Phosphorylase-catalyzed N-formyl-α-glucosaminylation of maltooligosaccharides

Article abstract of DOI:10.1016/j.carres.2010.01.001

This paper describes the phosphorylase-catalyzed enzymatic N-formyl-α-glucosaminylation of maltooligosaccharides for direct incorporation of 2-deoxy-2-formamido-α-d-glucopyranose units into maltooligosaccharides. When the reaction of 2-deoxy-2-formamido-α-d-glucopyranose-1-phosphate (GlcNF-1-P) as the glycosyl donor and maltotetraose as a glycosyl acceptor was performed in the presence of phosphorylase, the N-formyl-α-d-glucosaminylated pentasaccharide was produced, as confirmed by MALDI-TOF MS. Furthermore, the glucoamylase-catalyzed reaction of the crude products supported that the 2-deoxy-2-formamido-α-d-glucopyranoside unit was positioned at the non-reducing end of the pentasaccharide. The pentasaccharide was isolated from the crude products and its structure was further determined by the ¹H NMR analysis. On the other hand, when the phosphorylase-catalyzed reactions of maltotriose and maltopentaose using GlcNF-1-P were conducted, no N-formyl-α-glucosaminylation took place in the former system, whereas the latter system gave N-formyl-α-d-glucosaminylated oligosaccharides with various degrees of polymerization. These results could be explained by the recognition behavior of phosphorylase toward maltooligosaccharides.

Full text of DOI:10.1016/j.carres.2010.01.001

Carbohydrate Research 345 (2010) 631–636
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Phosphorylase-catalyzed N-formyl-a-glucosaminylation
of maltooligosaccharides
*
Satoshi Kawazoe, Hironori Izawa, Mutsuki Nawaji, Yoshiro Kaneko, Jun-ichi Kadokawa
Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto,
Kagoshima 890-0065, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the phosphorylase-catalyzed enzymatic N-formyl-
a
-glucosaminylation of maltool-
-glucopyranose units into maltooli-
-glucopyranose-1-phosphate (GlcNF-1-P)
as the glycosyl donor and maltotetraose as a glycosyl acceptor was performed in the presence of
phosphorylase, the N-formyl- -glucosaminylated pentasaccharide was produced, as confirmed by
MALDI-TOF MS. Furthermore, the glucoamylase-catalyzed reaction of the crude products supported that
the 2-deoxy-2-formamido- -glucopyranoside unit was positioned at the non-reducing end of the
Received 27 October 2009
Received in revised form 27 December 2009
Accepted 1 January 2010
igosaccharides for direct incorporation of 2-deoxy-2-formamido-
gosaccharides. When the reaction of 2-deoxy-2-formamido-
a-D
a-D
Available online 7 January 2010
a
-D
Keywords:
Enzymatic glycosylation
a
-D
pentasaccharide. The pentasaccharide was isolated from the crude products and its structure was further
N-Formyl-
a
-
-glucosaminylation
-glucosaminylated
determined by the 1H NMR analysis. On the other hand, when the phosphorylase-catalyzed reactions of
N-Formyl-
a
D
maltotriose and maltopentaose using GlcNF-1-P were conducted, no N-formyl-
a-glucosaminylation took
oligosaccharide
2-Deoxy-2-formamido-
1-phosphate
place in the former system, whereas the latter system gave N-formyl- -glucosaminylated oligosaccha-
a
-D
a-D-glucopyranose
rides with various degrees of polymerization. These results could be explained by the recognition behav-
ior of phosphorylase toward maltooligosaccharides.
