Acceptor specificity in the transglycosylation reaction using Endo-M

Article abstract of DOI:10.1016/j.carres.2010.08.022

To determine the structural specificity of the glycosyl acceptor of the transglycosylation reaction using endo-β-N-acetylglucosaminidase (ENGase) (EC 3.2.1.96) from Mucor hiemalis (Endo-M), several acceptor derivatives were designed and synthesized. The narrow regions of the 1,3-diol structure from the 4- to 6-hydroxy functions of GlcNAc were found to be essential for the transglycosylation reaction using Endo-M. Furthermore, it was determined that Endo-M strictly recognizes a 1,3-diol structure consisting of primary and secondary hydroxyl groups.

Full text of DOI:10.1016/j.carres.2010.08.022

Carbohydrate Research 345 (2010) 2458–2463
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Acceptor specificity in the transglycosylation reaction using Endo-M
Yusuke Tomabechi a,b, Yuki Odate a, Ryuko Izumi c, Katsuji Haneda a, Toshiyuki Inazu a,b,
⇑
a Department of Applied Chemistry, School of Engineering, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
b Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
c Research Association for Biotechnology, 3-9, Nishi-Shinbashi 2-chome, Minato-ku, Tokyo 105-0003, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2010
Received in revised form 21 August 2010
Accepted 26 August 2010
Available online 17 September 2010
To determine the structural specificity of the glycosyl acceptor of the transglycosylation reaction using
endo-b-N-acetylglucosaminidase (ENGase) (EC 3.2.1.96) from Mucor hiemalis (Endo-M), several acceptor
derivatives were designed and synthesized. The narrow regions of the 1,3-diol structure from the 4- to 6-
hydroxy functions of GlcNAc were found to be essential for the transglycosylation reaction using Endo-M.
Furthermore, it was determined that Endo-M strictly recognizes a 1,3-diol structure consisting of primary
and secondary hydroxyl groups.
Keywords:
Endo-b-N-acetylglucosaminidase
Endo-M
Ó 2010 Elsevier Ltd. All rights reserved.
Transglycosylation reaction
Recognition site
1. Introduction
group of the sugar was suggested from reported data1 indicating
that a transglycosylation reaction does not occur with glucuronic
Endo-b-N-acetylglucosaminidase (ENGase, EC 3.2.1.96) cata-
lyzes the hydrolysis of the b-(1?4)-glycosidic linkage between
the N,N0-diacetylchitobiose moiety of N-glycans.1 This enzyme
from Mucor hiemalis (Endo-M), is unique in that it can act on all
three types of N-glycans, that is, high-mannose-type, complex-
type, and hybrid-type oligosaccharides, for the hydrolysis and
transglycosylation of the appropriate acceptors containing the
N-acetylglucosamine (GlcNAc) residue (Scheme 1).2–5 Endo-M is
an effective tool for the reconstruction and remodeling of glyco-
peptides and glycoproteins.6–10
acid and xylose. The acceptor specificity of Endo-M was examined
in this study, with focus on the C-5 hydroxymethyl group of the
glycosyl acceptor and oxygen atom of the pyranose ring.
2. Results and discussion
First, cyclohexyl asparagines 2 and 3 were synthesized and
designed based on the structure of Fmoc-Asn(GlcNAc)-OH
(1, Fig. 1). Fmoc-Asn(c-Hex(OH))-OH (2) possessing only a mono-
However, rapid hydrolysis of the product with N-glycan by the
wild-type endoglycosidases is difficult to avoid, thus limiting its
wide application. Development of unique ENGase-based glyco-
synthases that cannot hydrolyze the product but are still able to
take highly activated species as a donor substrate is a solution to
this problem.11,12 Umekawa et al. achieved site-directed mutagen-
esis of residues in the putative catalytic region of Endo-M mutants
with superior transglycosylation activity.5,13–15
Scheme 1. Transglycosylation reaction using Endo-M.
On the other hand, a summary of acceptor specificity of the
transglycosylation activity of Endo-M has been previously
reported.1 Endo-M transfers a sugar chain to glucose, mannose,
and other sugars as well as GlcNAc. It has been revealed that the
enzyme requires the C-4 equatorial hydroxy group of the carbohy-
drate, and the C-1 and C-2 of GlcNAc are not important for the trans-
glycosylation activity. The importance of the C-5 hydroxymethyl
⇑
Corresponding author. Tel.: +81 463 58 1211; fax: +81 463 50 2012.
