Enzymatic synthesis of two salicin analogues by reaction of salicyl alcohol with Bacillus macerans cyclomaltodextrin glucanyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase

Article abstract of DOI:10.1016/j.carres.2004.03.018

β-Salicin is a naturally occurring glycoside found in the bark of poplar and willow trees. Ancient man used it as an analgesic and antipyretic. It has a D-glucopyranose unit attached by a β-linkage to the phenolic hydroxyl of salicyl alcohol. Two new salicin analogues have been enzymatically synthesized by transglycosylation reactions: (a) by the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and salicyl alcohol, followed by reactions with alpha amylase and glucoamylase to give D-glucopyranose attached by an α-linkage to the phenolic hydroxyl of salicyl alcohol as the major product, α-salicin; and (b) by the reaction of Leuconostoc mesenteroides B-742CB dextransucrase with sucrose and salicyl alcohol, followed by reactions with dextranase and glucoamylase to give α-D-glucopyranose attached to the primary alcohol hydroxyl of salicyl alcohol as the major product, α-isosalicin.

Full text of DOI:10.1016/j.carres.2004.03.018

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1517–1529
Enzymatic synthesis of two salicin analogues by reaction of
salicyl alcohol with Bacillus macerans
cyclomaltodextrin glucanyltransferase and Leuconostoc
mesenteroides B-742CB dextransucrase
Seung-Heon Yoon,a,b D. Bruce Fultonb and John F. Robyta,b,
*
aLaboratory of Carbohydrate Chemistry and Enzymology, Iowa State University, Ames, IA 50011, USA
bDepartment of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
Received 25 August 2003; accepted 9 March 2004
Abstract—b-Salicin is a naturally occurring glycoside found in the bark of poplar and willow trees. Ancient man used it as an
analgesic and antipyretic. It has a -glucopyranose unit attached by a b-linkage to the phenolic hydroxyl of salicyl alcohol. Two new
D
salicin analogues have been enzymatically synthesized by transglycosylation reactions: (a) by the reaction of Bacillus macerans
cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and salicyl alcohol,followed by reactions with alpha amylase and
glucoamylase to give
salicin; and (b) by the reaction of Leuconostoc mesenteroides B-742CB dextransucrase with sucrose and salicyl alcohol,followed by
D
-glucopyranose attached by an a-linkage to the phenolic hydroxyl of salicyl alcohol as the major product, a-
reactions with dextranase and glucoamylase to give a-
as the major product, a-isosalicin.
D-glucopyranose attached to the primary alcohol hydroxyl of salicyl alcohol
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Salicyl alcohol; Salicin analogues; a-Salicin; a-Isosalicin; b-Salicin; Bacillus macerans cyclomaltodextrin glucanyltransferase; Cyclo-
maltohexaose; Dextransucrase
1. Introduction
willow (Salix) barks. It was used by ancient man as an
3;8
anti-inflammatory,analgesic,and antipyretic prodrug.
Many of the naturally occurring glycosides found in
plants have biological activities.1–7 The activities are
primarily due to the aglycone of the glycosides. The
water solubility of hydrophobic molecules,containing
one or more hydroxyl groups,can be increased by gly-
cosylation,which can influence their physicochemical
and pharmacological properties,such as transport
through membrane barriers and transport by body flu-
ids.2 Glycosylation also often reduces the irritability and
toxicity of the aglycone.2
Bacillus macerans cyclomaltodextrin glucanyltrans-
ferase (CGTase) catalyzes transglycosylation reactions
between cyclomaltohexaose and acceptors having a
hydroxyl group.9 It also catalyzes disproportion reac-
tions between two maltodextrin chains.9 Leuconostoc
mesenteroides B-742CB dextransucrase catalyzes the
transfer of D-glucose from sucrose primarily to a pri-
mary alcohol hydroxyl of various carbohydrate accep-
tors to give new dextrins.10
In the present study,we report two distinct glycosyl-
ation reactions of salicyl alcohol. In the first reaction,
B. macerans CGTase transferred maltohexaose from
cyclomaltohexaose to the primary alcohol hydroxyl of
salicyl alcohol. This was followed by treatments with
alpha amylase,glucoamylase,and immobilized yeast
Salicin [2-(hydroxymethyl)phenyl-b-D-glucopyrano-
side] is a plant glycoside found in poplar (Populus) and
* Corresponding author. Tel.: +1-515-294-1964; fax: +1-515-294-0453;
e-mail: jrobyt@iastate.edu
0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.03.018