MALDI-TOF MS
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Enzymatic glycosylation is a useful tool for the regio- and ster-
eoselective construction of glycosidic bonds under mild conditions,
In addition to naturally occurring oligo- and polysaccharide
structures displayed on cell surfaces that are involved in molecular
recognition processes mediated by binding events,1 unnatural sac-
charide structures can be expected to show new functions and
applications in glycoscience such as potential as drug candidates.2
Therefore, approaches for the efficient syntheses of unnatural
building blocks for the assembly of complex oligosaccharides com-
posed of multiple different monosaccharide units still receive sig-
nificant attention. Oligosaccharides containing 2-amino-2-deoxy-
where glycosyl donors and glycosyl acceptors can be employed in
their unprotected forms, leading to the direct formation of unpro-
tected saccharide chains in aqueous media.4 Among the enzymes
involved in the formation of glycosidic bonds, phosphorylases have
the potential to be employed in the practical synthesis of saccha-
ride chains.5
a
-Glucan phosphorylase (EC 2.4.1.1, simply phos-
phorylase) is the most extensively studied phosphorylase. This
enzyme catalyzes the reversible phosphorolysis of -(1?4)-glu-
cans such as glycogen and amylose, giving -glucose 1-phosphate
(Glc-1-P). Due to the reversibility of the reaction, -(1?4)-gluco-
a
a
D
-glucopyranose (D-glucosamine, GlcN) units and its derivatives,
a
such as 2-acetamido-2-deoxy-
D
-glucopyranose (N-acetyl- -gluco-
D
sidic linkages can be prepared by the phosphorylase-catalyzed
chain-elongation (glycosylation) using Glc-1-P as a glycosyl do-
nor.6 To initiate the reaction, a maltooligosaccharide as a glycosyl
acceptor is required.
The smallest substrates typically accepted for phosphorolysis
and glycosylation by phosphorylase are maltopentaose (Glc5) and
maltotetraose (Glc4), respectively. Because enzymes often express
loose specificity for substrate structures, extension of the
enzymatic chain-elongation or glycosylation using different sub-
strates is useful to obtain unnatural oligosaccharides. Previously,
samine, GlcNAc), serve key functions in living organisms such as
in cell–cell recognition and immune responses. The preparation
of saccharide chains containing GlcN derivatives, therefore, has
been frequently required for the various studies in glycoscience.
Because highly selective glycosylations are promising approaches
to supply such substrates with well-defined structures, much effort
has been focused on glycosylation using glycosyl donors derived
from GlcNAc and other N-substituted GlcN residues, such as the
oxazoline glycosylation.3
a
-
D
-mannopyranose-1-phosphate, 2-deoxy-
phosphate, and -xylopyranose-1-phosphate were used as the
glycosyl donors for phosphorylase-catalyzed glycosylation, giving
-mannosylated,7a 2-deoxy- -glucosylated,7b and -xylosylated7c
a-D-glucopyranose-1-
a-D
* Corresponding author. Tel.: +81 99 285 7743; fax: +81 99 285 3253.
E-mail address: kadokawa@eng.kagoshima-u.ac.jp (J. Kadokawa).
a
a
a
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.01.001

632
S. Kawazoe et al. / Carbohydrate Research 345 (2010) 631–636
oligosaccharides, respectively. These results indicate that phos-
phorylase does not strictly recognize the hydroxy groups at posi-
tions 2 and 6 in the donor.
amido group prevented approach to the phosphorylase active site.
We have continued to study the extension of the phosphorylase-
catalyzed a-glucosaminylation by investigating the use of 1-phos-
On the basis of the above-mentioned considerations, we have
focused on the use of enzymatic glycosylation to incorporate
phates of other GlcN derivatives as glycosyl donors. Construction of
the new saccharide chains containing different GlcN units has
the potential to lead to the development of new applications of
unnatural substrates, such as drug candidates. In this paper, we re-
a-GlcN derivatives directly into oligosaccharide chains, catalyzed
by phosphorylase. The aforementioned previous studies on the
extension of the phosphorylase-catalyzed glycosylation using dif-
ferent substrates inspired us to examine the possibility of the rec-
ognition of 1-phosphates of GlcN derivatives as the glycosyl donor
by this enzyme, because GlcN is a sugar having an amino group at
position 2 in the place of a hydroxy group of a glucose. This would
allow the facile and highly selective glycosylation for the formation
port that 2-deoxy-2-formamido-a-D-glucopyranose-1-phosphate
(GlcNF-1-P), which has a smaller substituent, that is, a formamide
group rather than the acetamide of GlcNAc-1-P, is recognized as a
glycosyl donor by phosphorylase. This allows the glycosylation
of maltooligosaccharides to give N-formyl-
a-
D-glucosaminylated
maltooligosaccharides.
