, Takeru Kamiya,
and Hatsuo Yamamura
Cite This: https://dx.doi.org/10.1021/acs.joc.0c01943
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The chemical synthesis of sugar nucleotides requires
a multistep procedure to ensure a selective reaction. Herein, sugar
nucleotides were synthesized in one step using 2-chloro-1,3-
dimethylimidazolinium chloride as the condensation reagent. The
products were obtained in yields of 12−30%, and the yields were
increased to 35−47% by the addition of a tuning reagent. NMR
identification of the sugar nucleotides showed that mainly 1,2-trans-
glycosides were present. The reported method represents a one-
step route to sugar nucleotides from commercially available
materials.
ligosaccharides, polysaccharides, and glycoconjugates
have attracted the attention of biologists due to their
raphy because the dimer exhibits a polarity similar to that of
the desired sugar nucleotide. Moreover, the latter technique
requires the use of an unstable glycosyl halide and strict
anhydrous conditions, and it leads to an anomeric mixture of
sugar nucleotides. However, the resulting products can be
easily separated.
O
broad structural diversities and their roles in the modulation of
a variety of biological processes.1−3 The biosyntheses of these
compounds are catalyzed by glycosyltransferases, which
transfer monosaccharide residues in a step-by-step manner,
both regio- and stereoselectively.4,5 Such enzymatic reactions
are extremely useful in the syntheses of oligosaccharides
because they do not require multistep reactions based on
chemical protection, deprotection, and selective glycosylation.
To date, over 100 families of glycosyltransferases have been
identified and classified based on the sequence similarities of
their amino acids. In terms of the glycosyl donor substrates for
glycosyltransferases, sugar nucleotides (for example, uridine 5′-
diphosphate(UDP)-glucose and UDP-galactose) are con-
sumed as the primary building blocks to build complex
oligosaccharides on glycoconjugates. Therefore, sugar nucleo-
tides are essential in the enzymatic glycosylation process
catalyzed by glycosyltransferase.6−8
To date, the preparation of sugar nucleotides has been
conducted via both chemical and enzymatic routes. More
specifically, the chemical methods can be divided into two
types, viz. pyrophosphate formation by the condensation of a
sugar-1-phosphate with an activated nucleoside monophos-
phate (phosphormorpholidate or diisopropylphosphorami-
dite)9−16 and the direct coupling of an activated sugar (mainly
a bromide) with a nucleoside diphosphate.17−20 Although the
former reaction is particularly slow and gives poor product
yields, these obstacles have been overcome by the addition of
1H-tetrazole10 and metals.21 However, the nucleoside mono-
phosphate dimer formed as a byproduct in the condensation
stage causes issues during purification by silica gel chromatog-
In contrast, enzymatic methods can yield the desired sugar
nucleotides in a one-pot reaction using multiple enzymes.22−26
First, a glycokinase catalyzes the formation of a sugar-1-
phosphate from the corresponding sugar and a nucleoside
triphosphate. Subsequently, a sugar nucleotide pyrophosphor-
ylase constructs the desired sugar nucleoside diphosphate
using the sugar-1-phosphate and a nucleoside triphosphate. An
inorganic pyrophosphatase, which degrades the pyrophosphate
formed in these reactions, is then employed to drive the overall
equilibrium of the multienzymatic reaction toward the
formation of the desired sugar nucleotide. This system
exclusively gives the product exhibiting only the natural-type
anomer configuration. However, issues regarding obtaining
suitable enzymes, optimization of the enzymatic reactions, and
isolation/purification of the products from the reaction
mixture remain.
To address these issues related to tedious synthetic methods,
the direct reaction of a monosaccharide with a nucleotide using
Received: August 11, 2020
https://dx.doi.org/10.1021/acs.joc.0c01943
© XXXX American Chemical Society
J. Org. Chem. XXXX, XXX, XXX−XXX
A

The Journal of Organic Chemistry
pubs.acs.org/joc
Note
Scheme 1. Synthesis of UDP-Glucose 1 via a One-Step Reaction
a
Table 1. Effect of the Base on the Condensation Reaction
b
entry
base
solvent
yield (%)
1
2
3
4
5
6
7
8
TEA
DIPEA
TMA
imidazole
proton sponge
DMAP
H2O/CH3CN (1:1)
28
17
12
4.5
4.2
1.3
nd
nd
nd
nd
nd
nd
nd
nd
DBU
piperidine
2,6-lutidine
NaHCO3
CsCO3
K2SO4
CaCl2
9
10
11
12
13
14
H2O/CH3CN (2:1)
CH3COONa
a
Reaction conditions: glucose (11 μmol), UDP (55 μmol), DMC (36 μmol), and base (220 μmol) in H2O/CH3CN (66 μL) were allowed to react
b
4 °C for 1 h. Determined by HPLC analysis; nd = not detected.
a
Table 2. Effects of the Reaction Solvent and Temperature on the Condensation Reaction
b
entry
solvent
temperature (°C)
yield (%)
1
2
3
4
5
6
7
8
9
10
H2O
4
4
4
4
25
4
4
4
4
25
23
24
29
27
21
24
31
6.8
0.4
nd
H2O/CH3CN (6:1)
H2O/CH3CN (2:1)
H2O/CH3CN (3:2)
H2O/CH3CN (2:1)
H2O/THF (1:1)
H2O/acetone (1:1)
H2O/2-propanol (1:1)
DMF
DMSO
a
Reaction conditions: glucose (11 μmol), UDP (55 μmol), DMC (36 μmol), and TEA (220 μmol) in solvent (66 μL) were allowed to react for 1
b
h. Determined by HPLC analysis; nd = not detected.