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S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
to give a new salicin derivative, a-salicin,with glucose
attached to the phenolic hydroxyl group. In the sec-
ond reaction, L. mesenteroides B-742CB dextransucrase
transferred glucose from sucrose to the primary
hydroxyl group of salicyl alcohol. This was followed
by treatments with dextranase and glucoamylase to give
a second new salicin derivative, a-isosalicin,with
glucose attached to the primary alcohol group of salicyl
alcohol.
lized yeast was used to remove fermentable saccha-
rides,15 which were produced as by-products from the
enzyme reactions. Wet immobilized yeast (18 g) was
added to the reaction digest and the mixture was kept at
37 °C for 24 h. The immobilized yeast was removed by
filtration,and washed three times with 6 mL each of
deionized water. The washings were added to the su-
pernatants of the digests. The supernatants were then
concentrated to about 2 mL by vacuum rotary evapo-
ration. The insoluble material in the concentrated
solution was removed by centrifugation at 4400g for
10 min. The reaction products were analyzed by TLC.
An appropriate amount (1–5 lL) of sample was loaded
onto a 10 Â 20 cm Whatman K5 silica gel plate (Fisher
Scientific,Chicago,IL). The plate was irrigated two
times with 85:15 volume parts of acetonitrile–water at
21 °C,with an 18 cm irrigation path length. The carbo-
hydrates on the TLC plate were visualized by dipping
the plate into a methanol solution containing 0.3% (w/v)
N-(1-naphthyl)ethylenediamine and 5% (v/v) sulfuric
acid,followed by heating at 120 °C for 10 min.16 The
relative percents of the carbohydrates were determined
by TLC densitometry,using a Bio-Rad scanning densi-
tometer (GS-670,Bio-Rad Laboratories,Hercules,
CA).16;17
2. Experimental
2.1. Materials
Salicyl alcohol was obtained from Aldrich (Milwaukee,
WI). Bacillus macerans CGTase [EC 2.4.1.19] was
obtained by growing B. macerans ATCC 8517 on wheat
bran media and purification by a modification of the
method of Kobayashi et al.11 as previously described.12
Porcine pancreatic alpha amylase (PPA) was obtained
from Boehringer Mannheim (Indianapolis,IN). Asper-
gillus niger glucoamylase (GA) was obtained from
Megazyme International (Wicklow,Ireland). Dextran-
sucrase [EC 2.4.1.5] from L. mesenteroides B-742CB was
prepared in our laboratory by previously reported pro-
cedures.13;14 Dextranase from Penicillium sp. was
obtained from Sigma (St. Louis,MO). Cyclomalto-
hexaose (a-CD) was obtained from Ensuiko Sugar
Refining Co. (Yokohama,Japan). Immobilized Sac-
charomyces cerevisiae was prepared by the procedure
previously described.15
2.3. Preparation of salicyl alcohol analogues by reaction
with dextransucrase
L. mesenteroides B-742CB dextransucrase [EC 2.4.1.5]
(12 IU) was added to 12 mL of substrate solution,
containing 200 mM sucrose and 100 mM salicyl alcohol
in 20 mM pyridinium acetate buffer (pH 5.2). The
enzyme reaction was carried out at 25 °C for 24 h with
magnetic stirring,and was stopped by heating in boiling
water for 5 min. Dextranase (DA,120 units) and gluco-
amylase (GA,120 units) were added to 12 mL of the
dextransucrase reaction mixture and incubated at 37 °C
for 24 h. DA and GA were used to increase the amount
of glucosyl salicyl alcohol by hydrolyzing isomalto-
dextrins that were conjugated to salicyl alcohol. The DA
and GA enzyme reactions were stopped by heating in
boiling water for 5 min and cooled down to 20 °C.
Immobilized yeast (18 g) was used as described above.
The supernatants were concentrated to about 2.4 mL by
vacuum rotary evaporation. Insoluble dextran in the
concentrated reaction mixture was removed by centri-
fugation at 9000g for 10 min. The enzyme reaction
products were analyzed by TLC as described above.
2.2. Preparation of salicyl alcohol analogues by reaction
with CGTase
B. macerans CGTase (240 IU,1 IU is the amount of
enzyme that will produce 1 lmol of cyclomaltodextrin
per min at pH 6.0 and 37 °C) was added to 12 mL of
substrate solution,containing 100 mM
a-CD and
100 mM salicyl alcohol in 25 mM imidazolium HCl
buffer (pH 6.0). The enzyme reaction was carried out at
37 °C for 72 h with stirring,and the reaction was stop-
ped by heating in boiling water for 5 min. Porcine pan-
creatic alpha amylase (PPA,120 IU,1 IU is that amount
of enzyme that will hydrolyze 1 lmol of a-(1 ! 4) glu-
cosidic linkages per min at pH 6.0 and 37 °C) and glu-
coamylase (GA,120 IU,1 IU is the amount of enzyme
that will produce 1 lmol of glucose from waxy maize
starch per min at pH 6.0 and 37 °C) were added to 12 mL
of the CGTase reaction mixture and incubated at 37 °C
for 24 h. These two enzymes were used to produce glu-
cosyl salicyl alcohol from maltodextrin conjugated sal-
icyl alcohol by hydrolyzing the maltodextrins. The PPA
and GA enzyme reactions were stopped by heating in
boiling water for 5 min and cooled to 21 °C. Immobi-
2.4. Fractionation of the enzyme products by BioGel P2
column chromatography
About 0.6–0.8 mL of the concentrated digests were
loaded onto BioGel P2 (fine) column (1.5 Â 115 cm),and
eluted with deionized water at a flow rate of 3.78 mL/h,

S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
1519
collecting 1.0 mL fractions. This procedure was repeated
3. Results
several times to fractionate the entire concentrated
reaction digest. The carbohydrate content of each frac-
tion was determined by the micro phenol–H2SO4
method,18 and the composition of the fractions were
analyzed by TLC as described above.
3.1. Reaction of CGTase with cyclomaltohexaose and
salicyl alcohol followed by alpha amylase and gluco-
amylase
B. macerans CGTase catalyzed a transglycosylation
reaction between salicyl alcohol and cyclomaltohexaose,
giving maltohexaosyl salicyl alcohol. This was followed
by a series of transglycosylation reactions (dispropor-
tionation reactions) between two maltodextrin units at-
tached to two salicyl alcohol molecules to give several
2.5. Purification of the enzyme products by descending
paper chromatography
Each of the reaction products obtained from the BioGel
P2 column chromatography were further purified by
preparative descending paper chromatography:19
an appropriate amount (50–180 lL) of the reaction
products (pooled fractions 1–7) were loaded onto
Whatman 3 mm paper (23 Â 56 cm). The paper was
irrigated with 10:4:3 volume parts of EtOAc–pyridine–
water for 5.5–36 h. Strips (2.54 cm) were cut on both
sides of the paper and the products on the paper were
visualized by the silver nitrate method and used to
locate the products,19 which were cut from the paper,
eluted with deionized water,and concentrated to
about 0.5 mL by vacuum rotary evaporation for further
study.
salicyl alcohol analogues with
D-glucose and malto-
dextrins of different sizes attached to salicyl alcohol (Fig.
1A,lane 3). When PPA and GA were added to these
products,several CGTase reaction products (PC3,PC4,
PC6,PC7,PC8,PC9,maltodextrins,and higher
molecular weight products) were removed or signifi-
cantly decreased,while the amounts of PC1,PC2,and
D
-glucose were increased and new products (PC5,PC10,
PC12,and PC13) were produced (Fig. 1A,lane 4). Table
1 gives the relative percents of the compounds in the
reaction digest obtained by TLC determinations. When
the PPA and GA treated products were treated with
immobilized yeast,most of the free
D-glucose was
2.6. Analysis of the products by matrix-assisted laser
desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS)
removed and PC2 resulted as the major product (Fig.
1A,lane 5 and Table 1).
3.2. Reaction of L. mesenteroides B-742CB dextransu-
crase with sucrose and salicyl alcohol followed by
dextranase and glucoamylase
Salicyl alcohol (1 lL,1 mg/mL) and the purified reaction
products (about 1 mg/mL) were transferred to the probe,
and mixed with 1.0 lL of 0.1 M 2,5-dihydroxybenzoic
acid (DHB) in 70% (v/v) acetonitrile. The solvent was
evaporated under a stream of cold air or by vacuum.
The masses of the samples were analyzed by MALDI-
TOF MS,13;20 using a Dynamo 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.
L. mesenteroides B-742CB dextransucrase catalyzed the
transfer of
produce several salicyl alcohol analogues,
glucose,leucrose,and isomaltodextrins (Fig. 1B,lane 2).
D
-glucose from sucrose to salicyl alcohol to
D
-fructose, D-
When the dextransucrase reaction was treated with
dextranase and glucoamylase,several products (PD7,
PD8,PD10,and high molecular weight products)
were significantly or completely removed,and at the
same time the relative amounts of PD1,PD2,PD3,
2.7. NMR analysis of the products
PD5, D-glucose,and isomaltose were increased (Fig. 1B,
lane 3,and Table 2). When the dextranase- and gluco-
amylase-treated digest was incubated with immobi-
About 50 mg of salicyl alcohol and 5–50 mg of the
purified products were exchanged three to four times
with 600 lL of D2O and then dissolved in 600 lL of pure
D2O,and placed into 5 mm NMR tubes. NMR spectra
were obtained on a Bruker DRX 500 spectrometer,
lized yeast,the fermentable saccharides,mainly
glucose and
Table 2).
D-
D-fructose,were removed (Fig. 1B,lane 4,
1
operating at 500 MHz for H and 125 MHz for 13C at
25 °C. COSY (homonuclear correlation spectroscopy)
and HMQC (heteronuclear multiple quantum coherence
spectroscopy) spectra were recorded,following standard
experimental protocols,and analyzed with the
XWINNMR (Bruker) program.13 Sodium 4,4-dimethyl-
4-sila propanesulfonate (DDS) was used as a H-chem-
ical shift standard and 1,4-dioxane was used as the 13C
standard.
3.3. Purification of the reaction products on BioGel P2
The immobilized yeast-treated CGTase reaction prod-
ucts were fractionated into seven groups by BioGel P2
gel permeation column chromatography (data
not shown). PC4 was a single compound on TLC,but
was resolved into two compounds (PC41 and PC42)
on the BioGel P2 column. The compounds that were
1