of
a-(1?4)-glucosaminyl linkages. In our previous study in this
area, we found that 2-amino-2-deoxy-
a-D
-glucopyranose-1-phos-
2. Results and discussion
phate (GlcN-1-P) was recognized as the glycosyl donor by phos-
phorylase, leading to direct incorporation of a GlcN unit at the
non-reducing end of maltooligosaccharides.8 To the best of our
We examined the enzymatic N-formyl-
a-glucosaminylation
using GlcNF-1-P as the glycosyl donor and Glc4 as the glycosyl
acceptor catalyzed by phosphorylase (from potato) in sodium ace-
tate buffer solution (pH 6.2) at 40 °C for 4 days. Maltotetraose
(Glc4) is the smallest glycosyl acceptor for the phosphorylase-cat-
alyzed glycosylation and five equivalents of GlcNF-1-P was used
relative to Glc4. After the reaction mixture was lyophilized, the
knowledge, this report was the first example of the enzymatic
a-
glycosaminylation using the GlcN donor with a free amino group.
However, we also confirmed that phosphorylase did not recognize
2-acetamido-2-deoxy-a-D-glucopyranose- 1-phosphate (GlcNAc-
1-P) as the glycosyl donor. This is probably because the bulky acet-
Figure 1. MALDI-TOF MS spectra of the crude products by enzymatic reaction of maltotetraose (a) and the hydrolyzed products by glucoamylase (b).

S. Kawazoe et al. / Carbohydrate Research 345 (2010) 631–636
633
MALDI-TOF MS measurement was conducted to confirm the trans-
fer of the GlcNF unit to the maltooligosaccharides.
In the MALDI-TOF MS spectrum of the crude products (Fig. 1a),
only a significant peak corresponding to the mass of a pentasaccha-
ride containing one GlcNF unit (GlcNF-Glc4) is observed (m/z
878.9). This data indicates that the transfer of one GlcNF residue
to Glc4 from GlcNF-1-P occurred under these conditions. However,
it was not evident from this data that the GlcNF unit was posi-
tioned at the non-reducing end of the produced pentasaccharide
as shown in Scheme 1. Consequently, the hydrolysis of the crude
products was performed using glucoamylase (GA, EC 3.2.1.3, Wako
Pure Industries) to reveal whether the GlcNF unit was positioned
at the non-reducing end. Glucoamylase catalyzes the exo-type
hydrolysis from the non-reducing end of
a-(1?4)-glucans. In the
Figure 2. Total yield of glycosylated products versus reaction time in the
phosphorylase-catalyzed glycosylation using GlcNF-1-P, GlcN-1-P, and Glc-1-P as
MALDI-TOF MS spectrum of the hydrolyzed products (Fig. 1b),
the peak assigned to the molecular mass of the produced pentasac-
charide remained intact, supporting that the GlcNF unit is posi-
tioned at the non-reducing end. If the transfer of one GlcNF
residue to Glc4 from GlcNF-1-P proceeds once, further N-formyl-
the glycosyl donors and Glc4 as
a glycosyl acceptor (glycosyl donor–glycosyl
acceptor 5:1): d; GlcNF-1-P, N; GlcN-1-P, j; Glc-1-P.
2-formamido-2-deoxy-
The integrated ratio of the signals due to H-1
a
-
D
-glucopyranoside residues, respectively.
+ b:H-10:H-100 is
a
-glucosaminylation is probably suppressed because GlcNF-Glc4
is not recognized as a glycosyl acceptor by phosphorylase.
