Efficient method for the elongation of the N-acetylglucosamine unit by combined use of chitinase and β-galactosidase

Article abstract of DOI:10.1002/1522-2675(200211)85:11<3919::AID-HLCA3919>3.0.CO;2-P

A novel strategy for the regio- and stereoselective synthesis by two enzymatic steps of oligosaccharides having an N-acetylglucosamine unit at the nonreducing end was developed. The first step involves a chitinase-catalyzed highly selective β-N-acetyllactosamination of an oligosaccharide acceptor with a 4,5-dihydrooxazole derivative of N-acetyllactosamine as the glycosyl donor. The usage of a transition-state-analogue substrate for the chitinase under basic conditions allows the reaction to proceed only in the synthetic direction while suppressing hydrolysis of the product in aqueous media. Several chitinase mutants also catalyzed the glycosylation efficiently under neutral conditions, The second step is a regioselective cleavage of the glycosidic bond between the terminal galactose unit and the adjacent N-acetylglucosamine unit by the action of a β- galactosidase. This constitutes a very useful method to add an N-acetylglucosamine unit to the nonreducing end of chito- and cello-oligosaccharide derivatives in a regio- and stereoselective manner.

Full text of DOI:10.1002/1522-2675(200211)85:11<3919::AID-HLCA3919>3.0.CO;2-P

Helvetica Chimica Acta ± Vol. 85 (2002)
3919
Efficient Method for the Elongation of the N-Acetylglucosamine Unit by
Combined Use of Chitinase and b-Galactosidase
by Shin-ichiro Shoda*, Masaya Fujita, Chakapan Lohavisavapanichi, Yoshinori Misawa, Koushin Ushizaki,
Yukiko Tawata, Mao Kuriyama, Michinari Kohri, and Hideyuki Kuwata
Department of Materials Chemistry, Graduate School of Engineering, Tohoku University, Aoba 07,
Aoba-ku, 980-8579 Sendai, Japan
(tel: ‡81-22-217-7230; fax: ‡81-22-217-7293; e-mail: shoda@poly.che.tohoku.ac.jp)
and
Takeshi Watanabe
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2,
Niigata 950-2181, Japan (tel: ‡81-25-262-6647; fax: ‡81-25-262-6854; e-mail:wata@agr.niigata-u.ac.jp)
Dedicated to Professor Dieter Seebach on the occasion of his 65th birthday
A novel strategy for the regio- and stereoselective synthesis by two enzymatic steps of oligosaccharides
having an N-acetylglucosamine unit at the nonreducing end was developed. The first step involves a chitinase-
catalyzed highly selective b-N-acetyllactosamination of an oligosaccharide acceptor with a 4,5-dihydrooxazole
derivative of N-acetyllactosamine as the glycosyl donor. The usage of a transition-state-analogue substrate for
the chitinase under basic conditions allows the reaction to proceed only in the synthetic direction while
suppressing hydrolysis of the product in aqueous media. Several chitinase mutants also catalyzed the
glycosylation efficiently under neutral conditions. The second step is a regioselective cleavage of the glycosidic
bond between the terminal galactose unit and the adjacent N-acetylglucosamine unit by the action of a b-
galactosidase. This constitutes a very useful method to add an N-acetylglucosamine unit to the nonreducing end
of chito- and cello-oligosaccharide derivatives in a regio- and stereoselective manner.
1. Introduction. ± Invention of new synthetic tools for a highly selective glycosidic-
bond formation is one of the most lively topics in glycotechnology [1]. To develop a
highly regio- and stereoselective glycosylation, several requirements concerning the
chemical properties of the leaving group on the glycosyl donor, the protecting groups,
as well as the nature of the catalysts must be fulfilled. However, chemical approaches
thus far reported for the regio- and stereoselective construction of a glycosidic bond
have always required blocking and deblocking of at least some OH groups in the
glycosyl donor and glycosyl acceptor. In addition, despite the numerous efforts toward
development of new glycosylation reactions, complete stereocontrol of the glycosidic-
bond formation has not been achieved [2].
Enzymatic glycosylation is a useful tool for the regio- and stereoselective
construction of a glycosidic bond under mild reaction conditions where glycosyl
donors and glycosyl acceptors can be employed in their unprotected forms, leading to
the direct formation of unprotected oligosaccharides in aqueous media [3]. There are
many possibilities concerning the combination of glycosyl donor, glycosyl acceptor, and
enzyme catalyst. Since it was found that a dihydrooxazole (ˆ−oxazoline×) derivative of

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N-acetylglucosamine was recognized by a glycosidase, several studies on the enzymatic
polyaddition reaction of sugar dihydrooxazole derivatives have been demonstrated.
For example, the enzymatic glycosylation by the dihydrooxazole of N,N'-diacetylchi-
tobiose or of N-acetylglucosamine catalyzed by a chitinase was reported [4]. However,
in these reactions, it was impossible to achieve the exclusive elongation by a single N-
acetylglucosamine unit. This is because the sugar dihydrooxazole derivative behaved as
glycosyl donor and as glycosyl acceptor at the same time, leading to the formation of a
mixture of self-coupling products; a selective cross-coupling between the N-acetylglu-
cosamine moiety with another saccharide moiety could, therefore, not be achieved.
We postulated that, if the 4-OH group of the N-acetylglucosamine could be
protected by an appropriate H O-soluble group, an enzymatic cross-coupling would be
2
possible provided the protected substrate was recognized by the chitinase effectively,
resulting in the formation of glycosyl-enzyme intermediate. The present paper
describes a novel synthetic process for the preparation of oligosaccharides having an
N-acetylglucosamine unit at the nonreducing end, with d-galactose as the protecting
group for the glycosyl donor (Scheme 1). The reaction involves a chitinase-catalyzed,
highly selective transglycosylation of the 4,5-dihydrooxazole derivative of N-acetyl-
lactosamine to an oligosaccharide (step 1), followed by the enzymatic cleavage of the
d-galactose unit from the nonreducing end of the product by the action of b-
galactosidase (step 2). As a result of these enzymatic processes, an N-acetylglucos-
amine unit can be added in a defined way to the nonreducing end of the starting
oligosaccharide without accompanying self-coupling reactions.
Scheme 1. Strategy for Elongation of an N-Acetylglucosamine Unit by the Combined Use of Chitinase and
b-Galactosidase. The 4-OH group of the N-acetylglucosamine unit is protected by introducing a b-d-galactose
moiety, avoiding self-coupling of the substrate.
2
. Results and Discussion. ± 2.1. Design and Synthesis of the Glycosyl Donor, the
Dihydrooxazole Derivative of N-Acetyllactosamine. ± First, a new glycosyl donor for
the transglycosylation to transfer an N-acetylglucosamine unit was designed on the