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S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
Figure 1. Thin-layer chromatogram of the cyclomaltodextrin glucanyltransferase (CGTase) reaction products from salicyl alcohol (SA) and
cyclomaltohexaose (a-CD) (A) and dextransucrase B-742CB reaction products from SA and sucrose (B). (A) Lane 1,maltodextrin standard; lane 2,
a-CD; lane 3,CGTase reaction products before porcine pancreatic alpha amylase (PPA) and glucoamylase (GA) treatment; lane 4,CGTase reaction
products after PPA and GA treatment; lane 5,enzyme reaction products after immobilized yeast treatment. G denotes maltodextrins having n
n
number of D-glucose units. (B) Lane 1,salicyl alcohol and carbohydrate standards; lane 2,dextransucrase reaction products before dextranase (DA)
and glucoamylase (GA) treatment; lane 3,dextransucrase reaction products after DA and GA treatment; lane 4,enzyme reaction products after
immobilized yeast treatment. IGn denotes isomaltodextrins having n units of
D-glucose.
Table 1. The relative percent of the products from the CGTase reac-
tion of cyclomaltohexaose with salicyl alcohol
Table 2. The relative percent of the products from the reaction of
dextransucrase with sucrose and salicyl alcohol
Reaction
productsb
Rxn 1a w CGTase Rxn 2a
Rxn 3a ImYeast
PPAc þ GAc
3.03
Reaction
productsb
Rxn 1a w DS Rxn 2a DAc þ GAc Rxn 3a ImYeast
PC1
PC2
PC3
PC4
PC5
PC6
PC7
G1
1.07
6.26
1.54
7.89
0
6.21
31.24
0
12.69
0
PD1
PD2
4.94
2.58
3.12
3.82
3.37
27.57
9.14
2.12
2.44
5.49
3.49
7.82
3.65
2.84
1.79
15.83
8.07
3.87
3.92
3.72
4.70
29.76
10.9
2.8
10.35
4.72
4.65
5.58
7.23
0
1.04
1.98
0
2.25
1.78
0
PD3
PD4
2.10
PD5
Tr
Tr
0
Fructose
Glucose
PD6
14.93
1.25
6.27
11.43
0
64.5
0
11.94
0
0
PC8
PC9
G2
4.61
0
0
0
PD7
PD8
0
3.76
2.17
Tr
6.09
6.73
1.58
3.17
0
2.61
2.82
7.43
7.13
0
3.43
5.19
14.32
15.87
0
PC10
PC11
PC12
G3
PD9
6.98
0
Leucrose
Isomaltose
PD10
PD11
HMWPb
1.04
0
9.90
0
PC13
HMWPb
3.94
5.87
8.68
21.1
2.72
9.58
3.19
20.87
30.38
aSalicyl alcohol is not included; Rxn 1,CGTase reaction products
from 100 mM salicyl alcohol and 100 mM cyclomaltohexaose; Rxn 2
is Rxn 1 þ PPA þ GA; Rxn 3 is Rxn 2 treated with immobilized yeast.
aSalicyl alcohol was not included; Rxn 1,dextransucrase (DS) reaction
products from 100 mM salicyl alcohol and 200 mM sucrose; Rxn 2 is
Rxn 1 þ DA þ GA; Rxn 3 is Rxn 2 treated with immobilized yeast.
bPD ¼ dextransucrase reaction product; HMWP ¼ high molecular
weight products.
bPC ¼ CGTase reaction product; G1, G2, G3 ¼
D-glucose,maltose,and
maltotriose; HMWP ¼ high molecular weight products; Tr ¼ trace.
cPPA ¼ porcine pancreatic alpha amylase; GA ¼ glucoamylase.
cDA ¼ dextranase; GA ¼ glucoamylase.

