Synthesis of acarbose analogues by transglycosylation reactions of Leuconostoc mesenteroides B-512FMC and B-742CB dextransucrases.

Article abstract of DOI:10.1016/S0008-6215(02)00350-6

Two new acarbose analogues were synthesized by the reaction of acarbose with sucrose and dextransucrases from Leuconostoc mesenteroides B-512FMC and B-742CB. The major products for each reaction were subjected to yeast fermentation, and then separated and

Full text of DOI:10.1016/S0008-6215(02)00350-6

Carbohydrate Research 337 (2002) 2427–2435
www.elsevier.com/locate/carres
Synthesis of acarbose analogues by transglycosylation reactions of
Leuconostoc mesenteroides B-512FMC and B-742CB
dextransucrases
Seung-Heon Yoon, John F. Robyt*
Laboratory of Carbohydrate Chemistry and Enzymology, 4252 Molecular Biology BLDG, Iowa State Uni6ersity, Ames, IA 50011, USA
Received 10 June 2002; accepted 12 September 2002
Abstract
Two new acarbose analogues were synthesized by the reaction of acarbose with sucrose and dextransucrases from Leuconostoc
mesenteroides B-512FMC and B-742CB. The major products for each reaction were subjected to yeast fermentation, and then
separated and purified by Bio-Gel P2 gel permeation chromatography and descending paper chromatography. The structures of
the products were determined by one- and two-dimensional 1H and 13C NMR spectroscopy and by matrix-assisted laser
desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). B-512FMC-dextransucrase produced one major
acarbose product, 2I-a-
D
-glucopyranosylacarbose and B-742CB-dextransucrase produced two major acarbose products, 2I-a-
-glucopyranosylacarbose. © 2002 Elsevier Science Ltd. All rights reserved.
D-
glucopyranosylacarbose and 3IV-a-
D
Keywords: Acarbose; Acarbose analogues; Dextransucrase; L. mesenteroides B-512FMC; L. mesenteroides B-742CB; Transglycosylation reactions
products from maltotriose.3 On the other hand, mu-
tansucrase (GTF-I) from S. mutans gives 3III-O-a-
D-
1. Introduction
glucopyranosylmaltotirose and 3I-O-a-
D-glucopyrano-
Dextransucrases [EC 2.4.1.5] elaborated by some
Leuconostoc mesenteroides strains and Streptococcus
species catalyze the syntheses of various kinds of dex-
trans from sucrose.1 It is also well known that these
enzymes catalyze the transfer of glucosyl units from
sucrose to other carbohydrates. This reaction is the
so-called acceptor reaction, and the added carbohy-
drates are called acceptors.1 More than 30 different
carbohydrates are known to act as acceptors, so that it
is possible to utilize dextransucrases to synthesize new
kinds of carbohydrates by the acceptor reaction.1,2 Fur-
ther, different kinds of acceptor products are obtained
from the same acceptor by changing glucansucrases
elaborated by different species of microorganisms. Dex-
transucrase from L. mesenteroides B-512F produces
sylmaltotirose in addition to the above two acceptor
reaction products from maltotriose produced by L.
mesenteroides B-512F dextransucrase.4
Acarbose is a pseudotetrasaccharide with an unsatu-
rated cyclitol [2,3,4-trihydroxy-5-(hydroxymethyl)-5,6-
cyclohexene in a
D-gluco-configuration] attached to the
nitrogen of 4-amino-4, 6-dideoxy-
D-glucopyranose,
which is linked a-(1“4) to maltose. Acarbose is a
strong competitive inhibitor of a-glucosidase,5–7 alpha-
amylase,5–8 cyclomaltodextrin glucanyltransferase (CG-
Tase),7,9,10 glucoamylase,11,12 and glucansucrases.13,14
The mechanism of inhibition for these enzymes has
been postulated to be due to the cyclohexene ring and
the nitrogen linkage that mimics the transition state for
the enzymatic cleavage of glycosidic linkages.6,15
6III-O-a-
D
-glucopyranosylmaltotriose and 6I-O-a-
D-
Some naturally occurring acarbose analogues have
glucopyranosylmaltotriose as the acceptor reaction
been found that have several
D-glucose residues at-
tached to the nonreducing-end of acarbose.5 Yoon and
Robyt16 modified the nonreducing-end of acarbose
with specific maltodextrin chains, maltohexaose (G6),
maltododecaose (G12), and maltooctadecaose (G18),
* Corresponding author. Tel.: +1-515-294-1964; fax: +1-
515-294-0453
E-mail address: jrobyt@iastate.edu (J.F. Robyt).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00350-6

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S.-H. Yoon, J.F. Robyt / Carbohydrate Research 337 (2002) 2427–2435
which were added to the C-4-hydroxyl group of the
cyclohexene ring by the reaction of acarbose with cyclo-
maltohexaose in a coupling reaction catalyzed by Bacil-
lus macerans CGTase.