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basis of 1) the retro-synthetic analysis of an (N-acetylglucosamin)ide, and 2) a new
mechanism for a chitinase-catalyzed hydrolysis of N-acetylglucosamine oligomers.
When the (N-acetylglucosamin)ide unit is retro-synthetically disconnected, a sugar
dihydrooxazole derivative is formed as a reasonable synthon. Dihydrooxazole
derivatives of N-acetylglucosamine whose OH groups are protected have indeed been
utilized as glycosyl donors for the preparation of various (N-acetylglucosamin)ides.
The −oxazoline method× found by Micheel et al. in 1958 [5] has been employed
extensively in the stereoselective synthesis of 1,2-trans-2-amino-glycosides promoted
by various Broensted or Lewis acids [6].
In addition, a new mechanism for hydrolysis of chitin by chitinase has recently been
proposed [7]. According to this mechanism, a dihydrooxazolium ion intermediate is
formed in the catalytic site of chitinase as a result of the neighboring participation of
the 2-acetamido group. These reports prompted us to investigate the chemical behavior
of a sugar dihydrooxazole derivative, the neutral form of the dihydrooxazolium ion
intermediate, as a potential glycosyl-donor substrate for chitinases.
A novel irreversible chitinase-catalyzed glycosylation reaction can be designed
based on the hypothesis that the usage of a distorted glycosyl-donor substrate lowers
the free energy of activation significantly. By choosing basic conditions where the
chitinase shows almost no hydrolytic activity, an irreversible glycosylation would be
possible. The combined use of a transition-state-analogue substrate [8] and a
deactivated hydrolytic enzyme would allow the reaction to proceed only in the
direction of the transglycosylation because under basic conditions, the chitinase will not
hydrolyze the product (irreversible glycosylation catalyzed by a glycosyl hydrolase).
The substrate, the dihydrooxazole derivative 1 of N-acetyllactosamine, was
prepared from N-acetyllactosamine by acetylation followed by treating the acetate
with trimethylsilyl trifluoromethanesulfonate [9]. Finally, the acetyl groups were
removed by NaOMe in MeOH.
The 1H-NMR spectrum of 1 shows a signal at d 6.10due to the anomeric proton at the reducing end
(
Fig. 1,a). The J(1,2) ˆ 7.32 Hz and a signal at d 2.07 for the Me group clearly indicate that the dihydrooxazole
13
ring was formed. The C-NMR spectrum shows signals at d 104.8 and 78.3 due to C(1') and C(4), respectively,
indicating that the b-glycosidic bond of the Gal-GlcNAc moiety was retained during the synthesis (Fig. 1,b).
2
.2. Hydrolysis of 1 Catalyzed by Chitinase A1 from Bacillus circulans WL-12.
Chitinases (EC 3.2.1.14) cleave the b-1,4-glycosidic linkage in chitin, a homopolymer of
N-acetylglucosamine. Based on the amino acid sequence similarities, chitinases are
classified mostly into two families, Families 18 and 19 in the classification scheme of
Henrissat [10]. Recently, the hydrolysis mechanism of Family 18 chitinases was found
to be different from that of Family 19 chitinases [7]. The mechanism involves a
dihydrooxazolium ion as intermediate, as supported by several studies including an
inhibition experiment [11] and by structural studies of a chitinase in complex with
allosamidin, a natural product that mimics the dihydrooxazole moiety [7a].
On the basis of these reports, we decided to start our investigation by testing
whether the dihydrooxazole derivative 1 is recognized by the active site of a Family 18
chitinase. We used a cloned enzyme, chitinase A1 (from Bacillus circulans WL-12) for
hydrolytic experiments [12]. The consumption of the dihydrooxazole substrate was
monitored by means of high-performance liquid chromatography (HPLC). When the

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Fig. 1. a) H-NMR and b) 13C-NMR spectra (D
1
O) of glycosyl donor 1 (LacNAc-oxa)
2
substrate 1 was kept at 378 in solution in a phosphate buffer (pH 6.0), only very slow
hydrolysis (ring opening of the dihydrooxazole moiety) was observed. Addition of the
chitinase A1 caused rapid cleavage of the anomeric CÀO bond, yielding the
corresponding N-acetyllactosamine. The commercially available crude chitinase from
Bacillus sp., chitinase C1 (Bacillus circulans WL-12), and chitinase D1 (Bacillus
circulans WL-12) also hydrolyzed 1 to N-acetyllactosamine. Neither lysozyme nor
cellulase catalyzed the hydrolysis of 1.
As a reference, hydrolysis of 4-nitrophenyl (N-acetyllactosamin)ide (Ga1(b1 ±
4
)GlcNAcb-O4NP) was carried out with chitinase A1. The hydrolysis rate of

Helvetica Chimica Acta ± Vol. 85 (2002)
3923
Gal(b1 ± 4)GlcNAcb-O4NP (liberation of 4-nitrophenol(HO4NP)) was much slower
than that of 1. These results indicate that compound 1, having a distorted conformation
close to the dihydrooxazolium-ion intermediate and an axial 4'-OH group, is accepted
by the catalytic site of the chitinase A1 to form an enzyme-substrate complex whose
anomeric center is attacked by H O. The use of such an artificial substrate having a
2
higher free energy as a glycosyl donor promised to be very suitable for the irreversible
transglycosylation catalyzed by a chitinase.
2
.3. Transglycosylation. The glycosyl donor 1 was successfully transglycosylated to
methyl (N-acetyl-b-d-glucosamin)ide 2a (GlcNAcb-OMe) (Fig. 2) catalyzed by
chitinase (Bacillus sp.) to afford trisaccharide 3a (Gal(b1 ± 4)GlcNAc(b1 ±
4
)GlcNAcb-OMe). The reaction was performed under basic conditions in citrate
buffer (0.04m, pH 9.0) to favor transglycosylation over hydrolysis of the product 3a.
HPLC Monitoring of the mixture showed rapid consumption of the starting material 1
and the appearance of a new peak corresponding to trisaccharide 3a. The mixture was
Fig. 2. Structure of glycosyl acceptors, transglycosylated products, and degalactosylated products

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Helvetica Chimica Acta ± Vol. 85 (2002)
heated for 20min at 90 8 to deactivate the enzyme at the point of maximum formation of
a. The yield of the product was determined to be 55% after 30min by comparing the
3
peak area of the product with that of the other sugar units. The crude product was
purified by prep. HPLC, and the structure of the resulting trisaccharide determined by
1
13
means of H- and C-NMR spectroscopy. All anomeric protons were assigned on the
basis of the reported chemical shift of methyl (N-acetyl-b-d-glucosamin)ide 2a and N-
13
acetyllactosamine (Gal(b-1 ± 4)GlcNAc). The C-NMR spectrum also supported the
structure of trisaccharide 3a.
It may be noted that, with methyl (N-acetyl-a-d-glucosamin)ide, the anomer of 2a,
as the glycosyl acceptor, no addition product was obtained, and hydrolysis of 1 to N-
acetyllactosamine occurred quantitatively. These results indicate that the configuration
at C(1) of the glycosyl acceptor is an important factor that controls the course of
transglycosylation. These results may be explained by a steric repulsion between the
anomeric MeO group of (N-acetyl-a-d-glucosamin)ide and an amino acid residue at
the catalytic site of chitinase.
This transglycosylation was successfully applied to the 1-thioglycoside derivative 2b
(GlcNAcb-SMe) as well as to the 1-thioglycoside 2c having a polymerizable group at
the reducing end (GlcNAcb-SCH CH CONHCH NHCOCHˆCH ) [19] as glycosyl
2
2
2
2
acceptors (Fig. 2). A chitinase-catalyzed transglycosylation between 1 and 2b was
performed in a carbonate buffer (0.05m, pH 9.0) at 408, giving rise to the trisaccharide
3b (Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-SMe). HPLC Monitoring of the mixture
showed after 3 h a decrease of the starting glycosyl donor 1, and a new peak of the
product 3b appeared. The product yields as a function of reaction time at pH 9.0as
monitored by HPLC is shown in Fig. 3. During the course of the reaction, the yield of
the product did not decrease at longer reaction times establishing that the newly
formed glycosidic bonds of the obtained trisaccharide are not cleaved by the enzyme.
These results clearly show that the addition reaction proceeded irreversibly under basic
1
reaction conditions (pH 9.0). The structure of product 3b was determined by H- and
13
C-NMR spectroscopy (Fig. 4).
Fig. 3. Product yields vs. reaction time for the chitinase-catalyzed addition reaction of GlcNAcb-SMe 2b to
LacNAc-oxa 1 (408 in carbonate buffer (pH 9.0))