B
https://dx.doi.org/10.1021/acs.joc.0c01943
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Note
a
Table 3. Effect of the Number of UDP Equivalents on the Condensation Reaction
b
entry
UDP (equiv)
TEA (equiv)
yield (%)
1
2
3
4
5
3
1
0.2
20
15
12
10
29
25
28
18
a
Reaction conditions: glucose (11 μmol), DMC (36 μmol), UDP, and TEA in H2O/CH3CN (2:1, 66 μL) were allowed to react at 4 °C for 1 h.
b
Determined by HPLC analysis.
Table 4. Preparation of Sugar Nucleotides Using the Optimized Reaction Conditions
a
The J1,2 value of the broadened peak could not be determined.
a condensing agent was investigated and optimized. Herein, we
report a simple and novel method for the synthesis of sugar
nucleotides in a one-step reaction using commercially available
reagents in H2O/CH3CN at 4 °C, followed by subsequent
silica gel chromatographic separation of the desired products
(Scheme 1).
Initially, glucose and UDP were treated with two different
condensation reagents, viz. 4-(4,6-dimethoxy-1,3,5-triazin-2-
C
https://dx.doi.org/10.1021/acs.joc.0c01943
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Note
yl)-4-methylmorpholinium chloride (DMT-MM)27,28 and 2-
chloro-1,3-dimethylimidazolinium chloride (DMC),28−31 to
promote the nonprotecting glycosidation reaction. These
condensation reagents have previously been used for the
synthesis of glycosides using unprotected sugars in aqueous
solution; thus, they were selected for this condensation
reaction, which was also carried out in an aqueous solution
to ensure the dissolution of UDP. As indicated by HPLC
analysis, the coupling reaction employing DMC proceeded to
afford a condensation product, whereas the reaction carried
out in the presence of DMT-MM appeared unsuccessful, with
no product peak being observed. DMC was therefore selected
for use in further reactions.
was condensed with glucose to afford UMP-glucose 2 with the
β-configuration in 18% yield (Table 4, entry 2). This result
indicated that monophosphates can also be coupled easily with
sugars using our procedure. Subsequently, galactose was
condensed with UDP to give an α- and β-mixture of UDP-
galactose 3 in 21% yield, which was a higher α ratio than that
obtained for UDP-glucose 1 (Table 4, entry 3). In contrast,
UDP-mannose 4, bearing the same α-linkage as the natural
product,32 was synthesized through the formation of a 1,2-
trans-glycosidic bond, and guanosine 5′-diphosphate (GDP)
was successfully coupled with mannose to afford the natural
conformation of GDP-mannose 5,33 thereby indicating that
GDP was also suitable for application in the condensation
reaction.
Subsequently, the effect of the base (20 equiv) was
examined, whereby the condensation reaction was conducted
using 3 equiv of DMC in H2O/CH3CN at 4 °C (Table 1).
Due to a constant yield being obtained beyond a reaction time
of 1 h, a comparison of the reaction yields was carried out after
1 h. More specially, the reaction using triethylamine (TEA)
proceeded most efficiently to give UDP-glucose 1 in 28% yield
(Table 1, entry 1). Diisopropyl ethylamine (DIPEA) and
trimethylamine (TMA) gave the desired product in moderate
yields (Table 1, entries 2 and 3), while the coupling reactions
carried out using heterocyclic amines gave low yields (Table 1,
entries 4−6), and inorganic bases produced no trace of the
product (Table 1, entries 10−14). As a result, TEA was
considered the preferred base for this condensation reaction.
The effect of the reaction solvent was then examined, as
summarized in Table 2. Due to the presence of highly polar
phosphate groups in the structure of UDP, it was poorly
soluble in organic solvents, and so the use of DMF, DMSO,
and H2O mixed with various organic solvents was examined.
Based on the obtained product yields, a mixture of H2O/
CH3CN was found to be superior, with a ratio of 2:1 being
optimal. In terms of the reaction temperature, the reaction
carried out at 4 °C gave improved yields compared to those
obtained at 25 °C.
Finally, to improve the yield of this reaction, DMC was
added to the reaction mixture at hourly intervals. The resulting
yields of UDP-glucose and GDP-mannose gradually increased
from 17 and 10%, respectively, at 30 min, to 47 and 35%,
respectively, after 4 h, as shown in Figure 1. The obtained
results indicate that the nucleotides are not consumed by side
reactions but rather that DMC or activated sugars are
hydrolyzed in the aqueous reaction solvent prior to reacting
with the nucleotides.
Figure 1. Synthesis of UDP-glucose and GDP-mannose with the
stepwise addition of DMC. For UDP-glucose: glucose (12 μmol),
UDP (4.6 equiv), DMC (3.0 equiv), and TEA (18.5 equiv) in H2O/
CH3CN (2:1) were allowed to react at 4 °C. To the resulting solution
were added DMC (3.0 equiv) and TEA (5.0 equiv) in H2O/CH3CN
(2:1) at each step. For GDP-mannose: mannose (12 μmol), GDP
(4.1 equiv), DMC (3.2 equiv), and TEA (18.5 equiv) in H2O/
CH3CN (2:1) were allowed to react at 4 °C. To the solution were
added DMC (3.0 equiv) and TEA (5.0 equiv) in H2O/CH3CN (2:1)
at each step.