E-mail address: inz@keyaki.cc.u-tokai.ac.jp (T. Inazu).
Figure 1. Structure of glycosyl acceptors.
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.08.022

Y. Tomabechi et al. / Carbohydrate Research 345 (2010) 2458–2463
2459
The transglycosylation reaction of one of the two isomers did not
proceed at all. It was assumed that the -GlcNAc mimetic did not
L
react. However, the transglycosylation of the other isomer using
Endo-M yielded the desired transglycosylated product. The RP-HPLC
profile of the transglycosylation of SGP to 3 after a reaction time of
4 h is shown in Figure 2. Characterization of the new product was
performed using matrix-assisted laser desorption/ionization time-
of-flightmass spectrometry(MALDI-TOFMS). The m/z of theproduct
Scheme 2. Reagents and conditions: (a) MptCl, Et3N, 1-amino-4-hydroxycyclohex-
ane or 3-hydroxymethyl-4-hydroxycyclohexylamine, CH2Cl2; (b) H2 gas, AcOH, Pd/
C, MeOH; (c) Fmoc-OSu, Na2CO3, dioxane–H2O (1:1).
hydroxyl functionality, a mimic of the equatorial hydroxyl group at
C-4 of GlcNAc, was synthesized as follows: Benzyloxycarbonyl
aspartic acid
a-benzyl ester and trans-4-aminocyclohexanol were
synthesized using the dimethylphosphinothioic mixed anhydride
(Mpt-MA) method16 producing the amide derivative 4a in 75%
yield (Scheme 2).
The benzyland benzyloxycarbonylgroups of 4a were removedby
hydrogenation in the presence of 10% Pd–C, the Fmoc group was
reacted with the amino group by the reaction of the N-(9-fluorenyl-
methoxycarbonyl) succinimide reagent, to obtain the desired prod-
uct 2 in 85% yield. Next, a transglycosylation of the complex-type
oligosaccharide block of 2 was attempted using Endo-M. Using 2
as the glycosyl acceptor, the transglycosylation of a complex-type
oligosaccharide block [(NeuAc-Gal-GlcNAc-Man)2-Man-GlcNAc-]
with sialylglycopeptide (SGP)17 as the glycosyl donor was examined
in the presence of Endo-M under previously described reaction
conditions.18 Under these conditions, the transglycosylation reac-
tion did not proceed at all. This result showed that Endo-M does
not recognize only 4-OH of GlcNAc.
Next, Fmoc-Asn(c-Hex(OH)(CH2OH))-OH (3), which is a mimic
of the C-4 equatorial hydroxyl group and C-5 hydroxymethyl group
of GlcNAc, was synthesized. Using a method similar to that
described for the synthesis of compound 2, compound 3 was ob-
tained from 3-hydroxymethyl-4-hydroxycyclohexylamine, (which
has been previously described)19 and benzyloxycarbonyl aspartic
Figure 3. Time course of the transglycosylation reaction using Endo-M. j: Fmoc-
Asn(GlcNAc)-OH (1), d: Fmoc-Asn(c-Hex(OH)(CH2OH))-OH (3).
acid
a-benzyl ester. Although this product was a mixture of
diastereomers, each isomer was isolated using reversed-phase
high-performance liquid chromatography (RP-HPLC). It was as-
sumed that one isomer was in the same conformation as
and the other was an -isomer.
D-GlcNAc,
L
Figure 4. Structure of new glycosyl acceptors.
Figure 2. RP-HPLC profile of the reaction mixture incubated with cyclohexanediol derivative 3 and SGP in the presence of Endo-M after a reaction time of 4 h, as detected by
absorbance at 280 nm. The large peak at the retention time of 31–32 min corresponds to remaining amounts of acceptor 3.

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Y. Tomabechi et al. / Carbohydrate Research 345 (2010) 2458–2463
Scheme 3. Reagents and conditions: (a) BnBr, NaH, DMF, rt; (b) 80%-AcOH, 40 °C.
was 2484.5 whereas the calculated m/z was 2482.9. The time course
of the transglycosylation reaction of 3 is shown in Figure 3.