Helvetica Chimica Acta ± Vol. 85 (2002)
3925
Fig. 4. a) 1H-NMR and b) 13C-NMR spectra (D
)GlcNAcb-SMe)
O) of addition product 3b (Gal(b1 ± 4)GlcNAc(b1 ±
2
4
1
The H-NMR spectrum of 3b showed a d with J ˆ 7.89 Hz at d 4.57 for HÀC(1'), indicating that the newly
1
3
formed glycosidic bond in 3b is of the b-d-type (Fig. 4,a). In the C-NMR, two peaks at d 101.5 due to the C(1')
and at d 79.3 due to the C(4) clearly indicate the regio- and stereoselective formation of the b-d-1,4 glycosidic
linkage (Fig. 4,b).
The thioglycoside derivative 2c with a polymerizable group at the reducing end was
similarly glycosylated under basic conditions, giving rise to the trisaccharide derivative
3c with a polymerizable group at the reducing end (Gal(b1 ± 4)GlcNAc(b1 ±
4
)GlcNAcb-SCH CH CONHCH NHCOCHˆCH ) whose structure was confirmed
2
2
2
2
1
13
by H- and C-NMR spectroscopy.

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1
The H-NMR spectrum of 3c showed the olefinic protons of the acrylamide group (d 5.74 ± 6.19 (3 H)) and
three anomeric protons (d 4.56 (HÀC(1)), 4.53 (HÀC(1')), and 4.41 (HÀC(1''))). The configuration of the
resulting glycosidic bond was determined to be b-d, judging from the coupling constant of the signal assigned to
the anomeric proton of the internal GlcNAc unit (J(1',2') ˆ 7.14 Hz). These data show that the trans-
glycosylation reaction of 1 to 2c proceeds in a regio- and stereoselective manner, giving rise to the trisaccharide
derivative 3c.
The transglycosylation was monitored by HPLC. After the addition of chitinase, a
peak corresponding to the glycosyl acceptor 2c became smaller and a new peak
attributed to 3c appeared. Product 3c was purified by prep. HPLC and freeze-dried. To
optimize reaction conditions, the effect of pH on the transglycosylation yield was
investigated at pH 9.0, 9.8, 10.4, and 11.0 (Fig. 5). When the reaction was carried out at
pH 9.0and 9.8, the yield of the product increased to a maximum after 2 h and then
slowly decreased as the reaction proceeded. This behavior may be explained by the
hydrolysis of product 3c by the action of chitinase. When the reaction was performed at
pH 10.4, the yield gradually increased as the reaction proceeded and became constant
after 20h, indicating that the addition reaction was irreversible; the newly produced
glycosidic bond of 3c was not cleaved by the catalyst chitinase. The reaction did not
take place at pH 11.0.
Fig. 5. The effect of pH on the yields of 3c in the chitinase-catalyzed addition reaction of 2c (GlcNAcb-
SCH
2
CH
2
CONHCH
2
NHCOCHˆCH
2
) to LacNAc-oxa 1
A chitobiose derivative 4c having a polymerizable group at the reducing end
GlcNAc(b1 ± 4)GlcNAcb-SCH CH CONHCH NHCOCHˆCH ) was also subjected
(
2
2
2
2
to the addition reaction with 1 in a buffer solution to give the corresponding
tetrasaccharide derivative 5c (Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-
SCH CH CONHCH NHCOCHˆCH ) (Scheme 2). The HPLC trace of the reaction
2
2
2
2

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3927
mixture showed that the peak of the glycosyl acceptor 4c had completely disappeared
after 2 h, and a new peak of the resulting tetrasaccharide 5c was detected as a main
product, indicating that the desired addition took place almost quantitatively (Fig. 6,a).
However, in addition to the desired product 5c, a small peak of 2c (GlcNAcb-
SCH CH CONHCH NHCOCHˆCH ) appeared as a by-product. Since the chitinase
2
2
2
2
is known to be inactive towards the glycosidic bond between two GlcNAc units of the
glycosyl acceptor 4c, the formation of this side product is attributed to the fact that the
chitinase cleaved the glycosidic bond between the GlcNAc unit at the reducing end and
the adjacent GlcNAc unit of the resulting tetrasaccharide derivative 5c. A prolonged
reaction time led to a considerable amount of by-product 2c. To minimize this side
reaction and to improve the yield, we stopped the reaction after 2 h.
Scheme 2
Interestingly, the chitinase from Bacillus sp. can also catalyze the N-acetyllac-
tosamination of glycosyl acceptors that differ in structure from the natural substrate of
chitinases, viz. cellooligosaccharides with a degree of polymerization (DP) 2 ± 6. The
formation of the glycosylation products was confirmed by measuring their molec-
ular masses by means of MALDI-TOF mass spectroscopy. Cellobiose derivative
7
having
a
polymerizable group at the reducing end (Glc(b1 ± 4)Glcb-
SCH CH CONHCH NHCOCHˆCH ) (Fig. 2) can also be N-acetyllactosaminated
2
2
2
2
to give the corresponding tetrasaccharide derivative 8 (Gal(b1 ± 4)GlcNAc(b1 ±
)Glc(b1 ± 4)Glcb-SCH CH CONHCH NHCOCHˆCH ). These results indicate that
4
2
2
2
2
cellooligomers are recognized efficiently by the catalytic center as glycosyl acceptors.