Food Science and Technology, Seoul National Univer-
sity, Suwon, Korea).
The substitution of different saccharides for the mal-
tose unit at the reducing-end of acarbose has given
analogues that have significantly increased inhibition
and/or altered enzyme specificity.7,17,18 Park et al.7,17
reported the formation of several acarbose analogues
modified at the reducing-end by the transglycosylation
reaction between acarbose and various carbohydrate
acceptors catalyzed by Bacillus stearothermophilus mal-
togenic amylase (BSMA). They found that the removal
2.2. Removal of carbohydrates from reaction mixtures
by fermentation, using immobilized yeast
One gram of Saccharomyces cere6isiae (commercial
Fleishman’s bread yeast) was swollen in 5 mL of sterile
water for 15 h, and then mixed well with 50 mL of 2.5%
(w/v) medium viscosity sodium alginate (Sigma Chemi-
cal Co, St. Louis, MO). The mixture was dropped from
a tube with a 0.2-0.5 mm orifice into 300 mL of 4%
(w/v) CaCl2 solution with stirring and allowed to stand
for 3 h at 4 °C to harden the alginate/yeast beads,
which were then washed with 300 mL of 20 mM
pyridinium acetate buffer (pH 5.2) three times to re-
move soluble, unwanted material. The immobilized
yeast was kept in buffer at 4 °C until use.
of one
D-glucose residue from the reducing-end of
acarbose produced acarviosine-glucose, which inhibited
yeast a-glucosidase 430-times better than acarbose.
They also found that the replacement of the maltose
unit by isomaltose gave an inhibitor that inhibited
porcine pancreatic alpha-amylase 15 times better than
acarbose. Lee et al.18 found that when the maltose unit
of acarbose was replaced by cellobiose or lactose, the
acarbose analogues were potent inhibitors for b-glu-
cosidase and b-galactosidase, whereas acarbose was not
an inhibitor at all.
2.3. Preparation of dextransucrase acceptor reaction
products
In the present study, we report the enzymatic synthe-
Twenty IU of B-512FMC-dextransucrase were added
to 2.0 mL of substrate mixture composed of 100 mM
acarbose and 100 mM sucrose in 20 mM pyridinium
acetate buffer (pH 5.2). The enzyme reaction was car-
ried out at 27 °C for 5 days with the periodic addition
of 0.5 mL of 400 mM sucrose solution every 24 h. For
B-742CB-dextansucrase, 4 IU of enzyme were added to
4.0 mL of 100 mM acarbose and 100 mM sucrose in 20
mM pyridinium acetate buffer (pH 5.2) buffer, and the
reaction was carried out at 37 °C for 3 days. The
enzyme reactions were stopped by heating in boiling
water for 5 min. The fermentable carbohydrates,
ses of new acarbose analogues in which a
D-glucopy-
ranosyl residue is added to the 3-hydroxyl group of the
cyclohexene ring or to the 2-hydroxyl group of the
reducing-end,
D-glucose unit by the reaction of acar-
bose with sucrose in an acceptor reaction catalyzed by
dextansucrases from L. mesenteroides B-512FMC and
B-742CB.