When the GlcNAc derivative 1 was used as an acceptor, the
yield of the transglycosylated product reached 18% after 1 h. There-
after, the product was promptly hydrolyzed. On the other hand, the
highest yield resulting from using cyclohexanediol derivative 3 as
an acceptor was 16% after 4 h. Cyclohexanediol derivative 3 was
found to have transglycosylation activity similar to that of GlcNAc
derivatives 1. However, the hydrolysis rate of cyclohexanediol
derivative 3 was considerably slower compared to the hydrolysis
rate of GlcNAc derivative 1. Therefore, it was determined that the
oxygen atom on the pyranose ring was not essential; however, a
narrow region of the acceptor—the area from 4-OH to 6-OH of
GlcNAc—was essential for the transglycosylation reaction using
Endo-M. From these results it was assumed that 6-OH of GlcNAc
would be important for the transglycosylation reaction, which
was similar to the reported study.15,20,21
To narrow down the structural specificity areas of the glycosyl
acceptor in this reaction, several acceptor derivatives with a 1,3-
diol structure were designed and synthesized (Fig. 4). Furthermore,
linear 1,3-diol structures, such as erythritol derivatives 5 and 6,
and threoninol derivative 722–24 were also designed.
Figure 5. RP-HPLC profile of the reaction mixture incubated with 2-O-benzyl
erythritol (5) and SGP in the presence of Endo-M after a reaction time of 1 h as
detected by absorbance at 254 nm. The large peak at the retention time of 18–
19 min corresponds to remaining amounts of acceptor 5.
For the preparation of erythritol derivatives 5 and 6, 1,3-O-ben-
zylidene-L
-erythritol25,26 was benzylated using 1.2 equiv benzyl
On the other hand, erythritol derivative 11 and 2-O-benzyl glyc-
erolà yielded no transglycosylated products. From erythritol deriva-
tive 11, it was hypothesized that the free hydroxyl group, which
exists in the position corresponding to C-5 of GlcNAc, would
interrupt the transglycosylation reaction. For confirmation, the
transglycosylation reaction was attempted with several 1,2,3-triol
derivatives, but the reaction did not proceed (data is not shown).
In spite of having an essential 1,3-diol structure, 2-O-benzyl glycerol,
which consists of two primary hydroxyl groups, did not function as
an acceptor. Based on these results, the two hydroxyl groups need to
act as primary and secondary hydroxyl groups.
bromide to yield 4-O-benzyl erythritol derivative 8 (13%), 2-O-
benzyl erythritol derivative 9 (41%), and 2,4-di-O-benzyl erythritol
derivative 10 (25%) (Scheme 3). After deprotection of the benzyli-
dene group using 80% acetic acid, erythritol derivatives 11, 5, and 6
were obtained in 87%, 89%, and 75% yields, respectively.
Next, the transglycosylation reaction was carried out using 1,3-
diol derivatives 5–7 as acceptors, SGP as a glycosyl donor, and
Endo-M. Surprisingly, Endo-M recognized linear 1,3-diol deriva-
tives, such as erythritol derivatives 5 and 6, and threoninol deriv-
ative 7 (Fig. 5). In the case of monobenzylated erythritol 5, which
was the most superior acceptor, the yield of the transglycosylated
product reached 26% after 1 h (Fig. 6). Dibenzylated erythritol 6
and threoninol derivative 7 also demonstrated transglycosylation
activity (15% and 7%, respectively). The hydrolysis rate of these
compounds and the cyclohexanediol derivatives 3 were slow com-
pared to that of GlcNAc derivative 1. These data suggest that the C-
3 hydroxyl group of GlcNAc may be related to hydrolysis activity.
To determine the transglycosylated position of these non-natural
compounds using NMR, transglycosylated dibenzylated erythritol
6 was treated with sialidase from Arthrobacter ureafaciens and b-
galactosidase from Jack bean and b-N-acetylhexosaminidase from
Jack bean. Long-range correlations were detected between the
NMR signals of the anomer protons of GlcNAc and the C-2 methine
carbon of erythritol in HMBC experiments. As a result, the transgly-
cosylated position of the secondary hydroxyl group was observed,
similar to the transglycosylation reaction of GlcNAc.