S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
1521
Figure 2. (A) Thin-layer chromatogram of the purified cyclomaltodextrin glucanyltransferase (CGTase) reaction products by BioGel P2 column
chromatography and preparative descending paper chromatography. Lane 1,maltodextrin standard; lane 2 and 12,CGTase reaction products before
purification; lane 3,PC1; lane 4,PC2; lane 5,PC4 1; lane 6,PC4 2; lane 7,PC5; lane 8,PC10; lane 9,PC11; lane 10,PC12; lane 11,PC13. G
denotes
n
maltodextrins having n units of -glucose. (B) Thin-layer chromatogram of the L. mesenteroides B-742CB dextransucrase reaction products after by
D
BioGel P2 column chromatography and preparative descending paper chromatography. Lane 1,carbohydrate standards; lane 2 and 11,dextran-
sucrase reaction products before purification; lane 3,PD1; lane 4,PD2; lane 5,PD3; lane 6,PD4; lane 7,PD5; lane 8,PD6; lane 9,PD9; lane 10,
PD11. IGn denotes isomaltodextrins having n units of
D-glucose.
fractionated on the BioGel P2 column were further
purified by descending paper chromatography and all of
the products were purified to single compounds (Fig.
2A).
The dextransucrase reaction products,remaining after
yeast fermentation,were fractionated into nine groups
by BioGel P2 column chromatography (data not
shown). The fractionated products (groups 1–7) were
further purified by preparative,descending paper chro-
matography. All reaction products except PD8 and
PD11 were highly purified (Fig. 2B).
3.4. Determination of the molecular weights of the purified
products by MALDI-TOF MS
The molecular weights of the CGTase and the dex-
transucrase reaction products were determined by mass
spectrometry (MALDI-TOF MS). The MS-spectral
results of the substrate,salicyl alcohol,and the major
reaction products from the CGTase and dextransucrase
reactions,PC1 and PD1,and PC2 and PD2,are given in
Table 3. The purified products fell into two groups: (a)
those with carbohydrate attached to salicyl alcohol
Table 3. The theoretical and MALDI-TOF MS determined molecular weights of salicyl alcohol and its synthesized salicyl alcohol analogues and the
synthesized carbohydrates,resulting from the reaction of CGTase and dextransucrase with salicyl alcohol
Compound
Mass number (m=z)
CGTase reaction products Dextransucrase reaction
products
Calculateda [MþNa]þ
124.1
Determinedb [MþNa]þ
124.2
309.2
Salicyl alcohol (SA)
––
PC1,PC2
––
PD1,PD2
Salicyl alcohol with one glucose 309.1
unit (G1–SA)
Salicyl alcohol with two glucose 471.2
units (G1–G1–SA)
471.3
633.2
PC41,PC4 2,PC5
––
PD3,PD4,PD5
PD6
Salicyl alcohol with three glucose 633.2
units (G1–G1–G1–SA)
Disaccharide (G1–G1)
Trisaccharide (G1–G1–G1)
365.1
527.2
365.2
527.2
PC10,PC11
PC12,PC13
PD9
––
aMass numbers were calculated from C ¼ 12.000,H ¼ 1.0078,O ¼ 15.9949,N ¼ 14.0031,and Na ¼ 22.9898; mass number of SA was from [M]þ.
b[MþNa]þ and [M]þ (for SA) were determined by MALDI-TOF MS.

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S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
Table 4. 1H NMR chemical shiftsa for salicyl alcohol and its analogues produced by the reaction of CGTase with cyclomaltohexaose and salicyl
alcohol
Positionb
Salicyl alcohol
PC1c
PC2c
PC41
PC42
PC5c
c
c
Salicyl alcohol (SA)
H-1
H-2
H-3
H-4
H-5
H-6
H-7
H-70
––
––
––
––
––
––
––
––
––
––
––
––
7.24
6.89
7.18
6.86
4.59
4.59
7.36
6.96
7.28
6.94
4.63
4.73
7.45
7.07
7.31
7.19
4.60
4.75
7.41
7.12
7.37
7.24
4.65
4.80
7.40
7.13
7.37
7.25
4.65
4.80
7.42
7.14
7.39
7.25
4.66
4.79
Glc(I)d
H-1
5.03
(3.4)
3.53
3.69
3.41
3.67
3.71
3.71
5.68
(3.1)
3.72
3.95
3.50
3.68
3.69
3.69
5.76
(3.4)
3.85
4.10
3.76
3.79
3.79
3.85
5.73
(3.1)
3.79
4.24
3.75
3.83
3.76
3.81
5.75
(3.4)
3.78
3.96
3.59
3.92
3.65
3.91
H-2
H-3
H-4
H-5
H-6
H-60
Glc(II)e
H-1
5.45
(3.4)
3.58
3.77
3.46
4.06
3.73
3.78
5.42
(3.4)
3.58
3.68
3.40
3.69
3.76
3.76
4.85
(3.1)
3.48
3.63
3.40
3.68
3.72
3.81
H-2
H-3
H-4
H-5
H-6
H-60
aChemical shifts and coupling constants (in parentheses) were measured at 500 MHz in D2O at 25 °C,using sodium 44, -dimethyl-4-silapentane-1-
sulfonate (DSS) as a standard.
bChemical structures and numbers refer to Figure 3.
cPC indicates a product from the reaction; PC1,product 1; PC2,product 2; and so forth.
dFor the glucose unit attached to salicyl alcohol.
eFor the glucose unit of the disaccharide attached to salicyl alcohol.
Table 5. 13C NMR chemical shiftsa for salicyl alcohol and its analogues produced by the reaction of CGTase with cyclomaltohexaose and salicyl
alcohol
Positionb
Salicyl alcohol
PC1c
PC2c
PC41
PC42
PC5c
c
c
Salicyl alcohol (SA)
C-1
C-2
C-3
C-4
C-5
C-6
C-7
154.3
126.7
130.0
121.0
129.8
115.8
60.1
154.9
124.0
131.4
121.0
130.5
116.2
66.0
154.2
129.7
130.1
123.3
130.0
115.4
59.9
154.1
129.7
130.2
120.2
130.0
115.2
59.7
154.2
129.8
130.1
123.3
130.0
115.3
59.7
154.0
129.7
130.1
123.4
130.0
115.5
59.8
Glc(I)d
C-1
C-2
C-3
C-4
C-5
C-6
98.3
71.8
73.6
69.9
72.3
60.7
97.0
71.8
73.5
69.8
73.1
60.8
97.0
70.2
79.6
70.2
73.3
60.7
96.6
71.5
73.8
77.1
71.4
60.8
96.8
71.6
74.0
70.0
71.7
65.9
Glc(II)e
C-1
C-2
C-3
C-4
C-5
C-6
99.4
72.1
72.9
69.7
72.1
60.4
100.2
72.1
73.2
69.7
73.0
60.6
98.2
71.8
73.6
69.9
72.3
60.9
aChemical shifts were measured at 125 MHz in D2O at 25 °C,using 14, -dioxane as a standard.
bChemical structures and numbers refer to Figure 3.
cPC indicates products; PC1,product 1; PC2,product 2; and so forth.
dFor the glucose unit attached to salicyl alcohol.
eFor the second glucose unit of the disaccharide attached to salicyl alcohol.