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Fig. 6. a) HPLCTrace of chitinase-catalyzed addition reaction of
SCH CH CONHCH ) to LacNAc-oxa 1, after 2 h. b) HPLCTrace of b-galactosidase-
NHCOCHˆCH
catalyzed degalactosylation reaction of 5c (Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-
SCH CH CONHCH ).
NHCOCHˆCH
4c (GlcNAc(b1 ± 4)GlcNAcb-
2
2
2
2
2
2
2
2
1
The H-NMR spectrum of 8 shows a d for the anomeric proton (HÀC(1''), J(1'',2'') ˆ 7.82 Hz) at d ca. 4.4,
1
3
indicating the stereoselective formation of a b-d glycosidic linkage. The C-NMR spectrum shows signals at
d 101.5 and 79.1 attributed to C(1'') and C(4'), respectively. No signals typical for b-d-1,3 and b-d-1,6 glycosidic
bonds were detected. These data show that the enzymatic addition of 1 to 7 took place regio- and
stereoselectively, leading to the exclusive formation of the b-d-1,4 linkage.
In the reaction presented involving 1 as a transition-state-analogue substrate, the N-
atom of the dihydrooxazole ring may be protonated by an acidic amino acid located in
the À1 subsite. The nucleophilic attack of the 4-OH group of the glycosyl acceptor
occurs by the assistance of the carboxylate ion of the acidic amino acid that behaves as a
general base. The a-d-glycosidic bond of 1 is then cleaved and the CˆO bond is formed
simultaneously, leading to the formation of the 2-acetamido group in the product
oligosaccharides.
Chemical modification of the reducing end of oligosaccharides is an important topic
in the field of glycotechnology, because these functionalized, end-reactive oligosac-
charides can be used as precursors for the production of various glycoconjugate
polymers. For example, oligosaccharides having a polymerizable group at the reducing
end can be copolymerized with various vinyl monomers by a radical initiator to give
copolymers having oligosaccharide graft chains [13]. However, it is extremely difficult
to prepare such modified oligosaccharides by the conventional chemical approach,
because it requires the introduction of several kinds of protecting groups into the
oligosaccharide moiety. As the DP of an oligosaccharide increases, the chemical
modification of the oligosaccharide becomes more difficult due to its low solubility in

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organic solvents. The present enzymatic method of using an unprotected glycosyl donor
and an unprotected glycosyl acceptor makes it possible to synthesize a functional
oligosaccharide without employing laborious blocking and deblocking procedures.
2
.4. Degalactosylation. In the above described reactions, the galactosyl group
protects the N-acetylglucosamine unit and prevents self-coupling. The transgly-
cosylation products possess a galactose unit at the nonreducing end (Fig. 2).
We now discuss the b-galactosidase-catalyzed deprotection of these products. The
trisaccharides 3b and 3c and the tetrasaccharide 8 were converted to the
disaccharides 4b (GlcNAc(b1 ± 4)GlcNAcb-SMe) and 4c (GlcNAc(b1 ± 4)GlcNAcb-
SCH CH CONHCH NHCOCHˆCH ) and the trisaccharide
9
(GlcNAc(b1 ±
2
2
2
2
4
)Glc(b1 ± 4)Glcb-SCH CH CONHCH NHCOCHˆCH ), respectively, by selective
2
2
2
2
cleavage of the b-d glycosidic bond between the terminal d-galactose unit and the
adjacent N-acetylglucosamine unit. The tetrasaccharide 5c prepared by the enzymatic
addition of 1 and 4c was also converted to the corresponding trisaccharide 6c
(
GlcNAc(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-SCH CH CONHCH NHCOCHˆCH ) by
2
2
2
2
enzymatic removal of the d-galactose unit by the same procedure (Scheme 2). When 5c
was treated with b-galactosidase (Streptococcus pneumoniae) in phosphate buffer
solution (0.02m, pH 6.0), the peak of 5c disappeared completely after 92 h. A new peak
attributed to product 6c and two peaks due to the a-d and b-d isomers of the liberated
d-galactose appeared (Fig. 6,b). The product 6c was purified by HPLC and freeze-
dried. The structures of all degalactosylated products were confirmed by comparing
1
13
their H- and C-NMR spectra with those of GlcNAc-SR, chitobiose (GlcNAc(b1 ±
)GlcNAc), and chitotriose (GlcNAc(b1 ± 4)GlcNAc(b1 ± 4)GlcNAc). Thus, a new
4
route for the preparation of chitooligosaccharides or cellooligosaccharides having an
N-acetylglucosamine unit at the nonreducing end has been developed by the
combination of a chitinase-catalyzed addition reaction and a b-galactosidase-catalyzed
selective degalactosylation reaction.
The present method including the elongation of a single N-acetylglucosamine unit
will become a convenient tool for the synthesis of various oligosaccharides useful in
glycobiology. This method also makes it possible to prepare specifically labeled
oligosaccharide derivatives that can be utilized for the elucidation of the roles of
oligosaccharides in vivo, for example, the elicitor activity of oligosaccharides for higher
plants [14]. The use of a thioglycoside moiety at the reducing end enables us to convert
the products to free chitooligosaccharides or free hybrid-type oligosaccharides using an
appropriate reagent without damaging the inner glycosidic bonds. Furthermore,
thioglycosides can be converted to the corresponding sulfoxides that can be used as a
glycosyl donor in acid-catalyzed glycosylations [15].
2
.5. Transglycosylation Catalyzed by Mutants of Chitinase A1. So far, we had to
carry out the chitinase-catalyzed addition reactions under basic conditions to ensure
the irreversibility of the glycosylating process. To improve the presented glycosylation
reaction so that it proceeds at a different pH, especially under neutral conditions, it is
necessary to modify the catalyst enzyme. In Sect. 2.5, we wish to report that the
transglycosylation can also be carried out under neutral conditions with several
mutants of chitinase A1 from Bacillus circulans WL-12.

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Recently, the three-dimensional structure of the catalytic domain of chitinase A1
from Bacillus circulans WL-12 was determined by X-ray analysis [16]. The study
exhibited the amino acid residues that participate in hydrolysis of chitin as well as their
peripheral residues. On the basis of this analysis, we investigated the addition reaction
using several mutant chitinases as catalysts, the dihydrooxazole derivative 1 as glycosyl
donor, and chitobiose (GlcNAc(b1 ± 4)GlcNAc) or methyl (N-acetyl-b-d-glucosami-
n)ide (2a; GlcNAcb-OMe) as glycosyl acceptor. The effect of several amino acids on
the transglycosylating ability of these mutant chitinases was examined. The enzymes
employed were mutant chitinases from Bacillus circulans WL-12, where the glutamic
acid residue 204 was changed to a glutamine residue (E204Q), the aspartic acid residue
202 to an asparagine residue (D202N), the aspartic acid residue 200 to an asparagine
residue (D200N), the tyrosine residue 279 to a phenylalanine residue (Y279F), the
aspartic acid residue 280to a asparagine residue (D280N), and the tryptophan residue
433 a to phenylalanine residue (W433F).
The enzymatic addition reaction was carried out under neutral conditions (pH 7.0)
as well as under basic conditions (pH 10.4), and the transglycosylating activity was
evaluated by tracing the formed tetrasaccharide (Gal(b1 ± 4)GlcNAc(b1 ±
4
)GlcNAc(b1 ± 4)GlcNAc) or 3a (Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-OMe) by
means of HPLC. Interestingly, when the reaction was carried out with D202N,
Y279F, D280N, and W433F as catalysts at pH 7.0, the yields were better than those
obtained by wild-type chitinase. Fig. 7 shows the trace of the addition product from
chitobiose as glycosyl acceptor catalyzed by the wild-type chitinase A1 and D202N at
Fig. 7. Product yields vs. reaction time for chitinase-catalyzed addition reaction of chitobiose (GlcNAc(b1 ±
4
)GlcNAc) to LacNAc-oxa 1. ~: catalyst, mutant enzyme D202N; pH 7.0. .: catalyst, wild-type enzyme; pH 7.0.
~
: catalyst, mutant enzyme D202N; pH 10.4. *: catalyst, wild-type enzyme; pH 10.4.