3. Conclusions
To determine the structural specificity of the glycosyl acceptor
in the transglycosylation reaction using Endo-M, several acceptor
derivatives were synthesized. It was determined that the oxygen
atom on the pyranose ring was not essential; however, the narrow
regions of the 1,3-diol structure from the 4-OH to 6-OH of GlcNAc
were considered essential for the transglycosylation reaction using
Endo-M. The enzyme recognizes not only cyclic 1,3-diol structures
such as GlcNAc but also linear 1,3-diol structures such as erythritol
and threoninol. The reaction did not proceed to glycerol using two
primary hydroxyl groups: Endo-M strictly recognizes the 1,3-diol
structures consisting of primary and a secondary hydroxyl groups.
Based on these data, new effective acceptors for Endo-M are
currently being designed and synthesized.
à
These results will be described elsewhere.
2-Benzyloxy glycerol was available from Tokyo Chemical Industry Co., Ltd.

Y. Tomabechi et al. / Carbohydrate Research 345 (2010) 2458–2463
2461
c-Hex-CH), 3.55 (1H, m, c-Hex-CH), 2.78 (2H, dd, J = 4.4, 4.0 Hz,
Asn-b-CH2), 1.94–1.85 (4H, m, c-Hex), 1.52 (1H, br s, OH), 1.36–
1.33 (2H, m, c-Hex), 1.11–1.05 (2H, m, c-Hex). 13C NMR (DMSO-
d6): d 171.5, 167.8, 155.8, 136.9, 135.9, 128.3–127.6 (aromatic car-
bon), 68.1, 66.0, 65.5, 50.9, 47.3, 37.1, 33.9, 30.1. HRMS calcd for
C
25H30N2O6ꢁK+ [M+K]+ 493.1741; found m/z 493.1741.
a
4.3. N -9-Fluorenylmethoxycarbonyl-Nb-4-hydroxycyclohexyl-
L
-
asparagine, Fmoc-Asn(c-Hex(OH))-OH (2)
Compound 4a (1.97 g, 4.34 mmol) and acetic acid (261 mg,
4.34 mmol) in MeOH (80 mL) were stirred under an H2 atmosphere
in the presence of 10% Pd–C (394 mg) at room temperature for 2 h.
The mixture was filtered through filter paper and concentrated in
vacuo. A solution of Fmoc succinimide (1.36 g, 4.02 mmol) in diox-
ane (20 mL) was added to a stirred solution of the compound in
10% sodium carbonate aq (20 mL) at room temperature. The reac-
tion mixture was stirred for 24 h. EtOAc was added to the reaction
mixture and washed with water. The water layer was adjusted to
pH 2–3 using 1 N HCl; white solids were separated out. The solids
were washed with water, EtOAc, and Et2O. The solid when dried in
vacuo yielded 2 (1.41 g, 72% in two steps). 1H NMR (CD3OD): d
7.80–7.29 (8H, m, Fmoc), 4.52–4.45 (1H, m, Asn-a-CH),
4.34ꢂ4.32 (2H, m, OCH2), 4.23 (1H, t, J = 6.8 Hz, CH), 3.63ꢂ3.53
(1H, m, c-Hex-CH), 3.52ꢂ3.43 (1H, m, c-Hex-CH), 2.66 (2H, m,
Asn-b-CH2), 1.95–1.82 (4H, m, c-Hex), 1.33ꢂ1.23 (4H, m, c-Hex).
13C NMR (DMSO-d6): d 173.1, 168.2, 155.8, 143.8, 140.7, 127.6–
120.1 (aromatic carbon), 68.1, 65.7, 50.8, 47.3, 46.6, 37.2, 33.9,
30.2. HRMS calcd for C25H27N2O6 [MꢂH+]ꢂ 451.1869; found m/z
451.1856.
Figure 6. Time course of the transglycosylation reaction using Endo-M. j: Fmoc-
Asn(GlcNAc)-OH (1), d: Fmoc-Asn(c-Hex(OH)(CH2OH))-OH (3), ꢀ: 2-O-Benzyl
erythritol (5), }: 1,3-O-dibenzyl-erythritol (6), N: Fmoc-Thr-ol (7).