Subsequently, the ratio of glucose to UDP was varied (Table
3), whereby the number of equivalents of UDP to glucose
ranged from 5 to 0.2. Using 5, 3, or 1 equiv of UDP, similar
product yields were obtained (25−29%), while 0.2 equiv of
UDP gave a yield of only 18%. However, HPLC analysis
indicated the presence of various byproducts, derived from
UDP, which causes issues during purification. Based on the
obtained results, the use of 1 equiv of UDP per glucose
molecule was considered optimal.
Purification of the reaction mixture was then attempted
using silica gel column chromatography as per a previous
report.16 Thus, ethyl acetate/methanol/H2O was employed as
the solvent system, and the fractions were collected. All
fractions exhibiting UV absorbance originating from the uracil
moiety were analyzed by HPLC, and the fractions containing
only the desired product were evaporated and freeze-dried to
afford a white powder. Analysis of this powder by NMR
spectroscopy indicated that the anomeric configuration of the
obtained UDP-glucose 1 was that of the unnatural β-
configuration (J = 7.8 Hz). Since the α-isomer is commercially
available, it was possible to compare its spectral properties (i.e.,
1H, 13C, and 31P NMR spectra) with those of the obtained
product.
In conclusion, a facile synthetic route to sugar nucleotides
was developed based on a simple condensation of sugars with
nucleotides using DMC as the condensing reagent. The
synthesized sugar nucleotides mainly exhibited 1,2-trans-
glycosidic bonds, which differed from the configuration of
natural sugar nucleotides. Although the reaction initially gave a
low yield, this was improved by the stepwise addition of DMC
to afford UDP-glucose 1 and GDP-mannose 5 in 47 and 35%
yields, respectively. Further studies will focus on the
application of our developed method for the formation of
the 1,2-cis bond, which remains an ongoing challenge for
carbohydrate chemists.
EXPERIMENTAL SECTION
The optimized procedure was then applied to a range of
substrates to determine the substrate scope of the reaction
(Table 4). For example, uridine 5′-monophosphate (UMP)
■
1
General. H NMR spectra were recorded in D2O at 400 MHz
using a Bruker AVANCE 400 Plus Nanobay instrument. 13C and 31P
D
https://dx.doi.org/10.1021/acs.joc.0c01943
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Note
NMR spectra were recorded in D2O at 101 and 162 MHz using a
Bruker AVANCE 400 Plus Nanobay instrument and a Bruker
AVANCE 500 instrument fitted with a cryoprobe, respectively.
Chemical shifts (δ) are given in ppm and referenced to the internal
solvent signal used as an internal standard. Assignments in the NMR
spectra were made by first-order analysis of the spectra and were
supported by 1H−1H COSY, 1H−13C HMQC correlation results.
High-resolution mass spectrometry was performed on a Waters
Synapt G2-S HDMS spectrometer. Optical rotation measurements
were carried out on a Horiba SEPA-300 instrument. Unless otherwise
stated, all of the commercially available solvents and reagents were
purchased from FUJIFILM Wako Pure Chemical Corporation and
Merck KGaA without further purification. During purification by silica
gel column chromatography, the absorbances of all fractions were
measured at 262 nm using a Shimadzu UV-1280 detector. 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). The HPLC system consisted of a system controller (CBM-20A),
a diode array detector (SPD-M20A), a pump (LC-20AD), an
autosampler (SIL-20AC), a column oven (CTO-20AC), and a
degasser (DGU-20As).
General Procedure for Investigating the Effect of Different
Bases (Table 1). The sugar (11 μmol) was dissolved in H2O/
CH3CN (2:1, 44 μL), and 3.3 equiv of DMC (36 μmol) dissolved in
H2O/CH3CN (2:1, 22 μL) was added. Subsequently, 10 equiv of the
base (220 μmol) and 5.0 equiv of UDP (55 μmol) were added to the
solution, and the mixture was stirred until all components had
completely dissolved. After stirring at 4 °C for 1 h, the reaction
mixture was collected and diluted 100 times with water. The diluted
solution was analyzed using HPLC to calculate the product yields.
General Procedure for Investigating the Effect of Different
Reaction Solvents (Table 2). The sugar (11 μmol) was dissolved in
the desired solvent (2:1, 44 μL), and 3.3 equiv of DMC (36 μmol)
dissolved in the desired solvent (2:1, 22 μL) was added. Subsequently,
10 equiv of Et3N (220 μmol) and 5.0 equiv of UDP (55 μmol) were
added to the solution, and the mixture was stirred until all of the
components had completely dissolved. After stirring at 4 or 25 °C for
1 h, the reaction mixture was collected and diluted 100 times with
water. The diluted solution was analyzed using HPLC to calculate the
product yields.
73.7 (C-2′), 73.6 (C-2), 69.7 (C-3′), 69.4 (C-4), 65.0 (C-5′), 60.8
(C-6); 31P NMR (162 MHz, D2O) δ −11.37 (d, JP,P = 16.2 Hz),
−13.11 (d); HRMS (ESI/Q-TOF) m/z [M − H]− calcd for
−
C15H23N2O17P2 565.0477, found 565.0475; [α]2D6.4 −11.7 (10−1 deg
cm2 g−1) (c 3.216 × 10−3, H2O).