For further analysis, the formation of N-formyl- -glucosami-
a
1:3:1, supporting the pentasaccharide structure. Furthermore,
there is no signal (d 3.40 ppm) assigned to the H-4 position of a
glucose residue at the non-reducing end of Glc4, whereas there is
a signal (H-400, d 3.51 ppm) ascribed to the free H-4 position of
GlcNF. This observation indicates that the GlcNF unit is positioned
a
-D
nylated oligosaccharides versus reaction time in the phosphory-
lase-catalyzed glycosylation using GlcNF-1-P and Glc4 (5:1) was
evaluated and compared with that using GlcN-1-P as well as Glc-
1-P under the same conditions. The total yields of the glycosylated
products were calculated on the basis of the amounts of inorganic
phosphate produced from the glycosyl donor by the phosphory-
lase-catalyzed glycosylation, which were measured by a modified
Fiske–Subbarow assay.9 A reaction time of 4 days in the glycosyla-
tion using GlcNF-1-P gave 33% yield of the products based on the
amount of Glc4, whereas the yield was 72% in the glycosylation
using GlcN-1-P under the same conditions (Fig. 2). These data indi-
cate that phosphorylase recognizes GlcN-1-P more efficiently than
GlcNF-1-P. However, the reaction using Glc-1-P was much faster
than such two reactions and the yield reached almost 100% in
45 min, because Glc-1-P is the native substrate for phosphorylase.
Moreover, GlcNF-Glc4 was the nearly sole product, as judged by
the MALDI-TOF MS spectra (Fig. 1). This compound was isolated
from the hydrolyzed crude products by the following subsequent
procedures; acetylation, column chromatography, and deacetyla-
tion. Thus, the isolated product was analyzed by the 1H NMR mea-
surement. Besides the signals due to the anomeric proton of the
at the non-reducing end, probably bound with the
a-(1?4)-link-
age. Moreover, the two signals at d 8.04 and 8.17 ppm are ob-
served, which are assigned to E- and Z-N-formyl groups due to
hindered rotation of the formyl C–N bond (two rotamers).10 The
above-mentioned NMR analysis fully supports the GlcNF-Glc4
structure of the isolated oligosaccharide.
We also examined the phosphorylase-catalyzed reaction using
GlcNF-1-P as a glycosyl donor and maltotriose (Glc3) or maltopen-
taose (Glc5) as a glycosyl acceptor under the same conditions. In
the MALDI-TOF MS spectrum of the crude products using Glc3,
no peaks assignable to the molecular masses of oligosaccharides
having a GlcNF unit are observed (Fig. 4a), indicating no occurrence
of N-formyl-a-glucosaminylation of Glc3 by GlcNF-1-P. Because
the smallest glycosyl acceptor for the phosphorylase-catalyzed gly-
cosylation is Glc4, Glc3 was not recognized by phosphorylase. In the
MALDI-TOF MS spectrum of the crude products using Glc5, on the
other hand, several peaks separated by m/z = 162 are observed
(Fig. 4b), which correspond to the molecular masses of pentasac-
charides–octasaccharides containing one GlcNF unit. This finding
reducing ends at d 5.22 ppm (H-1a) and d 4.64 ppm (H-1b) in the
1H NMR spectrum (Fig. 3), two kinds of the anomeric signals are
observed at around d 5.34–5.37 (H-10) and d 5.39 ppm (H-100,
indicates occurrence of both the N-formyl-
a-glucosaminylation
by GlcNF-1-P and glucosylation by Glc-1-P when Glc5 was used
J = 3.7 Hz), which are assignable to the
a-D-glucopyranoside and
Scheme 1. Enzymatic N-formyl-a-glucosaminylation of maltotetraose using GlcNF-1-P.

634
S. Kawazoe et al. / Carbohydrate Research 345 (2010) 631–636
Figure 3. 1H NMR spectrum of the isolated material (D2O).
Figure 4. MALDI-TOF MS spectra of the crude products by enzymatic reaction of maltotriose (a) and maltopentaose (b).
as the glycosyl acceptor. Because Glc5 is the smallest substrate for
the phosphorolysis in the presence of inorganic phosphate, Glc-1-P
is possibly produced by phosphorolysis of Glc5 with simulta-
neously production of Glc4 at an early stage of the reaction, where
inorganic phosphate is formed by the transfer of a GlcNF unit to
Glc5. Then, Glc-1-P is recognized more efficiently by the enzyme
than GlcNF-1-P. Thus, the maltooligosaccharides with larger de-
grees of polymerization (DPs), such as Glc6 and Glc7, are produced
by the transfer of the glucose residues to Glc4 or Glc5 from Glc-1-P.