S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
1523
Table 6. 1H NMR chemical shiftsa for salicyl alcohol and its analogues produced from the reaction of dextransucrase with sucrose and salicyl alcohol
Positionb
PD1c
PD2c
PD3c
PD4c
PD5c
PD6c
Salicyl alcohol (SA)
H-1
H-2
H-3
H-4
H-5
H-6
H-7
H-70
––
––
––
––
––
––
––
––
––
––
––
––
7.36
6.96
7.28
6.94
4.63
4.73
7.45
7.07
7.31
7.19
4.60
4.75
7.38
6.96
7.28
6.93
4.66
4.75
7.38
6.95
7.29
6.93
4.69
4.75
7.42
7.14
7.39
7.25
4.66
4.79
7.39
6.99
7.30
6.94
4.70
4.76
Glc(I)d
H-1
5.03
(3.4)
3.53
3.69
3.41
3.67
3.71
3.71
5.68
(3.1)
3.72
3.95
3.50
3.68
3.69
3.69
5.04
(3.4)
3.62
3.82
3.64
3.69
3.70
5.01
(3.4)
3.55
3.67
3.52
3.84
3.48
3.92
5.75
(3.4)
3.78
3.96
3.59
3.92
3.65
3.91
5.05
(3.4)
3.57
3.68
3.56
3.81
3.54
3.92
H-2
H-3
H-4
H-5
H-6
H-60
Glc(II)e
H-1
5.31
(3.4)
3.52
3.72
3.41
3.97
3.75
3.78
4.90
(3.1)
3.52
3.72
3.41
3.69
3.76
3.83
4.85
(3.1)
3.48
3.63
3.40
3.68
3.72
3.81
5.12
(3.1)
3.65
3.83
3.48
3.72
3.76
3.82
H-2
H-3
H-4
H-5
H-6
H-60
Glc(III)f
H-1
5.09
(3.4)
3.53
3.75
3.44
3.89
3.76
3.82
H-2
H-3
H-4
H-5
H-6
H-60
aChemical shifts and coupling constants (in parentheses) were measured at 500 MHz in D2O at 25 °C with sodium 3-(trimethylsilyl)-1-propane
sulfonate as a standard.
bChemical structures and numbers refer to Figure 3.
cPD indicates products; PD1,product 1; PD2,product 2; and so forth.
dFor the glucose unit attached to salicyl alcohol.
eFor the second glucose unit of the disaccharide attached to salicyl alcohol.
fFor the third glucose unit of the trisaccharide attached to salicyl alcohol.
(PC1,PC2,PC4 1,PC4 2,and PC5 from the CGTase
reaction and PD1,PD2,PD3,PD4,PD5,and PD6 from
the dextransucrase reaction) and (b) those that only
contained carbohydrate and no salicyl alcohol (PC10,
PC11,PC12,and PC13 from the CGTase reaction and
PD9 from dextransucrase reaction) are also given in
Table 3.
3.5. Determination of the structures of the products by
NMR
The structures of the products were determined by 1D
1
1H,1D 13C, H/1H COSY,and 1H/13C HMQC NMR.
The major product from the dextransucrase reaction
(PD1) and the minor product from the CGTase reaction
(PC1) were identical and their 1H- and 13C-chemical
shifts are given in Tables 4–7. The major product from
the CGTase reaction (PC2) and the minor product from
the dextransucrase reaction (PD2) were identical and
their 1H- and 13C-chemical shifts are given in Tables 4–7.
For PD1 and PC1 there is only one anomeric proton
(H-1) and one anomeric carbon (C-1) for the bound
glucose unit. The H-1 chemical shift was at 5.03 ppm,
with a coupling constant of 3.4 Hz and the 13C-chemical
shift for C-1 was at 98.3 ppm (Tables 4–7). These results
PC1,PC2,PD1,and PD2 each had
[MþNa]þ ¼ 309,indicating a single
m=z
D-glucopyranosyl
unit was attached to salicyl alcohol (Table 3). PC41,
PC42,PC5,PD3,PD4,and PD5 had an
m=z
[MþNa]þ ¼ 471,indicating that two glucose units were
attached to salicyl alcohol to give salicyl alcohol ana-
logue isomers (Table 3). PD9,PC10,and PC11 each had
m=z 365 and were disaccharides,such as maltose or
isomaltose (Table 3). PC12 and PC13,each with m=z
527,were trisaccharides (Table 3).