Uridine 5′-Monophospho-β-D-glucopyranose, 2. D-Glucose (16
mg, 87 μmol) was dissolved in H2O/CH3CN (2:1, 200 μL), and
DMC (46 mg, 269 μmol) dissolved in H2O/CH3CN (2:1, 150 μL)
was added to the resulting solution. Subsequently, Et3N (148 μL, 1.06
mmol) and UMP (32 mg, 87 μmol) were added to the solution, and
the mixture was stirred at 4 °C for 1 day. The reaction mixture was
then purified by silica gel chromatography using a solvent
composition and gradient of 9:2:1 → 8:2:1 → 6:2:1 → 5:2:1 →
4:2:1 → 3:2:1 (v/v/v) ethyl acetate/methanol/H2O. The absorbance
of each fraction was measured at 262 nm, and the combined fractions
were evaporated to give UMP-glucose 2 (9 mg, 18%): 1H NMR (400
MHz, D2O) δ 7.95 (d, 1.0H, J5″,6′′ = 8.1 Hz, H-6″), 6.00−5.95 (m,
2.0H, H-1′, H-5′′), 4.93 (dd, 1.0H, J1,P = 7.6 Hz, J1,2 = 7.6 Hz, H-1),
4.39−4.35 (m, 2.0H, H-2′, H-3′), 4.29−4.14 (m, 3.0H, H-4′, H-5a′,
H-5b′), 3.92 (dd, 1.0H, J6a,6b = 12.5 Hz, J5,6a = 2.0 Hz, H-6a), 3.74
(dd, 1.0H, J5,6b = 5.6 Hz, H-6b), 3.56−3.49 (m, 2.0H, H-3, H-5),
3.44−3.34 (m, 2.0H, H-2, H-4); 13C{1H} NMR (101 MHz, D2O) δ
166.2 (C-4″), 151.8 (C-2′′), 141.6 (C-6′′), 102.5 (C-5′′), 97.8 (C-1),
88.5 (C-1′), 83.2 (C-4′), 76.4, 75.3 (C-3, C-5), 73.7 (C-2′), 73.5 (C-
2), 69.7 (C-3′), 69.3 (C-4), 64.8 (C-5′), 60.6 (C-6); 31P NMR (162
MHz, D2O) δ −1.81 (d, JP,P = 4.86 Hz); HRMS (ESI/Q-TOF) m/z
−
[M − H]− calcd for C15H22N2O14P1 485.0814, found 485.0802.
Uridine 5′-Diphospho-αβ-D-galactose, 3. D-Galactose (25 mg,
139 μmol) was dissolved in H2O/CH3CN (2:1, 600 μL), and DMC
(70 mg, 416 μmol) dissolved in H2O/CH3CN (2:1, 50 μL) was
added to the resulting solution. Subsequently, Et3N (232 μL, 1.80
mmol) and UDP (62 mg, 139 μmol) were added to the solution, and
the mixture was stirred at 4 °C for 1 d. The reaction mixture was
purified by silica gel chromatography using a solvent composition and
gradient of 9:2:1 → 8:2:1 → 7:2:1 → 6:2:1 (v/v/v) ethyl acetate/
methanol/H2O. The absorbance of each fraction was measured at 262
nm, and the combined fractions were evaporated. The obtained
powders were identified and then collected to afford UDP-galactose 3
(16 mg, 21%, α/β = 1:1.9). UDP-galactose 3 (α:β = 1:13): 1H NMR
(400 MHz, D2O) δ 7.96 (d, 1.1H, J5″,6′′ = 8.1 Hz, H-6″), 6.00−5.98
(m, 2.2H, H-1′, H-5′′), 5.66 (dd, 0.1H, J1,P = 7.2 Hz, J1,2 = 3.6 Hz, H-
1α), 4.97 (br d, 1.0H, J1,P = 7.8 Hz, J1,2 = 7.8 Hz, H-1β), 4.41−4.38
(m, 2.2H, H-2′, H-3′), 4.30−4.20 (m, 3.3H, H-4′, H-5a′, H-5b′),
4.04−4.03 (br d, 0.1H, H-4α), 3.94−3.93 (br d, 1.0H, H-4β), 3.85−
3.69 (m, 4.2H, H-3β, H-5β, H-6aβ, H-6bβ, H-2α, H-3α), 3.63 (dd,
1.2H, J2,3 = 10.0 Hz, H-2β, H-6aα, H-6bα); 13C{1H} NMR (101
MHz, D2O) δ 166.3 (C-4″), 151.9 (C-2′′), 141.6 (C-6′′), 102.7 (C-
5′′), 98.5 (C-1β), 88.3 (C-1′), 83.3, 83.2 (C-4′), 75.9 (C-5), 73.7 (C-
2′), 72.3 (C-3), 71.3, 71.2 (C-2), 69.7 (C-3′), 68.6 (C-4), 65.1, 65.0
(C-5′), 61.2 (C-6), 46.7 (NCH2), 8.2 (CH3); 31P NMR (162 MHz,
D2O) δ −11.51 (d, JP,P = 14.6 Hz), −13.82 (d); HRMS (ESI/Q-
General Procedure for Investigating of the Effect of UDP
Equivalents (Table 3). The sugar (11 μmol) was dissolved in H2O/
CH3CN (2:1, 44 μL), and 3.3 equiv of DMC (36 μmol) dissolved in
H2O/CH3CN (2:1, 22 μL) was added. Subsequently, Et3N (220, 165,
or 136 μmol) and UDP (55, 34, or 11 μmol) were added to the
solution, and the mixture was stirred until all of the components had
completely dissolved. After stirring at 4 °C for 1 h, the reaction
mixture was collected and diluted 100 times with water. The diluted
solution was analyzed using HPLC to calculate the product yields.
