Enzymatic synthesis of galactosylated 1D-chiro-inositol and 1D-pinitol derivatives using the β-galactosidase from Bacillus circulans

Article abstract of DOI:10.1055/s-2001-11388

The β-D-galactosidase from Bacillus circulans regioselectively galactosylates 1D-chiro-inositol and 1D-pinitol (1D-3-O-methyl-chiro-inositol) to give mono- and digalactosylated inositols in yields of up to 45%. The enzyme does not galactosylate 1L-chiro-i

Full text of DOI:10.1055/s-2001-11388

LETTER
329
Enzymatic Synthesis of Galactosylated 1D-Chiro-Inositol and 1D-Pinitol
Derivatives Using the -Galactosidase from Bacillus circulans
Joanne Hart,a Andrew Falshaw,a Erzsébet Farkas,b Joachim Thiemb*
aCarbohydrate Chemistry Team, Industrial Research Ltd., PO Box 31-310, Lower Hutt, New Zealand
bInstitut für Organische Chemie, Universität Hamburg, Martin-Luther-Platz 6, 20146 Hamburg, Germany
Fax +49(0)40428384325; E-mail: thiem@chemie.uni-hamburg.de
Received 15 November 2000
Dedicated to Professor András Lipták on his 65th birthday
Glycoside hydrolases (glycosidases) are responsible for
the cleavage of oligosaccharides into constituent
monosaccharide units in vivo. In vitro, it is possible to
drive the reaction in the reverse direction, i.e. to form in-
Abstract: The -D-galactosidase from Bacillus circulans regiose-
lectively galactosylates 1D-chiro-inositol and 1D-pinitol (1D-3-O-
methyl-chiro-inositol) to give mono- and digalactosylated inositols
in yields of up to 45%. The enzyme does not galactosylate 1L-chiro-
inositol or 1L-quebrachitol (1L-2-O-methyl-chiro-inositol). This is terglycosidic linkages from monomeric sugar units. By
the first report of enzymatic glycosylation of chiro-inositol and its
derivatives.
employing glycosidases in this manner it is possible to cir-
cumvent classical organic synthesis methods and tradi-
Key words: carbohydrates, enzymatic glycosylations, Bacillus
circulans, -D-galactosidase, inositol oligosaccharides
tional labour-intensive extraction procedures in the
production of oligosaccharides.
The -D-galactosidase from Bacillus circulans (EC
3.2.1.23) has been reported as being an excellent, high
yielding catalyst for the formation of di- and trisaccha-
rides with Gal 1 4 linkages.9-13 This enzyme catalyzes
the transfer of Gal from p-nitrophenyl -D-galactopyrano-
side (Gal OpNP) or lactose to Glc, GlcNAc, Gal and Gal-
NAc, predominantly to the OH 4 position.13
Oligosaccharides containing inositol sugars such as 1D-
chiro-inositol (DCI) (1) and 1D-pinitol (2) are the focus of
current interest for their putative role as secondary mes-
sengers of insulin and their associated implications in the
etiology of Type 2 diabetes.1-5 Although the detailed
structures of these secondary messengers remain elusive,
they are believed to contain an amino sugar such as galac-
tosamine linked to a phosphorylated inositol sugar such as
1, 2 or myo-inositol.6 In addition, DCI and pinitol-based
oligosaccharides have been isolated in high levels from
the seeds of leguminous plants and are thought to provide
protection for the organelles, enzymes, proteins etc. dur-
ing dessiccation.7,8
Herein we describe the enzymatic synthesis and charac-
terisation of the products from the enzyme catalyzed reac-
tions of Gal OpNP with 1 and 2 employing the -D-
galactosidase from B. circulans (Figures 1 and 2).14 To
our knowledge, this is the first report describing an enzy-
matic synthesis to prepare inositol containing oligosac-
charides.