1524
S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
Table 7. 13C NMR chemical shiftsa for salicyl alcohol and its analogues produced by the reaction of dextransucrase with sucrose and salicyl alcohol
Positionb
PD1c
PD2c
PD3c
PD4c
PD5c
PD6c
Salicyl alcohol (SA)
C-1
C-2
C-3
C-4
C-5
C-6
C-7
154.9
124.0
131.4
121.0
130.5
116.2
66.0
154.2
129.7
130.1
123.3
130.0
115.4
59.9
155.0
124.1
131.6
120.9
130.5
115.3
65.9
155.2
124.1
131.7
120.8
130.7
116.3
66.4
154.0
129.7
130.1
123.4
130.0
115.5
59.8
154.9
124.1
131.5
121.0
130.8
116.2
66.4
Glc(I)d
C-1
C-2
C-3
C-4
C-5
C-6
98.3
71.8
73.6
69.9
72.3
60.7
97.0
71.8
73.5
69.8
73.1
60.8
98.4
70.4
80.2
70.2
72.1
60.4
98.4
71.7
73.8
69.6
70.7
65.7
96.8
71.6
74.0
70.0
71.7
65.9
98.6
71.8
73.9
70.0
70.9
65.9
Glc(II)e
C-1
C-2
C-3
C-4
C-5
C-6
99.5
72.1
73.2
69.9
72.1
60.6
98.3
71.8
73.4
69.7
72.1
60.8
98.2
71.8
73.6
69.9
72.3
60.9
95.9
76.0
71.8
69.9
72.1
60.9
Glc(III)f
C-1
C-2
C-3
C-4
C-5
C-6
96.8
71.8
73.4
69.9
72.3
60.9
aChemical shifts were measured at 125 MHz in D2O at 25 °C,using 14, -dioxane as a standard.
bFor positions and chemical structures refer to Figure 3.
cPD indicates product; PD1,product 1; PD2,product 2; and so forth.
dFor the glucose unit attached to salicyl alcohol.
eFor the second glucose unit of the disaccharide attached to salicyl alcohol.
fFor the third glucose unit of the trisaccharide attached to salicyl alcohol.
indicate that one
D
-glucopyranose was attached to sal-
was a significant downfield shift of 0.4 ppm for the
proton on C-6 of salicyl alcohol and a significant
downfield 13C shift of 4.8 ppm for the C-2 of salicyl
alcohol. In our experiments,there was no significant
shift for C-1 of salicyl alcohol (Table 5),but there was a
significant downfield shift of about 0.33 ppm for the
proton at the C-6 of salicyl alcohol and a significant
downfield 13C shift of 3.0 ppm for the C-2 of salicyl
alcohol (Tables 4–7). The NMR results for PC2 and
icyl alcohol by an a-linkage.21;22 When the 13C NMR
spectra of PD1 and PC1 was compared with that of
salicyl alcohol,there was a significant change from 60.1
to 66.0 ppm for C-7 carbon of salicyl alcohol,with only
very small chemical shifts for the other salicyl alcohol
carbons (Tables 5 and 7). These results show that the D-
glucopyranose was attached to the hydroxyl group on
C-7 of salicyl alcohol by an a-linkage.13;23 PD1 and PC1,
thus,have
alcohol hydroxyl group of salicyl alcohol to give 7-a-
glucopyranosyl salicyl alcohol (see Fig. 3B for the
structure).
D
-glucopyranose attached to the primary
D
PD2,thus,indicate that one
attached to the C-1-phenolic hydroxyl group of salicyl
alcohol,giving 1- a- -glucopyranosyl salicyl alcohol.
PD3 and PD4 had spectra that were nearly identical
to the spectra of PC1 and PD1 (7-a- -glucopyranosyl
D-glucopyranose unit is
-
D
For the HMQC-spectra for PC2 and PD2 only one
anomeric proton (H-1) and carbon (C-1) peak appeared
D
salicyl alcohol),but both of these compounds also had
two glucose units instead of one. PD3 differed from PD1
in the C-3 position of the glucose unit attached to salicyl
alcohol. The 13C-chemical shift for C-3 changed from
73.6 to 80.2 ppm (Tables 5 and 7). The C-1 of the second
glucose unit had a 13C-chemical shift of 99.5 ppm and a
1H-chemical shift of 5.31 ppm,with a coupling constant
of 3.4 Hz (Tables 4–7),indicating that it was a-linked.
Thus,the second glucose unit was a-(1 ! 3) linked to the
1
for the bound glucose unit and the H-chemical shift
was at 5.03 ppm,with a coupling constant of 3.4 Hz and
the 13C-chemical shift was at 97.0 ppm (Tables 4–7).
These results indicate that one D-glucopyranose was
attached to salicyl alcohol by an a-linkage,similar to
PC1 and PD1.21;22 Previous studies24;25 showed that
when carbohydrate is bound to the phenolic hydroxyl of
salicyl alcohol,there was no significant deviation of the
chemical shift for the C-1 of salicyl alcohol,but there
first glucose unit,giving 7- a-D-nigerosyl salicyl alcohol.

S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
1525
Figure 3. The chemical structures of b-salicin and the reaction products, a-salicin and a-isosalicin analogues,produced by the reactions of CGTase
and B-742CB dextransucrase.
PD4 differed from PD3 by the C-6 carbon of the
glucose unit attached to salicyl alcohol,which had a
downfield shift of 5.0 ppm,from 60.7 to 65.7 ppm (Ta-
bles 4–7). PD4,thus,had the second glucose unit linked
a-(1 ! 6) to the first glucose unit,giving 7- a-isomaltosyl
salicyl alcohol.
pling constant of 3.4 Hz (Tables 6 and 7),indicating that
it was a-linked. The third glucose unit is,thus,linked a-
(1 ! 2) to the second glucose of the nigerosyl unit to give
2II-(a-
-glucopyranosyl)-7-a-nigerosyl salicyl alcohol.
D
PC41,PC4 ,and PC5 are products produced by the
2
CGTase reaction and PD5 is a product produced by the
dextransucrase reaction. All four products have two
glucose residues and the 1H- and 13C NMR spectra were
very similar to the spectrum of PC2.
The spectrum of PC41 differed from PC2 by having a
downfield 13C shift from 73.5 to 79.6 ppm for the C-3 of
the glucose attached to salicyl alcohol,and the C-1 of
PD6 has three glucose units and its 1H- and 13C-
chemical shifts were nearly identical to those of PD4,
with the exception of the C-2 position of the second
glucose unit in which there was a 13C shift of 72.1–
76.0 ppm,a downfield shift of 3.9 ppm. C-1 of the third
1
glucose unit had H-chemical shift of 5.09,with a cou-