Preparation of the Sugar Nucleotides. Uridine 5′-Diphospho-
αβ-D-glucopyranose, 1. D-Glucose (27 mg, 150 μmol) was dissolved
in D2O/CH3CN (2:1, 400 μL), and DMC (76 mg, 450 μmol)
dissolved in D2O/CH3CN (2:1, 200 μL) was added to the resulting
solution. Subsequently, Et3N (251 μL, 1.80 mmol) and UDP (68 mg,
152 μmol) were added to the solution, and the mixture was stirred at
4 °C for 3 days. The reaction mixture was then divided into 351 μL
portions for analysis and 500 μL for separation. The 500 μL aliquot
was purified by silica gel chromatography using a solvent composition
and gradient of 9:2:1 → 8:2:1 → 6:2:1 (v/v/v) ethyl acetate/
methanol/H2O. The absorbance of each fraction was measured at 262
nm, and the combined fractions were evaporated to give UDP-glucose
−
TOF) m/z [M − H]− calcd for C15H23N2O17P2 565.0477, found
565.0478.
Uridine 5′-Diphospho-α-D-mannopyranose, 4. D-Mannose (27
mg, 149 μmol) was dissolved in H2O/CH3CN (2:1, 400 μL), and
DMC (77 mg, 454 μmol) dissolved in H2O/CH3CN (2:1, 200 μL)
was added to the resulting solution. Subsequently, Et3N (251 μL, 1.80
mmol) and UDP (67 mg, 150 μmol) were added to the solution, and
the mixture was stirred at 4 °C for 1 day. The reaction mixture was
then divided into 351 μL and 500 μL portions for analysis and
separation, respectively. The 500 μL portion was purified by silica gel
chromatography using a solvent composition and gradient of 9:2:1 →
8:2:1 → 6:2:1 → 5:2:1 → 4:2:1 → 3:2:1 → 2:2:1 (v/v/v) ethyl
acetate/methanol/H2O. The absorbance of each fraction was
measured at 262 nm, and the combined fractions were evaporated
to give UDP-mannose 4 (10 mg, 20%): 1H NMR (400 MHz, D2O) δ
7.96 (d, 1.0H, J5″,6′′ = 8.0 Hz, H-6″), 6.00−5.97 (m, 2.0H, H-1′, H-
5′′), 5.53 (dd, 1.0H, J1,2 = 1.6 Hz, J1,P = 7.6 Hz, H-1), 4.39−4.35 (m,
2.0H, H-2′, H-3′), 4.30−4.17 (m, 3.0H, H-4′, H-5a′, H-5b′), 4.06−
4.04 (m, 1.0H, H-2), 3.94−3.84 (m, 3.0H, H-3, H-5, H-6a), 3.77 (dd,
1
1 (15 mg, 30%, α/β = 1:17): H NMR (400 MHz, D2O) δ 7.95 (d,
1.0H, J5″,6′′ = 8.0 Hz, H-6″), 6.00−5.98 (m, 2.1H, H-1′, H-5′′), 5.62
(dd, 0.1H, J1α,P = 7.2 Hz, J1α,2 = 3.6 Hz, H-1α), 5.03 (dd, 1.0H, J1β,P
=
7.9 Hz, J1β,2 = 7.9 Hz, H-1β), 4.40−4.36 (m, 2.1H, H-2′, H-3′), 4.29−
4.18 (m, 3.2H, H-4′, H-5a′, H-5b′), 3.92 (dd, 1.0H, J6a,6b = 12.4 Hz,
J
5,6a = 2.0 Hz, H-6a), 3.72 (dd, 1.0H, J5,6b = 6.4 Hz, H-6b), 3.57−3.50
(m, 2.1H, H-3, H-5), 3.42−3.37 (m, 2.1H, H-2, H-4); 13C{1H} NMR
(101 MHz, D2O) δ 166.2 (C-4″), 151.8 (C-2′′), 141.6 (C-6′′), 102.7
(C-5′′), 97.9 (C-1), 88.4 (C-1′), 83.2 (C-4′), 76.5, 75.2 (C-3, C-5),
E
https://dx.doi.org/10.1021/acs.joc.0c01943
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Note
1.0H, J5,6b = 5.6 Hz, J6a,6b = 12.4 Hz, H-6b), 3.69 (dd, 1.0H, J5,6b = 9.6
Hz, H-4); 13C{1H} NMR (101 MHz, D2O) δ 166.3 (C-4″), 151.9
(C-2′′), 141.6 (C-6′′), 102.6 (C-5′′), 96.5 (C-1), 88.5 (C-1′), 83.2,
83.1 (C-4′), 73.7 (C-2′, C-5), 70.2 (C-2), 69.8 (C-3), 69.6 (C-3′),
66.4 (C-4), 64.9 (C-5′), 60.8 (C-6); 31P NMR (162 MHz, D2O) δ
−11.56 (d, JP,P = 13.0 Hz), −13.91 (d); HRMS (ESI/Q-TOF) m/z
reaction mixture was collected for analysis by HPLC and yield
determination. To the remaining reaction mixture was added DMC
(6.7 mg, 40 μmol) dissolved in H2O/CH3CN (2:1, 10 μL) and Et3N
(9.3 μL, 67 μmol), and the mixture was allowed to stir at 4 °C for a
further 60 min. After this time, an aliquot (10 μL) of the reaction
mixture was collected for analysis by HPLC and yield determination.