4. Experimental methods
4.1. General methods
a
4.4. N -Benzyloxycarbonyl-Nb-3-hydroxymethyl-4-hydroxycy-
1H NMR and 13C NMR spectra were recorded at 400 MHz on a
JEOL JNM-400. MALDI-TOF mass spectra were obtained using a
Daltonix-Bruker AUTOFLEX. The structure of the acceptors was
confirmed by MALDI-TOF MS in linear and negative ion modes
using 2,5-dihydroxy-benzoic acid as a matrix, unless otherwise
described. HRMS data were obtained on an LC–MS-IT-TOF (Shima-
dzu Corporation). Reactions were monitored using thin-layer chro-
matography on a pre-coated plate of Silica Gel 60F254 (Merck) and/
or RP-HPLC (HPLC system equipped with a Delta 600 series
interface). Flash chromatography was performed on Silica Gel
clohexyl-
(CH2OH))-OBn (4b)
L-asparagine a-benzyl ester, Z-Asn(c-Hex(OH)
Triethylamine (198 mg, 1.93 mmol) and dimethylphosphi-
nothioyl chloride (150 mg, 1.17 mmol) were added to a solution
a
of N -benzyloxycarbonyl-
L-aspartic acid
a-benzyl ester (Z-Asp-
OBn) (353 mg, 0.988 mmol) in CH2Cl2 (3 mL) and the mixture
was stirred at 0 °C to prepare the corresponding Mpt-MA. After
stirring at room temperature for 1 h, the reaction mixture was
added to
a stirred solution of 1-amino-3-hydroxymethyl-4-
(Silica Gel 60N, 40–50
l
m; Kanto Chemical Co., Inc.). The bian-
hydroxycyclohexane (110 mg, 0.758 mmol) in MeOH (2 mL). After
stirring at room temperature for 3 h, the reaction mixture was
washed with 1 N HCl, water, saturated NaHCO3, water, and brine.
The organic layer was dried over anhydrous Na2SO4 and concen-
trated in vacuo. Purification by flash column chromatography
(CHCl3–MeOH–AcOH 85:25:20) yielded 4b (291 mg, 79%) as color-
less amorphous crystals. 1H NMR (CDCl3): d 7.32–7.26 (10H, m,
Ph ꢀ 2), 6.20 (1H, br d, J = 7.2 Hz, NH), 6.11 (1H, br d, J = 7.2 Hz,
NH), 5.18 (2H, dd, J = 13.2, 12.8 Hz, OCH2), 5.10 (2H, br s, OCH2),
tennary complex-type sialylglycopeptide (SGP), Endo-M, and
Lys-Val-Ala-Asn[(NeuAc-Gal-GlcNAc-Man)2 Man-GlcNAc2]-Lys-Thr
were obtained from Tokyo Chemical Industry Co., Ltd.
a
4.2. N -Benzyloxycarbonyl-Nb-4-hydroxycyclohexyl-
L-
asparagine a-benzyl ester, Z-Asn(c-Hex(OH))-OBn (4a)
Triethylamine (610 mg, 6.03 mmol) and dimethylphosphi-
nothioyl chloride (390 mg, 3.03 mmol) were added to a solution
4.62 (1H, t, J = 4.4 Hz, Asn-a-CH), 4.08 (1H, br d, J = 4.0 Hz, c-Hex-
a
of N -benzyloxycarbonyl-
L
-aspartic acid
a
-benzyl ester (Z-Asp-
CH), 3.63–3.60 (2H, m, CH2–OH), 3.56–3.51 (1H, m, c-Hex-CH),
2.93–2.72 (2H, m, Asn-b-CH2), 2.04 (1H, br s, OH), 1.25 (1H, br s,
OH), 1.81–1.11 (7H, m, c-Hex). 13C NMR (CDCl3): d 174.1, 170.5,
163.0, 136.0, 135.1, 128.4–128.0 (aromatic carbon), 67.5, 67.1,
50.4, 38.7, 36.8, 36.4, 31.7, 28.9, 23.0, 14.1. HRMS calcd for
OBn) (1.07 g, 2.99 mmol) in CH2Cl2 (15 mL), and the mixture was
stirred at 0 °C to prepare the corresponding Mpt-MA. After stirring
at room temperature for 30 min, the reaction mixture was added to
a stirred solution of trans-4-aminocyclohexanol (350 mg, 3.04
mmol) in CH2Cl2 (30 mL). After stirring at room temperature for
1 h, the reaction mixture was washed with 1 N HCl, water, 5% aq
NaHCO3, water, and brine. The organic layer was dried over anhy-
drous Na2SO4, and concentrated in vacuo. Purification by flash col-
umn chromatography (CH2Cl2–MeOH 50:1) yielded 4a (1.03 g,
75%) as colorless amorphous crystals. 1H NMR (CDCl3): d 7.36–
7.30 (10H, m, Ph ꢀ 2), 6.06 (1H, br d, J = 8.4 Hz, NH), 5.40 (1H, br
d, J = 6.8 Hz, NH), 5.18 (2H, dd, J = 12.4, 12.4 Hz, OCH2), 5.11 (2H,
C
26H33N2O7 [M+H+]+ 485.2288; found m/z 485.2289.
