Enzyme-catalysed synthesis of galactosylated 1D- and 1L-chiro-inositol, 1D-pinitol, myo-inositol and selected derivatives using the β-galactosidase from the thermophile Thermoanaerobacter sp. strain TP6-B1

Carbohydrate

RESEARCH

Carbohydrate Research 339 (2004) 1857–1871

Enzyme-catalysed synthesis of galactosylated 1D- and

-chiro-inositol, 1D-pinitol, myo-inositol and selected derivatives

1

L

using the b-galactosidase from the thermophile

Thermoanaerobacter sp. strain TP6-B1

b

a

b

Joanne B. Hart,^a,Lars Kroger, Andrew Falshaw, Ruth Falshaw, Erzsebet Farkas,

*

€

ꢀ

Joachim Thiem^b,*and Anna L. Win^a

^aCarbohydrate Chemistry Team, Industrial Research Ltd, PO Box 31-310, Lower Hutt, New Zealand

^bInstitut f €ur Organische Chemie, Universit€at Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany

Received 27 April 2004; accepted 8 May 2004

Available online 25 June 2004

Abstract—The products from the enzymatic b-

chiro-inositol, 1 -3,4-di-O-methyl-chiro-inositol, 1

have been obtained using the b-galactosidase isolated from an anaerobic extreme thermophile, Thermoanaerobacter sp. strain TP6-

B1 and p-nitrophenyl b- -galactopyranoside as the donor. Analysis of the products from these reactions reveals information about

D

-galactopyranosylation of 1

D-chiro-inositol, 1D-pinitol, 1D-3-O-allyl-4-O-methyl-

D

L

-chiro-inositol and myo-inositol in combined yields ranging from 46% to 64%

D

the acceptor preferences of the enzyme.

Keywords: Glycosidases; Thermostable enzymes; Inositol oligosaccharides; Glycosylations; Thermoanaerobacter sp.

1. Introduction

and a lack of these second messengers has been impli-

cated in the etiology of Type 2 diabetes in humans.⁷

Recently, Larner and co-workers reported the ﬁrst full

characterisation of a putative insulin mediator from

insulin sensitive tissue consisting of galactosamine b-

linked to the 4-position of 2.¹⁴However, most previous

eﬀorts at identifying the mediator largely focused on

synthesising candidate structures and assessing their

insulin mimetic ability.^12;16–18

Oligosaccharides containing 1

3-O-methyl ether 1

D

-chiro-inositol (1) and its

-pinitol (2) have previously been

D

isolated from the seeds of leguminous plants such as

buckwheat^1–3and jojoba beans.⁴They are thought to

play a role in protecting the organelles and proteins of

the seeds during desiccation and storage.^3;5In addition,

disaccharides consisting of 1, 2 or myo-inositol coupled

to a sugar such as galactosamine are the focus of

attention for their putative role as second messengers of

insulin in vivo.^6–14It has been proposed that these di-

saccharides are released from glycosyl phosphatidyl

inositols anchored to the outer surface of insulin sensi-

tive cells, in response to insulin-receptor binding^{10;11;13–15}

Recently, considerable interest has focussed on the

use of glycoside hydrolase enzymes (glycosidases) to

synthesise di- and oligo-saccharides in only one step.^19–24

The particular characteristics of the glycosidase em-

ployed determines, which transglycosylation products, if

any, are formed and the products formed are a function

of the enzyme’s acceptor and donor preferences as well

as its glycosylation regio- and stereoselectivity. How-

ever, these selectivities need not be regarded as limita-

tions to the use of glycosidases in oligosaccharide

synthesis. Instead, the approach can be thought of as

* Corresponding authors. Tel.: +64-4-9313-064; fax: +64-4-9313-055

(J.B.H.); tel.: +49-040-42838-4241; fax: +49-040-42838-4325 (J.T.);

e-mail addresses: j.hart@irl.cri.nz; thiem@chemie.uni-hamburg.de

doi:10.1016/j.carres.2004.05.016

1858

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

being directed and biomimetic, because the regioselec-

tivity encountered is more likely to be of biological

signiﬁcance than a corresponding random chemical ap-

proach.

OH

MeO

OH

1

2

OH

OMe

4

OMe

HO

OH

4

2

4

2

OH

OMe

OH

OAll

OH

4

5

3

1

OH

HO

OH

4

2

1

2

OH

OMe

OH

HO

1

2

4

OH

We have previously reported the galactosylation of

-chiro-inositol 1 and 1 -pinitol 2, using the b-galac-

6

7

1

D

tosidase from Bacillus circulans,¹⁹the ﬁrst report of the

enzymatic glycosylation of inositols. An analysis of the

products obtained from these glycosylations allowed us

to gain some insight into the hydroxyl group conﬁgu-

rations key to the recognition of the acceptor as a sub-

strate in the active site of the enzyme.

A thermostable b-galactosidase isolated from an

anaerobic extreme thermomophile, Thermoanaerobacter

sp. strain TP6-B1 (henceforth referred to as TP6-B1)

from a New Zealand geothermal source,²⁵displayed a

preference for hydrolysing b-linked galactopyranosides

2. Results and discussion

2.1. Galactosylation of selected inositols using TP6-B1

b-galactosidase

The galactosylation products obtained from the selected

inositol acceptors 1–7, employing the b-galactosidase

from TP6-B1 are summarised in Table 1, and the posi-

tions on the substrates where galactosylation has oc-

curred are depicted in Figure 1. All reactions were

conducted using a moderate excess of the inositol

acceptor and the yields reported are based on the lim-

such as o- and p-nitrophenyl b-D-galactopyranoside and

lactose as opposed to a-linked galactopyranosides and

glucopyranosides.²⁵As part of an investigation into

the transglycosylation activity of various glycosidases,

we report the results of the TP6-B1 b-galactosidase-

catalysed galactosylations of a number of inositols:

iting reagent, namely p-nitrophenyl b-D-galactopyrano-

side.

1

D

-chiro-inositol 1, 1

chiro-inositol 3, 1 -3-O-allyl-4-O-methyl-chiro-inositol

4, 1 -quebrachitol 5, 1 -chiro-inositol 6 and myo-inosi-

D

-pinitol 2, 1

D

-3,4-di-O-methyl-

In contrast to the single disaccharide reaction product

observed for the galactosylation of 1D-chiro-inositol 1

by the b-galactosidase from B. circulans,¹⁹two disac-

charide products were obtained in a 10:3 ratio from the

D

L

tol 7. In conjunction with NMR analysis, GC–MS

analysis was used to characterise and/or conﬁrm the

identity of the glycosylation products. These results are

described, along with the synthetic routes to a series of

mono-O-acetyl-penta-O-methylated chiro- and myo-

inositol standards required for the GC–MS identiﬁca-

tion of the individual product components. In addition,

the acceptor preferences of the enzyme have been

deduced on the basis of the products formed.

galactosylation of 1 -chiro-inositol 1 using the b-

D

galactosidase from TP6-B1, in a combined yield of 46%.

An analysis of the product mixture by ¹H and ¹³C NMR

spectroscopy revealed that the resonances of the major

component were identical to those of the disaccharide

product observed from the enzymatic galactosylation of

1 using the b-galactosidase from B. circulans, namely

1

D

-1-O-(b-D

-galactopyranosyl)-chiro-inositol 8.¹⁹The

Table 1. Summary of galactosylation products obtained using TP6-B1 b-galactosidase with p-nitrophenyl b-

Acceptor Products

D

-galactopyranoside as the donor

Isolated yield (%)^a

1

D

-chiro-Inositol 1

-Pinitol 2

-3,4-Di-O-methyl-chiro-inositol 3

b-

D

-Galp-(1 ﬁ 1)-DCI 8 and b- -Galp-(1 ﬁ 3)-DCI 9

D

46^b

-Galp-(1 ﬁ 1)-4-OMe-DCI 10 and b-

-Galp-(1 ﬁ 1)-3,4-di-OMe-DCI 12

D

-Galp-(1 ﬁ 1)-3-OMe-DCI 11

50^b

52

-3-O-Allyl-4-O-methyl-chiro-inositol 4 b-

b-

-Galp-(1 ﬁ 1)-4-O-All-3-OMe-DCI 13 and

-Galp-(1 ﬁ 1)-3-O-All-4-OMe-DCI 14

60^b

1

L

-Quebrachitol 5

-chiro-Inositol 6

n.r.

64^b

1

b-

D

-Galp-(1 ﬁ 3)-LCI 15 and b-

-Galp-(1 ﬁ 2)-myo-inositol 17 and b-

-Galp-(1 ﬁ 5)-myo-inositol 19 and b-

D

-Galp- (1 ﬁ 1)-LCI 16

myo-Inositol 7

D

-Galp-(1 ﬁ 4/6)-myo-inositol 18 and 63^b

-Galp-(1 ﬁ 1/3)-myo-inositol 20

D

n.r. No reaction.

^aYields based on limiting reagent, that is p-nitrophenyl b-

D-galactopyranoside.

^bCombined yield.

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

1859

* Percentages

represent the

proportion of

each product

formed

OH

77%* 8

OH

β

-Galactosidase

from TP6-B1

1

OH

HO

OH

2

4

OH

23% 9

1

GalβOpNP

HOpNP

OH

50% 11

β

-Galactosidase

from TP6-B1

1

OH

HO

OH

2

4

OH

OMe

4

50% 10

OMe

2

GalβOpNP

HOpNP

OH

100% 12

β

-Galactosidase

1

OH

OMe

from TP6-B1

1

HO

OH

OMe

HO

OH

2

4

OMe

2

OH

4

OMe

3

GalβOpNP

HOpNP

OH

64% 13

OH

β

-Galactosidase

1

OH

OAll

from TP6-B1

OH

OMe

HO

OH

2

4

OMe

OH

2

4

OAll

OH

36% 14

4

GalβOpNP

HOpNP

OH

MeO

β

-Galactosidase

from TP6-B1

1

2

OH

4

OH

5

GalβOpNP

HOpNP

OH

HO

β

-Galactosidase

from TP6-B1

OH

25% 16

1

2

HO

OH

1

2

75% 15

OH

4

OH

4

OH

6

OH

GalβOpNP

HOpNP

OH

HO

β

-Galactosidase

from TP6-B1

OH

57% 17

10% 20

2

1

OH

HO

1

HO

2

OH

4

OH

14% 19

HO

OH

4

7

OH

GalβOpNP

HOpNP

19% 18

Figure 1. Products from galactosylation of various inositols catalysed by TP6-B1 b-galactosidase.