Helvetica Chimica Acta ± Vol. 85 (2002)
3931
pH 7.0and 1 0. 4. The addition reaction of chitobiose to the sugar dihydrooxazole 1 was
smoothly catalyzed by the mutant D202N, giving rise to the corresponding tetrasac-
charide (Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAc(b1 ± 4)GlcNAc). The yield of the formed
tetrasaccharide was found to exceed 40% and did not decrease at a prolonged reaction
time even at pH 7.0( ~ in Fig. 7). When the reaction was performed under basic
conditions (pH 10.4), the yields obtained with wild-type chitinase and mutant chitinase
were almost the same at a prolonged reaction time (* and ~ in Fig. 7). The wild-type
chitinase gave the addition product in quite good yield at the initial stage of the
reaction; however, the resulting tetrasaccharide was immediately hydrolyzed at pH 7.0
(. in Fig. 7). These results indicate that the addition reaction catalyzed by the mutant
enzyme D202N proceeded irreversibly at pH 7.0 where the wild-type enzyme shows
strong hydrolysis activity. Thus, the hydrolyzing activity toward the resulting
oligosaccharides was reduced by changing an amino acid of chitinase A1, while
retaining their transglycosylating activities even under neutral conditions.
That the reaction catalyzed by these mutant enzymes is irreversible can be
explained by their effective recognition of the transition-state analogue 1, whereas the
mutants show very low affinity toward the (N-acetylglucosamin)ide moiety of the
product. It was assumed that the carboxy group of the aspartic acid residue 202 of the
wild-type chitinase A1 interacts with the C(2) amide carbonyl of the substrate in the À1
subsite and serves to direct the carbonyl group toward the anomeric C-atom to assist
the formation of the dihydrooxazole ring (Scheme 3). The replacement of the aspartic
acid residue 202 by asparagine may cause a significant decrease of hydrolysis activity
toward an (N-acetylglucosamin)ide unit of the formed tetrasaccharide.
The irreversible formation of the product with W433F as catalyst may also be
explained by the decrease of hydrolytic activity of the mutant at pH 7.0. The specific
activity of W433F toward 4-nitrophenyl chitobioside decreased to 0.02 U/mg whereas
that of the wild type was 0.11U/mg, indicating that the W433F has only 20% of
hydrolytic activity compared to that of wild-type chitinase. These results are consistent
with the decrease of hydrolytic activity observed in the enzymatic addition reaction
catalyzed by W433F. It is well known that the tryptophane residue 433 of chitinase A1
assists the incorporation of the substrate into the À2 subsite of the catalytic site through
a hydrophobic interaction. The replacement of the tryptophane residue 433 by the
phenylalanine residue may cause the decrease of the interaction described above,
leading to the significant deactivation of hydrolysis activity.
In contrast, the mutant E204Q where the essential glutamic acid residue is changed
to glutamine could not promote the transglycosylation. The glutamic acid residue 204
plays a very important role for hydrolysis and transglycosylation of a (N-acetylgluco-
samin)ide unit [17]. It has been established that the carboxylate derived from the
glutamic acid residue 204 acts as a general base to enhance the nucleophilic attack of
the H O molecule or one of the OH groups of the glycosyl acceptor in the hydrolysis or
2
transglycosylation, respectively (Scheme 3). Consequently, the lack of the glutamic
acid residue 204 caused almost complete disappearance of hydrolytic activity as well as
transglycosylating activity.
Although the detailed mechanism of the transglycosylation reaction catalyzed by
these mutant enzymes has not been made clear, the usage of such modified catalysts
will widen the scope of the enzymatic glycosylation based on the site-directed

3
932
Helvetica Chimica Acta ± Vol. 85 (2002)
Scheme 3. Roles of aspartic acid residue 202 (D202), glutamic acid residue 204 (E204), and tryptophane residue
33 (W433) in the catalytic site of wild-type chitinase A1 from Bacillus circulans WL-12
4
mutagenesis technique. It is to be noted that some experimental evidence has been
obtained that suggests that the transglycosylation yield can be optimized, depending
upon various reaction conditions, by changing the nature of catalyst enzyme. Such
optimizations will be extremely important for the development of a new multi-enzyme
reaction system in the synthesis of complex oligosaccharides in the future.
Experimental Part
General. Commercially available trimethylsilyl trifluoromethanesulfonate was used without purification.
The 3-[(acrylamidomethyl)amino]-3-oxopropyl 2-acetamido-2-deoxy-1-thio-b-d-glucopyranoside (2c) was pre-
pared according to [18]. MeCN was distilled from CaH . Chitinase (Bacillus sp.) (0.04 U/mg) was purchased
2
from Wako Chemicals (Osaka). b-Galactosidase (Streptococcus pneumoniae) (1.27 U/mg) was obtained from
Calbiochem. The purity of wild-type chitinase A1, the corresponding mutants, chitinase C1, and chitinase D1
was confirmed by 10% SDS polyacrylamide-gel electrophoresis (SDS-PAGE). Sequence determination was
TM
carried out with ALF express DNA sequencer (Amercham Pharmacia Biotech). HPLC: Inertsil ODS-3 and
TSK-gel-Amide-80 columns. UV Spectra: Beckman DU-70 and Shimadzu UV-160 spectrophotometers. NMR
Spectra: Bruker AC-250T, Bruker DPX-400, and Bruker ORX-500 spectrometers; d in ppm, J in Hz.