To the remaining reaction mixture was added DMC (6.4 mg, 38
μmol) dissolved in H2O/CH3CN (2:1, 10 μL) and Et3N (9.3 μL, 67
μmol), and the mixture was allowed to stir at 4 °C for a further 60
min. After this time, an aliquot (10 μL) of the reaction mixture was
collected for analysis by HPLC and yield determination. To the
remaining reaction mixture was added DMC (6.7 mg, 40 μmol)
dissolved in H2O/CH3CN (2:1, 10 μL) and Et3N (9.3 μL, 67 μmol),
and the mixture was allowed to stir at 4 °C for a further 60 min. After
this time, an aliquot (10 μL) of the reaction mixture was collected for
analysis by HPLC and yield determination. More specifically, the
sample aliquots for HPLC analysis were diluted with H2O/CH3CN
(2:1, 240 μL) prior to injection. The analytical results gave the
following yields 30 min = 10%; 90 min = 18%; 150 min = 22%; 210
min = 26%; 270 min = 35%.
−
[M − H]− calcd for C15H23N2O17P2 565.0477, found 565.0466.
Guanosine 5′-Diphospho-α-D-mannopyranose, 5. D-Mannose
(13 mg, 72 μmol) was dissolved in H2O/CH3CN (2:1, 400 μL),
and DMC (35 mg, 207 μmol) dissolved in H2O/CH3CN (2:1, 60
μL) was added to the resulting solution. Subsequently, Et3N (110 μL,
789 μmol) and GDP (33 mg, 67 μmol) were added to the solution,
and the mixture was stirred at 4 °C for 1 h. The reaction mixture was
then purified by silica gel chromatography using a solvent
composition and gradient of 9:2:1 → 8:2:1 → 7:2:1 → 6:2:1 →
5:2:1 → 4:2:1 → 3:2:1 (v/v/v) ethyl acetate/methanol/H2O. The
absorbance of each fraction was measured at 262 nm, and the
combined fractions were evaporated to give GDP-mannose 5 (5 mg,
1
12%): H NMR (400 MHz, D2O) δ 8.13 (s, 1.0H, H-8″), 5.92 (d,
1.0H, J1′,2′ = 5.9 Hz, H-1′), 5.48 (br s, 1.0H, H-1), 4.48 (br t, 1.0H,
J3′,4′ = 3.8 Hz, H-3′), 4.33 (br s, 1.0H, H-4′), 4.18 (br s, 2.0H, H-5a′,
H-5b′), 4.01 (br s, 1.0H, H-2), 3.90−3.82 (m, 3.0H, H-3, H-5, H-6a),
3.74−3.62 (m, 2.0H, H-4, H-6b); 13C{1H} NMR (101 MHz, D2O) δ
154.0 (C-2″), 96.5 (C-1), 86.9 (C-1′), 83.8 (C-4′), 73.7 (C-2′), 73.6
(C-5), 70.4 (C-3′), 70.2 (C-2), 69.8 (C-4), 66.4 (C-3), 65.3 (C-5′),
60.8 (C-6); 31P NMR (162 MHz, D2O) δ −11.57 (d, JP,P = 13.0 Hz),
−13.92 (dd); HRMS (ESI/Q-TOF) m/z [M − H]− calcd for
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c01943.
1H, 13C, and 31P NMR, 1H−1H COSY, 1H−13C HMQC,
and ESI-MS spectra for all products (PDF)
−
C16H24N5O16P2 604.0699, found 604.0684.
Effect of the Interval Addition of DMC. Uridine 5′-
Diphospho-αβ-D-glucopyranose. D-Glucose (2.2 mg, 12 μmol) was
dissolved in H2O/CH3CN (2:1, 44 μL), and DMC (6.0 mg, 36 μmol)
dissolved in H2O/CH3CN (2:1, 22 μL) was added to the resulting
solution. Subsequently, Et3N (31 μL, 222 μmol) and UDP (25 mg, 56
μmol) were added to the solution, and the mixture was stirred at 4 °C
for 30 min. After this time, an aliquot (5.0 μL) of the reaction mixture
was collected for analysis by HPLC and yield determination. To the
remaining reaction mixture was added DMC (5.4 mg, 32 μmol)
dissolved in H2O/CH3CN (2:1, 10 μL) and Et3N (9.3 μL, 67 μmol),
and the mixture was allowed to stir at 4 °C for a further 60 min. After
this time, an aliquot (5.0 μL) of the reaction mixture was collected for
analysis by HPLC and yield determination. To the remaining reaction
mixture was added DMC (6.1 mg, 36 μmol) dissolved in H2O/
CH3CN (2:1, 10 μL) and Et3N (9.3 μL, 67 μmol), and the mixture
was allowed to stir at 4 °C for a further 60 min. After this time, an
aliquot (5.0 μL) of the reaction mixture was collected for analysis by
HPLC and yield determination. To the remaining reaction mixture
was added DMC (5.6 mg, 33 μmol) dissolved in H2O/CH3CN (2:1,
10 μL) and Et3N (9.3 μL, 67 μmol), and the mixture was allowed to
stir at 4 °C for a further 60 min. After this time, an aliquot (5 μL) of
the reaction mixture was collected for analysis by HPLC and yield
determination. To the remaining reaction mixture was added DMC
(5.2 mg, 31 μmol) dissolved in H2O/CH3CN (2:1, 10 μL) and Et3N
(9.3 μL, 67 μmol), and the mixture was allowed to stir at 4 °C for a
further 60 min. After this time, an aliquot (5 μL) of the reaction
mixture was collected for analysis by HPLC and yield determination.
More specifically, the sample aliquots for HPLC analysis were diluted
with H2O/CH3CN (2:1, 245 μL) prior to injection. The analytical
results gave the following yields 30 min = 17%; 90 min = 25%; 150
min = 34%; 210 min = 41%; 270 min = 47%.