1

second, minor, component was shown by H and ¹³C

occurred on the axial position remote from the methyl

group.¹⁹NMR spectroscopic analysis of the product

mixture indicated that one of the disaccharide prod-

NMR analysis to be a disaccharide with a b-linkage to

1D-chiro-inositol at a diﬀerent position to that observed

for compound 8. However, it was not possible to deﬁne

the attachment point on the inositol due to the complex

nature of the NMR spectra. The structure of the minor

ucts was that observed previously, namely 1

D

-1-O-(b-D-

galactopyranosyl)-4-O-methyl-chiro-inositol 10.¹⁹

A

detailed analysis of the unassigned NMR correlations in

the 2D-NMR spectra of the product mixture revealed

component was shown to be 1

D

-3-O-(b-D-galactopyr-

anosyl)-chiro-inositol 9 by methylation analysis of the

product mixture (vide infra).

A 1:1 mixture of two products was obtained from the

that the second product was 1

D

-1-O-(b-D-galactopyr-

anosyl)-3-O-methyl-chiro-inositol 11. It is apparent that

disaccharide 10 is the result of galactosylation of 2 at

the axial hydroxyl position remote from that of the

methyl group of 2, that is OH-6, whereas 11 is the result

of galactosylation at the other axial hydroxyl of 2,

OH-1, two positions removed from that of the methyl

group.

galactosylation of 1

from TP6-B1, in a combined yield of 50%. Previously,

we reported that the galactosylation of 1 -pinitol 2 by

D

-pinitol 2 using the b-galactosidase

D

the b-galactosidase from B. circulans gave only one

disaccharide product, 10, whereby galactosylation

1860

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

For the b-galactosidase from TP6-B1, the presence of

naming of inositols: the highest priority is given to the

galactosyl-substituent attached to the inositol sugar,

with lower priority given to the methyl group. This

results in an apparent change in the position of the

methyl group of 10 (OH-4) relative to those of 2 and 11

(OH-3), as the galactosyl substituent is deﬁned in both

compounds 10 and 11 as being at the 1-position. Closer

inspection reveals that the galactosyl substituent is

attached at the axial hydroxyl remote to that of the

methyl group in 10 (numbered as the 6-position of 2),

and in 11 it is attached at the other axial hydroxyl

(numbered as the 1-position of 2), and the methyl group

has not changed position.

the methyl group in 2 does not appear to have a direct

inﬂuence on the direction of galactosylation towards

either of the two axial hydroxyl groups at positions OH-

1 and OH-6 of 2 as both regioisomers 10 and 11 were

observed in equal amounts as reaction products. This

result is in contrast with that obtained for the B. circu-

lans b-galactosidase whereby galactosylation occurred

only at the OH-6 of 2 to give disaccharide 10, that is

only at the position remote from that of the methyl

group (Fig. 2).

It is worth noting that the numbering of the products

10 and 11 follows the IUPAC rules applicable to the

OH

4

O

HO

OH

Acceptor

Acceptor in galactose configuration

OH

major

product

minor

product

B. circulans

TP6-B1

OH

3

HO

1D-chiro-inositol

HO

1

OH

1

HO

OH

1D-pinitol

OH

OMe

OH

2

HO

OH

OMe

OH

3

1

HO

OMe

OH

OMe

minor

product

major

product

OH

4

OH

OMe

OH

OAll

HO

OH

OAll

OMe

HO

OH

HO

3

OH

MeO

3

1L-quebrachitol

1

OH

1

HO

MeO

OH

5

1

3

OH

OMe

HO

3

OH

HO

OH

1L-chiro-inositol

3

HO

1

OH

1

6

OH

1

OH

3

HO

4

HO

2

OH

HO

OH

HO

OH

6

OH

5

myo-inositol

OH

and/or:

7

6

5

3

HO

OH

4

HO

OH

HO

OH

HO

1

2

OH

Figure 2. Galactosylation patterns for B. circulans and TP6-B1 b-galactosidases with various inositols.

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

1861

O

OH

O

OH

4

OH

MeO

RO

1

2

MeO

RO

MeO

HO

2

1

O

HO

4

HO

OH

R

21

H

c

3

4

Me

All

a

b

d

22 Me

23 All

1

D-pinitol 2

Scheme 1. Synthesis of inositol derivatives 3 and 4. Reagents and conditions: (a) cat. pTSA, Me₂CO, dimethyoxypropane, DMF; (b) NaH, MeI,

DMF; (c) TFA–H₂O (1:1); (d) NaH, AllBr, DMF.

In order to investigate the inﬂuence of additional

groups on 1 -pinitol 2 with regard to the galactosyl-

for galactosylation towards the hydroxyl position re-

mote from that of the allyl group was observed. If the

D

ation pattern observed, two inositol derivatives (3 and 4)

were synthesised (Scheme 1). The allyl group was chosen

because of its increased size relative to the methyl group

and its potential for further synthetic utility. The C₂-

inositol derivatives are depicted in a ‘b-

conﬁguration’ so that the structures have the same

conﬁguration as seen at C-1–C-4 of b- -galactose (Fig.

2) it can be seen that the TP6-B1 b-galactosidase has a

higher degree of tolerance for larger groups at the po-

sition, which corresponds to the C-2 of galactose than

the B. circulans b-galactosidase, which displays a strong

preference for a hydroxyl group at this position.

D-galactose

D

symmetric inositol 1D-3,4-di-O-methyl-chiro-inositol 3

with methyl groups at the OH-3 and OH-4 positions of

1, and the mono-allyl derivative 4, were synthesised

from a common intermediate, pinitol diacetonide 21, by

performing standard methylation and allylation reac-

tions, respectively. Removal of the acetonide groups

under acid conditions yielded the desired derivatives 3²⁶

and 4, respectively.

Galactosylation of the C₂-symmetrical dimethylated

inositol derivative 3 using the b-galactosidase from TP6-

B1 yielded only one product, in an isolated yield of 52%.

Analysis of the NMR spectra obtained for this product

revealed the galactose was attached at either of the

equivalent OH-1 or OH-6 positions of 3, to give the b-

linked disaccharide 12. The b-galactosidase from TP6-

B1 therefore displays an apparent tolerance for methyl

Galactosylation of 1 -chiro-inositol 6 using the b-

L

galactosidase from TP6-B1 gave two separable products

in a 3:1 ratio with a combined yield of 64%. The major

component was shown by an analysis of the 1D- and

2D-NMR data from the mixture to contain a galactose

moiety b-linked at the OH-3 of 6 that is 1

galactopyranosyl)-chiro-inositol 15. Similarly, a detailed

L

-3-O-(b-D-

analysis of the 2D-NMR spectra of the minor reaction

product revealed that 1

chiro-inositol 16 was the disaccharide formed, that is a

L

-1-O-(b-D-galactopyranosyl)-

galactose b-linked to the OH-1 position of 6. In con-

trast, 1L-quebrachitol 5, the 2-O-methyl ether of 6, was

groups at both the OH-3 and OH-4 positions of 1

D

-

not glycosylated by the b-galactosidase from TP6-B1.

Four inseparable products were obtained from the

galactosylation of myo-inositol 7 using the b-galactosi-

dase from TP6-B1, in a combined yield of 63%. Analysis

of the product mixture by GC–MS was required to

determine the linkage patterns on the myo-inositol (vide

infra).

chiro-inositol 1, as seen by the occurrence of galactosy-

lation at either of the two equivalent axial hydroxyl

groups of 3, namely OH-1 or OH-6.

Galactosylation of the mono-allylated inositol deriv-

ative 4 by the TP6-B1 b-galactosidase resulted in two

products in a 16:9 ratio (as determined by H NMR

1

spectroscopy), isolated in a combined yield of 60%. For

the major component, analysis of the NMR data ob-

tained from the product mixture revealed that the

galactose was b-linked to the axial hydroxyl of 4 that is

remote from the allyl group to give disaccharide 13. A

corresponding analysis of the unassigned correlations in

the NMR spectra of the product mixture showed that

the minor product was the result of b-galactosylation

at the axial hydroxyl of 4 remote from the methyl group

to give disaccharide 14 (Fig. 1).

2.2. Preparation of inositol standards for GC–MS anal-

ysis

To identify the disaccharide products that were obtained

as mixtures of inseparable components that could not be

established by NMR analysis alone, and to conﬁrm the

identities of those products for which the NMR data

were suﬃcient to enable a full characterisation, meth-

ylation analyses employing GC–MS were undertaken on

the enzymatic products (Fig. 3). In order to identify the

derivatised inositol components by GC–MS, it was ﬁrst

necessary to synthesise the set of penta-O-methylated

The presence of the allyl group in 4 inﬂuenced the

product distribution of the reaction catalysed by the

TP6-B1 b-galactosidase such that a 1.8-fold preference

1862

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

mono-O-acetyl standards for chiro-inositol and myo-

inositol viz. compounds 24–30, as shown in Schemes

2–5. Because of the C₂-symmetry inherent in the chiro-

inositol molecule, only three pentamethylated deriva-

tives need to be synthesised to obtain the full set of

chiro-inositol standards, viz. the 1- (or 6-), the 2- (or 5-)

and the 3- (or 4-) O-acetyl-penta-O-methyl-chiro-inosi-

tols. For myo-inositol, a meso compound, acetylations at

the 2- or the 5-positions also give rise to meso-com-

pounds. However, for acetylations at either of the

enantiotopic 1- or 3-hydroxyl groups, the possibility of

enantiomers arises; acetylation of the 1-hydroxyl pro-

duces the 1

hydroxyl gives the 1

D

-enantiomer, whereas acetylation of the 3-

-enantiomer. A similar situation

L

exists for the enantiotopic 4- and 6-hydroxyl groups.