OH
OH
6'
O
HO
OH
O
3'
β-Galactosidase
from Bacillus circulans
1'
OH
1
1
OH
OH
OH
OH
HO
HO
OH
2
4
HO
2
OH
OH
4
OH
OH
1
3
6''
3''
O
GalβOpNP
HOpNP
HO
O
OH
1''
6'
OH
O
HO
O
3'
1'
OH
1
OH
OH
HO
5
OH
2
OH
4
Figure 1 Galactosylation Reaction with DCI as Acceptor
Synlett 2001 No. 3, 329–332 ISSN 0936-5214 © Thieme Stuttgart · New York

330
J. Hart et al.
LETTER
OH
OH
6'
O
HO
OH
O
3'
β-Galactosidase
from Bacillus circulans
1'
OH
1
1
OH
OH
OH
OMe
HO
HO
OH
2
4
HO
OMe
2
OH
4
OH
OH
2
4
6''
O
GalβOpNP
HOpNP
HO
O
OH
3''
1''
6'
3'
OH
O
HO
O
1'
OH
1
OH
HO
6
OMe
OH
2
OH
4
Figure 2 Galactosylation Reaction with Pinitol as Acceptor
Galactosylations using Gal OpNP14 as the donor were D-galactopyranosyl)-4-O-methyl-chiro-inositol
(4),16
performed in sodium acetate buffer (pH 5.0) at 50 °C with were obtained in 43% and 45% respective yields and the
4.4 eq. of the acceptor 1.15 Galactosylations using 4.4 eq. digalactosylated products, 1D-1-O-( -D-galactopyrano-
of 2 were performed at 37 °C in sodium phosphate buffer syl-(1 4)-O- -D-galactopyranosyl)-chiro-inositol
(5)
(pH 7.0) with acetonitrile as a co-solvent.15 These are op- and 1D-1-O-( -D-galactopyranosyl-(1 4)-O- -D-galac-
timized reaction conditions. Previous results obtained us- topyranosyl)-4-O-methyl-chiro-inositol (6),16 were ob-
ing acetonitrile as a co-solvent in glycosylation reactions tained in 9% and 7% respective yields (Table). The
with glycoside acceptors (eg. OAll and SPh) improved the products (3-6) were fully characterized by 1D and 2D
acceptor solubility and the overall reaction yields.13 The NMR spectroscopy (HH-COSY, HMQC, HMBC, TOC-
reactions were worked up after 2 to 3 days and the prod- SY, HMQC-TOCSY and ROESY experiments) and ES-
ucts isolated and purified by column chromatography MS.17-20 Key correlations in the NMR spectra were ob-
(Biogel P2). The monogalactosylated products, 1D-1-O- served across the glycosidic bonds, clearly defining the
( -D-galactopyranosyl)-chiro-inositol (3) and 1D-1-O-( - inositol-Gal and Gal-Gal linkages. For example, in com-
Table Summary of Enzymatic Galactosylation Results with the -Galactosidase from B. circulans
nr = no reaction
Synlett 2001, No. 3, 329–332 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Enzymatic Synthesis of Galactosylated 1D-Chiro-Inositol
331
Galactosylation
OH
OH
OH
OH
6
O
1
OH
OMe
OH
OH
HO
HO
HO
OH
OH
OH
3
2
OH
1
4
OH
OH
2
Gal
1
OH
OH
HO
HO
OH
RO
OH
4
HO
RO
OH
2
1
1
6
OH
HO
1
6
OR
OH
2
OH
4
2
4
OH
OH
OH
C
B
A
7 R:H
8 R:Me
Figure 3 Structure Comparison of Selected Inositol Sugars with Galactose
pound 3, HMBC correlations were observed from the C
1’ of Gal to H 1 of DCI and from C 1 of DCI to the -
linked H 1’ proton of Gal.
References and Notes
(1) Fonteles, M.C.; Huang, L.C.; Larner, J. Diabetologia 1996,
39, 731-734.
(2) Kennington, A.S.; Hill, C.R.; Craig, J.; Bogardus, C.; Raz, I.;
Ortmeyer, H.K.; Hansen, B.C.; Romero, G.; Larner, J. N.
Engl. J. Med. 1990, 323, 373-378.
(3) Garegg, P.J.; Konradsson, P.; Oscarson, S.; Ruda, K.
Tetrahedron 1997, 53, 17727-17734.