AUTHOR INFORMATION
■
Corresponding Author
Atsushi Miyagawa − Department of Materials Science and
Engineering and Department of Life Science and Applied
Chemistry, Nagoya Institute of Technology, Nagoya 466-
8555, Japan; orcid.org/0000-0003-0809-0912;
Email: miyagawa.atsushi@nitech.ac.jp
Authors
Sanami Toyama − Department of Materials Science and
Engineering, Nagoya Institute of Technology, Nagoya 466-
8555, Japan
Ippei Ohmura − Department of Life Science and Applied
Chemistry, Nagoya Institute of Technology, Nagoya 466-
8555, Japan
Shun Miyazaki − Department of Life Science and Applied
Chemistry, Nagoya Institute of Technology, Nagoya 466-
8555, Japan
Takeru Kamiya − Department of Life Science and Applied
Chemistry, Nagoya Institute of Technology, Nagoya 466-
8555, Japan
Hatsuo Yamamura − Department of Materials Science and
Engineering and Department of Life Science and Applied
Chemistry, Nagoya Institute of Technology, Nagoya 466-
8555, Japan
Guanosine 5′-Diphospho-α-D-mannopyranose. D-Mannose (2.2
mg, 12 μmol) was dissolved in H2O/CH3CN (2:1, 132 μL), and
DMC (6.4 mg, 38 μmol) dissolved in H2O/CH3CN (2:1, 66 μL) was
added to the resulting solution. Subsequently, Et3N (31 μL, 222
μmol) and GDP (24 mg, 49 μmol) were added to the solution, and
the mixture was stirred at 4 °C for 30 min. After this time, an aliquot
(10 μL) of the reaction mixture was collected for analysis by HPLC
and yield determination. To the remaining reaction mixture was
added DMC (6.6 mg, 39 μmol) dissolved in H2O/CH3CN (2:1, 10
μL) and Et3N (9.3 μL, 67 μmol), and the mixture was allowed to stir
at 4 °C for a further 60 min. After this time, an aliquot (10 μL) of the
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c01943
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
F
https://dx.doi.org/10.1021/acs.joc.0c01943
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Note
(19) Timmons, S. C.; Jakeman, D. L. Stereoselective chemical
synthesis of sugar nucleotides via direct displacement of acylated
glycosyl bromides. Org. Lett. 2007, 9, 1227−1230.
ACKNOWLEDGMENTS
■
This work was supported by JSPS KAKENHI grant nos.
JP20K05602 (A.M.).
̀
(20) Peltier, P.; Daniellou, R.; Nugier-Chauvin, C.; Ferrieres, V.
Versatile synthesis of rare nucleotide furanoses. Org. Lett. 2007, 9,
5227−5230.
(21) Dabrowski-Tumanski, P.; Kowalska, J.; Jemielity, J. Efficient
and Rapid Synthesis of Nucleoside Diphosphate Sugars from
Nucleoside Phosphorimidazolides. Eur. J. Org. Chem. 2013, 11,
2147−2154.
ABBREVIATIONS
■
DMC, 2-chloro-1,3-dimethylimidazolinium chloride; UDP,
uridine 5′-diphosphate; UMP, uridine 5′-monophosphate;
GDP, guanosine 5′-diphosphate
(22) Roseman, S. Enzymatic synthesis of cytidine 5′-monophospho-
sialic acids. Proc. Natl. Acad. Sci. U. S. A. 1962, 48, 437−441.
(23) Jiang, J.; Biggins, J. B.; Thorson, J. S. A general enzymatic
method for the synthesis of natural and “unnatural” UDP-and TDP-
nucleotide sugars. J. Am. Chem. Soc. 2000, 122, 6803−6804.
(24) Oka, T.; Jigami, Y. Reconstruction of de novo pathway for
synthesis of UDP-glucuronic acid and UDP-xylose from intrinsic
UDP-glucose in Saccharomyces cerevisiae. FEBS J. 2006, 273, 2645−
2657.
(25) Chen, Y.; Thon, V.; Li, Y.; Yu, H.; Ding, L.; Lau, K.; Qu, J.;
Hie, L.; Chen, X. One-pot three-enzyme synthesis of UDP-GlcNAc
derivatives. Chem. Commun. 2011, 47, 10815−10817.
(26) Muthana, M. M.; Qu, J.; Li, Y.; Zhang, L.; Yu, H.; Ding, L.;
Malekan, H.; Chen, X. Efficient one-pot multienzyme synthesis of
UDP-sugars using a promiscuous UDP-sugar pyrophosphorylase from
Bifidobacterium longum (BLUSP). Chem. Commun. 2012, 48, 2728−
2730.
REFERENCES
■
(1) Bertozzi, C. R.; Kiessling, L. L. Chemical Glycobiology. Science
2001, 291, 2357−2364.
(2) Gabius, H.-J. The Sugar Code: Fundamentals of Glycosciences;
Wiley-VCH: Weinheim, Germany, 2011.
(3) Varki, A.; Cummings, R. D.; Esko, J. D.; Stanley, P.; Hart, G. W.;
Aebi, M.; Darvill, A. G.; Kinoshita, T.; Packer, N. H.; Prestegard, J.
H.; Schnaar, R. L.; Seeberger, P. H. Essentials of Glycobiology, 3rd ed.;
Cold Spring Harbor Laboratory Press: New York, 2017.
̌
́
̌
(4) Breton, C.; Snajdrova, L.; Jeanneau, C.; Koca, J.; Imberty, A.
Structures and mechanisms of glycosyltransferases. Glycobiology 2006,
16, 29R−37R.
(5) Lairson, L. L.; Henrissat, B.; Davies, G. J.; Withers, S. G.
Glycosyltransferases: structures, functions, and mechanisms. Annu.