However, for the sake of simplicity we decided to pro-

duce only the four myo-inositol derivatives 25, 27, 28

and 29 arising from acetylation at the 1-, 2-, 4- and 5-

hydroxyls of myo-inositol, respectively. It is worth not-

ing that the four penta-O-methylated myo-inositols 41,

51, 56 and 61 have been reported previously,²⁷however

the syntheses of these compounds involved, as the ﬁrst

step, the separation of three myo-inositol dicyclohexyl-

idene adducts and this approach resulted in the gener-

ation of the 1/3- and 4/6-myo-inositol penta-O-methyl

derivatives as racemates. More recently, the individual

(OMe)₅

(OR)₅

R=H,Me

Disaccharide

(MeO)₄-Gal-O

Gal-O

enantiomeric 1

myo-inositols and the corresponding 1

acetyl-enantiomers were reported.^28;29The 1

O-acetyl-penta-O-methyl-myo-inositols were obtained

from a chiral resolution of -(+)-O-acetylmandelate

dicyclohexylidene myo-inositol derivatives and the 1

and 1 -1-O-acetyl-penta-O-methyl-myo-inositol enantio-

mers were obtained from a chiral resolution of the

isopropyl 2,3-di-O-benzoyl- -(+)-tartrate dicyclohexyl-

idene myo-inositol derivatives.

The standard compounds were prepared as shown

D

- and 1

L

-1-O-acetyl-penta-O-methyl-

- and 1 -4-O-

- and 1 -4-

2M TFA

50°C

MeI

NaOH/DMSO

D

L

D

L

(MeO)₄-Galactitol-(OAc)₂

(MeO)₄-Gal

HO

OH

1. NaBH₄

1M NH₄OH 50°C

2. HClO₄, AcOH,

Ac₂O, EtOAc

D

-

(OMe)₅

AcO

(OMe)₅

L

GC-MS analysis

from 1

L

-quebrachitol 5 (24, 25, 26, 27) in Schemes 2 and

Figure 3. The derivatisation method for methylation analysis.

OR

OAc

OR¹

MeO

1

MeO

2

OH

O

1

2

MeO

1

OMe

MeO

2

OR²

1

2

OH

O

4

OMe

O

OR²

4

OMe

OR²

OH

4

OH

R¹

33 Bn

R²

24

R

H

d

e

c

f

a

31 H

34 Bn Me

35 OH Me

b

1L

-Quebrachitol 5

32 Bn

1

MeO

O

RO

O

1

MeO

R¹O

1

O

OR²

4

2

O

4

2

O

2

O

4

OR²

O

R¹

R²

H

R

g

h

39 Bn

40 Bn

i

31

41

37 H

38 Bn

36

d

j

b

Me

41 OH Me

1

MeO

AcO

OMe

2

OMe

4

OMe

f

25

Scheme 2. Syntheses of the methylation analysis standards 1-OAc-chiro-(OMe)₅(24) and 1-OAc-myo-(OMe)₅(25). Reagents and conditions: (a) cat.

H₂SO₄, cyclohexanone, benzene, DMF; (b) NaH, BnBr, DMF; (c) TFA–H₂O (1:1); (d) NaH, MeI, DMF; (e) Pd/C, H₂, EtOH, AcOH; (f) Ac₂O, py;

(g) RuCl₃, NaIO₄, K₂CO₃, MeCN, DCM, H₂O; (h) NaBH₄, H₂O, THF; (i) AcOH–H₂O (4:1); (j) Pd/C, H₂, TFA, EtOH.

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

1863

OR

MeO

1

2

OMe

MeO

OR

OMe

4

MeO

OMe

1

2

O

4

OMe

O

2

1

OAc

OR

4

OMe

1

OR

R

H

R

R¹

H

26

a

31

c

42

44

H

d

e

f

b

43 Me

g

45 Bn

H

46 Bn Me

47

H

Me

OMe

MeO

2

OMe

MeO

OMe

1

OMe

MeO

4

2

MeO

2

1

OMe

4

OMe

OAc

RO

1

4

OBn

OR

O

27

h

R

H

R¹

Bn

i

45

48

49

g

b

f

50 Me Bn

51 Me

H

Scheme 3. Syntheses of the methylation analysis standards 2-OAc-chiro-(OMe)₅(26) and 4-OAc-myo-(OMe)₅(27). Reagents and conditions: (a)

H₂O–AcOH (1:6); (b) NaH, MeI, DMF; (c) TFA–H₂O (1:1); (d) (Bu₃Sn)₂O, Bu₄NBr, BnBr, toluene, reﬂux; (e) NaH, MeI, DMF; (f) Pd/C, H₂, TFA,

EtOH; (g) Ac₂O, py; (h) RuCl₃, NaIO₄, K₂CO₃, MeCN, DCM, H₂O; (i) NaBH₄, H₂O, THF.

OMe

2

OR

OAc

2

O

OR

1

OR

OMe

OH

2

OMe

2

1

OH

R O

MeO

1

O

R O

OR

1

OMe

O

R O

1

HO

OH

1

5

R O

MeO

5

HO

O

OMe

R¹R²

Bn

28

R

H

R¹

a

54

H

myo-inositol 7

f

c

52

53 Bn

H

d

e

b

55 Me Bn

56 Me

H

OMe

2

OR

O

OR

OMe

OR

2

OMe

2

MeO

RO

O

OMe

O

OR

1

AcO

R O

5

MeO

5

RO

O

OMe

R¹

29

R

H

R¹

Bn

R

H

59

52

H

f

g

b

d

i

h

60 Me Bn

57 All

61 Me

H

58 All Bn

Scheme 4. Syntheses of the methylation analysis standards 2-OAc-myo-(OMe)₅(28) and 5-OAc-myo-(OMe)₅(29). Reagents and conditions: (a) 2,3-

butadione, CH(OMe)₃, CSA, MeOH; (b) NaH, BnBr, DMF; (c) TFA–H₂O (1:1); (d) NaH, MeI, DMF; (e) Pd/C, H₂, TFA, H₂O; (F) Ac₂O, py; (g)

NaH, AllBr, DMF; (h) Pd/C, pTSA, H₂O, MeOH; (i) Pd/C, H₂, TFA, EtOH.

O

OR¹

OMe

OH

OR¹

O

OMe

3

OH

5

3

MeO

RO

MeO

AcO

MeO

R²O

MeO

HO

5

O

5

3

R¹O

MeO

O

OR¹

OMe

HO

OH

R¹R²

Bn

64 Me Bn

65 Me

R

30

63

H

c

d

e

21

H

a

f

b

62 Bn

1D-pinitol 2

H

Scheme 5. Synthesis of the methylation analysis standard 3-OAc-chiro-(OMe)₅(30). Reagents and conditions: (a) cat. pTSA, Me₂CO, dimethoxy-

propane, DMF; (b) NaH, BnBr, DMF; (c) TFA–H₂O (1:1); (d) NaH, MeI, DMF; (e) Pd/C, H₂, AcOH, EtOH; (f) Ac₂O, py.

1864

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

3, myo-inositol 7 (28, 29) in Scheme 4, and 1

(30) in Scheme 5.

D

-pinitol 2

the 3-hydroxyl groups of 7 to give 1

D

- or 1

L

-1-O-(b-D-

galactopyranosyl)-myo-inositol (20), respectively, but

without chiral GC analysis it was not possible to

distinguish whether only one, or both disaccharides

were produced. The second largest peak correspond-

ing to galactosylation at the 4- or 6-hydroxyl groups of

In the future the synthesis of the full set of six possible

mono-O-acetyl penta-O-methyl myo-inositols in con-

junction with analysis by chiral GC–MS could be used

to reveal additional information regarding the stereo-

preferences of the enzyme.

7 to give 1

D

- or 1

L

-4-O-(b-D-galactopyranosyl)-myo-

inositol (18), respectively, could also be attributed

to the presence of one or both of the disaccharide

products.

2.3. Methylation analysis of the enzyme products using

GC–MS

The chiro- and separately the myo-series of standards

gave rise to chromatographic peaks that were well re-

solved by GC–MS (Table 2). The derivatives generated

from the various enzyme-catalysed glycosylation prod-

ucts by methylation analysis (methylation, reductive

hydrolysis, acetylation (Fig. 3)) were analysed by GC–

MS and the retention times were compared with those of

the standards to reveal the identities of the individual

component products. As an illustration of the power of

this method, Figure 4 shows the GC–MS trace of the

chiro-inositol standards and of derivatives obtained

2.4. Analysis of enzyme speciﬁcity

The TP6-B1 b-galactosidase is reported to hydrolyse

lactose as well as b-galactosides such as p-nitrophenyl b-

D

-galactopyranoside²⁵therefore it is likely that galactose

and glucose can be accommodated into the acceptor site

of the enzyme for reactions occurring in the transgly-

cosylation direction. An assessment of the acceptor

preferences of the enzyme, as detailed in Figure 2,

revealed that TP6-B1 is relatively nonselective with

regard to substitution of the inositol acceptor in com-

parison with the results obtained with the B. circulans b-

galactosidase.¹⁹The structures in Figure 2 are drawn as

if the enzyme were forming a b-(1 ! 4)-linked disac-

charide. The enzyme galactosylated hydroxyl groups in

an equatorial (i.e., gluco-) or an axial (i.e., galacto-)

conﬁguration at the 4-position.

It is apparent that the key determinant for transglyco-

sylation to occur is the conﬁgurations of the 2- and

3-positions, namely the requirement that either C-2 or

C-3 possess an equatorial hydroxyl substituent for

galactosylation to occur at C-4 (galactose number-

ing). Galactosylation of the inositol acceptor was

achieved with a wide range of inositol substitution pat-

terns and conﬁgurations, for example, for the 2-position

(galactose numbering) the enzyme generally accommo-

dated equatorial hydroxyl, methoxyl and allyl groups.

from the 1D-chiro-inositol monogalactoside product

mixture. It can be seen that the major product is 1-O-

linked to galactose and the minor component is 3-O-

linked to galactose. The galactosyl moiety is derivatised

to tetra-O-methyl galactitol diacetate. Employing this

method, the identities of the components in the reaction

mixtures were successfully established and this infor-

mation is given in Table 1.