2. Experimental
2.1. Materials
mainly unreacted sucrose,
D-fructose and D-glucose, in
the enzyme reaction digest were removed by yeast (S.
cere6isiae) fermentation at 37 °C for 24 h, followed by
concentration to about 2.0 mL by rotary vacuum evap-
oration. Any soluble dextran that was in the reaction
mixture was removed by adding an equal volume of
ethanol (4 °C), followed by centrifugation at 6,000×g
for 10 min and the supernatant was concentrated to
about 1.2 mL by rotary vacuum evaporation. The
reaction products were analyzed by TLC; an appropri-
ate amount (1-5 mL) of sample was spotted onto a
10×20 cm Whatman K5 or K6 silica gel plate (Fisher
Scientific, Chicago, IL). The plate was irrigated 2–3
times with 85:20:50:50 (v/v) of acetonitrile–ethyl ace-
tate–1-propanol–water with an 18 cm irrigation path
length. The carbohydrates on the TLC plate were visu-
alized by dipping the plate into a MeOH solution
containing 0.3% (w/v) N-(1-naphthyl)ethylenediamine
and 5% (v/v) H2SO4, followed by heating at 12 °C for
10 min.21
Dextransucrases [EC 2.4.1.5] from L. mesenteroides B-
512FMC and B-742CB were prepared in our laboratory
by previously reported procedures.19,20 The constitutive
mutant (B-742CB/AE4B6) was cultivated statically in a
D-glucose medium composed of Bactopeptone (4.4 g/
L), yeast extract (4.4 g/L), K2HPO4 (20 g/L), glucose
(20 g/L), MgSO4·7H2O (0.17 g/L), NaCl (0.08 g/L),
FeSO4·7H2O (0.08 g/L), MnSO4·H2O (0.072 g/L), and
CaCl2·2H2O (0.011 g/L) at 21 °C for 24 h. Cells were
removed by using a hollow fiber cartridge with a 0.1 mm
cutoff (H5MP01-43, Amicon, Inc., Beverly, MA) and
the culture supernatant was concentrated and dialyzed
against 20 mM pyridinium acetate buffer (pH 5.2) by
using a hollow fiber cartridge with a 100 K MW cut-off
(Amicon, H5P100-43). The activities of B-512FMC-
dextransucrase and B-742CB-dextransucrase were 259
and 13 IU/mL, respectively, using a 14C-sucrose assay.19
Acarbose was a gift from Dr. K.-H. Park (Dept. of

S.-H. Yoon, J.F. Robyt / Carbohydrate Research 337 (2002) 2427–2435
2429
2.4. Fractionation of the dextransucrase reaction prod-
ucts by Bio-Gel P2 column chromatography
The purified carbohydrates on the paper were located
using a AgNO3 reagent, the paper was sectioned, eluted
with deionized water, and concentrated to 1 mL by
rotary evaporation.23
About 1.2 mL of the reaction digest was loaded onto
Bio-Gel P2 (fine) column (1.5×115 cm), and eluted
with deionized water at a flow rate of 0.063 mL/min,
collecting 1.0 mL fractions. The total carbohydrate
content of each fraction was determined by the micro
phenol–H2SO4 method,22 and the carbohydrate compo-
sition of the fractions was analyzed by TLC as de-
scribed above with Whatman K6 plates.
2.6. Analysis of reaction products by matrix-assisted
laser desorption ionization-time of flightmass spec-
trometry (MALDI-TOF MS)24
One mg of acarbose and 1.5 mg of the two reaction
products (P71 and P72=P51) were dissolved in 1.0 mL
of pure water and filtered through a 0.2 mm membrane;
10 mL of the filtered solution were mixed with 10 mL of
0.1 M 2,5-dihydroxybenzoic acid in MeCN; 1.0 mL of
the mixture was transferred to the probe and the sol-
vent was evaporated under 6acuum. The masses of the
compounds in the samples were analyzed by MALDI-
TOF MS, using a Dyanmo instrument (Thermo Bio-
Analysis, Ltd., Paradise, UK) with a nitrogen laser (337
nm). Ions were detected in a positive mode at an
acceleration voltage of 20 kV.
2.5. Purification of the dextransucrase reaction prod-
ucts by descending paper chromatography
About 250 mL of the concentrated enzyme reaction
products, which were fractionated by BioGel P2
column chromatography, were loaded onto Whatman 3
MM paper (23×56 cm).23 The paper was irrigated with
10:4:3 (v/v) of ethyl acetate–pyridine–water for 36 h to
purify B-512FMC-dextransucrase reaction products or
72 h for B-742CB-dextransucrase reaction products.