a
4.5. N -9-Fluorenylmethoxycarbonyl-Nb-3-hydroxymetyl-4-
hydroxy-cyclohexyl-L-asparagine, Fmoc-Asn(c-Hex(OH)
(CH2OH))-OH (3)
Compound 4b (195 mg, 0.403 mmol) and acetic acid (48.0 mg,
0.799 mmol) in MeOH (5 mL) were stirred overnight under a H2
atmosphere in the presence of 10% Pd–C (28 mg) at room temper-
br s, OCH2), 4.59 (1H, t, J = 4.0 Hz, Asn-a-CH), 3.65 (1H, m,

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Y. Tomabechi et al. / Carbohydrate Research 345 (2010) 2458–2463
ature. The mixture was filtered through filter paper and concen-
trated in vacuo. solution of Fmoc succinimide (140 mg,
cool, washed with saturated NaHCO3, water, and brine, and then
dried over anhydrous Na2SO4. The organic solution was concen-
trated in vacuo. Purification by flash column chromatography (hex-
ane–EtOAc gradient elution 1:2?100% EtOAc) yielded 5 (55.0 mg,
87%) as a colorless oil. 1H NMR (CDCl3): d 7.38–7.30 (5H, m, Ph),
4.66 (1H, d, J = 11.6 Hz, Ph-CH2a), 4.59 (1H, d, J = 11.6 Hz, Ph-
CH2b), 3.92–3.41 (6H, m, H-1, H-2, H-3, H-4), 2.50-2.10 (3H, br,
OH ꢀ 3). 13C NMR (CD3OD) d 139.8, 129.2–128.5 (aromatic carbon),
81.4, 73.2, 72.6, 64.3, 61.8. HRMS calcd for C11H16O4ꢁNa [M+Na+]+
235.0946; found m/z 235.0989.
A
0.415 mmol) in dioxane (5 mL) was added to a stirred solution of
the compound in 10% sodium carbonate aq (5 mL) at room temper-
ature. The reaction mixture was stirred for 18 h, to which Et2O was
added and it was extracted with water. The water layer was
washed with Et2O, adjusted to pH 1 using 1 N HCl, and was
extracted with EtOAc. The organic layer was washed with brine
and dried over anhydrous Na2SO4. The organic solution was con-
centrated in vacuo. Purification by flash column chromatography
(CHCl3–MeOH 5:1) yielded 3 (127 mg, 65% in two steps) as a color-
less amorphous crystal. 1H NMR (CD3OD): d 7.79–7.31 (8H, m,
4.7.1. 1,3-Di-O-benzyl-D-erythritol (6)
Fmoc), 4.58–4.55 (1H, m, AsnH
a
), 4.37–4.29 (2H, m, Fmoc-
75% yield as a colorless oil. 1H NMR (CDCl3): d 7.38–7.28
(10H, m, Ph ꢀ 2), 4.64–4.50 (4H, m, CH2 ꢀ 2), 3.96 (1H, dt,
J = 6.3, 3.9 Hz, CH–OH), 3.84 (1H, dd, J = 12.2, 4.4 Hz, CH2a–OH),
3.79 (1H, dd, J = 12.2, 4.4 Hz, CH2b–OH), 3.66 (1H, dd, J = 9.3,
3.9 Hz, CH2a–OBn), 3.58 (1H, dd, J = 9.3, 6.3 Hz, CH2b–OBn), and
3.54 (1H, td, J = 6.3, 4.4, 4.4 Hz, CH–OBn). 13C NMR (CDCl3): d
137.9, 128.5–127.9 (aromatic carbon), 78.8, 73.5, 72.3, 70.9,
61.5. HRMS calcd for C18H22O4ꢁNa [M+Na+]+ 325.1416; found
m/z 325.1399.