Furthermore, if N-formyl-
maltooligosaccharides by GlcNF-1-P proceeds, subsequent glyco-
sylation is probably suppressed because the N-formyl- -glucosa-
a-glucosaminylation of the formed
a
-D
minylated oligosaccharides are less efficiently recognized by
phosphorylase.

S. Kawazoe et al. / Carbohydrate Research 345 (2010) 631–636
635
In the reaction using Glc5, therefore, multiple oligosaccharides
of the GlcNF-Glc4–7 are produced in contrast to the reaction with
Glc4. The total yield of the multiple N-formyl-a-glucosaminylated
ride (5.00 g, 14.2 mmol) in 40% yield. 1H NMR (CDCl3) d 2.04, 2.06,
2.11 (3s, 9H, CH3), 4.14 (d, J = 10.5 Hz, 1H, H-6a), 4.28–4.32 (m, 2H,
H-5,6b), 4.63 (ddd, J = 4.1, 8.7, 10.6 Hz, 1H, H-2), 5.23 (t, J = 10.1 Hz,
1H, H-4), 5.37 (t, J = 10.6 Hz, 1H, H-3), 5.95 (d, J = 8.7 Hz, 1H, NH),
oligosaccharides in the reaction using Glc5 after ꢀ50 h was esti-
mated as 16% based on the determination of produced inorganic
phosphate during the reaction. Then, the obtained crude products
were subjected to high performance anion exchange chromatogra-
phy (HPAEC) analysis after glucoamylase-catalyzed hydrolysis
(40 °C for 18 h) to determine the ratio of each oligosaccharide.
The ratio of peak areas corresponding to GlcNF-G4, GlcNF-G6, and
GlcNF-G7 in the HPAEC chart was 1:0.30:0.15. However, a peak
area of GlcNF-G5 could not be calculated under this HPAEC condi-
tions because the corresponding peak was completely overlapped
with a peak due to residual GlcNF-1-P.
6.20 (d, J = 4.1 Hz, 1H, H-1
a), 8.18 (s, 1H, HCO).
To a solution of 3,4,6-tri-O-acetyl-2-deoxy-2-formamido-
a-D-
glucopyranosyl chloride (2.18 g, 6.20 mmol) in CHCl3 (10.0 mL)
were added pyridine (0.500 mL, 6.20 mmol) and water (0.110 mL,
620 mmol) at room temperature and the mixture was stirred over-
night. The reaction mixture was concentrated and the residue was
subjected to column chromatography on silica gel (CHCl3–CH3OH
80:1 (v/v)) to give 3,4,6-tri-O-acetyl-2-deoxy-2-formamido-a-D-
glucopyranose (1.03 g, 3.12 mmol) in 50.3% yield. 1H NMR (CDCl3)
d 2.05, 2.10, 2.18 (3s, 9H, CH3), 3.67 (br s, 1H, OH), 4.13–4.27 (m,
3H, H-5,6), 4.40 (dt, J = 3.6, 10.1 Hz, 1H, H-2), 5.15 (t, J = 9.6 Hz,
In conclusion, we examined the phosphorylase-catalyzed enzy-
matic N-formyl-
using GlcNF-1-P. The results of the reactions depended on the
DPs of maltooligosaccharides used as the glycosyl acceptor. When
a
-glucosaminylation of maltooligosaccharides
1H, H-4), 5.30–5.36 (m, 2H, H-1
8.17 (s, 1H, HCO).
a,3), 6.01 (d, J = 9.6 Hz, 1H, NH),
Dibenzyl N,N-diethylphosphoramidite (3.23 g, 10.2 mmol) was
added to a solution of 3,4,6-tri-O-acetyl-2-deoxy-2-formamido-
-glucopyranose (0.680 g, 2.04 mmol) and 1,2,4-triazole (1.12 g,
Glc4 and Glc5 were used as the glycosyl acceptor, N-formyl-
a
-glu-
a-
cosaminylation occurred to produce the sole product of GlcNF-Glc4
and the multiple products of GlcNF-Glc4–7, respectively. The former
product was isolated and the structure was further determined by
the 1H NMR analysis. On the other hand, the use of Glc3 as the gly-
D
16.3 mmol) in dry CH2Cl2 (20.0 mL) under argon at room tempera-
ture. The mixture was allowed to stir at room temperature for 5 h.