2.7. NMR analysis
About 50 mg of acarbose and about 10–20 mg of the
purified enzyme reaction products were exchanged three
times with D2O and were dissolved in 0.5 mL of pure
D2O, and then placed into 5 mm NMR tubes. NMR
spectra were obtained on a Bruker DRX 500 spectrom-
1
eter, operating at 500 MHz for H and at 125 MHz for
13C at 25 °C. Spectra of homonuclear correlation spec-
troscopy (COSY) and heteronuclear multiple quantum
coherence spectroscopy (HMQC) were recorded and
analyzed with XWINNMR (Bruker) or NMRVIEW
programs.25
3. Results
Dextransucrase from L. mesenteroides B-512FMC cata-
lyzed an acceptor reaction between acarbose and su-
crose, and gave one major product (P51), several minor
products (P52–P55),
glucopyranosyl -fructopyranose), and isomaltose (Fig.
1, lane 3). When the reaction products were treated
with yeast, the fermentable carbohydrates, -fructose
D-fructose, leucrose (5-a-D-
D
D
and sucrose, were removed (Fig. 1, lane 4). The remain-
ing reaction products were fractionated by Bio-Gel P2
gel permeation chromatography (Fig. 2). Unreacted
acarbose and minor reaction products (P52–P55) were
well separated, but the major reaction product, P51,
was co-eluted with isomaltose (IG2) and leucrose (Leu).
A51 was separated from IG2 and Leu by descending
paper chromatography (Fig. 1, lane 5). The minor
reaction products (P52–P55) were determined to be
isomaltodextrins by TLC RG values (Fig. 1, lanes 6 and
7).
Fig. 1. Thin-layer chromatogram of the dextransucrase (from
L. mesenteroides B-512FMC) reaction products from sucrose
and acarbose. Whatman K5 TLC plates, irrigated three times
(path length 18 cm each) with 85:20:50:50 (v/v) MeCN–
EtOH–1-propanol–water. Lanes 1 and 8, isomaltodextrin
standards; lane 2, carbohydrate standards; lane 3, enzyme
reaction products before yeast fermentation; lane 4, enzyme
reaction products after yeast fermentation; lane 5, P51; lane 6,
P52 (isomaltotriose), lane 7, P53-P55 (isomaltodextrins). Aca
designates acarbose and IGn designates isomaltodextrins hav-
ing n
D-glucose residues.

2430
S.-H. Yoon, J.F. Robyt / Carbohydrate Research 337 (2002) 2427–2435
Fig. 2. Bio-Gel P2 gel permeation column chromatogram (1.5×115 cm; flow rate 0.063 mL/min; fraction size, 1.0 mL) of the L.
mesenteroides B-512FMC dextransucrase reaction products.
1
The H/13C-HMQC NMR spectrum of P71 (Fig. 7B)
Dextransucrase from L. mesenteroides B-742CB gave
was almost identical to that of acarbose (Fig. 7A) with
the exception of the 5-hydroxymethyl cyclohexene ring
(unit IV in Fig. 7A and B) and the D-glucopyranosyl
two major acceptor reaction products (P71 and P72),
along with other minor products, and -fructose and
-glucose (Fig. 3, lane 3), which were removed by yeast
D
D
fermentation (Fig. 3, lane 4). The products remaining
after yeast fermentation were fractionated by Bio-Gel
P2 gel permeation chromatography. Acarbose was sep-
arated from the two major reaction products, P71, P72,
and leucrose, which eluted together. P71, P72, and
leucrose were separated and purified by a descending
paper chromatography (Fig. 3, lanes 5 and 6).
unit (unit V in Fig. 7B). The changes of the carbon-
chemical shifts of the 5-hydroxymethyl-cyclohexene
ring before and after the addition of
D-glucopyranose
to acarbose were, respectively, as follows; C-1, 56.5–
56.2; C-2, 71.5–69.0; C-3, 73.3–79.5; C-4, 71.1–70.1;
C-5, 139.4–139.0; C-6, 124.0–124.6; C-7, 62.0–62.0
ppm (Fig. 6A and B) with a significant downfield
chemical shift of 6.2 ppm for C-3 and a relatively small
upfield chemical shift at C-2 of 2.5 ppm, and 1.1 ppm
for C-4 of the 5-hydroxymethyl cyclohexene ring. These
chemical shifts are characteristic of the attachment of a
The number of
D-glucose units that were added to
acarbose for products P71 and P72 were determined by
using MALDI-TOF MS. The results are shown in Fig.
