4,6-Dimethoxy-1,3,5-triazine oligoxyloglucans: Novel one-step preparable substrates for studying action of endo-β-1,4-glucanase III from Trichoderma reesei

Article abstract of DOI:10.1016/j.bmcl.2010.04.122

Two kinds of 4,6-dimethoxy-1,3,5-triazine (DMT) oligoxyloglucans, DMT-β-XXXG and DMT-β-XLLG, have been synthesized via one-step procedure starting from the corresponding unprotected oligoxyloglucans in water. The resulting DMT derivatives were found to be hydrolyzed by endo-β-1,4-d-glucanase III from Trichoderma reesei (EGIII) and utilized as substrates for determination of the kinetic parameters of EGIII. The present DMT-method would be a convenient analytical tool for studying the action of glycosyl hydrolases due to the extremely simple synthetic process of DMT-glycosides without using protecting groups.

Full text of DOI:10.1016/j.bmcl.2010.04.122

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3588–3591
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4,6-Dimethoxy-1,3,5-triazine oligoxyloglucans: Novel one-step preparable
substrates for studying action of endo-b-1,4-glucanase III
from Trichoderma reesei
Atsushi Kobayashi a, Tomonari Tanaka a, Kazuhito Watanabe a, Masaki Ishihara a, Masato Noguchi a,
Hirofumi Okada b, Yasushi Morikawa b, Shin-ichiro Shoda a,
*
a Graduate School of Engineering, Tohoku University, 6-6-11-514 Aoba, Sendai 980-8579, Japan
b Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Two kinds of 4,6-dimethoxy-1,3,5-triazine (DMT) oligoxyloglucans, DMT-b-XXXG and DMT-b-XLLG, have
Received 10 February 2010
Revised 23 April 2010
Accepted 27 April 2010
Available online 22 May 2010
been synthesized via one-step procedure starting from the corresponding unprotected oligoxyloglucans
in water. The resulting DMT derivatives were found to be hydrolyzed by endo-b-1,4-D-glucanase III from
Trichoderma reesei (EGIII) and utilized as substrates for determination of the kinetic parameters of EGIII.
The present DMT-method would be a convenient analytical tool for studying the action of glycosyl hydro-
lases due to the extremely simple synthetic process of DMT-glycosides without using protecting groups.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Oligosaccharide
Glycosidase
Substrate
DMT-MM
The determination of kinetic parameters for endo-glycanases, a
hydrolytic enzyme of polysaccharides, is extremely important for
evaluating their hydrolysis patterns in the field of food science
and woody biomass utilization. There is a drawback to employ nat-
urally occurring polysaccharides as substrates for studying the ac-
tion of endo-type glycanases that the structure and the molecular
weight of these substrates change during an enzymatic hydrolysis
reaction. Therefore, the development of an artificial sugar substrate
with a definite leaving group at the reducing end has long been a
hot topic in polysaccharide chemistry.
amylase in serum, pNP maltopentaoside, can be prepared by con-
necting a maltotetraose moiety to pNP glucoside by using an amy-
lase as catalyst under mild reaction conditions.5 However, these
methods are highly restricted to the synthesis of specific oligosac-
charides, its applicability in principle being dominated by the class
of the enzyme catalyst employed. A novel substrate having other
leaving group than nitrophenyl has, therefore, strongly been de-
manded in order to carry out kinetic analyses of glycosidases, par-
ticularly in case of using substrates whose glycon parts possess
acid or base-labile functions.
Nitrophenyl glycosides are known to be useful compounds for
kinetic studies of glycosidases, because the liberated nitrophenol
derivatives show yellowish colors in aqueous solutions (pH
>7.5).1 However, the synthesis of nitrophenyl glycosides normally
requires multi-step reactions including the usage of strong acids
or bases as well as the protection and deprotection of the hydroxyl
groups (Scheme 1(A)).2 For example, in order to activate the ano-
meric center, severe acidic reaction conditions by using hydrogen
bromide/acetic acid is necessary, and protecting groups like acetyl
groups must be removed under a basic conditions by using sodium
methoxide at the final stage of the synthesis.3,4
Recently, we have found that 4,6-dimethoxy-1,3,5-triazin-2-yl b-
lactoside (DMT-b-Lac), prepared by the reaction of 4-(4,6-dime-
thoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride (DMT-
MM)6 and lactose, was hydrolyzed by a cellulase, giving rise to lac-
tose and 2-hydroxy-4,6-dimethoxy-1,3,5-triazine.7 The synthesis
of DMT-b-Lac could be achieved in water without requiring any pro-
tection–deprotection process as well as any severe reaction condi-
tions (Scheme 1(B)). These results prompted us to investigate a
kinetic study on the enzymatic hydrolysis of DMT-oligosaccharides
derived from naturally occurring xyloglucan (Tamarindus seeds).
It is well known that endo-b-1,4-glucanase III from Trichoderma
reesei (EGIII) is able to hydrolyze naturally occurring xyloglucan
into the oligoxyloglucan (XXXG or XLLG).8 In order to know sub-
strate recognition of EGIII in more detail, kinetic analysis using oli-
goxyloglucans carrying a chromophore as their aglycon moiety is
ideal. In this paper, we demonstrated a kinetic analysis of EGIII
An enzymatic approach to the production of p-nitrophenyl
(pNP) oligosaccharides has been reported. A substrate for human
* Corresponding author.
E-mail address: shoda@poly.che.tohoku.ac.jp (S. Shoda).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.122

A. Kobayashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3588–3591
3589
Scheme 1. Comparison of chromogenic substrates: conventional pNP-glycoside versus one-step preparable substrate DMT-glycoside.
Scheme 2. DMT-glycosides used in this study. The backbone structure of naturally occurring xyloglucans from Tamarindus seeds is a mixture of at least two oligosaccharide
units denoted by XXXG and XLLG, where a capital G represents an unsubstituted glucopyranose residue, a capital X represents a glucopyranose residue substituted with a
xylopyranose through an
glycosidic bond.
a-1,6-glycosidic bond, and a capital L represents a glucopyranose residue substituted with a galactopyranose b(1–2)xylopyranose through an a-1,6-
by using two kinds of DMT-oligoxyloglucans, DMT-b-XXXG and
DMT-b-XLLG, as novel sugar substrates (Scheme 2). The results of
enzyme kinetics employing DMT-b-Lac have also been discussed.
The substrates, DMT-b-Lac, DMT-b-XXXG, and DMT-b-XLLG,
were directly prepared starting from lactose,7 XXXG,9 and XLLG,9
respectively, by the action of DMT-MM in the presence of 2,6-luti-
dine in water (yields: 61%, 44%, 36%, respectively). The enzyme
EGIII was expressed and purified according to the previous re-
port.10 The reagent DMT-MM was synthesized from 2-chloro-4,6-
dimethoxy 1,3,5-triazine and N-methylmorpholine.6 Deuterium
oxide was obtained by Acros.
NMR spectra (1H, 13C) were recorded on a Bruker DRX500 spec-
trometer. The samples were dissolved in D2O, and acetone was
used as an internal standard. HPLC analysis was carried out using