For the galactosylation of myo-inositol 7, the GC

trace obtained for the derivatised product mixture

revealed four separate inositol-related peaks with

retention times corresponding to the four myo-inositol

standards. The major component corresponded to

galactosylation at the 2-hydroxyl group of 7 to give 2-O-

(b-D-galactopyranosyl)-myo-inositol (17), while the

third largest peak corresponded to galactosylation at the

other meso-position of 7, the 5-position, to give 5-O-(b-

D

-galactopyranosyl)-myo-inositol (19). The smallest

peak was attributed to galactosylation at either the 1- or

The exception to this is seen for 1L-quebrachitol 5

where a methoxyl group is in the C-2 position and no

glycosylation occurs. An axial hydroxyl group at C-2

was also accommodated for some ‘gluco-type’ systems

(i.e., an equatorial 4-OH) such as 1L-chiro-inositol 6 but

not for others, for example, 1 -quebrachitol 5, which

L

Table 2. Relative GC–MS retention times for methylation analysis

standards

Methylation analysis standard^a

Relative R_t^b

has an equatorial methoxyl group at C-3 (galactose

numbering). For the 3-, 4- and 5-positions (galactose

numbering) the enzyme tolerated the presence of axial or

equatorial hydroxyl groups. However, it did not galac-

tosylate at the 4-position if there were methoxyl or

methylene hydroxy (CH₂OH) groups present at the

5-position (since the TP6-B1 enzyme did not appear to

1-OAc-chiro-(OMe)₅24

2-OAc-chiro-(OMe)₅26

3-OAc-chiro-(OMe)₅30

2-OAc-myo-(OMe)₅28

1-OAc-myo-(OMe)₅25

5-OAc-myo-(OMe)₅29

4-OAc-myo-(OMe)₅27

0.538

0.608

0.641

0.539

0.581

0.658

0.668

galactosylate galactose) or

the 3-position (as is the case with 1

a

methoxyl group at

-quebrachitol 5).

^aAbbreviated compound names used, for example, ‘1-OAc-chiro-

(OMe)₅’ is shorthand for 1L-1-O-acetyl-2,3,4,5,6-penta-O-methyl-

L

The information described above is summarised in

Figure 5.

chiro-inositol.

^bRelative to hexa-O-acetyl-myo-inositol ¼ 1.00.

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

1865

Chiro-Inositol Methylation Standards*

1-OAc-CI-(OMe)₅

2-OAc-CI-(OMe)₅

3-OAc-CI-(OMe)₅

* Abbreviated compound names used

eg "1-OAc-DCI-(OMe)₅" is shorthand for

1D-1-O-acetyl-2,3,4,5,6-penta-O-methyl-

chiro-inositol.

16

20

24

28

12

Time (min)

32

1D-Chiro-Inositol Galactosylation Products*

1-OAc-DCI-(OMe)₅

Galactitol- (OMe)₄(OAc)₂

3-OAc-DCI-(OMe)₅

Time (min)

28

12

16

20

24

32

OH

HO

OH

O

HO

3

+

2

O

HO

OH

1

5

OH

HO

OH

2

4

β-D-Galp-(1-3)-DCI (9)

OH

β-D-Galp-(1-1)-DCI (8)

Figure 4. GC–MS analysis of 1D-chiro-inositol (1) reaction products and standards.

tosidase from B. circulans. Speciﬁcally, TP6-B1 requires

an equatorial hydroxyl group at either C-2 or C-3 for

glycosylation to occur at C-4 (galactose numbering) of

the acceptor moiety.

Galactosylation

point

Axial/equatorial -OH

-OR, -OMe, -CH₂OH

Axial/equatorial -OH

-OMe, -OAll

One must be an equatorial -OH

4. Experimental

4.1. General methods

Figure 5. Summary of the acceptor speciﬁcity of TP6-B1.

NMR spectra were acquired either on a Bruker Advance

1

3. Summary

NMR Spectrometer operating at 300 MHz for H and

75 MHz for ¹³C nuclei or on a Varian Unity 500 NMR

Spectrometer operating at 500 MHz for ¹H and

126 MHz for ¹³C nuclei, and chemical shifts are listed in

ppm. NMR experiments performed in CDCl₃are ref-

erenced to Me₄Si (0 ppm), those in D₂O to external

acetone (d 218.1 ppm (C@O) and d 33.2 ppm (Me) for

The b-galactosidase isolated from the extreme anaerobic

thermophile, Thermoanaerobacter sp. strain TP6-B1

galactosylated 1

chiro-inositol 6, myo-inositol 7 and two related 1

inositol derivatives, 3 and 4. In contrast, it did not

galactosylate 1 -quebrachitol 5. The products from the

D

-chiro-inositol 1, 1

D

-pinitol 2, 1

L-

D

-chiro-

1

¹³C and d 2.22 ppm for H) and those in CD₃OD to

L

external MeOH (d 49.0 ppm for ¹³C and d 3.30 ppm

(Me) for H). Chemical shift assignments were made on

galactosylation reactions were identiﬁed and an analysis

of the products revealed that the enzyme displayed a

greater tolerance towards substitution of the inositol

acceptor than was observed previously for the b-galac-

1

the basis of ¹³C-DEPT, DQF HH-COSY, HC-COSY or

HMQC, HMBC, ROESY and HMQC-TOCSY spectral

1866

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

analysis as required. Melting points were determined

using a Reichert hot stage microscope apparatus. All

reagents used were AR grade and solvents used were AR

grade or were distilled, unless otherwise stated. Flash

chromatography (FC) was performed using Scharlau

silica gel 60 (0.04–0.06 mm, 230–400 mesh ASTM).

Puriﬁcation of the enzyme reaction products was

achieved using a Bio-Rad Bio-Gel P-2 (45–90 lm) col-

umn (1100 · 25 mm) equipped with a peristaltic pump

and a fraction collector. Optical rotations were acquired

with a Perkin Elmer 241 Polarimeter, and mass spectra

were obtained on either a VG70-250S Double Focusing

Magnetic Sector Mass Spectrometer or a MARINER

Biospectrometer for electrospray mass spectrometry re-

sults. GC–MS was performed by GC separation on a

H₂O (1:1, 20 mL) was kept at room temperature for

2.5 h. Removal of the solvents followed by FC per-

formed on the residue (EtOAc) gave the title compound

as a white foam (1.35 g, 95%); ½aꢀ²⁰+46.9 (c 1.0, MeOH);

D

¹H NMR (300 MHz, CD₃OD): d 6.02 (m, 1H, CH@),

5.28 (d, 1H, J 17.3 Hz, CH_trans@), 5.14 (d, 1H, J 10.4 Hz,

CH_cis@), 4.30 (m, 2H, CH₂), 3.88 (d, 2H, J 2.4 Hz, H-1,

H-6), 3.76 (dt, 2H, J 10.2, 2.5, H-2, H-5), 3.60 (s, 3H,

CH₃), 3.42 (t, 1H, J 9.3 Hz, H-3 or H-4), 3.31 (t, 1H, J

9.4 Hz, H-3 or H-4); ¹³C NMR (75 MHz, CDCl₃): d

137.6 (CH@), 116.8 (CH₂@), 85.4, 83.4 (C-3, C-4), 75.4

(CH₂), 74.1, 74.0 (C-1, C-6), 72.8, 72.6 (C-2, C-5), 61.6

(CH₃); HR ES-MS: Calcd for C₁₀H₁₉O₆(M+H^þ) m=z

235.1176. Found 235.1183.

Hewlett Packard (HP) Ultra

2

capillary column

4.3. TP6-B1 b-galactosidase-catalysed reactions

(50 m · 0.2 mm i.d., 0.33 lm ﬁlm thickness) with the

oven programmed from 75 ꢁC (held for 1 min) to 130 ꢁC

at a rate of 40 ꢁC/min then at a rate of 4 ꢁC/min to

250 ꢁC followed by detection using a HP 5970 MSD.

Yields for enzymatic reactions are reported relative to

the amount of p-nitrophenyl galactopyranoside as the

donor. Pinitol was supplied by New Zealand Pharma-

ceuticals Ltd. The b-galactosidase from Thermoanaero-

bacter sp. strain TP6-B1 was supplied by Prof. Roy

Daniel, Waikato University, Hamilton, New Zealand

with an activity of 5500 MU/mg.

4.3.1. Galactosylation of 1

phenyl b- -galactopyranoside (555 mg, 1.8 mmol) and

-chiro-inositol 1 (1.99 g, 11.1 mmol, 6.2 equiv) were

D

-chiro-inositol (1). p-Nitro-

D

1

D

incubated in 20 mL phosphate buﬀer (pH 7.0, 0.1 M

NaH₂PO₄) with the b-galactosidase from TP6-B1

(900 mg, 4950 MU) at 37 ꢁC. After 3 days the enzyme

was denaturated by heating at 95 ꢁC for 10 min. The

solution was extracted with EtOAc to remove p-nitro-

phenol and lyophilised. The residue was applied to a

mixed-bed ion exchange column (Ionac resin NM-60)

eluting with H₂O and the eluent lyophilised. The solid

was re-dissolved in H₂O and applied to a Bio-Gel P-2

column eluting with Millipore H₂O and the disacchar-

ide-containing fractions (by TLC) were lyophilised to

4.2. Synthesis of substrates

4.2.1.

1D-3-O-Allyl-1,2:5,6-di-O-isopropylidene-4-O-

methyl-chiro-inositol (23). NaH (0.32 g of 60% w/w in

oil, 8.0 mmol) was added to a stirred solution of 21²⁶

(2.0 g, 7.3 mmol) in DMF (20 mL) under Ar at 0 ꢁC.