In contrast to the reactions observed for the D-enanti-
omers, no reaction was observed when either 1L-chiro-
inositol (LCI) (7) or 1L-quebrachitol (1L-2-O-methyl-chi-
ro-inositol) (8) were employed as acceptors (Table). From
these results it appears that DCI (1) and 1D-pinitol (2) are
able to mimic the hydroxyl configuration of the natural
acceptor, Gal, resulting in galactosylation at the axial
OH 1 or OH 6 groups of 1 and the axial OH 6 group of
216 which is remote from that of the methyl group (Figure
3). This is consistent with the predominant OH galac-
tosylation pattern observed for the -galactosidase from
B. circulans.13 1L-chiro-inositol (7), and its 2-O-methyl
ether quebrachitol (8), are not substrates for the enzyme,
despite containing arrangements of OH groups (A, B, C
Figure 3) which mimic Glc and Gal. The presence of axial
OH groups in 7 and 8 (denoted as circled OH’s in Figure
3), appears to prevent their binding as acceptors in the en-
zyme catalyzed reaction.
(4) Asplin, A.; Galasko, G.; Larner, J. Proc. Natl. Acad. Sci. 1993,
90, 5924-5928.
(5) Larner, J.; Huang, L.C.; Schwartz, C.F.W.; Oswald, A.S.;
Shen, T.-Y.; Kinter, M.; Tang, G.; Zeller, K. Biochem.
Biophys. Res. Commun. 1988, 151, 1416-1426.
(6) Field, M.C. Glycobiology 1997, 7, 161-168.
(7) Szczecinski, P.; Gryff-Keller, A.; Horbowicz, M.; Lahuta,
L.B. J. Agric. Food Chem. 2000, 48, 2717-2720.
(8) Ogawa, K; Watanabe, T.; Ikeda, Y.; Kondo, S. Carbohydr.
Res. 1997, 302, 219-222.
(9) Vic, G.; Hastings, J.J.; Howarth, O.W.; Crout, D.H.G.
Tetrahedron: Asymmetry 1996, 7, 709-720.
(10) Singh, S.; Scigelova, M.; Vic, G.; Crout, D.H.G. J. Chem.
Soc., Perkin Trans. 1 1996, 1921-1926.
(11) Usui, T.; Kubota, S.; Ohi, H. Carbohydr. Res. 1996, 285, 29-
39.
(12) Kimura, T.; Takayama, S.; Huang, H.; Wong, C.-W. Angew.
Chem., Int. Ed. Engl. 1996, 35, 2348-2350.
Acknowledgement
(13) Farkas, E.; Thiem, J. Eur. J. Org. Chem. 1999, 11, 3073-3078.
(14) -Galactosidase from Bacillus circulans (EC 3.2.1.23) was
purchased from “Biolacta” Daiwa Kasei Co., Ltd., Osaka,
Japan, and p-nitrophenyl -D-galactopyranoside from Sigma,
Germany.
(15) Experimental procedure: Compounds 3 and 5: Gal pNP (20
mg, 0.066 mmol) and 1D-chiro-inositol (53 mg, 0.29 mmol,
4.4 eq.) were incubated in 1 mL sodium acetate buffer (pH 5.0,
50 mM) with 0.5 U -galactosidase from Bacillus circulans at
50 °C. After 5 h the donor was not completely hydrolyzed so
the reaction was further incubated at 37 °C. After 2 days the
enzyme was denaturated by heating at 90 °C for 10 min. The
solution was extracted with ethyl acetate to remove p-nitro-
Support of this study by the European Comission (FAIR CT 97-
3142, programme NOFA) and the BMBF (NZ 951007) is gratefully
acknowledged. The New Zealand Foundation for Research, Science
and Technology provided Public Good Science Funding for this re-
search through Industrial Research Ltd. as a CEO NSOF grant (con-
tract number CO8X0004-8).
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LETTER
phenol and lyophilized. The residue was applied to a Biogel
P2 column and eluted with water to give compound 3 (9 mg,
43%) and compound 5 (2 mg, 9%);
H 4); 13C NMR (125 MHz, D2O): = 105.4 (C 1’), 104.7
(C 1’’), 82.2 (C 1), 77.6 (C 4’), 75.7 (C 5’), 74.9 (C 5’’),
73.4 (C 3’), 73.3 (C 3), 73.2 (C 3’’), 73.2 (C 4), 72.0 (C
2’), 71.9 (C 2’’), 71.1 (C 6), 70.8 (C 5), 70.8 (C 2), 69.1
(C 4’’), 61.5 (C 6’’), 61.0 (C 6’); HR ES-MS: Calcd. for
C18H32O16ONa [M+Na]+ 527.1583, found 527.1578.