4 in which the masses of P71 and P72 have been
increased over that of acarbose by exactly a single
D-glucopyranosyl unit to the C-3 position of 5-hydrox-
D
-glucose residue.
Acarbose and the acceptor reaction products, P51,
ylmethyl cyclohexene ring (unit IV in Fig. 7B) of acar-
bose.16 Thus, the NMR results indicate that the
P71, and P72, were analyzed by H, 13C, H-COSY and
1H/13C-HMQC NMR.16 The analysis of the anomeric
proton (H-1) of P71 displayed a new anomeric proton
(V-1 in Fig. 5B) at 5.27 ppm, which was very close to
the III-1 proton (5.24 ppm) of acarbose (Fig. 5A). The
Roman numerals refer to the particular carbohydrate
residue, starting with the reducing-end and the Arabic
numerals refer to the particular carbon on the residue,
as shown in Fig. 7. When the integral of IV-6 proton
was made 1, the total integrals of III-1 proton and V-1
proton was 2. The 13C chemical shift of the new
anomeric carbon (C-1) appeared at 99.2 ppm (data not
shown). These results indicate that one molecule of
D-glucopyranose unit was attached to the 5-hydrox-
1
1
ymethyl cyclohexene ring by an a-(1“3) linkage.
P72 and P51 were the same compounds, because they
1
both gave the same H and 13C NMR spectra. When
the anomeric proton of P51 was analyzed, two new
anomeric proton peaks appeared at 5.03 and 5.33 ppm
chemical shifts, V(I(a))-1 and V(I(b))-1 in Fig. 5(C).
This indicates that a new anomeric proton is present on
acarbose, which has an a-configuration of the
residue that is attached to the reducing-end
D
-glucose
-glucose
D
unit (I in Fig. 7C), as compared with unsubstituted
acarbose (Fig. 5A). When the integral of the IV-6
proton (the chemical structures refer to those in Fig. 7)
was 1, the integrals of the new anomeric proton peaks
D
-glucose was added to acarbose with an a-linkage.26,27

S.-H. Yoon, J.F. Robyt / Carbohydrate Research 337 (2002) 2427–2435
2431
were 0.5 for each proton. This indicates that one
glucopyranosyl unit was attached to acarbose, but that
there are two forms of the newly bound -glucopyran-
D-
D
osyl unit due to the a- and b-anomeric structures at the
reducing-end. The 13C chemical shifts of the new
anomeric carbons, V(I(a))-1 and V(I(b))-1, appeared at
96.6 and 98.1 ppm (data not shown).
1
The H/13C-HMQC NMR spectrum of P51 (Fig. 6C)
was almost identical to that of acarbose (Fig. 6A) with
the exception of the reducing-end
D-glucose (unit I in
Fig. 7A and C) and the -glucose unit (unit V in Fig.
D
7C). The changes of the carbon-chemical shifts of the
reducing-end a-glucose residue (unit I) before and after
the addition of
D-glucopyranose to acarbose were re-
spectively as follows; C-1, 92.3–89.6; C-2, 71.9–76.0;
C-3, 73.7–71.8; C-4, 77.5–76.5; C-5, 70.3–70.1; C-6,
61.0–61.0 ppm (Fig. 5A and C). And those of the
reducing-end b-glucose residue (unit I) before and after
the addition of
D-glucopyranose were, respectively, as
follows; C-1, 96.1–96.5; C-2, 74.3–78.8; C-3, 76.6–
75.3; C-4, 77.5–76.5; C-5, 75.0–74.8; C-6, 61.0–61.0
Fig. 4. Matrix-assisted laser desorption ionization-time of
flight mass spectrometric (MALDI-TOF MS) analysis of the
number of
D-glucose units added to acarbose by the dextran-
sucrase transglycosylation reactions. A, spectrum of acarbose;
B, spectrum of P71 product; C, spectrum of P72/P51 prod-
ucts.
ppm (Fig. 6A and C). There were relatively large
downfield chemical shifts of 4.1 ppm for C(a)-2 and 4.5
ppm for C(b)-2, respectively, and relatively small
changes for the chemical shifts at C(a and b)-1 and at
C(a and b)-3 of the reducing-end glucose unit. These
chemical shifts are characteristic of the attachment of a
D-glucopyranosyl unit to the C-2 position of the reduc-
ing-end glucose residue (unit I in Fig. 7C) of acarbose.