CH2CONH), 4.22 (1H, t, J = 6.8 Hz, Fmoc-CH), 3.95 (1H, br, CHNH),
3.61 (2H, d, J = 5.4 Hz, CH2OH), 3.52 (1H, m, CHOH), 2.82–2.69
(2H, m, AsnHb), 1.82–1.40 (7H, m, c-Hex). 13C NMR (DMSO-d6): d
169.6, 155.6, 143.9, 140.7, 127.6–120.1 (aromatic carbon), 69.1,
65.6, 63.1, 52.5, 46.7, 43.5, 41.5, 38.8, 31.0, 29.2, 28.0. HRMS calcd
for C26H29N2O7 [MꢂH+]ꢂ481.1975; found m/z 481.1976.
4.6. Preparation of benzylidene erythritol derivatives 8–10
Sodium hydride (140 mg, 5.83 mmol) was added to a stirred
4.7.2. 1-O-Benzyl-D-erythritol (11)
solution of 1,3-O-benzylidene-
L
-erythritol (500 mg, 2.38 mmol) in
87% yield as a colorless oil. 1H NMR (CDCl3): d 7.38–7.24 (5H,
m, Ph), 4.88 (2H, s, PhCH2), 3.75–3.67 (3H, m, H-1a, H-3, H-4a),
3.62–3.54 (3H, m, H-1b, H-2, H-4b). 13C NMR (CDCl3): d 139.7,
129.3–128.6 (aromatic carbon), 74.3, 73.7, 73.0, 72.4, 64.6.
DMF (20 mL) at 0 °C. After the suspension was stirred for 40 min,
benzyl bromide (485 mg, 2.84 mmol) was added dropwise over
5 min period and stirred for 30 min. The reaction temperature
was then allowed to increase to room temperature for 1.5 h, MeOH
was then slowly added to react with the excess sodium hydride,
and water was added to the reaction. The reaction mixture was
extracted with EtOAc, and the organic layer was washed with
water and brine, and then dried over anhydrous Na2SO4. The
organic solution was concentrated in vacuo. Purification by flash
column chromatography (hexane–EtOAc gradient elution 9:1?
2:1) yielded 8 (95.0 mg, 13%), 9 (295 mg, 41%), and 10 (232 mg,
25%) as colorless amorphous crystals.
HRMS calcd for
235.0921.
C
11H16O4ꢁNa [M+Na+]+ 235.0946; found m/z
4.8. Transglycosylation reaction with Endo-M
The transglycosylation reaction was performed with a reaction
mixture composed of 10 mM 3, 20 mM SGP, and 0.2 U/mL Endo-
M in a total volume of 20 mL of 0.4 M phosphate buffer (pH 6.25)
containing 30% DMSO (v/v). After incubation for 0–24 h at 25 °C,
aliquots (2 lL) were added to 98 lL of 0.2% trifluoroacetic acid
4.6.1. 1,3-O-Benzylidene-4-O-benzyl-
L
-erythritol (8)
(TFA) solution to finish the reaction. Analyses of the transglycosy-
lation products were performed using RP-HPLC. The elution was
carried out using a linear gradient of acetonitrile (20–45%) contain-
ing 0.1% aq TFA for 35 min at a flow rate of 1 mL/min. The reaction
products were monitored at an absorption of 280 nm. The yields of
the transglycosylation products were calculated from the ratio of
the peaks of the transglycosylation products to the glycosyl accep-
tors. This was based on the assumption that the absorption wave-
length of the transglycosylation products and those of the
acceptors would be approximately the same. The purification of
the transglycosylation products was performed using RP-HPLC,
using the same procedure described in the methods of analysis
section.