The resulting solution was diluted with CH2Cl2 and then washed
with saturated aq NaHCO3, saturated aq NaCl, and water. The or-
ganic layer was dried over anhydrous Na2SO4 and concentrated.
The residue was dissolved in dry CH2Cl2 (50.0 mL) and aq H2O2
(30%, 13.0 mL, 450 mmol) was added dropwise to the solution at
ꢁ10 °C. After the mixture was stirred at room temperature for
1 h, the reaction mixture was diluted with CH2Cl2 and washed with
saturated aq NaHCO3, saturated aq NaCl, and water. The organic
layer was dried over anhydrous Na2SO4 and concentrated. The res-
idue was chromatographed by silica gel with hexane–EtOAc–trieth-
ylamine (500:400:1 (v/v/v)) to give dibenzyl 3,4,6-tri-O-acetyl-2-
cosyl acceptor did not afford any N-formyl-a-glucosaminylated
products. As reported in our previous paper,8 it should be noted
that GlcNAc-1-P was not recognized by phosphorylase. The smaller
substituent of the formamido group in GlcNF-1-P, compared with
the more bulky acetamido group in GlcNAc-1-P, probably allows
the approach of the substrate to the active site of the enzyme.
3. Experimental
3.1. Materials
deoxy-2-formamido-a-D-glucopyranose 1-phosphate (0.140 g,
0.230 mmol) in 11% yield. 1H NMR (CDCl3) d 2.00, 2.01, 2.18 (3s,
9H, CH3), 3.87 (dd, J = 2.3, 12.6 Hz, 1H, H-6a), 3.98–4.01 (m, 1H,
H-5), 4.12 (dd, J = 4.1, 12.6 Hz, 1H, H-6b), 4.39–4.41 (m, 1H, H-2),
5.06–5.14 (m, 6H, H-3,4, CH2–C6H5), 5.34 (d, J = 9.2 Hz, 1H, NH),
Phosphorylase (from potato) was supplied by Ezaki Glico Co.
Ltd.11 Glucoamylase was purchased from Wako Pure Chemical
Industries (Osaka, Japan). Other reagents and solvents were used
as received.
5.63 (dd, J = 3.2, 5.5 Hz, 1H, H-1a), 7.36–7.41 (m, 10H, C6H5), 7.81
(s, 1H, HCO); 31P NMR (CDCl3) d ꢁ1.98.
3.2. Synthesis of 2-deoxy-2-formamido-
a-D-glucopyranose 1-
Dibenzyl 3,4,6-tri-O-acetyl-2-deoxy-2-formamido-a-d-gluco-
phosphate disodium salt (GlcNF-1-P)12
pyranose 1-phosphate (0.137 g, 0.230 mmol) was hydrogenated
over 10% Pd–C (27.4 mg) in dry CH3OH (5.0 mL) for 1 h at room
temperature. After the mixture was filtered, 1 M aq NaOH was
added dropwise to the filtrate until it was turbid. The resulting
mixture was poured into ethanol to precipitate the product, which
was isolated by filtration and dried under reduced pressure to give
Under argon,
66.6 mmol) in CH3OH (100 mL) was added to a suspension of 2-
amino-2-deoxy- -glucopyranose hydrochloride (10.0 g, 46.3
a solution of sodium methoxide (3.60 g,
D
mmol) in CH3OH (100 mL) at room temperature and the mixture
was stirred for 10 min. After triethylamine (6.00 mL, 46.3 mmol)
was added to the mixture, methyl formate (40.0 mL, 324 mmol)
was added with vigorous stirring at 0 °C. After the mixture was
stirred at room temperature overnight, the resulting precipitate
was isolated by filtration, washed with CH3OH, and dried under re-
2-deoxy-2-formamido-a-D-glucopyranose-1-phosphate disodium
salt (GlcNF-1-P, 0.0505 g, 0.150 mmol) in 65% yield. 1H NMR
(D2O) d 3.48 (t, J = 9.6 Hz, 1H, H-4), 3.74–3.82 (m, 2H, H-3,6a),
3.88 (dd, J = 1.4, 8.2 Hz, 1H, H-6a), 3.93–4.01 (m, 2H, H-2,5), 5.36,
5.45 (dd, J = 3.2, 7.8 and 3.5, 7.5 Hz, respectively, 1H, H-1
a (two
rotamers), 8.02, 8.17 (2s, 1H, HCO (two rotamers); 31P NMR
duced pressure to give 2-deoxy-2-formamido-
D
-glucopyranose
:b = 0.6:0.4) d
(7.46 g, 36.9 mmol) in 80% yield. 1H NMR (D2O,
a
(D2O) d 2.20.