Helvetica Chimica Acta ± Vol. 85 (2002)
3933
4
,5-Dihydro-2-methyl{1,2-dideoxy-4-O-(b-d-galactopyranosyl)-a-d-glucopyranoso}[2,1-d]oxazole (1). A
mixture of N-acetyllactosamine (500 mg, 1.32 mmol), Ac O (2.7 ml), and pyridine (1.3 ml) was stirred at r.t.
for 22 h. After diluting with CHCl , the resulting soln. was washed with H O, and sat. aq. NaHCO soln., dried
Na SO ), and evaporated: crude lactosamine peracetate that was used for the following experiment without
2
3
2
3
(
2
4
further purification. To a soln. of lactosamine peracetate (948 mg, 1.40mmol) in 1,2-dichloroethane (17 ml),
trimethylsilyl trifluoromethanesulfonate (0.3 ml, 1.66 mmol) was added dropwise, and the mixture was heated at
5
08 for 5 h. Me
3
N (0.6 ml) was added and the mixture stirred for 10 min. After diluting with CHCl
3
, the mixture
was washed with cold H
2
O, the org. layer dried (Na SO ) and evaporated, and the residue submitted to column
2
4
chromatography (Merck silica gel 60 (0.040 ± 0.063 mm), AcOEt/hexane 4 :1) 520 mg of 4,5-dihydro-2-
methyl{1,2-dideoxy-4-O-(2,3,4,6-tetra-O-acetyl-b-d-galactopyranosyl)-a-d-glucopyranoso}[2,1-d]oxazole (61%).
To a soln. of this oxazole derivative (498 mg, 0.81 mmol) in MeOH (50 ml) was added a solution of 0.5m
NaOMe in MeOH (1.0ml) at 0 8, and the mixture was stirred at r.t. for 1 h. After neutralizing the mixture with
‡
1
Amberlite IR-120 H , the solvent was evaporated: 1 (quant.). White solid. H-NMR (D
2
O, 400 MHz): 2.07
O, 100 MHz): 13.1 (NˆC(Me)O); 61.3 (C(6));
1.8 (C(6')); 65.3 (C(2)); 68.7 (C(4')); 69.4 (C(5)); 71.0(C(3)); 71.1 (C(2 ')); 72.6 (C(3')); 75.4 (C(5')); 78.3
1
3
(
s, NˆC(Me)O); 6.10( d, J(1,2) ˆ 7.31, HÀC(1)). C-NMR (D
2
6
(
C(4)); 100.0 (C(1)); 104.8 (C(1')); 168.4 (NˆC(Me)O).
Methyl O-b-d-Galactopyranosyl-(1 ! 4)-O-2-acetamido-2-deoxy-b-d-glucopyranosyl(1 ! 4)-2-acetamido-
2
-deoxy-b-d-glucopyranoside (Gal(b1 ±4)GlcNAc(b1 ±4)GlcNAcb-OMe; 3a). Glycosyl donor 1 (48 mg,
0
.13 mmol) and methyl (N-acetyl-b-d-glucosamin)ide (2a; GlcNAcb-OMe; 92 mg, 0.39 mmol) were dissolved
in 150 ml of 0.05m citric buffer (pH 9.0). To this mixture, chitinase (Bacillus sp., 10wt.-% for 1) dissolved in 80 ml
of 0.01m citric buffer (pH 9.0) was added, and the mixture was stirred for 0.5 h at 308. Yields were determined by
taking aliquots of the sample and evaporation of the solvent prior to HPLC analysis. For final workup, the
mixture was treated with THF to inactivate the enzyme. To obtain samples for NMR spectral analysis, semi-
prep. HPLC was performed. The HPLC fraction containing the product was chromatographically homogeneous.
1
H-NMR (D
2
O, 400 MHz): 3.49 (s, MeO); 4.41 (d, J(1,2) ˆ 7.85, HÀC(1)); 4.44 (d, J(1'',2'') ˆ 8.14, HÀC(1''));
13
4
(
.58 (d, J(1',2') ˆ 7.48, HÀC(1')). C-NMR (D
2
O, 100 MHz): 58.0 (MeO); 80.2 (C(4)); 102.2 (C(1')); 102.7
‡
C(1)); 103.7 (C(1'')). MALDI-TOF-MS: 601.3 ([M ‡ Na] ; calc. 600.6).
Methyl O-b-d-Galactopyranosyl-(1 ! 4)-O-2-acetamido-2-deoxy-b-d-glucopyranosyl-(1 ! 4)-2-acetamido-
2
-deoxy-1-thio-b-d-glucopyranoside (Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-SMe; 3b. To 1 (18.5 mg, 0.05 mmol)
in a microtube was added a soln. of methyl 2-acetamido-2-deoxy-1-thio-b-d-glucopyranoside (2b) in 0.5 ml of
.05m carbonate buffer (pH 9.0), and the resulting mixture was incubated at 408. After adding an excess of THF,
0
the mixture was heated at 908 for 20min to deactivate the enzyme. The solvent was evaporated and the residue
1
dissolved in H
2
O and separated by prep. HPLC: 3b (13.0mg, 64%). H-NMR (D
2
O, 500 MHz): 2.01 (2 COMe);
2
.15 (s, MeS); 4.43 (d, J(1'',2'') ˆ 7.43, HÀC(1'')); 4.49 (d, J(1,2) ˆ 10.0, HÀC(1)); 4.56 (d, J(1',2') ˆ 7.89,
13
HÀC(1')). C-NMR (D
0.4 (C(6')); 61.2 (C(6'')); 68.7 (C(4'')); 71.1 (C(2'')); 72.3 (C(3)); 72.6 (C(3'')); 73.9 (C(3')); 75.0(C(5 ')); 75.5
C(5'')); 78.3 (C(5)); 78.6 (C(4')); 79.3 (C(4)); 84.7 (C(1)); 101.5 (C(1')); 103.0 (C(1'')); 174.6, 174.7 (2 COMe).
Trisaccharide Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-SCH CH CONHCH (3c). To 1
2
O, 125 MHz): 12.1 (MeS); 22.3 (2 NˆC(Me
3
)O); 53.9 (C(2)); 55.3 (C(2')); 60.0 (C(6));
6
(
2
2
2
NHCOCHˆCH
2
(
(
73 mg, 0.2 mmol) in a microtube was added a soln. of GlcNAc derivative 2c (26 mg, 66.7 mmol) and chitinase
Bacillus sp.; 7.3 mg, 292 mU) in 2.0 ml of 0.05m Tris buffer (pH 9.0), and the resulting mixture was incubated in
a dry thermo bath at 408. After adding an excess of THF, the mixture was heated at 908 for 20min to deactivate
the enzyme, and the solvent was evaporated. The residue was dissolved in H
2
O and separated by prep. HPLC
1
(
Inertsil-ODS, H
.54, 2.9 (m, SCH
0.0, HÀC(1)); 4.60( s, NCH
6.4, 36.0(SCH CH ); 44.4 (NCH
2
O/MeOH, 3.0ml/min): 3c (35 mg, 69%). H-NMR (D
2
O, 500 MHz): 1.96, 2.02 (s, 2 COMe);
2
1
2
2
CH
2
); 4.41 (d, J(1'',2'') ˆ 7.82, HÀC(1'')); 4.53 (d, J(1',2') ˆ 7.14, HÀC(1')); 4.56 (d, J(1,2) ˆ
13
2
N); 5.79 ± 6.19 (m, CHˆCH
2
). C-NMR (D
2
O, 125 MHz): 22.3 (2 NˆC(Me)O);
2
2
2
N); 54.2 (C(2)); 55.3 (C(2')); 60.0 (C(6')); 60.5 (C(6')); 61.2 (C(6'')); 68.7
(C(4'')); 71.1 (C(2'')); 72.2 (C(3')); 72.6 (C(3'')); 73.8 (C(3)); 74.9 (C(5')); 75.5 (C(5'')); 78.3 (C(4')); 78.5 (C(5));
7
1
9.4 (C(4)); 84.6 (C(1)); 101.5 (C(1')); 103.0 (C(1'')); 128.4 (ˆCH
2
); 129.7 (CHˆ); 168.8 (NHCOCHˆ); 174.5 ±
‡
74.8 (2 COMe). MALDI-TOF-MS: 778.8 ([M ‡ Na] ; calc. 779.9).
Tetrasaccharide Gal(b1 ± 4)GlcNAc(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-SCH
2
CH
2
CONHCH
2
NHCOCHˆCH
2
(
5c). To 1 (18 mg, 48 mmol) in a microtube was added a solution of GlcNAc derivative 4c (19 mg, 32 mmol) and
chitinase (Bacillus sp., 70.4 mU) in 2.0 ml of 0.05m carbonate buffer (pH 10.4), and the resulting mixture was
incubated at 408 for 2 h. The mixture was heated at 908 for 20min to deactivate the enzyme, and the solvent was
evaporated. The residue was dissolved in H
2
O and separated by prep. HPLC (Inertsil-ODS, H
O, 400 MHz): 1.90 ± 1.96 (s, 3 COMe); 2.48, 2.83 (m, SCH
N); 5.68 ± 6.15
2
O/MeOH 900 :7,
1
5
4
.0ml/min): 5c (17 mg, 54%). H-NMR (D
.35 (d, J(1''',2''') ˆ 7.76); 4.45 ± 4.51 (HÀC(1), HÀC(1'), HÀC(1'')); 4.55 (s, NCH
2
2
CH );
2
2