Compounds 4 and 6: Gal pNP (17 mg, 0.056 mmol) and 1D-
pinitol (52 mg, 0.27 mmol, 4.8 eq.) were incubated in a 1:1
mixture of sodium phosphate buffer (0.4 mL, pH 7.0, 0.1 M)
and acetonitrile with 0.5 U -galactosidase from Bacillus
circulans at 37 °C. After 3 days the enzyme was denaturated
by heating at 90 °C for 10 min. The solution was extracted
with ethyl acetate to remove p-nitrophenol and lyophilized.
The residue was applied to a Biogel P2 column and eluted
with water to give compound 4 (9 mg, 45%) and compound 6
(2 mg, 7%).
(19) 1D-1-O-( -D-galactopyranosyl)-4-O-methyl-chiro-inositol
(4): [ ]21D+38 (c 0.9, H2O), 1H NMR (500 MHz, D2O):
= 4.47 (d, 1 H, J1’,2’ = 7.8 Hz, H 1’), 4.27 (dd, 1 H, J6,1 and
J
6,5 = 3.7 Hz, H 6), 4.01 (dd, 1 H, J1,2 = 3.7 Hz, H 1), 3.88 (d,
1 H, J4’,3’ = 3.4 Hz, H 4’), 3.84 (dd, 1 H, J5,4 = 9.5 Hz, H 5),
3.77 (dd, 1 H, J2,3 = 9.5 Hz, H 2), 3.74 (dd, 1 H, J6’,5’ = 4.4 Hz,
J6’,6’ = 11.7 Hz, H 6’) 3.70 (dd, 1 H, J6’,5’ = 7.8 Hz, H 6’),
3.68 (dd, 1 H, J3,4 = 9.5 Hz, H 3), 3.65 (dd, 1 H, H 5’), 3.62
(dd, 1 H, J3’,2’ = 10.0 Hz, H 3’), 3.57 (s, 3 H, OCH3), 3.51 (dd,
1 H, H 2’), 3.31 (dd, 1 H, H 4); 13C NMR (125 MHz, D2O):
= 105.3 (C 1’), 83.1 (C 4), 81.6 (C 1), 75.6 (C 5’), 72.9
(C 3’), 72.7 (C 3), 71.6 (C 2’), 71.1 (C 6), 70.8 (C 2),
70.2 (C 5), 68.9 (C 4’), 61.4 (C 6’), 60.1 (OCH3); HR ES-
MS: Calcd. for C13H24O11Na [M+Na]+ 379.1216, found
379.1211.
(16) Note on numbering: Pinitol is formally described as 1D-3-O-
methyl-chiro-inositol under the IUPAC rules applicable to the
naming of inositols. Galactosylation occurs at the 6 position
of pinitol, giving a molecule for which the correct numbering
places the galactosyl substituent on the O-1-position of the
chiro-inositol molecule, renumbering the methyl at the 4
position i.e. the correct numbering is 1-O-galactosyl-4-O-
methyl- and the methyl group has not changed position.
(17) 1D-1-O-( -D-galactopyranosyl)-chiro-inositol (3): [ ]21
(20) 1D-1-O-( -D-galactopyranosyl-(1 4)-O- -D-
galactopyranosyl)-4-O-methyl-chiro-inositol (6): 1H NMR
(500 MHz, D2O): = 4.57 (d, 1 H, J1’’,2’’ = 7.7 Hz, H 1’’),
4.52 (d, 1 H, J1’,2’ = 7.8 Hz, H 1’), 4.22 (dd, 1 H, J6,1 and
D
+39 (c 0.9, H2O); 1H NMR (500 MHz, D2O): = 4.48 (d, 1
H, J1’2’ = 7.8 Hz, H 1’), 4.27 (dd, 1 H, J6,1 and J6,5 = 3.3 Hz,
H 6), 4.02 (dd, 1 H, J1,2 = 3.3 Hz, H 1), 3.88 (d, 1 H,
J
4’,3’ = 3.4 Hz, H 4’), 3.79 (dd, 1 H, J5,4 = 10.0 Hz, H 5), 3.77
J
J
6,5 = 3.7 Hz, H 6), 4.14 (d, J4’,3’ = 3.3 Hz, H 4’), 4.02 (dd,
1,2 = 3.7 Hz, H 1), 3.88 (d, J4’’,3’’ = 2.7 Hz, H 4’’), 3.84
(dd, 1 H, J2,3 = 10.0 Hz, H 2), 3.72 (m, 2 H, H6’/H6’), 3.65
(dd, 1 H, J5’6’/6’ = 4.2, 7.8 Hz, H 5’), 3.62 (dd, 1 H, J3’2’ = 9.5
Hz, H 3’), 3.60 (dd, 1 H, J3,4 = 10.0 Hz, H 3), 3.53 (dd, 1 H,
H 4), 3.52 (dd, 1 H, H 2’); 13C NMR (125 MHz, D2O):
= 105.3 (C 1’), 81.9 (C 1), 75.6 (C 5’), 73.3 (C 3), 73.2
(C 4), 73.0 (C 3’), 71.6 (C 2’), 71.1 (C 6), 70.9 (C 5),
70.8 (C 2), 68.9 (C 4’), 61.4 (C 6’); HR ES-MS: Calcd. for
C12H23O11 [M+H]+ 343.1240, found 343.1235.