3.17–3.98 (m, 6H, H-2,3,4,5,6), 4.72, 4.75 (2d, J = 8.3, 8.2 Hz,
respectively, 0.4H, H-1(b)), 5.21, 5.25 (2d, J = 3.6, 3.7 Hz, respec-
3.3. Enzymatic N-formyl-a-glucosaminylation of maltooligosac-
charides
tively, 0.6H, H-1(
of rotamers of and b anomers)).
Under argon, acetyl chloride (26.0 mL, 366 mmol) was added to
2-deoxy-2-formamido- -glucopyranose (7.30 g, 36.1 mmol) at 0 °C
a)), 8.02, 8.04, 8.17, 8.21 (4s, 1H, HCO (two pairs
a
A typical experimental procedure for N-formyl-a-glucosaminy-
D
lation was as follows. mixture of GlcNF-1-P (0.070 g,
A
and the mixture was stirred at room temperature overnight. After
the reaction mixture was diluted with CHCl3, the solution was
washed with saturated aq NaHCO3, dried over anhydrous Na2SO4,
filtered, and concentrated. The residue was subjected to column
chromatography on silica gel (hexane–EtOAc 3:2 (v/v)) to give
0.210 mmol) and maltotetraose (0.028 g, 0.042 mmol) in 200 mM
sodium acetate buffer solution (7.0 mL, pH 6.2) was incubated in
the presence of phosphorylase (42 U) at 40 °C for 4 days. After
the reaction mixture was heated at 100 °C for 15 min and filtered,
the filtrate was lyophilized. The residue was applied for MALDI-
TOF MS analysis.
3,4,6-tri-O-acetyl-2-deoxy-2-formamido-a-D-glucopyranosyl chlo-

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S. Kawazoe et al. / Carbohydrate Research 345 (2010) 631–636
3.4. Estimation of the total yield of glycosylated products
5.22 (d, J = 3.7 Hz, 0.5H, H-1(a)), 5.34–5.37 (m, 3H, Glc-H-1), 5.39
(d, J = 3.7 Hz, 1H, GlcNF-H-1), 8.04, 8.17 (2s, 1H, HCO (two
The determination of produced inorganic phosphate in the
phosphorylase-catalyzed glycosylation was as follows. The phos-
phorylase-catalyzed enzymatic reaction was carried out according
to a similar experimental manner as that described in Section 3.3
rotamers)).