After stirring at room temperature for 15 min allyl

bromide (0.69 mL, 8.0 mmol) was added dropwise at

0 ꢁC, the reaction was allowed to warm to room tem-

perature and stirring was continued overnight. EtOH

(2 mL) was added, the solvents removed and FC per-

give a mixture of 1

inositol (8)¹⁹and 1

D

-1-O-(b-

-3-O-(b-

D

-galactopyranosyl)-chiro-

D

inositol (9) in the ratio of 10:3. Yield: 292 mg, 46%;

Major product, 1 -1-O-(b- -galactopyranosyl)-chiro-

inositol (8): H and ¹³C NMR data were identical with

those previously obtained.¹⁹The minor product, 1

-3-

O-(b- -galactopyranosyl)-chiro-inositol 9, was identiﬁed

D

1

D

by methylation analysis using GC–MS. Partial NMR

data was obtained for 9: ¹H NMR (300 MHz, CD₃OD):

d 4.55 (d, 1H, J 7.8 Hz, H-1⁰), 3.85 (obsc., 1H, H-4⁰),

3.70 (obsc., 1H, H-3), 3.62 (obsc., 1H, H-3⁰), 3.54 (obsc.,

1H, H-2⁰); ¹³C NMR (75 MHz, CDCl₃): d 104.2 (C-1⁰),

83.3 (C-3), 61.8 (C-6⁰); For the mixture, LR ES-MS

(isobaric mixture of 8 and 9): Calcd for C₁₂H₂₂O₁₁

(M+H^þ) m=z 343. Found 343.

formed on the residue (EtOAc–hexanes 1:3) gave the

20

title compound as a colourless oil (2.04 g, 88%); ½aꢀ

D

1

)18.5 (c 1.2, CHCl₃); H NMR (300 MHz, CD₃OD): d

5.95 (m, 1H, CH@), 5.32 (dd, 1H, J 1.4, 17.3 Hz,

CH_trans@), 5.17 (d, 1H, J 10.5 Hz, CH_cis@), 4.30–4.17

(m, 6H, H-1–H-6), 3.60 (s, 3H, OCH₃), 3.42 (m, 2H,

CH₂), 1.51 (s, 3H, CH₃), 1.50 (s, 3H, CH₃), 1.35 (s, 6H,

CH₃); ¹³C NMR (75 MHz, CDCl₃): d 135.4 (CH@),

117.1 (CH₂@), 109.9 (C(CH₃)₂), 82.0, 79.8, 79.6, 79.2,

77.9, 76.9 (C-1–C-6), 72.8 (CH₂), 60.4 (OCH₃), 28.1

(CH₃), 25.6 (CH₃); HR FAB^þ-MS (NBA/CH₂Cl₂):

Calcd for C₁₆H₂₆O₆(M+H^þ) m=z 315.1808. Found:

315.1798; Anal. Calcd for C₁₆H₂₆O₆: C, 61.13; H, 8.34.

Found: C, 61.27; H, 8.08.

4.3.2. Galactosylation of 1 -pinitol (2). p-Nitrophenyl b-

D

-galactopyranoside (23.0 mg, 0.076 mmol) and 1D-

D

pinitol 2 (91 mg, 0.47 mmol, 6.2 equiv) were incubated

in 1 mL phosphate buﬀer (pH 7.0, 0.1 M NaH₂PO₄)

with the b-galactosidase from TP6-B1 (20 mg, 110 MU)

at 37 ꢁC. After 3 days the reaction was processed as for

the galactosylation of 1 above to give a 1:1 mixture of

4.2.2.

solution of 23 (1.90 g, 6.1 mmol) in triﬂuoroacetic acid:

1

D

-3-O-Allyl-4-O-methyl-chiro-inositol (4).

A

two disaccharides, 1

methyl-chiro-inositol (10) and 1

D

-1-O-(b-

D

-galactopyranosyl)-4-O-

-1-O-(b- -galactopyr-

D

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

1867

anosyl)-3-O-methyl-chiro-inositol (11) (13.5 mg, 50%).

¹H and ¹³C NMR data for one of the products, 10, were

identical with those obtained previously.¹⁹For the sec-

ond product, 11, the following NMR data were

obtained; ¹H NMR (500 MHz, D₂O): d 4.51 (d, 1H,

J 5.1 Hz, H-1⁰), 4.30 (dd, 1H, J 3.9, 3.8 Hz, H-6), 4.03

(dd, 1H, J 3.7, 3.7 Hz, H-1), 3.91 (obsc., 1H, H-4⁰), 3.87

(obsc., 1H, H-2), 3.81 (obsc., 1H, H-5), 3.74 (obsc., 2H,

H6⁰/H6⁰), 3.68 (obsc., 1H, H-5⁰), 3.63 (obsc., 1H, H-4),

3.62 (obsc., 1H, H-3⁰), 3.69 (s, 3H, OCH₃), 3.54 (obsc.,

1H, H-2⁰), 3.40 (dd, 1H, J 9.7, 9.7 Hz, H-3); ¹³C NMR

(126 MHz, D₂O): d 106.0 (C-1⁰), 83.9 (C-3), 82.7 (C-1),

76.3 (C-5⁰), 73.6 (C-3⁰), 73.2 (C-4), 72.3 (C-2⁰), 71.5 (C-

6), 71.5 (C-5), 70.9 (C-2), 69.6 (C-4⁰), 62.1 (C-6⁰), 60.8

(OCH₃); For the mixture, HR ES-MS (isobaric mixture

of 10 and 11): Calcd for C₁₃H₂₈NO₁₁(M+H^þ) m=z

374.1657. Found: 374.1659.

4.47 (d, 1H, J 7.8 Hz, H-1⁰), 4.28 (m, 2H, CH₂), 4.26

(obsc., 1H, H-6), 4.00 (obsc., 1H, H-1), 3.88 (obsc., 1H,

H-4⁰), 3.86 (obsc., 1H, H-2), 3.84 (obsc., 1H, H-5), 3.72

(m, 2H, H-6⁰/H-6⁰), 3.65 (obsc., 1H, H-5⁰), 3.62 (obsc.,

1H, H-3⁰), 3.60 (s, 3H, OCH₃), 3.52 (obsc., 1H, H-2⁰),

3.49 (obsc., 1H, H-4), 3.42 (dd, 1H, J 9.5, 9.5 Hz, H-3);

¹³C NMR (126 MHz, D₂O): d 134.7 (CH@), 119.0

(CH₂@), 105.3 (C-1⁰), 83.2 (C-3), 81.7 (C-1), 81.1 (C-4),

75.6 (C-5⁰), 74.4 (CH₂), 73.0 (C-3⁰), 71.6 (C-6), 71.6 (C-

2⁰), 70.6, 70.4 (C-2, C-5), 68.9 (C-4⁰), 61.4 (C-6⁰), 60.6

(OCH₃); Minor product, 1

D

-3-O-allyl-1-O-(b-D-galac-

topyranosyl)-4-O-methyl-chiro-inositol (14): ¹H NMR

(500 MHz, D₂O): d 6.00 (m, 1H, CH@), 5.34 (dd, 1H, J

1.2, 17.1 Hz, CH_trans@), 5.24 (d, 1H, J 10.3 Hz, CH_cis@),

4.47 (d, 1H, J 7.8 Hz, H-1⁰), 4.28 (m, 2H, CH₂), 4.26

(obsc., 1H, H-6), 4.00 (obsc., 1H, H-1), 3.88 (obsc., 1H,

H-4⁰), 3.88 (obsc., 1H, H-2), 3.84 (obsc., 1H, H-5), 3.72

(m, 2H, H-6⁰/H-6⁰), 3.65 (obsc., 1H, H-5⁰), 3.62 (obsc.,

1H, H-3⁰), 3.60 (s, 3H, OCH₃), 3.56 (obsc., 1H, H-3),

3.52 (obsc., 1H, H-2⁰), 3.35 (dd, 1H, J 9.5, 9.5 Hz, H-4);

¹³C NMR (126 MHz, D₂O): d 134.7 (CH@), 119.0 (CH₂

@), 105.3 (C-1⁰), 83.1 (C-4), 82.0 (C-1), 81.3 (C-3), 75.6

(C-5⁰), 74.5 (CH₂), 73.0 (C-3⁰), 71.6 (C-2⁰), 70.8 (C-6),

70.6, 70.4 (C-2, C-5), 68.9 (C-4⁰), 61.4 (C-6⁰), 60.6

(OCH₃); For the mixture, HR ES-MS (isobaric mixture

of 13 and 14): Calcd for C₁₆H₂₈O₁₁Na (M+Na^þ) m=z

397.1737. Found 397.1704.

4.3.3. Galactosylation of 1

tol (3). p-Nitrophenyl b-

0.085 mmol) and 1

-3,4-di-O-methyl-chiro-inositol 3²⁶

D

-3,4-di-O-methyl-chiro-inosi-

D

-galactopyranoside (25.5 mg,

D

(99 mg, 0.42 mmol, 5.0 equiv) were incubated in 1 mL

phosphate buﬀer (pH 7.0, 0.1 M NaH₂PO₄) with the b-

galactosidase from TP6-B1 (20 mg, 110 MU) at 37 ꢁC.

After 3 days the reaction was processed as for the ga-

lactosylation of 1 above to give 1

pyranosyl)-3,4-di-O-methyl-chiro-inositol (12) (17.6 mg,

D

-1-O-(b-D-galacto-

20

52%); ½aꢀ +32 (c 1.0, H₂O); ¹H NMR (500 MHz, D₂O):

D

d 4.47 (d, 1H, J 7.8 Hz, H-1⁰), 4.26 (dd, 1H, J 3.4,

3.4 Hz, H-6), 3.99 (dd, 1H, J 3.6, 3.6 Hz, H-1), 3.88

(obsc., 1H, H-4⁰), 3.86 (obsc., 1H, H-2), 3.84 (dd, 1H, J

3.3, 9.7 Hz, H-5), 3.75 (dd, 1H, J 7.8, 8.5 Hz, H-6⁰), 3.70

(dd, 1H, J 3.6, 7.8 Hz, H-6⁰), 3.65 (obsc., 1H, H-5⁰), 3.62

(dd, 1H, J 3.3, 9.8 Hz, H-3⁰), 3.57 (s, 3H, 3-OCH₃), 3.57

(s, 3H, 4-OCH₃), 3.52 (dd, 1H, J 7.8, 9.8 Hz, H-2⁰), 3.46

(dd, 1H, J 9.7, 9.7 Hz, H-3), 3.35 (dd, 1H, J 9.7, 9.7 Hz,

H-4); ¹³C NMR (126 MHz, D₂O): d 105.3 (C-1⁰), 82.9

(C-3), 82.8 (C-4), 81.7 (C-1), 75.6 (C-5⁰), 72.9 (C-3⁰), 71.6

(C-2⁰), 70.8 (C-6⁰), 70.3 (C-5), 70.3 (C-2), 68.9 (C-4⁰),

61.4 (C-6⁰), 60.19, 60.15 (4-OCH₃and 3-OCH₃); HR ES-

MS: Calcd for C₁₄H₂₆O₁₁Na (M+Na^þ) m=z 393.1367.

Found 393.1388.