1526
S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
the second glucose unit had a 13C-chemical shift of
99.4 ppm and a H-chemical shift of 5.45 ppm,with a
coupling constant of 3.4 Hz (Tables 4 and 5). These re-
sults indicate that the second glucose unit is attached to
the first glucose unit by an a-(1 ! 3) linkage,giving 1- a-
nigerosyl salicyl alcohol.
70.3 ppm26;28 to 76.2 ppm (Table 9) and from
70.1 ppm26;28 to 76.1 ppm (Table 9),respectively. P13
1
was,thus,identified as 4 I-a-
D
-glucopyranosyl kojibiose
or 2I-a-
D-glucopyranosyl maltose.
The H- (Table 8) and 13C NMR results (Table 9) of
PD9 were identical to those of previously reported
a-isomaltulose or palatinose.29;30 Therefore,PD9 was
1
The spectrum for PC42 differed from PC2 by having a
downfield 13C shift from 69.8 to 77.1 ppm (Tables 4 and
5) for the C-4 of the first glucose unit attached to salicyl
alcohol. The C-1 of the second glucose unit had a 13C-
identified as 6-a-
or a-isomaltulose.
D
-glucopyranosyl-a-D-fructofuranose
1
chemical shift of 98.2 ppm and a H-chemical shift of
5.42 ppm,with a coupling constant of 3.4 Hz (Tables 4
and 5),indicating that the second glucose unit was at-
tached to the first glucose unit by an a-(1 ! 4) linkage of
PC2 to give,1- a-maltosyl salicyl alcohol.
4. Discussion
Two kinds of reactions were used to synthesize salicin
analogues. In the first synthesis, B. macerans CGTase
The spectra of PC5 and PD5 were identical and dif-
fered from PC2 by having a downfield 13C shift from
60.8 to 65.9 ppm for the C-6 of the first glucose attached
to salicyl alcohol (Tables 5 and 7). The C-1 of the second
glucose unit had a 13C-chemical shift of 98.2 ppm and
1H-chemical shift of 4.85 ppm,with a coupling constant
catalyzed
a
transglycosylation reaction between
cyclomaltohexaose and salicyl alcohol to give two
products,1- a-maltohexaosyl salicyl alcohol (major
product) and 7-a-maltohexaosyl salicyl alcohol (minor
product). The enzyme also catalyzed transglycosylation/
disproportionation reactions with the maltohexaosyl
unit attached to salicyl alcohol. To reduce the number of
of 3.1 Hz (Tables 4–7). PC5 and PD5,thus,had a
D-
glucose unit attached a-(1 ! 6) to the glucose unit of
PC2 structure,to give 1- a-isomaltosyl salicyl alcohol
(Tables 4–7).
salicyl products and give a single D-glucose unit
attached to salicyl alcohol,the reaction mixture was
reacted with alpha amylase and glucoamylase,and then
the free sugars were removed by fermentation with
immobilized S. cerevisiae. The major product has a
structure analogous to natural salicin,but with the
3.6. NMR determination of the structures of carbohy-
drates (PC10–PC13, and PD9) not attached to salicyl
alcohol
D
-glucopyranosyl moiety a-linked to the phenolic
hydroxyl group of salicyl alcohol. The minor product
has the -glucopyranosyl moiety a-linked to the pri-
mary alcohol hydroxyl group of salicyl alcohol. Natu-
rally occurring salicin has the -glucopyranosyl b-linked
The carbohydrates,PC10–PC13,were produced by
CGTase reaction and PD9 was produced by dextran-
sucrase reaction during preparation of salicyl alcohol
analogues. These carbohydrates products were not at-
tached to salicyl alcohol. The 13C-chemical shifts of
PC10 and PC11 (Table 9) were identical to those pre-
viously reported for a,b-trehalose26 and isomaltose,27
respectively,and thus were identified as being a,b-tre-
halose and isomaltose.
D
D
to the phenolic group of salicyl alcohol. We have given
common names to the newly synthesized analogues:
a-salicin for the major analogue and a-isosalicin for the
minor analogue,and b-salicin for the naturally occur-
ring salicin (see Fig. 3A–C for the structures).
CGTase also synthesized three additional minor
products. Their structures were determined to be: 1-a-
nigerosyl salicyl alcohol,named a-nigerosalicin (Fig.
3D); 1-a-maltosyl salicyl alcohol,named a-maltosalicin
(Fig. 3E); and 1-a-isomaltosyl salicyl alcohol,named
a-isomaltosalicin (Fig. 3F).
In the first reaction with CGTase,before reaction
with alpha amylase and glucoamylase,three products,
resulting from the disproportionation reactions (PC3,
PC6,and PC8) are salicin analogues with maltose,
maltotriose,and maltotetraose attached to C-7 position
of salicyl alcohol. PPA and GA hydrolyzed the carbo-
hydrates on these analogues,leaving a-isosalicin.
The formation of nigerose and isomaltose attached to
C-1 of salicyl alcohol to give a-nigerosalicin and
a-isomaltosalicin were most probably formed by con-
The 13C shifts of PC12 were almost identical to those
of maltose,26;28 with the exception of the C-3 of glucose
(II) unit and a newly bound glucose (III) (Table 9). The
newly bound glucose (III) unit has a 13C-chemical shift
of 99.5 ppm for C-1 and a 1H-chemical shift of 5.33 ppm,
with 3.4 Hz coupling constant (Tables 8 and 9). The 13C-
chemical shift of C-3 position of glucose (II) was chan-
ged from 73.8 ppm26;28 to 79.7 ppm (Table 9). Thus,
PC12 was identified as 3II-a-
D-glucopyranosyl maltose.
The 13C shifts of P13 (Table 9) were very similar to
that of the kojibiose,26;28 with the exception of the C-4-
position of the reducing end glucose unit and a newly
bound glucose (II) (Table 9). The newly bound glucose
(II) unit had a C-1 13C-chemical shift of 99.5 ppm and a
1H-chemical shift of 5.44 ppm,with a coupling constant
of 3.0 Hz (Tables 8 and 9). The 13C-chemical shifts of the
C-4 position of the first glucose unit was changed from
densation reactions of
D
-glucose with a-salicin,cata-
lyzed by glucoamylase.31;32 Likewise,the formation of

S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
1527
Table 8. 1H NMR chemical shiftsa for carbohydrates produced from CGTase and dextransucrase reactions
Position
PC10b
PC11b
PC12b
PC13b
PD9b
b-D
-Glc(I)
a-
D
-Glc(I)
a-
D
-Glc(I)
a-D
-Glc(I)
a-D-Glc(I)
H-1
4.63
(7.9)
3.39
3.51
3.41
3.45
3.69
3.85
5.21
(3.4)
3.50
3.69
3.39
3.70
3.73
3.93
5.20
(3.4)
3.56
3.96
3.64
3.92
3.76
3.83
5.43
(3.1)
3.65
4.09
3.69
3.95
3.79
4.95
(<3.0)
3.55
3.73
3.39
3.69
3.75
3.85
H-2
H-3
H-4
H-5
H-6
H-60
a-D
-Glc(II)
b-
D
-Glc(I)
b-
D
-Glc(I)
b-D
-Glc(I)
b-D-Fru(II)
H-1
H-10
H-2
H-3
H-4
H-5
H-6
H-60
5.22
(3.4)
3.57
3.73
3.43
3.92
3.74
3.81
4.65
(7.9)
3.22
3.44
3.39
3.70
3.73
3.93
4.63
(7.9)
3.25
3.75
3.64
3.84
3.76
3.92
4.79
(7.9)
3.41
3.86
3.68
3.58
3.75
3.89
3.51
3.63
––
4.10
4.18
3.95
3.68
3.86
D
-Glc(II)
D
-Glc(II)
D
-Glc(II)
H-1
4.92
5.38
5.44
(3.1)
3.52
3.69
3.39
3.66
3.73
3.81
(3.4)
3.66
3.83
3.64
3.72
3.75
3.82
(<3.0)
3.54
3.65
3.40
3.69
3.77
3.86
H-2
H-3
H-4
H-5
H-6
H-60
D
-Glc(III)
D
-Glc(III)-a(I)c
H-1
5.33
5.09
(3.4)
3.54
3.72
3.40
3.99
3.77
3.89
(3.1)
3.53
3.77
3.43
3.89
3.73
3.83
H-2
H-3
H-4
H-5
H-6
H-60
D
-Glc(III)-b(I)c
H-1
5.39
(3.1)
3.52
3.73
3.42
3.98
3.78
3.85
H-2
H-3
H-4
H-5
H-6
H-60
aChemical shifts and coupling constants (in parentheses,Hz) were measured at 500 MHz in D 2O at 25 °C,using sodium 4,4-dimethyl-4-silapentane-1-
sulfonate (DSS) as a standard.
bPC ¼ products from CGTase reaction and PD ¼ product from dextransucrase reaction.
ca-
-Glucose unit connected to a- and b-anomer of reducing-end glucose,respectively.
D
a,b-trehalose,isomaltose,3 II-a-
ose,and 2 I-a-
-glucopyranosyl maltose saccharides
D
-glucopyranosyl malt-
were also most probably the results of glucoamylase
reversion reactions that give condensed saccharide
D