3
934
Helvetica Chimica Acta ± Vol. 85 (2002)
(
(
(
m, CHˆCH
2
). 13C-NMR (D
2
O, 100 MHz): 22.3 (3 NˆCO(Me)); 26.4, 36.0(SCH
2 2 2
CH ); 44.4 (NCH N); 54.3
C(2)); 55.2, (C(2')); 55.3 (C(2'')); 60.0 (C(6'), C(6'')); 60.5 (C(6)); 61.2 (C(6''')); 68.7 (C(4''')); 71.1 (C(2''')); 72.2
C(3'), C(3'')); 72.6 (C(3''')); 73.8 (C(3)); 74.7 (C(5')); 75.0(C(5 '')); 75.5 (C(5''')); 78.2 (C(4'')); 78.6 (C(5));
7
1
9.1 (C(4')); 79.2 (C(4)); 84.7 (C(1)); 101.4 (C(1')); 101.5 (C(1'')); 103.0 (C(1''')); 128.5 (ˆCH
2
); 129.7 (CHˆ);
‡
68.8 (NHCOCHˆ); 174.7 ± 174.8 (3 COMe). MALDI-TOF-MS: 981.6 ([M ‡ Na] ; calc. 983.1).
Hybrid-Type
NHCOCHˆCH
17 mg, 33 mmol) and chitinase (Bacillus sp., 325 mU) in 1.0 ml of 0.05m Tris buffer (pH 9.0), and the resulting
mixture was incubated at 408. The reaction was monitored by HPLC (MeCN/H O 3 :1). After 1 was completely
consumed, the mixture was heated at 908 for 20min to deactivate the enzyme, and the solvent was evaporated.
Tetrasaccharide
2 2 2
Gal(b1 ± 4)GlcNAc(b1 ± 4)Glc(b1 ± 4)Glcb-SCH CH CONHCH -
2
(8). To 1 (37 mg, 0.1 mmol) in a microtube was added a soln. of cellobioside derivative 7
(
2
The residue was dissolved in H
min): 8 (5 mg, 16%). H-NMR (D
2
O and separated by prep. HPLC (Inertsil-ODS, H
O, 400 MHz): 1.98 (s, COMe); 2.55, 2.91 (m, SCH
2
O/MeOH 900 :70, 3.0 ml/
CH
); 4.39 (d, J(1''',2''') ˆ
1
2
2
2
7
.8, H ÀC(1''')); 4.43 (d, J(1',2') ˆ 7.9, HÀC(1')); 4.47 (d, J(1,2) ˆ 10.0, HÀC(1)); 4.50( d, J(1'',2'') ˆ 7.8,
1
3
HÀC(1'')); 4.58 (s, NCH
2
N); 5.70± 6.16 ( m, CHˆCH
2
). C-NMR (D
2
O, 100 MHz): 22.3 (NˆCO(Me)); 26.1,
3
6.2 (SCH CH ); 44.5 (NCH N); 60.1 (C(6'), C(6'')); 60.3 (C(6)); 61.2 (C(6''')); 79.1 (C(4')); 85.6 (C(1)); 101.5
2
2
2
(
C(1'')); 102.4 (C(1')); 103.1 (C(1''')); 128.4 (ˆCH
2
); 129.7 (CHˆ); 168.8 (NHCOCHˆ); 174.7, 174.9 (COMe,
‡
COCH
2
). MALDI-TOF-MS: 901.0 ([M ‡ Na] ; calc. 900.9).
Methyl 2-Acetamido-4-O-(2-acetamido-2-deoxy-b-d-glucopyranosyl)-2-deoxy-1-thio-b-d-glucopyranoside
GlcNAc(b1 ± 4)GlcNAcb-SMe; 4b). A typical procedure for the degalactosylation of 3b by b-galactosidase
(
is as follows: to a soln. of 3b (10mg, 16.2 mmol) in 2.0 ml of 0.05m phosphate buffer (pH6.0) was added 0.2 ml of
.05m phosphate buffer (pH 6.0) soln. of b-galactosidase (Streptococcus pneumoniae) (5 wt.-% rel. to 3b), and
the mixture was shaken for 1 h at 408. After adding an excess of THF, the mixture was heated for 10min at 90 8 to
deactivate the enzyme, and the solvent was evaporated. The residue was dissolved in a minimum amount of H
0
2
O
and the H
H-NMR (D
2
O-insoluble material was filtered off. The filtrate was chromatographed by prep. HPLC: 4b (7 mg).
O, 500 MHz): 2.03, 2.06 (2 COMe); 2.19 (MeS); 4.52 (d, J(1,2) ˆ 10.4, HÀC(1)); 4.59 (d, J(1',2') ˆ
1
2
1
3
8
.45, HÀC(1')). C-NMR (D
2
O, 125 MHz): 12.0(MeS), 22.2, 22.3 (2 N ˆC(Me)O); 53.9 (C(2)); 55.7 (C(2'));
6
0.4 (C(6)); 60.7 (C(6')); 69.8 (C(4')); 73.6 (C(3')); 73.9 (C(3)); 76.0(C(5 ')); 78.6 (C(5)); 79.4 (C(4)); 84.6
(C(1)); 101.6 (C(1')); 174.6, 174.7 (2 COMe).
GlcNAc(b1 ± 4)GlcNAcb-SCH CH CONHCH
and b-galactosidase (Streptococcus pneumoniae) (39 mU) in 6.5 ml of 0.02m phosphate buffer (pH 6.0) was
incubated for 12 h at 378. The mixture was heated at 908 for 25 min to deactivate the enzyme and filtered. The
2
2
2
NHCOCHˆCH
2
(4c). A mixture of 3c (35 mg, 46 mmol)
filtrate was chromatographed by prep. HPLC (Inertsil ODS-3, H
2
O/MeOH 900 :70; flow rate 5.0 ml/min): 4c
CH
); 4.48 (d, J(1',2') ˆ 8.4,
N); 5.69 ± 6.15 (CHˆCH
); 44.4 (NCH N); 54.2 (C(2)); 55.7 (C(2')); 60.5
C(6)); 60.7 (C(6')); 69.8 (C(4')); 73.6 (C(3')); 73.8 (C(3)); 76.1 (C(5')); 78.5 (C(5)); 79.5 (C(4)); 84.7 (C(1));
1
(25 mg). H-NMR (D
2
O, 400 MHz): 1.91, 1.97 (2 COMe); 2.49 ± 2.88 (SCH
2
2
1
3
HÀC(1')); 4.54 (d, J(1,2) ˆ 10.2, HÀC(1)); 4.56 (s, NCH
2
2 2
). C-NMR (D O,
1
00 MHz): 22.3 (2 NˆCO(Me)); 26.4, 36.0(SCH CH
2
2
2
(
1
01.6 (C(1')); 128.5 (ˆCH
2
); 129.7 (CHˆ); 168.8 (NCOCHˆ); 174.5, 174.7, 174.8 (2 NCOMe, NCOCH
2
).
‡
MALDI-TOF-MS: 617.3 ([M ‡ Na] ; calc. 617.7).
GlcNAc(b1 ± 4)GlcNAc(b1 ± 4)GlcNAcb-SCH
2
CH
2
CONHCH
2
NHCOCHˆCH
2
(6c). As described for 3c,
1
with 5c (16 mg, 16 mmol), phosphate buffer (2.0ml), and b-galactosidase (21 mU): 11 mg (86%) of 6c. H-NMR
(
D
2
O, 400 MHz): 1.93 (s, COMe); 1.99 (s, 2 COMe); 2.51 ± 2.87 (SCH
2
CH
O, 100 MHz): 22.3 (NˆCO(Me)); 26.4,
N); 54.3 (C(2)); 55.2 (C(2')); 55.7 (C(2'')); 60.1 (C(6')); 60.5 (C(6)); 60.7 (C(6''));
9.8 (C(4'')); 72.3 (C(3')); 73.6 (C(3'')); 73.8 (C(3)); 74.7 (C(5')); 76.1 (C(5'')); 78.6 (C(5)); 79.2 (C(4), C(4'));
2
); 4.48 ± 4.54 (HÀC(1), HÀC(1'),
1
3
HÀC(1'')); 4.58 (s, NCH
2
N); 5.71 ± 6.17 (m, CHˆCH
2 2
). C-NMR (D
3
6
8
6.0(SCH
2
CH
2
); 44.5 (NCH
2
4.7 (C(1)); 101.4 (C(1')); 101.6 (C(1')); 128.4 (ˆCH
2
); 129.7 (CHˆ); 168.8 (NCOCHˆ); 174.5 ± 174.8 (3
‡
NCOMe, NCOCH
2
). MALDI-TOF-MS: 820.8 ([M ‡ Na] ; calc. 820.9).
GlcNAc(b1 ± 4)Glc(b1 ± 4)Glc-b-SCH
2
CH
2
CONHCH
2
NHCOCHˆCH
2
(9). As described for 3b, with 8
1
(
5 mg, 5 mmol), phosphate buffer (730 ml), and b-galactosidase (7 mU): 3 mg (63%) of 8. H-NMR (D
2
O,
4
00 MHz): 1.86 (s, COMe); 2.58, 2.95 (m, SCH
2
CH
2
); 4.46 ± 4.54 (HÀC(1), HÀC(1'), HÀC(1'')); 4.62
1
3
(
s, NCH
2
N); 5.75 ± 6.20( m, CHˆCH
2
). C-NMR (D
2
O, 100 MHz): 22.2 (NˆCO(Me)); 26.1, 36.2 (SCH
2 2
CH );
4
1
2
4.5 (NCH N), 55.7 (C(2'')); 60.1 ± 60.7 (C(6') or C(6'')); 60.3 (C(6)); 79.2 (C(4')); 85.6 (C(1)); 101.6 (C(1''));
02.3 (C(1')); 128.4 (ˆCH
2
); 129.7 (CHˆ); 168.8 (NHCOCHˆ), 174.7, 174.9 (COMe, COCH
2
). MALDI-TOF-
‡
MS: 739.1 ([M ‡ Na] , calc. 738.7).
Production and Purification of Wild-Type Chitinases and Their Mutants. Wild-type chitinase A1 and mutant
chitinases were produced in E. coli HB101 cells carrying pHT012 and their derivatives encoding corresponding
mutant chitinases, respectively. E. coli HB101 cells carrying a plasmid were grown in 100 ml of LB medium