4.3.5. Galactosylation of 1

phenyl b- -galactopyranoside (111 mg, 0.37 mmol) and

-chiro-inositol 6 (398 mg, 2.2 mmol, 6.0 equiv) were

L

-chiro-inositol (6). p-Nitro-

D

1

L

incubated in 4 mL phosphate buﬀer (pH 7.0, 0.1 M

NaH₂PO₄) with the b-galactosidase from TP6-B1

(80 mg, 440 MU) at 37 ꢁC. After 3 days the enzyme was

denaturated by heating at 95 ꢁC for 10 min. The solution

was extracted with EtOAc to remove p-nitrophenol and

lyophilised. The residue was redissolved in H₂O and

passed through Amberlyst-A21 resin, lyophilised, and

then re-dissolved in Millipore H₂O, applied to a Bio-Gel

P-2 column and eluted with Millipore H₂O and lyophi-

lised. The residue was redissolved in Millipore H₂O and

re-applied to a Bio-Gel P-2 column to give 1

galactopyranosyl)-chiro -inositol (15) (61 mg, 49%) and

a minor fraction that contained mainly 1 -1-O-(b-

galactopyranosyl)-chiro-inositol (16) (14 mg, 11%); Ma-

jor product, 1 -3-O-(b- -galactopyranosyl)-chiro-inosi-

L

-3-O-(b-

D-

4.3.4. Galactosylation of 1

inositol (4). p-Nitrophenyl b-

(28.6 mg, 0.095 mmol) and 1 -3-O-allyl-4-O-methyl-

D

-3-O-allyl-4-O-methyl-chiro-

L

D-

D

-galactopyranoside

D

L

D

20

D

1

chiro-inositol 4 (99 mg, 0.48 mmol, 5.0 equiv) were

incubated in 1 mL phosphate buﬀer (pH 7.0, 0.1 M

NaH₂PO₄) with the b-galactosidase from TP6-B1

(20 mg, 110 MU) at 37 ꢁC. After 3 days the reaction was

processed as for the galactosylation of 1 above to give a

mixture of two disaccharides in a 16:9 ratio (21.0 mg,

tol (15): ½aꢀ )27 (c 1.1, H₂O); H NMR (500 MHz,

D₂O): d 4.62 (dd, 1H, J 1.0, 7.9 Hz, H-1⁰), 4.04 (dd, 1H,

J 2.7, 2.7 Hz, H-1 or H-6), 4.00 (dd, 1H, J 2.7, 2.7 Hz,

H-1 or H-6), 3.89 (d, 1H, J 3.4 Hz, H-4⁰), 3.80 (obsc.,

1H, H-2 or H-5), 3.78 (obsc., 1H, H-3), 3.76 (obsc., 1H,

H-2 or H-5), 3.76 (obsc., 1H, H-4), 3.76 (obsc., 1H, H-

6⁰), 3.72 (obsc., 1H, H-6⁰), 3.70 (obsc., 1H, H-5⁰), 3.66

(ddd, 1H, J 1.0, 3.4, 9.9 Hz, H-3⁰), 3.57 (ddd, 1H, J 1.0,

7.9, 9.9 Hz, H-2⁰); ¹³C NMR (126 MHz, D₂O): d 104.0

(C-1⁰), 82.9 (C-3), 75.8 (C-5⁰), 73.0 (C-3⁰), 72.7 (C-4),

60%); Major product, 1

D

-4-O-allyl-1-O-(b-D-galacto-

pyranosyl)-3-O-methyl-chiro-inositol (13): ¹H NMR

(500 MHz, D₂O): d 6.00 (m, 1H, CH@), 5.34 (dd, 1H, J

1.2, 17.1 Hz, CH_trans@), 5.24 (d, 1H, J 10.3 Hz, CH_cis@),

1868

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

71.7 (C-1 or C-6), 71.6 (C1 or C-6), 70.6 (C-2 or C-5),

69.5 (C-2 or C-5), 69.1 (C-4⁰), 61.6 (C-2⁰), 61.6 (C-6⁰);

HR ES-MS: Calcd for C₁₂H₂₂O₁₁Na (M+Na^þ) m=z

ched with acetone (0.5 mL) and the sample was evapo-

rated to dryness. Perchloric acid-catalysed acetylation

was undertaken using the method of Falshaw and

Furneaux,³⁰except that the organic layer was washed

with water (4 mL), Na₂CO₃(0.5 M, 4 mL) then water

(4 mL). The partially methylated and acetylated deriv-

atives were analysed by GC–MS.

365.1054. Found 365.1060. Minor product, 1

D

L

-1-O-(b-

-galactopyranosyl)-chiro-inositol (16): d 4.42 (d, 1H, J

7.8 Hz, H-1⁰), 4.14 (dd, 1H, J 3.5, 3.5 Hz, H-1), 4.12 (dd,

1H, J 3.5, 3.5 Hz, H-6), 3.90 (d, 1H, J 3.4 Hz, H-4⁰), 3.78

(obsc., 1H, H-5), 4.75 (obsc., 1H, H-2), 3.74 (m, 2H, H-

6⁰/H-6⁰), 3.67 (dd, 1H, J 3.9, 8.3 Hz, H-5⁰), 3.62 (dd, 1H,

J 3.4, 10.0 Hz, H-3⁰), 3.57 (obsc., 1H, H-3), 3.57 (obsc.,

1H, H-4), 3.49 (dd, 1H, J 7.8 Hz, 10.0, H-2⁰); ¹³C NMR

(126 MHz, D₂O): d 102.3 (C-1⁰), 79.1 (C-1), 75.6 (C-5⁰),

73.5 (C-3 or C-4), 73.1 (C-3 or C-4), 73.1 (C-3⁰), 70.9 (C-

2⁰), 70.8 (C-5), 70.1 (C-2), 69.6 (C-6), 69.1 (C-4⁰), 61.6

(C-6⁰); HR ES-MS: Calcd for C₁₂H₂₂O₁₁Na (M+Na^þ)

m=z 365.1054. Found 365.1064.

4.5. Synthesis of GC–MS standards

4.5.1. Standard benzylation procedure. NaH (60% w/w in

oil, 0.6 mol equiv per OH) was added under Ar to the

compound in DMF at 0 ꢁC and the reaction mixture was

allowed to warm to rt. BnBr (0.55 mol equiv per OH)

was added and, after stirring for 1 h at rt, the mixture

was re-cooled (0 ꢁC) and the NaH and BnBr additions

were repeated. After stirring overnight at rt, EtOH was

added to quench and the mixture was poured into H₂O

and extracted with CHCl₃(3·). The combined organic

extracts were dried (MgSO₄), ﬁltered and the solvents

removed in vacuo to give the crude product.

4.3.6. Galactosylation of myo-inositol (7). p-Nitrophenyl

b-D-galactopyranoside (111 mg, 0.37 mmol) and myo-

inositol 7 (398 mg, 2.2 mmol, 6.0 equiv) were incubated

in 4 mL phosphate buﬀer (pH 7.0, 0.1 M NaH₂PO₄) with

the b-galactosidase from TP6-B1 (80 mg, 440 MU) at

37 ꢁC. After 3 days the enzyme was denaturated by

heating at 95 ꢁC for 10 min. The solution was extracted

with EtOAc to remove p-nitrophenol and lyophilised.

The residue was redissolved in H ₂O and passed through

a mixed bed ion exchange column (Ionaic resin NM-60),

lyophilised, re-dissolved in Millipore H₂O and applied

to a Bio-Gel P-2 column and eluted with Millipore H₂O

to give a mixture of disaccharides (80 mg, 63%). GC–MS

4.5.2. Standard acetylation procedure. The compound

dissolved in equal volumes of pyridine (excess) and

Ac₂O (excess) was stirred overnight at rt under Ar, then

diluted with CH₂Cl₂, washed with H₂O (2·), which was

back-extracted with CH₂Cl₂. The combined organic

extracts were dried (MgSO₄), ﬁltered and the solvents

removed in vacuo to give the crude product.

showed the mixture to contain 2-O-(b-

anosyl)-myo-inositol (17), 1 - and/or 1

lactopyranosyl)-myo-inositol (18), 5-O-(b-

pyranosyl)-myo-inositol (19) and 1 - and/or 1

(b- -galactopyranosyl)-myo-inositol (20) in a ratio of

12:4:3:2.

D

-galactopyr-

-4-O-(b- -ga-

-galacto-

-1-O-

4.5.3. Standard methylation procedure. The compound

was dissolved in DMF and NaH (60% w/w in oil,

0.55 mol equiv per OH) was added at 0 ꢁC followed by

MeI (0.55 mol equiv per OH) under Ar. The mixture was

stirred at rt for 1 h before cooling to 0 ꢁC and repeating

the NaH and MeI additions. After stirring overnight at

rt under Ar, the reaction was quenched with EtOH, and

concentrated to give the crude product.

D

L

D

L

D

4.4. Derivatisation of enzyme products for GC–MS

analysis

4.5.4. Standard hydrogenolysis procedure. The com-

pound in EtOH and acetic acid (6:1 v/v EtOH–AcOH)

was hydrogenolysed for 48 h at rt under an atm pressure

of H₂over 10% Pd/C catalyst (ca. 1:5 w/w cata-

lyst:starting material). After ﬁltration through Celite to

remove the catalyst, the solvents were removed in vacuo

and toluene was added and removed to remove traces of

AcOH, yielding the crude product.

All derivatisation reactions were performed in 5 mL

Teﬂon-lined screw-cap tubes. To each disaccharide

sample (500 lg) was added DMSO (200 lL) and MeI

(50 lL), followed by a slurry of powdered NaOH in

DMSO (4 pellets of NaOH to 2 mL DMSO; 200 lL).

The suspension was sonicated for 30 min, MeI (50 lL)

was added then the suspension was sonicated for a

further 30 min. The reaction was quenched with H₂O

(4 mL), extracted with CH₂Cl₂(2 mL), then the extract

washed with H₂O (2 · 1 mL) and dried at 30 ꢁC under a

stream of dry air. The samples were hydrolysed in TFA

(2 M, 0.25 mL, 1 h, 120 ꢁC). The sample was then evap-

orated at 40 ꢁC under a stream of dry air followed by the

addition and evaporation of toluene (1 mL). Reduction

was performed with NaBH₄(15 mg/mL in aqueous 1 M

NH₄OH, 0.25 mL, 1 h, 40 ꢁC). The reaction was quen-

4.5.5. Standard RuCl₃catalysed oxidation procedure.

Catalytic RuCl₃(0.02 mol equiv), K₂CO₃(1.2 mol equiv)

and NaIO₄(2.5 mol equiv) were added to the compound

in a mixture of CH₂Cl₂, MeCN and H₂O (1:1:1 v/v/v)

and stirred vigorously overnight at rt. After quenching

with propan-2-ol, the mixture was poured through a pad

of Celite, washing with CH₂Cl₂. The aqueous portion

was washed with CH₂Cl₂, the combined organic extracts

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

1869

were washed with brine, dried (MgSO₄), ﬁltered and the

solvents removed in vacuo to give the crude product.