1H NMR (CD3OD): d 7.49–7.23 (10H, m, Ph ꢀ 2), 5.52 (1H, s, Ph-
CH), 4.60 (2H, s, Ph-CH2), 4.20 (1H, dd, J = 10.4, 4.8 Hz, H-1b), 3.88–
3.56 (5H, m, H-1a, H-2, H-3, H-4). 13C NMR (CD3OD): d 139.6, 139.4,
129.8–127.4 (aromatic carbon), 102.3, 102.3, 83.1, 74.5, 72.3, 70.8,
62.8. HRMS calcd for C18H21O4 [M+H+]+ 301.1440; found m/z
301.1398.
4.6.2. 1,3-O-Benzylidene-2-O-benzyl-L-erythritol (9)
1H NMR (CD3OD): d 7.49–7.28 (10H, m, Ph ꢀ 2), 5.53 (1H, s, Ph-
CH), 4.61 (2H, s, Ph-CH2), 4.34–4.32 (1H, m, H-1a), 3.91–3.86 (1H,
m, H-4a), 3.77–3.70 (2H, m, H-2, H-4b), 3.67–3.60 (2H, m, H-1b,
H-3). 13C NMR (CD3OD): d 139.6, 139.4, 129.8-127.5 (aromatic
carbon), 102.4, 82.9, 73.4, 70.3, 69.9, 62.5. HRMS calcd for
C
18H20O4ꢁNa [M+Na+]+ 323.1259; found m/z 323.1249.
4.8.1. Glycosylated Fmoc-Asn(c-Hex(OH)(CH2OH))-OH
MALDI-TOF MS m/z ratio for C102H152N7O63 [MꢂH+]ꢂ calcd for
2482.9; found 2484.5.
4.6.3. 1,3-O-Benzylidene-2,4-di-O-benzyl-L-erythritol (10)
1H NMR (CD3OD): d 7.47–7.24 (15H, m, Ph ꢀ 3), 5.51 (1H, s, Ph-
CH), 4.61–4.49 (4H, m, Ph-CH2 ꢀ 2), 4.32 (1H, dd, J = 9.6, 3.6 Hz,
H-1eq), and 3.93–3.59 (5H, m, H-1ax, H-2, H-3, H-4). 13C NMR
(CD3OD): d 139.5, 139.5, 139.4, 129.8–127.5 (aromatic carbon),
102.4, 102.3, 81.7, 74.3, 73.3, 70.4, 70.3, 69.8. HRMS calcd for
4.8.2. Glycosylated Fmoc-Thr-ol
MALDI-TOF MS m/z ratio for C95H143N6O60 [MꢂH+]ꢂ calcd for
2327.8; found 2329.9.
C
25H26O4ꢁNa [M+Na+]+ 413.1729; found m/z 413.1714.
4.8.3. Glycosylated 2-O-benzyl-D-erythritol
MALDI-TOF MS m/z ratio for C87H138N5O60 [MꢂH+]ꢂ calcd for
4.7. General procedure for the preparation of erythritol
derivatives 5, 6, and 11
2212.8; found 2214.1.
4.8.4. Glycosylated 1,3-di-O-benzyl-D-erythritol
Compound 9 (92.0 mg, 0.307 mmol) in 80% acetic acid (2 mL)
was heated at 40 °C for 4 h. The reaction mixture was allowed to
MALDI-TOF MS m/z ratio for C94H144N5O60 [MꢂH+]ꢂ calcd for
2304.2; found 2303.7.

Y. Tomabechi et al. / Carbohydrate Research 345 (2010) 2458–2463
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Acknowledgments
The authors gratefully acknowledge Professor Kenji Yamamoto
from Ishikawa Prefectural University for valuable discussions of
this work. The authors also thank the Tokyo Chemical Industry
Co., Ltd for providing Endo-M and SGP, and Dr. Takashi Yamanoi
and Mr. Yoshiki Oda from the Noguchi Institute for participating
in structural NMR analyses of the transglycosylated products,
and Professor Akemi Suzuki from Institute of Glycoscience, Tokai
University for participating in HRMS analyses. Part of this work
was supported by a Grant for Hi-Tech Research from Tokai Univer-
sity, a Sasakawa Scientific Research Grant from The Japan Science
Society, and a Research and Study Project funding from the Tokai
University Educational System General Research Organization.
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