These results show the formation of two forms of
anomeric carbons and anomeric protons when the
D-
glucopyranosyl unit is attached to I-2 and would be
found for a-and b-kojibiose.28 The NMR results, thus,
indicate that the
D-glucopyranose is attached to the
reducing-end glucose unit of acarbose by an a-(1“2)
linkage (Fig. 7C).
Fig. 3. Thin-layer chromatogram of the L. mesenteroides
B-742CB dextransucrase reaction products from sucrose and
acarbose. Whatman K5 TLC plates, irrigated three times
(path length 18 cm each) with 85:20:50:50 (v/v) MeCN–
EtOH–1-propanol–water. Lanes 1 and 7, isomaltodextrin
standards; lane 2, carbohydrate standards; lane 3, enzyme
reaction products before yeast fermentation; lane 4, enzyme
reaction products after yeast fermentation; lane 5, P71; lane 6,
P72. Aca designates acarbose and IGn designates isoma-
4. Discussion
B-512FMC-dextransucrase produces a dextran com-
posed of 95% of a-(1“6) linkages (main chains) and
5% a-(1“3) branch linkages from sucrose.1 When ac-
ceptor molecules are present in the reaction mixture,
ltodextrins having n
D-glucose residues.

2432
S.-H. Yoon, J.F. Robyt / Carbohydrate Research 337 (2002) 2427–2435
unusual acceptor products when cellobiose, lactose, and
B-512FMC-dextransucrase primarily transfers the
D-
raffinose were the acceptors.1,29 The enzyme exclusively
glucose residue from sucrose to the 6-hydroxyl group of
monosaccharides and to the nonreducing-end 6-hy-
droxyl group of di- and higher-saccharides.1,29 The first
transferred
group of the reducing-end
lobiose and then transferred
group of the first transferred
D
-glucose from sucrose to the C-2-hydroxyl
-glucose moiety of cel-
-glucose to the 6-OH
-glucose moiety of cel-
D
D
acceptor product of D-glucose is isomaltose. Isomaltose
D
can also act as an acceptor to give isomaltotriose, and
isomaltotriose can also act as an acceptor to give
isomaltotetraose and eventually a series of isomaltodex-
trins are formed.1,29 When maltose is the acceptor, the
lobiose to eventually give a series of isomaltodextrins
attached a-(1“2) to the reducing-end moiety.1,29 When
lactose was the acceptor, the enzyme also transferred
D
-glucose from sucrose to the 2-OH of the
moiety at the reducing-end. This -glucose unit, how-
ever, was not elongated as it was for cellobiose. When
raffinose was the acceptor, the enzyme transferred
glucose from sucrose to the 2-OH group of the -glu-
D-glucose
trisaccharide, panose (6II-a-
D-glucopyranosylmaltose) is
D
the product; this trisaccharide is also an acceptor and
eventually a series is produced with chains of isoma-
ltodextrins attached to maltose at the 6-OH group of
D-
D
the nonreducing-end
Fu and Robyt3 found that B-512F dextransucrase
transferred -glucose from sucrose to the 6-OH groups
of both the nonreducing-end and the reducing-end
D-glucose.
cose residue and did not elongate it. It, thus, appears
that the presence of a b-glycosidic bond in the acceptor
changed the specificity of B-512F dextransucrase to give
transfer to the 2-OH group at the reducing-end glucose
rather than transfer to 6-OH at the nonreducing-end
D
D-
glucose moieties when maltotriose to maltooctaose were
the acceptors. The nonreducing-end products predomi-
nated and they were the only ones that were further
elongated, as the reducing-end product contained only
and when
and raffinose, only a single
ferred. B-512F dextransucrase and B-512FMC dextran-
sucrase, the enzyme from the constituent,
D-galactose was present, as it is for lactose
D-glucose unit is trans-
a single
D-glucose residue. B-512F dextransucrase gave
high-producing mutant, give identical dextrans and ac-
ceptor products.