(200 mg) was acetylated and FC (1:1 EtOAc–hexanes)

performed on the resulting residue gave the title com-

pound 25 (50 mg, 0.17 mmol, 6% from 31) as a colourless

23

D

4.5.6. Standard NaBH₄reduction procedure. To an ice-

cooled solution of the compound in THF and H₂O (5:1

v/v) was added NaBH₄(1.5 mol equiv) and the solution

was brought to rt and stirred for 3 h. The reaction

mixture was diluted with Et₂O, washed with H₂O and

the aqueous layer was backwashed with Et₂O and the

combined organic extracts were washed with brine,

dried (MgSO₄), ﬁltered and concentrated to give the

crude product.

oil; ½aꢀ +4.7 (c 0.9, CHCl₃); ¹H NMR (300 MHz,

CDCl₃): d 4.62 (dd, 1H, J 2.5, 7.8 Hz, H-1), 3.78 (dd,

1H, J 2.5, 2.5 Hz, H-2), 3.61 (s, 3H, OCH₃-5), 3.60 (s,

3H, OCH₃-4), 3.55 (s, 3H, OCH₃-6), 3.54 (s, 3H, OCH₃-

2), 3.52 (m, 1H, H-6), 3.47 (s, 3H, OCH₃-3), 3.43 (d, 1H,

J 9.5 Hz, H-4), 3.06 (dd, J 2.5, 9.5 Hz, H-3), 3.00 (dd,

1H, J 9.1, 9.1 Hz, H-5), 2.14 (s, 3H, CH₃); ¹³C NMR

(75 MHz, CDCl₃): d 170.9 (C@O), 85.6 (C-5), 83.2 (C-4),

82.4 (C-3), 81.2 (C-6), 77.2 (C-2), 74.4 (C-1), 61.6

(OCH₃-2), 61.3 (OCH₃-4 and OCH₃-5), 61.2 (OCH₃-6),

58.9 (OCH₃-3), 21.4 (CH₃); HR FAB^þ-MS (CS^þin

glycerol/MeOH): Calcd for C₁₃H₂₅O₇(M+H^þ) m=z

293.1600. Found 293.1607.

4.5.7. 1L-1-O-Acetyl-2,3,4,5,6-penta-O-methyl-chiro-ino-

sitol (24). The standard benzylation method applied to

31³¹(1.08 g, 3.0 mmol) gave 32 (310 mg, 0.7 mmol).

Compound 32 (290 mg, 0.6 mmol) was stirred in triﬂuoro-

acetic acid (5 mL) and H₂O (5 mL) overnight at rt. The

solvents were removed to give a syrup (250 mg) con-

taining 33, which was methylated using the standard

conditions and FC (1:4 EtOAc–hexanes) performed on

the crude product gave 34 (50 mg, 0.15 mmol) as an oil.

Compound 34 (50 mg, 0.15 mmol) was hydrogenolysed,

yielding crude 35 (40 mg). Crude 35 (40 mg) was acetyl-

ated and the crude residue obtained was subjected to

4.5.9. 1 -2-O-Acetyl-1,3,4,5,6-penta-O-methyl-chiro-ino-

L

sitol (26). Compound 31³¹(1.5 g, 4.2 mmol) was stirred

in acetic acid (7.5 mL) and H₂O (1.5 mL) for 4 h at rt.

After concentration, the reaction mixture was treated

with toluene (2 · 20 mL) and the solvents removed in

vacuo to give 42 (1.15 g, 4.2 mmol, quant.) as a white

solid. Methylation of 42 (1.15 g, 4.2 mmol) gave a resi-

due that was subjected to FC (1:2 EtOAc–hexanes) to

give 43 (1.39 g, 4.3 mmol) as an oil. Compound 43

(1.39 g, 4.3 mmol) in triﬂuoroacetic acid (7 mL) and H₂O

(35 mL) was stirred overnight at rt. The reaction mixture

was washed with hexanes (30 mL), concentrated to an

oil and toluene was added (30 mL) and the solvents were

removed in vacuo, the process being repeated with

MeCN (30 mL), to give 44 (0.96 g, 4.1 mmol) as an oil.

Compound 44 (390 mg, 1.7 mmol) was reﬂuxed with

bis(tributyltin) oxide (841 lL, 1.7 mmol) in toluene

(20 mL) under Dean Stark conditions for 1.5 h. The

reaction mixture was ice-cooled then BnBr (436 mg,

2.6 mmol) and tetrabutylammonium bromide (219 mg,

0.9 mmol) were added and the mixture was reﬂuxed

overnight under Ar. After cooling the solvents were re-

moved in vacuo and FC performed on the residue (1:2

EtOAc–hexanes) gave 45 (320 mg, 1.0 mmol). Methyl-

ation of 45 (150 mg, 0.46 mmol) gave a concentrate,

which was subjected to FC (1:2 EtOAc–hexanes) to give

46 along with traces of the tin reagent (190 mg) as an oil.

Crude 46 (190 mg) was subjected to hydrogenolysis to

give crude 47 (199 mg) as a yellow oil that was used

directly in the next step. Crude 47 (190 mg) was acetyl-

ated and FC performed on the residue obtained (1:2

FC (1:2 EtOAc–hexanes) to give the title compound 24

23

D

(27 mg, 0.09 mmol, 4% from 31) as an oil; ½aꢀ )41.0 (c

1

1.3, CHCl₃); H NMR (300 MHz, CDCl₃): d 5.45 (dd,

1H, J 3.7, 3.7 Hz, H-1), 3.66 (s, 3H, OCH₃-3), 3.61 (s,

3H, OCH₃-4), 3.51, 3.50 (s, 3H, OCH₃-2 and OCH₃-5),

3.43 (s, 3H, OCH₃-6), 3.37 (dd, 1H, J 3.4, 8.7 Hz, H-2),

3.31–3.24 (m, 4H, H3-H6), 2.10 (s, 3H, CH₃); ¹³C NMR

(75 MHz, CDCl₃): d 170.3 (C@O), 83.6 (C-3), 83.5 (C-4),

81.7 (C-5), 79.7 (C-2), 76.2 (C-6), 66.9 (C-1), 61.4

(OCH₃-3), 61.3 (OCH₃-4), 59.6, 59.2 (OCH₃-5 and

OCH₃-2), 58.8 (OCH₃-6), 21.4 (CH₃); HR FAB^þ-MS

(Cs^þin glycerol/MeOH): Calcd for C₁₃H₂₅O₇(M+H^þ)

m=z 293.1600. Found 293.1612.

4.5.8. 1 -1-O-Acetyl-2,3,4,5,6-penta-O-methyl-myo-ino-

L

sitol (25). RuCl₃catalysed oxidation of 31³¹(1.01 g,

2.8 mmol) gave 36 (710 mg, 2.0 mmol). NaBH₄reduction

of 36 (710 mg, 2.0 mmol) gave crude 37 (500 mg,

1.4 mmol) as an oil. Benzylation of 37 (500 mg,

1.4 mmol) gave crude 38 (500 mg) as an oil. Crude 38

(500 mg) in acetic acid (20 mL) and H₂O (5 mL) was

stirred at rt for 48 h. The reaction mixture was washed

with CH₂Cl₂and the organic extract was washed with

H₂O. The solvents were removed from the combined

aqueous extracts and then toluene was added and the

solvents removed to give crude 39 (300 mg). Crude 39

(300 mg, 1.1 mmol) was permethylated and FC (1:2

EtOAc–hexanes) performed on the residue obtained

gave 40 (130 mg, 0.46 mmol) as a colourless oil. Com-

pound 40 (130 mg, 0.46 mmol) was hydrogenolysed,

yielding crude 41 (200 mg). Crude compound 41

EtOAc–hexanes) gave the title compound 26 (127 mg,

23

0.43 mmol, 57% from 31) as an oil; ½aꢀ )52.4 (c 0.7,

D

CHCl₃); ¹H NMR (300 MHz, CDCl₃): d 5.00 (dd, 1H, J

3.0, 7.0 Hz, H-2), 3.78 (dd, 1H, J 3.0, 3.9 Hz, H-1), 3.70

(dd, 1H, J 2.6, 4.0 Hz, H-6), 3.61 (s, 3H, OCH₃-4), 3.54

(s, 3H, OCH₃-3), 3.51 (s, 3H, OCH₃-5), 3.47 (s, 3H,

OCH₃-6), 3.44 (s, 3H, OCH₃-1), 3.43 (m, 2H, H-4 and

H-3), 3.37 (m, 1H, H-5), 2.13 (s, 3H, CH₃); ¹³C NMR

1870

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

(75 MHz, CDCl₃): d 169.5 (C@O), 82.1 (C-4), 80.1 (C-3),

79.8 (C-5), 75.1 (C-1), 74.8 (C-6), 72.5 (C-2), 60.0

(OCH₃-4), 59.7 (OCH₃-3), 58.5 (OCH₃-1), 57.8 (OCH₃-

5), 57.7 (OCH₃-6), 20.2 (CH₃); HR FAB^þ-MS (Cs^þin

glycerol/MeOH): Calcd for C₁₃H₂₅O₇(M+H^þ) m=z

293.1600. Found 293.1609.

3.00 (dd, 1H, J 9.2, 9.2 Hz, H-5), 2.12 (s, 3H, CH₃); ¹³

C

NMR (75 MHz, CDCl₃): d 170.6 (C@O), 85.5 (C-5),

83.2 (C-4 and C-6), 80.6 (C-1 and C-3), 66.1 (C-2), 61.5

(OCH₃-5), 61.4 (OCH₃-6 and OCH₃-4), 58.4 (OCH₃-1

and OCH₃-3), 21.3 (CH₃); HR FAB^þ-MS (Cs^þin

glycerol/MeOH): Calcd for C₁₃H₂₅O₇(M+H^þ) m=z

293.1600. Found 293.1607.

4.5.10.