(obsc., 1 H, H 5), 3.82 (dd, 1 H, J2,3 = 9.6 Hz, H 2), 3.82
(obsc., 1 H, H 6’), 3.76 (obsc., 2 H, H 6’’/6’’), 3.75 (obsc.,
1 H, H 6’), 3.74 (obsc., 1 H, H 3’), 3.72 (obsc. 1 H, H 5’),
3.68 (dd, 1 H, J3,4 = 9.6 Hz, H 3), 3.66 (obsc., 1 H, H 5’’),
3.66 (s, 3 H, OCH3), 3.64 (obsc., 1 H, H 3’’), 3.60 (obsc., 1
H, H 2’), 3.57 (obsc., 1 H, H 2’’), 3.31(dd, 1 H, J4,5 = 9.6 Hz,
H 4); 13C NMR (125 MHz, D2O): = 105.4 (C 1’), 104.7
(C 1’’), 83.1 (C 4), 81.9 (C 1), 77.5 (C 4’), 75.6 (C 5’’),
74.9 (C 5’), 73.4 (C 3’), 73.2 (C 3’’), 72.6 (C 3), 72.0 (C
2’), 71.9 (C 2’’), 71.1 (C 6), 70.9 (C 2), 70.2 (C 5), 69.1
(C 4’’), 61.5 (C 6’’), 61.0 (C 6’), 60.1 (OCH3); HR ES-MS:
Calcd. for C19H34O16Na [M+Na]+ 541.1739, found 541.1750.
(18) 1D-1-O-( -D-galactopyranosyl-(1 4)-O- -D-
galactopyranosyl)-chiro-inositol (5): 1H NMR (500 MHz,
D2O): = 4.57 (d, 1 H, J1’’,2’’ = 7.8 Hz, H 1’’), 4.53 (d, 1 H,
J
1’,2’ = 8.0 Hz, H 1’), 4.23 (dd, 1 H, J6,5 and J6,1 = 3.5 Hz, H
6), 4.15 (d, 1 H, J4’,3’ = 3.4 Hz, H 4’), 4.04 (dd, 1 H, J1,2 = 3.5
Hz, H 1), 3.88 (d, 1 H, J4’’,3’’ = 3.4 Hz, H 4’’), 3.80 (obsc., 1
H, H 2), 3.79 (m, 2 H, H 6’’/6’’), 3.77 (obsc., 1 H, H 5’),
3.76 (obsc., 1 H, H 5), 3.74 (obsc., 1 H, H 3’), 3.72 (m, 2 H,
H 6’/6’), 3.64 (obsc., 1 H, H 3’’), 3.64 (obsc., 1 H, H 5’’),
3.61 (obsc., 1 H, H 2’), 3.60 (dd, 1 H, J3,2 and J3,4 = 9.7 Hz,
H 3), 3.57 (obsc., 1 H, H 2’’), 3.54 (dd, 1 H, J4,5 = 9.7 Hz,
Article Identifier:
1437-2096,E;2001,0,03,0329,0332,ftx,en;G23600ST.pdf
Synlett 2001, No. 3, 329–332 ISSN 0936-5214 © Thieme Stuttgart · New York