Rev. Biochem. 2008, 77, 521−555.
(27) Kunishima, M.; Kawachi, C.; Monta, J.; Terao, K.; Iwasaki, F.;
Tani, S. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium
chloride: an efficient condensing agent leading to the formation of
amides and esters. Tetrahedron 1999, 55, 13159−13170.
(28) Noguchi, M.; Tanaka, T.; Gyakushi, H.; Kobayashi, A.; Shoda,
S. I. Efficient synthesis of sugar oxazolines from unprotected N-acetyl-
2-amino sugars by using chloroformamidinium reagent in water. J.
Org. Chem. 2009, 74, 2210−2212.
(29) Tanaka, T.; Kikuta, N.; Kimura, Y.; Shoda, S. I. Metal-catalyzed
stereoselective and protecting-group-free synthesis of 1,2-cis-glyco-
sides using 4,6-dimethoxy-1,3,5-triazin-2-yl glycosides as glycosyl
donors. Chem. Lett. 2015, 44, 846−848.
(30) Tanaka, T.; Matsumoto, T.; Noguchi, M.; Kobayashi, A.;
Shoda, S. I. Direct transformation of unprotected sugars to aryl 1-thio-
β-glycosides in aqueous media using 2-chloro-1,3-dimethylimidazoli-
nium chloride. Chem. Lett. 2009, 38, 458−459.
(31) Novoa, A.; Barluenga, S.; Serba, C.; Winssinger, N. Solid phase
synthesis of glycopeptides using Shoda’s activation of unprotected
carbohydrates. Chem. Commun. 2013, 49, 7608−7610.
(32) Nakajima, K.; Kizuka, Y.; Yamaguchi, Y.; Hirabayashi, Y.;
Takahashi, K.; Yuzawa, Y.; Taniguchi, N. Identification and
characterization of UDP-mannose in human cell lines and mouse
organs: Differential distribution across brain regions and organs.
Biochem. Biophys. Res. Commun. 2018, 495, 401−407.
(33) Jensen, J. W.; Schutzbach, J. S. The biosynthesis of
oligosaccharide-lipids. Partial purification and characterization of
mannosyltransferase II. J. Biol. Chem. 1981, 256, 12899−12904.
(6) Sears, P.; Wong, C. H. Toward automated synthesis of
oligosaccharides and glycoproteins. Science 2001, 291, 2344−2350.
(7) Murata, T.; Usui, T. Enzymatic synthesis of oligosaccharides and
neoglycoconjugates. Biosci., Biotechnol., Biochem. 2006, 70, 1049−
1059.
(8) Villadsen, K.; Martos-Maldonado, M. C.; Jensen, K. J.; Thygesen,
M. B. Chemoselective reactions for the synthesis of glycoconjugates
from unprotected carbohydrates. ChemBioChem 2017, 18, 574−612.
(9) Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G.
Nucleoside Polyphosphates. XI. An Improved General Method for the
Synthesis of Nucleotide Coenzymes. Syntheses of Uridine-5′,
Cytidine-5′ and Guanosine-5′, Diphosphate Derivatives. J. Am.
Chem. Soc. 1961, 83, 659−663.
(10) Wittmann, V.; Wong, C.-H. 1H-Tetrazole as Catalyst in
Phosphomorpholidate Coupling Reactions: Efficient Synthesis of
GDP-Fucose, GDP-Mannose, and UDP-Galactose. J. Org. Chem.
1997, 62, 2144−2147.
(11) Marlow, A. L.; Kiessling, L. L. Improved Chemical Synthesis of
UDP-Galactofuranose. Org. Lett. 2001, 3, 2517−2519.
(12) Dinev, Z.; Wardak, A. Z.; Brownlee, R. T.; Williams, S. J. A
convenient gram-scale synthesis of uridine diphospho (13C6) glucose.
Carbohydr. Res. 2006, 341, 1743−1747.
(13) Gold, H.; van Delft, P.; Meeuwenoord, N.; Codee, J. D. C.;
Filippov, D. V.; Eggink, G.; Overkleeft, H. S.; van der Marel, G. A.
Synthesis of Sugar Nucleotides by Application of Phosphoramidites. J.
Org. Chem. 2008, 73, 9458−9460.
(14) Wagner, G. K.; Pesnot, T.; Field, R. A. A survey of chemical
methods for sugar-nucleotide synthesis. Nat. Prod. Rep. 2009, 26,
1172−1194.
(15) Tanaka, H.; Yoshimura; Jørgensen, M. R.; Cuesta-Seijo, J. A.;
Hindsgaul, O. A Simple Synthesis of Sugar Nucleoside Diphosphates
by Chemical Coupling in Water. Angew. Chem., Int. Ed. 2012, 51,
11531−11534.
(16) Miyagawa, A.; Takeuchi, S.; Itoda, S.; Toyama, S.; Kurimoto,
K.; Yamamura, H.; Ito, Y. Chemical synthesis and isolation of UDP-2-
deoxy glucose and galactose. Synth. Commun. 2016, 46, 1790−1795.
(17) Ernst, C.; Klaffke, W. Chemical Synthesis of Uridine
Diphospho-D-xylose and UDP-L-arabinose. J. Org. Chem. 2003, 68,
5780−5783.
(18) Arlt, M.; Hindsgaul, O. Rapid chemical synthesis of sugar
nucleotides in a form suitable for enzymic oligosaccharide synthesis. J.
Org. Chem. 1995, 60, 14−15.
G
https://dx.doi.org/10.1021/acs.joc.0c01943
J. Org. Chem. XXXX, XXX, XXX−XXX