In present study, we have found that B-512FMC-
dextransucrase also transferred
to the 2-OH of the reducing-end moiety of acarbose to
-glucopyranosylacarbose (P51 and P72 in
D-glucose from sucrose
give 2I-O-a-
D
Fig. 7C) as an acceptor reaction product. This was not
the expected product as acarbose does not have either a
b-glycosidic bond or a
ture. Acarbose contains a pseudocarbohydrate, [2,3,4-
trihydroxy-5-(hydroxymethyl)-5,6-cyclohexene in
D-galactose moiety in its struc-
a
D-gluco-configuration] that is attached to the nitrogen
of 4-amino-4,6-dideoxy-D-glucose. It is possible that
these unusual structures are not bound in the enzyme
acceptor-binding site and that only the maltose moiety
of acarbose at the reducing-end is bound, thus giving
transfer to the 2-OH group of the reducing-end
D-glu-
cose moiety of the maltose unit. Acarbose is also
known as an inhibitor of B-512FMC dextransucrase13
and this could be a determining factor on the type of
acceptor product that is formed.
B-742CB-dextransucrase catalyzes the synthesis of a
highly branched dextran that is composed of a-(1“6)
linked
D-glucose in the main chains with a major
amount of a-(1“2) linked single branch
D-glucose
residues and smaller amounts of a-(1“3), long-chain
branch linkages.19,30 In this study, B-742CB-dextransu-
crase produced two acceptor reaction products with
Fig. 5. 1H NMR spectra of anomeric proton regions. A,
acarbose; B, P71; C, P72 and P51. The NMR peaks are
designated first by the Roman numeral of the residue, starting
with the reducing residue at I, and followed by the particular
proton position on the residue. For example, I(a)-1 indicates
that the anomeric proton of unit I has the a-configuration.
acarbose, 3IV-O-a-
Fig. 7B) and 2I-O-a-
D-glucopyranosyl acarbose (P71 in
D-glucopyranosyl acarbose (P72 in
Fig. 7C). The second has the same structure as the
acceptor product (P51) produced by B-512FMC-dex-
transucrase. The first product (P71) had
D-glucose at-

S.-H. Yoon, J.F. Robyt / Carbohydrate Research 337 (2002) 2427–2435
2433
Fig. 6. HMQC NMR spectrum of acarbose (A), P71 (B) and P72 and P51, (C) recorded in D2O at 25 °C. The NMR peaks are
designated first by the Roman numeral of the residue, starting with the reducing residue at I, followed by the particular
carbon/proton position on the residue. Chemical structures of acarbose and its analogues refer to Fig. 7A, B, and C, respectively.
tached a-(1“3) to the 3-hydroxyl group of the nonre-
ducing-end, cyclohexene ring. Acceptor products of
B-742CB have not been studied extensively, so it is not
possible to say whether or not the formation of the
a-(1“3) linkage at the nonreducing-end of acarbose is
ture without the ring oxygen and with the C-5,6 unsat-
urated linkage at the nonreducing-end might be a
determining factor in the unusually linked acceptor
product.
Mutansucrase (glucosyltransferase-insoluble, GTF-I)
from S. mutans 6715 synthesizes an a-(1“3) linked
unusual. The presence of the pseudo-D-glucose struc-

2434
S.-H. Yoon, J.F. Robyt / Carbohydrate Research 337 (2002) 2427–2435
Fig. 7. Proposed chemical structures of (A) acarbose, (B) P71, and (C) P51 and P72. Each of the residues of acarbose is designated
by Roman numerals, starting with I at the reducing-end residue.
glucan that is not branched.1 It also produces four
kinds of acceptor reaction products from maltotriose,
Acknowledgements
transferring
or to the nonreducing-end, forming a-(1“3) and a-
(1“6) linkages at both ends:1,3 6III-O-a-
-glucopyran-
osyl maltotriose, 6I-O-a-
-glucopyranosylmaltotriose,
3III-O-a- -glucopyranosyl maltotriose and 3I-O-a-
D-
D
-glucose from sucrose to the reducing-end
The authors thank Professor K.-H. Park of Seoul
National University, Korea, for the kind gift of acar-
bose and Dr. Bruce Fulton of Iowa State University for
assistance in obtaining the NMR spectra.
D
D
D
glucopyranosylmaltotriose.4 The former two acceptor
reaction products are also made by B-512F-dextransu-
crase, but the latter two are only produced by mutansu-
crase. Besides mutansucrase, alternansucrase is the only
other glucansucrases that is known to form a-(1“3)
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