Helvetica Chimica Acta ± Vol. 85 (2002)
3935
containing 100 mg/ml of ampicillin for 20± 23 h at 30 8. The cells were collected by centrifugation, and chitinases
were extracted from the cells by a cold osmotic-shock procedure. (NH SO was added to the extract to achieve
0% saturation. The precipitate formed was dissolved in 5 mm phosphate buffer and lyophilized. Wild-type and
4
)
2
4
4
mutant chitinases were purified by a chitin affinity column (2.5 Â 40.0 cm) equilibrated with 20 mm phosphate
buffer. After washing with 200 ± 300 ml of 20 mm NaOAc buffer (pH 5.5), adsorbed proteins were eluted with
1
00 ml of 20 mm NaOAc buffer (pH 5.5). The fractions containing chitinase A1 or their mutants were collected
and lyophilized to give 5 ± 10mg of proteins. Wild-type chitinases C1 and D1 were produced in a manner similar
as described above with E. coli JM109 and HB101 carrying the recombinant plasmid that encodes wild-type
chitinase C1 and D1, respectively. After precipitating with (NH ) SO at 60% saturation, the precipitated
4 2 4
proteins were subjected to chromatography on a hydroxyapatite column (2.5 Â 25.0cm) equilibrated with 1 m m
phosphate buffer (pH 6.0) as follows. Chitinase C1: after washing with 1 mm phosphate buffer, adsorbed protein
containing chitinase C1 was eluted with a linear gradient (1 ± 400 mm) of phosphate buffer (pH 6.0), giving rise
to 1 mg of chitinase C1. Chitinase D1: proteins containing chitinase D1 were eluted with 1 mm phosphate buffer
for separation from other proteins adsorbed on hydroxyapatite; each peak fraction containing chitinase activity
was collected and lyophilized to give 0.5 mg of chitinase D1.
Site-Directed Mutagenesis for Construction of Plasmid Encoding Mutant Gene. The site-directed mutant
D200N, D202N, E204Q, Y279F, D280N, or W433F was made according to the procedure described by Kunkel et
al. [20]. Plasmids encoding D200N, D202N, E204Q, or D280N were constructed by previous methods [17][21].
TM
The mutants, Y279F and W433F, were constructed by using the Quick Change site-directed mutagenesis kit
(
Stratagene, CA). The recombinant plasmid pHT012, which carries the open reading frame of chitinase A1, was
used as a template DNA for PCR. The synthetic oligonucleotides used for site-directed mutagenesis were 5'-
GGATTAACATTATGACATACGATTTTAACG-3' (Y279 ! F) and 5'-AGCGATGTTCTTCGAGCT-
CAGCGGT-3' (W433 ! F). To ensure that the desired mutation was the only mutation in the sequence, the
entire region of the inserted DNA was sequenced.
Determination of Chitinase Activity. The activity of chitinases was evaluated by means of the amount of 4-
nitrophenol released as a result of enzymatic hydrolysis of 4-nitrophenyl N,N'-diacetylchitobioside. The relative
activity of chitinase A1, C1, and D1 was estimated to be 0.11, 3.93, and 5.11 U/mg, respectively. One unit of
activity is defined as the amount of enzyme liberating 1 mmol of 4-nitrophenol per min.
The present research is partially supported by the Nagase Science and Technology Foundation. We thank
Yaizu Suisankagaku Industry for providing us with N-acetyllactosamine.
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Received June 3, 2002