3590
A. Kobayashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3588–3591
Figure 2. Michaelis–Menten analysis of EGIII-catalyzed reaction using DMT-b-Lac.
13 mM and the first-order rate constants (kcat) for the substrate
was 5.4 sÀ1 (Fig. 2).
These results clearly indicate that the existence of the 4,6-dime-
thoxy-1,3,5-triazine moiety is essential for the terminal glycosidic
bond at the reducing end to be cleaved in the catalytic site of the
enzyme. It is well understood from the viewpoint of stereoelec-
tronic effect theory that the conformation of the pyranose ring, lo-
cated in the À1 subsite, with a 4C1 conformation must change to a
half boat conformation in order that the orientation of the cleaving
b-glycosidic bond becomes antiperiplanar to one of the lone pairs
on the ring oxygen atom of the pyranose. This conformation
change may be caused by a strong interaction between the dimeth-
oxytriazine ring and specific amino acids in the vicinity of the +1
subsite.
On the other hand, pNP-b-Lac cannot be hydrolyzed by the
same enzyme, clearly indicating that the conformation change of
the pyranose ring in À1 subsite does not take place. These results
shows that DMT-glycosides possess an advantageous character as
enzyme substrates for kinetic studies of glycosidases over conven-
tionally used pNP-glycosides.
Figure 1. Michaelis–Menten analysis of EGIII-catalyzed reaction using DMT-b-
XXXG and DMT-b-XLLG.
two type of columns (4.6 Â 250 mm, eluent: acetonitrile aqueous
solution); InertsilÒ ODS-3 (GL Science) was used for DMT-b-Lac
and TSK-gel Amide80 (Tosoh) was used for DMT-b-XXXG and
DMT-b-XLLG, respectively. The UV absorbance of the eluate was
monitored at 214 nm on a Shimadzu SPD-10AV detector. Molecu-
lar weight data were obtained using a Shimadzu AXIMA CFR plus
mass spectrometer (2,5-dihydroxybenzoic acid matrix).
EGIII-catalyzed hydrolysis of DMT-glycosides was performed in
50 mM acetate buffer (pH 5.5) at 30 °C and all the reactions were
monitored by quantitating the starting DMT-glycosides (UV at
214 nm) by HPLC. The kinetic parameters and their standard errors
were calculated using the nonlinear regression analysis program
‘KaleidaGraph 4.1 J’.
In the present Letter, we demonstrated that two kinds of DMT-
oligoxyloglucans, DMT-b-XXXG and DMT-b-XLLG, could be recog-
nized as efficient substrates for an endo-b-glucanase. Taking the
fact that preparation of DMT-glycosides is much easier than that
of pNP-glycosides into consideration, the feasibility of DMT-glyco-
sides as ‘general substrates’ for enzyme studies would emerge
soon. In spite of the drawback that the hydrolytic product of
DMT-glycosides, 2-hydroxy-4,6-dimethoxy-1,3,5-triazine (4,6-
dimethoxy-1,3,5-triazine-2-one), shows no colors like pNP-glyco-
side, DMT-glycosides are attractive substrates for kinetic studies
of glycosidases because of their easy-to-prepare and tailor-made
characters as well as their higher activities from the viewpoints
of synthetic organic chemistry and biomolecular engineering. Fur-
ther studies for elucidating the detailed hydrolysis mechanism of
the DMT-glycosidic bond in the catalytic site of a glycosidase is
now in progress.
The Michaelis constants (Km) for DMT-b-XXXG and DMT-b-
XLLG by EGIII were 13 and 3.3 mM, and the first-order rate con-
stants (kcat) for the substrate were 170 and 23 sÀ1, respectively
(Fig. 1.)
In order to clarify the merits of DMT-glycosides as substrates,
we compared the kinetic parameters of DMT-b-Lac with that of
p-nitrophenyl b-lactoside (pNP-b-Lac) for EGIII enzyme. It has been
found that DMT-b-Lac was smoothly hydrolyzed by EGIII, affording
lactose and 4,6-dimethoxy-1,3,5-triazine derivative. On the other
hand, it is well known that pNP-b-Lac cannot be recognized by
EGIII.11 The Michaelis constant (Km) for DMT-b-Lac by EGIII was
Acknowledgments
This work was supported by a Grant-in Aid for Scientific Re-
search from the Ministry of Education, Sports, Science and Technol-
ogy, and Industrial Technology Research Grant Program in 2006
from NEDO of Japan.

A. Kobayashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3588–3591
3591
6. Kunishima, M.; Kawachi, C.; Morita, J.; Terao, K.; Iwasaki, F.; Tani, S. Tetrahedron
1999, 55, 13159.
References and notes
7. Tanaka, T.; Noguchi, M.; Kobayashi, A.; Shoda, S. Chem. Commun. 2008,
2016.
8. Yuan, S.; Wu, Y.; Cosgrove, D. J. Plant Physiol. 2001, 127, 324.
9. Tanaka, T.; Noguchi, M.; Ishihara, M.; Kobayashi, A.; Shoda, S. Macromol. Symp.,
in press.
10. Okada, H.; Tada, K.; Sekiya, T.; Yokoyama, K.; Takahashi, A.; Tohda, H.;
Kumagai, H.; Morikawa, Y. Appl. Environ. Microbiol. 1998, 64, 555.
11. Claeyssens, M.; Henrissat, B. Protein Sci. 1992, 1, 1293.
1. Rauscher, E.. In Methods of Enzymatic Analysis; Bergmeyer, E., Ed.; VHC, 1984;
Vol. 4, p 157.
2. Tokutake, S.; Yamaji, N.; Kato, M. Chem. Pharm. Bull. 1990, 38, 13.
3. Ibatullin, F. M.; Baumann, M. J.; Greffe, L.; Brumer, H. Biochemistry 2008, 47, 7762.
4. Ibatullin, F. M.; Banasiak, A.; Baumann, M. J.; Greffe, L.; Takahashi, J.;
Mellerowicz, E. J.; Brumer, H. Plant Physiol. 2009, 151, 1741.
5. Usui, T.; Murata, T. J. Biochem. 1988, 103, 969.