1528
S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
Table 9. 13C NMR chemical shiftsa for the carbohydrates produced from the reactions of CGTase and dextransucrase
Position
PC10b
PC11b
PC12b
PC13b
PD9b
b-D
-Glc(I)
a-
D
-Glc(I)
a-
D
-Glc(I)
a-
D
-Glc(I)
a-D-Glc(I)
C-1
C-2
C-3
C-4
C-5
C-6
103.3
73.5
75.8
69.7
76.5
61.0
92.6
71.8
73.4
69.9
70.4
66.0
92.3
75.0
73.5
77.2
70.3
60.9
89.5
76.0
71.8
76.2
70.1
60.8
98.6
71.8
73.5
69.9
72.3
61.0
a-D
-Glc(II)
b-
D
-Glc(I)
b-
D
-Glc(I)
b-
D
-Glc(I)
b-
D
-Fru(II)
C-1
C-2
C-3
C-4
C-5
C-6
100.6
71.8
73.2
69.7
73.1
60.8
96.4
74.7
76.3
69.8
74.4
66.0
96.2
74.4
76.5
77.2
75.1
61.1
96.5
78.8
75.3
76.1
74.7
61.1
63.0
102.2
75.7
74.9
79.4
68.3
D
-Glc(II)
D
-Glc(II)
D
-Glc(II)
C-1
C-2
C-3
C-4
C-5
C-6
98.3
72.2
73.4
69.9
71.8
60.8
100.4
70.6
79.7
70.1
72.9
60.9
99.3/99.6
72.1
73.0
69.6
73.0
60.8
D
-Glc(III)
D
-Glc(III)-a(I)c
C-1
C-2
C-3
C-4
C-5
C-6
99.5
72.1
73.2
69.8
72.2
60.7
96.5
72.1
73.2
69.6
71.9
60.6
D
-Glc(III)-b(I)c
C-1
C-2
C-3
C-4
C-5
C-6
98.0
72.2
73.1
69.6
71.8
60.6
aChemical shifts were measured at 125 MHz in D2O at 25 °C,using 14, -dioxane as a standard.
bPC refers to products produced by CGTase and PD refers to the product produced by dextransucrase.
ca-
-Glucose unit connected to a- and b-anomer of reducing-end glucose,respectively.
D
products from the relatively high amounts of
D
-glucose
units are transferred to give isomaltodextrinyl chains
attached to salicyl alcohol. These chains were reduced to
a single glucose unit attached to the primary alcohol
hydroxyl of salicyl alcohol by the action of dextranase
and glucoamylase,which gave a-isosalicin as the major
product (see Fig. 3B).
that is formed by the action of glucoamylase on the
maltodextrin chains attached to salicyl alcohol and the
maltodextrins produced by the CGTase disproportion-
ation reactions.
In the second synthesis, L. mesenteroides B-742CB
dextransucrase catalyzed transglycosylation reactions in
which D-glucopyranose is transferred from sucrose to
B-742 Dextransucrase also formed three additional
minor a-isosalicin analogues by the acceptor reaction
the primary alcohol hydroxyl group to give a-isosalicin,
as the major product,in contrast to CGTase,which gave
a-salicin as the major product. This glucose unit also
acts as an acceptor and a sequential series of glucose
transfer of
D
-glucopyranose from sucrose to the
-
glucopyranosyl moiety of a-isosalicin to form a-(1 !D3)
and a-(1 ! 6) glycosidic linkages to give a-nigeroiso-
salicin (Fig. 3G) and a-isomaltoisosalicin (Fig. 3H),

S.-H. Yoon et al. / Carbohydrate Research 339 (2004) 1517–1529
1529
5. Arakawa,H.; Funayama,M.; Yamamoto,R.; Nishino,T.
respectively,and the transfer of D-glucopyranose from
sucrose to the nonreducing end of the isomaltose unit of
Jpn. Kokai Tokkyo Koho 06284897, 1994.
6. Shimoide,A.; Watanabe,M.; Donho,M.
Tokkyo Koho 08173183, 1996.
Jpn. Kokai
a-isomaltoisosalicin to form an a-(1 ! 2) glycosidic
linkage to give 2II-(a-
-glucopyranosyl)-a-isomaltoiso-
D
7. Okano,Y.; Masaki,H.; Ogawa,A.
Koho 11116426, 1999.
Jpn. Kokai Tokkyo
salicin (Fig. 3I). In addition to these a-isosalicin ana-
logues,B-742CB dextransucrase also formed minor
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amounts of a-salicin and transferred
from sucrose to form an a-(1 ! 6) linkage to the
D-glucopyranose
D
-
glucopyranosyl moiety of a-salicin to give 1-a-isomal-
tosyl salicyl alcohol or a-isomaltosalicin (Fig. 3F).
It is known that the major active metabolite from b-
salicin in serum is salicylic acid.3;7 b-Salicin is absorbed
only to a small extent in the small intestine after oral
administration.3 It is hydrolyzed to salicyl alcohol,
which is readily absorbed from the small intestine by the
action of b-glycosidases secreted by the intestinal mi-
croflora3;33–35 or by b-galactosidase in the mucous
membrane of the small intestine.35 The absorbed salicyl
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Although it has not yet been demonstrated,it might
be expected that a-salicin and its analogues, a-isosalicin,
a-nigerosalicin, a-maltosalicin,and a-isomaltosalicin
that have been synthesized in this study,would be
readily hydrolyzed by the a-glucosidases secreted by the
brush border cells that line the small intestinal wall.38
This would then facilitate the absorption of salicyl
alcohol and would make it a mild but effective antipy-
retic and analgesic prodrug with none of the gastric in-
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In conclusion,two types of salicin prodrugs have been
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CGTase with cyclomaltohexaose and salicyl alcohol
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L. mesenteroides B-742CB dextransucrase with sucrose
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uct.
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