1D-4-O-Acetyl-1,2,3,5,6-penta-O-methyl-myo-

inositol (27). RuCl₃catalysed oxidation of 45 (183 mg,

0.56 mmol) gave crude 48 (162 mg, 0.50 mmol). Crude 48

(162 mg, 0.5 mmol) was reduced with NaBH₄to give 49

(122 mg, 0.37 mmol) as colourless crystals. Methylation

of 49 (122 mg, 0.37 mmol) gave 50 (63 mg, 0.19 mmol),

which was obtained as white crystals. Hydrogenolysis of

50 (63 mg, 0.19 mmol) gave crude 51 (65 mg) as a brown

oil. Acetylation of 51 (65 mg) gave crude 27 and the

crude material was then subjected to FC (1:1 EtOAc–

hexanes). The resulting solid was extracted with CH₂Cl₂

several times and the solvents were removed from the

4.5.12. 5-O-Acetyl-1,2,3,4,6-penta-O-methyl-myo-inositol

(29). NaH (37 mg, 60% w/w in oil, 0.92 mmol) and allyl

bromide (80 lL, 0.92 mmol) were added to a solution of

52³²(311 mg, 0.76 mmol) in DMF (10 mL) at 0 ꢁC and

the reaction was stirred overnight at rt. EtOH (5 mL)

was added to quench the reaction, the reaction mixture

was concentrated and FC performed on the concentrate

(1:1 EtOAc–hexanes) to give 57 (50 mg, 0.11 mmol) as a

white solid. Benzylation of 57 (50 mg, 0.11 mmol) gave

crude 58 (80 mg). p-Toluenesulfonic acid (30 mg,

0.15 mmol) and 10% Pd/C (20 mg) were added to a

solution of crude 58 (80 mg) in MeOH (4.5 mL) and

H₂O (1 mL) and the solution was reﬂuxed for 6 h. After

ﬁltration though Celite the solvents were removed from

the ﬁltrate to give crude 59 (40 mg). Standard perme-

thylation conditions were employed in the synthesis of

60 from crude 59 (40 mg). The residue obtained was

subjected to FC (1:1 EtOAc–hexanes) to give 60 (10 mg,

0.03 mmol) as a white solid. Hydrogenolysis gave crude

61 (5 mg) as a white solid. Acetylation of crude 61

(10 mg) gave a residue that was subjected to FC (1:1

EtOAc–hexanes) to give the title compound 29 (4 mg,

combined washings to give the title compound 27 (46 mg,

23

0.16 mmol, 29% from 45) as a pale yellow oil; ½aꢀ +1.1

D

(c 1.2, CHCl₃); ¹H NMR (300 MHz, CDCl₃): d 5.24 (dd,

1H, J 9.0, 9.0 Hz, H-4), 3.82 (dd, 1H, J 2.3, 2.3 Hz, H-2),

3.53 (s, 3H, OCH₃-6), 3.52 (s, 3H, OCH₃-2), 3.48 (dd,

1H, J 9.6, 9.6 Hz, H-6), 3.44 (s, 3H, OCH₃-1), 3.42 (s,

3H, OCH₃-5), 3.34 (s, 3H, OCH₃-3), 2.99 (dd, 1H, J 9.6,

9.6 Hz, H-5), 2.98 (obsc., 1H, H-3), 2.93 (dd, 1H, J 2.3,

9.6 Hz, H-1), 2.03 (s, 3H, CH₃); ¹³C NMR (75 MHz,

CDCl₃): d 169.0 (C@O), 82.1 (C-5), 81.5 (C-6), 81.3 (C-

1), 79.7 (C-3), 73.9 (C-2), 71.9 (C-4), 60.0 (OCH₃-2),

59.9 (OCH₃-6), 59.0 (OCH₃-5), 57.5, 57.2 (OCH₃-1 and

OCH₃-3), 20.1 (CH₃); HR FAB^þ-MS (Cs^þin glycerol/

MeOH): Calcd for C₁₃H₂₅O₇(M+H^þ) m=z 293.1600.

Found 293.1610.

1

0.01 mmol, 1% from 52) as a colourless solid; H NMR

(300 MHz, CDCl₃): d 4.84 (dd, 1H, J 9.7, 9.7 Hz, H-5),

3.86 (dd, 1H, J 2.4, 2.4 Hz, H-2), 3.62 (s, 3H, OCH₃-2),

3.52 (dd, 2H, J 9.7, 9.7 Hz, H-4 and H-6), 3.50 (s, 6H,

OCH₃-1 and OCH₃-3), 3.40 (s, 6H, OCH₃-4 and OCH₃-

6), 3.06 (dd, 2H, J 9.7, 2.4 Hz, H-1 and H-3), 2.12 (s, 3H,

CH₃); ¹³C NMR (75 MHz, CDCl₃): d 170.7 (C@O), 82.6

(C-1 and C-3), 81.0 (C-4 and C-6), 76.2 (C-2), 74.8 (C-5),

61.7 (OCH₃-2), 61.0 (OCH₃-4 and OCH₃-6), 59.1

(OCH₃-1 and OCH₃-3), 21.5 (CH₃); HR FAB^þ-MS (Cs^þ

in glycerol/MeOH): Calcd for C₁₃H₂₅O₇(M+H^þ) m=z

293.1600. Found 293.1586.

4.5.11. 2-O-Acetyl-1,3,4,5,6-penta-O-methyl-myo-inositol

(28). Bis-butane-diyl acetal 52 was prepared according

to the literature.³²Benzylation of 52 and FC (1:1

EtOAc–hexanes) performed on the concentrated reac-

tion mixture gave 53 (180 mg, 0.36 mmol) as a white

solid. Compound 53 (180 mg, 0.36 mmol) was stirred in

triﬂuoroacetic acid (5 mL) and H₂O (5 mL) at rt over-

night. The reaction solution was washed with hexanes

(30 mL) and concentrated to an oil, toluene (30 mL) was

added and evaporated, followed by MeCN (30 mL) to

give crude 54 (100 mg) as a solid. Permethylation of

crude 54 (100 mg) gave 55 (40 mg, 0.12 mmol) as a

crystalline solid. Hydrogenolysis of 55 (40 mg,

0.12 mmol) gave crude 56 (50 mg) as a yellow oil.

Acetylation and FC performed on the crude product

(1:1 EtOAc–hexanes) gave the title compound 28 (28 mg,

0.10 mmol) as a white solid; ¹H NMR (300 MHz,

CDCl₃): d 5.69 (dd, 1H, J 2.8, 2.8 Hz, H-2), 3.66 (s, 3H,

OCH₃-5), 3.60 (s, 6H, OCH₃-4 and OCH₃-6), 3.42 (s,

6H, OCH₃-1 and OCH₃-3), 3.34 (dd, 2H, J 9.2, 9.2 Hz,

H-4 and H-6), 3.04 (dd, 2H, J 2.8, 9.2 Hz, H-1 and H-3),

4.5.13.

1D-3-O-Acetyl-1,2,4,5,6-penta-O-methyl-chiro-

inositol (30). Benzylation of 21 (2.0 g, 7.3 mmol) gave a

residue that was subjected to FC (1:30 EtOAc–hexanes)

to give 62 (2.4 g, 6.7 mmol). Compound 62 (2.03 g,

5.6 mmol) was kept in triﬂuoroacetic acid (10 mL) and

H₂O (10 mL) at rt for 6 h. The solvents were removed in

vacuo and co-evaporated with toluene and the residue

was subjected to FC (EtOAc) to give 63 (1.2 g, 4.6 mmol)

as a white solid. Permethylation of 63 (600 mg,

2.1 mmol) gave a crude product, which was subjected to

FC (1:3 EtOAc–hexanes) to give 64 (560 mg, 1.6 mmol)

as an oil. Hydrogenolysis of 64 (560 mg, 1.6 mmol) gave

65 (ca. 700 mg) as an orange oil. Acetylation gave the

J. B. Hart et al. / Carbohydrate Research 339 (2004) 1857–1871

1871

7. Kennington, A. S.; Hill, C. R.; Craig, J.; Bogardus, C.;

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1426.

crude product, which was subjected to FC (1:2 EtOAc–

hexanes), redissolved in CH₂Cl₂and passed through a

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pound 30 (290 mg, 0.99 mmol, 36% from 21) as a col-

23

D

ourless oil; ½aꢀ +43.5 (c 1.0, CHCl₃); ¹H NMR

(300 MHz, CDCl₃): d 5.19 (dd, 1H, J 9.4, 9.4 Hz, H-3),

3.78 (dd, 1H, J 2.9, 4.0 Hz, H-1 or H-6), 3.73 (dd, J 2.9,

4.0 Hz, H-6 or H-1), 3.51, 3.50, 3.49, 3.48, 3.41 (OCH₃-

6, OCH₃-5, OCH₃-4, OCH₃-2 and OCH₃-1), 3.78–3.46

(m, 3H, H-4, H-5 and H-2), 2.10 (s, 3H, CH₃); ¹³C NMR

(75 MHz, CDCl₃): d 170.3 (C@O), 81.4 (C-4), 81.2 (C-5

or C-2), 79.9 (C-2 or C-5), 76.6 (C-6 or C-1), 75.4 (C-1

or C-6), 73.1 (C-3), 60.4, 59.1, 58.8 (OCH₃-4, OCH₃-2

and OCH₃-5), 59.9, 59.6 (OCH₃-1 and OCH₃-6), 21.4

(CH₃); HR FAB^þ-MS (Cs^þin glycerol/MeOH): Calcd

for C₁₃H₂₅O₇(M+H^þ) m=z 293.1600. Found 293.1595.

11. Field, M. C. Glycobiology 1997, 7, 161–168.

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Support of this study by the European Commission

(FAIR CT 97-3142, programme NOFA), the BMBF

(NZL 95/007) and the Fonds der Chemischen Industrie

is gratefully acknowledged. New Zealand Pharmaceuti-

cals Ltd. is thanked for the donation of pinitol and Prof.

Roy Daniel from Waikato University for the gift of the

TP6-B1 b-galactosidase used for this study. The New

Zealand Foundation for Research, Science and Tech-

nology provided Public Good Science Funding for this

research through Industrial Research Ltd. as a CEO

NSOF grant (contract number CO8X0004-8) and

through the GlycoTherapeutics programme: A Diabetes

Therapeutic objective (contract number CO8X0209).

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Article Doi

Enzyme-catalysed synthesis of galactosylated 1D- and 1L-chiro-inositol, 1D-pinitol, myo-inositol and selected derivatives using the β-galactosidase from the thermophile Thermoanaerobacter sp. strain TP6-B1

DOI: 10.1016/j.carres.2004.05.016

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