3.7. Measurements
1H, and 31P NMR spectra were recorded at 400 and 162 MHz,
respectively, on a JEOL ECX400 spectrometer, and chemical shifts
were referenced to internal sodium 2,2-dimethyl-2-silapentane-
5-sulfonate (DSS, d = 0.0 ppm) or tetramethylsilane (TMS,
d = 0.0 ppm) for 1H NMR, and External 85% H3PO4 (d = 0.0 ppm)
for 31P NMR. MALDI-TOF MS measurements were carried out by
using Shimadzu Voyager Biospectrometry Workstation Ver.5.1
with 2.5-dihydroxybenzoic acid as matrix containing 0.05% triflu-
oroacetic acid under positive ion mode. HPAEC analysis was car-
ried out with a DIONEX DX-300 system (Dionex, CA, USA) with a
pulsed amperometric detector (model PAD-II, Dionex) and a Carb-
using GlcNF-1-P, GlcN-1-P, or Glc-1-P, and the sample (6
taken out at each reaction time. The mixture was diluted with
water (194 L) and 800 L of molybdate reagent (15 mM ammo-
nium molybdate, 100 mM zinc acetate, pH 5.0) and 200 L of
lL) was
l
l
l
ascorbic acid reagent (10% (w/v), pH 5.0) were added to the solu-
tion. The mixture was incubated at 30 °C for 20 min, and the absor-
bance was measured at 850 nm. The sodium acetate buffer
solution of GlcNF-1-P, GlcN-1-P, or Glc-1-P and phosphorylase
was used as a blank.
3.5. Glucoamylase-catalyzed hydrolysis of the crude products
opac PA-100 column (4 mm ꢂ 250 mm). A sample (25
lL) was
A solution of glucoamylase (20 U) in 200 mM sodium acetate
buffer solution (7.0 mL, pH 6.2) was added to the crude residue ob-
tained by the above-mentioned enzymatic N-formyl-a-glucosami-
nylation (Section 3.3) and the mixture was incubated at 40 °C for
1 h. After the reaction solution was heated at 100 °C for 20 min,
the precipitate was filtered. The filtrate was used for the MALDI-
TOF MS analysis.
injected and eluted with a gradient of sodium acetate (0–1 min,
50 mM; 1–23 min, increasing from 50 mM to 350 mM with the
installed gradient program 3; 23–25 min, 850 mM) in 150 mM
NaOH with a flow rate of 1.0 mL/min. For Fiske-Subbarow meth-
od, absorbance was measured at 850 nm using a JASCO V-650Q1
spectrometer.
Acknowledgment
3.6. Isolation of 2-deoxy-2-formamido-a-D-glucopyranosyl-
(1?4)-maltotetraose
The authors thank Ezaki Glico Co. Ltd, Osaka for the gift of phos-
phorylase and for performing the HPAEC measurement.
The crude residue (0.280 g) obtained by the above-mentioned
enzymatic hydrolysis by glucoamylase (Section 3.5) was dissolved
in a mixed solvent of pyridine (4.0 mL) and DMF (4.0 mL). Acetic
anhydride (4.70 mL, 49.5 mmol) was added to the solution and
the mixture was allowed to stir at 60 °C overnight. The reaction
mixture was diluted with CHCl3, washed with 1 M aq H2SO4 satu-
rated aq NaHCO3 and water. The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated. Thus the residue
was subjected to column chromatography on silica gel (hexane–
EtOAc 7:1 (v/v)) to give the pure acetylated product (0.0289 g).
The resulting product was dissolved in CH3OH (3.0 mL) and a solu-
tion of sodium methoxide (0.00102 g, 0.0189 mmol) in CH3OH
(2.0 mL) was added. After the mixture was stirred at room
temperature for 1 h, the reaction mixture was neutralized with
Dowex 50 W (H+ form). The mixture was filtered and the filtrate
was concentrated and dried under reduced pressure. This proce-
dure was repeated twice for completion of deacetylation to give
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2-formamido-2-deoxy-
(0.00710 g, 0.0083 mmol) in 44% yield (based on the amount of
Glc4 used for the enzymatic N-formyl-
-glucosaminylation). 1H
NMR (D2O, :b = 0.5:0.5) d 3.27 (dd, J = 9.6, 7.8 Hz, 0.5H, Glc-H-
a-D-glucopyranosyl-(1?4)-maltotetraose
a
a
2(b)), 3.51 (t, J = 9.2 Hz, 1H, GlcNF-H-4), 3.55–4.08 (m, 28.5H,
Glc-H-2 (expect a proton of H-2(b) corresponding to the signal at
d 3.27),3,4,5,6, GlcNF-H-2,3,5,6), 4.64 (d, J = 7.8 Hz, 0.5H, H-1(b)),
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