Enzymatic procedure catalysed by lipase from Candida antarctica for the regioprotection-deprotection of glucosamine

Article abstract of DOI:10.1016/S0957-4166(99)00292-X

Both peracetyl α-glucosamine 3 and its β-epimer 4 undergo alcoholysis with butanol in THF, catalysed by lipase from Candida antarctica (Novozym 435) to afford the triester 6 and diester 9, respectively. Esterification of these compounds, using vinyl acetate in the presence of Novozym 435, gives the partial esters 10 and 11, bearing a deprotected hydroxyl function at C-4, ready for inversion of configuration to open a route in the preparation of galactosamine.

Full text of DOI:10.1016/S0957-4166(99)00292-X

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2891–2897
Enzymatic procedure catalysed by lipase from Candida
antarctica for the regioprotection–deprotection of glucosamine
a,∗
b
b
Giovanni Nicolosi, Carmela Spatafora and Corrado Tringali
a
†
Istituto CNR Studio Sostanze Naturali di Interesse Alimentare e Chimico-Farmaceutico, Via del Santuario 110,
9
5028 Valverde CT, Italy
b
Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 Catania, Italy
Received 8 June 1999; accepted 12 July 1999
Abstract
Both peracetyl α-glucosamine 3 and its β-epimer 4 undergo alcoholysis with butanol in THF, catalysed by lipase
from Candida antarctica (Novozym 435) to afford the triester 6 and diester 9, respectively. Esterification of these
compounds, using vinyl acetate in the presence of Novozym 435, gives the partial esters 10 and 11, bearing a
deprotected hydroxyl function at C-4, ready for inversion of configuration to open a route in the preparation of
galactosamine. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
Aminosugars are an important class of natural compounds occurring as subunits in many biological
1
mediators, including heparin, an anticoagulant agent present in blood, or endotoxins, lipopolysacchari-
2
des of bacterial origin responsible for pathological effects, such as the ‘lethal septic shock’. The naturally
occurring 2-aminosugars are glucosamine 1 and the less widespread galactosamine 2.
∗
†
Corresponding author. Tel: 0039-0957212136; fax: 0039-0957212141; e-mail: nicolosi@issn.ct.cnr.it
Associated to the National Institute for the Chemistry of Biological Systems, CNR.
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00292-X

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Hydrolases are known to be valid catalysts in the selective hydrolysis or esterification of
3
carbohydrates. Nevertheless, only in a few cases has this methodology been applied to aminosugars.
Among their successful uses, we recall here the hydrolysis of the ester function at C-1 for both peracetyl
β-glucosamine and peracetyl β-mannosamine by the use of porcine pancreas lipase in DMF/phosphate
4
buffer. Hydrolysis of the acetate group at C-6 has been obtained for peracetyl α-glucosamine, in the
5
presence of an esterase obtained from Rodosporidium toruloides but not commercially available. On the
other hand, esterification at the same position has been obtained for N-acetyl-D-mannosamine working
6
with various proteases in organic solvents.
In order to have access to a series of selectively protected derivatives of glucosamine 1 we examined
alcoholysis of the relevant peracetates using Novozym 435, a commercially available lipase from
Candida antarctica immobilized on matrix, possessing good stereoselectivity.
2. Results and discussion
In a preliminary screening, peracetyl glucosamine [mixture of (+)-α-(3) and (+)-β-(4)] was subjected
to alcoholysis with butanol in THF for 24 h, in the presence of one of the following lipases, namely from
Aspergillus niger, Aspergillus orizae, Candida antarctica type A, Candida rugosa, Mucor javanicus,
Mucor miehei, Pseudomonas cepacia, Rizophus javanicus and porcine pancreas lipase. Among them,
only lipase from Candida antarctica (Novozym 435) proved to be active and was therefore selected
for a closer examination of the regioselectivity of the reaction on the glucosamine epimers, peracetyl
α-glucosamine 3 and peracetyl β-glucosamine 4.
2.1. Enzymatic alcoholysis of peracetyl α-glucosamine 3
Ester 3, subjected to Novozym catalyzed alcoholysis with butanol, afforded a single product after
1
4
8 h which was isolated by chromatography in 35% yield, and subsequently identified by H NMR
spectroscopy as 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-glucopyranoside 5.
When alcoholysis was protracted for 96 h, along with 5 (yield 36%), a second compound was isolated
(yield 24%), and identified as 2-acetamido-1,3-di-O-acetyl-2-deoxy-α-D-glucopyranoside 6. Since we
have never observed the presence, among the reaction products, of the partial ester deriving from
hydrolysis at C-4, it is apparent that 6 originates from partial ester 5, in which position C-4 is more
accessible following the removal of the acetate group at C-6.

G. Nicolosi et al. / Tetrahedron: Asymmetry 10 (1999) 2891–2897
2893
Further prolongation of the reaction time (10 days) resulted in the complete conversion of the starting
material into 6, isolated by column chromatography in 96% yield. Hydrolysis does not proceed beyond
the formation of 6 even after longer incubation.
2.2. Enzymatic alcoholysis of peracetyl β-glucosamine 4
When the alcoholysis was carried out on 4 under the same conditions used for the epimer 3, compound
7
was isolated after 48 h incubation as the main product (yield 32%), along with trace amounts of minor
1
products. The H NMR spectrum of 7, whose FABMS spectrum shows a quasi-molecular ion at 348 m/z,
contained signals for four acetate groups. The H-1 resonance showed a remarkable shift (∆δ=0.43 ppm),
as compared to the corresponding signal of 4; in addition, a coupling constant value of J =3.4 Hz, only
compatible with an axial–equatorial coupling, was measured, thus indicating that an inversion of the
configuration at C-1 had occurred. These data revealed a regioselective deprotection at C-1, establishing
1,2
7
7
as 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranoside.
When the incubation time was prolonged to four days, two additional compounds, 8 (yield 10%) and 9
(
yield 10%), were isolated in addition to 7 (yield 31%) from the reaction mixture. Spectroscopic analysis
1
(FABMS and H NMR) established 8 as an isomer of 5, arising from deprotection of the ester functions
at C-4 and C-6 of the parent compound, and therefore it was assigned the structure of 2-acetamido-1,3-
di-O-acetyl-2-deoxy-β-D-glucopyranoside.
1
In the H NMR spectrum of the second minor product, 9, decidedly more polar than 7 and 8, only
two signals for acetate groups were present and in the low-field region significant shifts were seen
for the signals of H-1 (∆δ=0.61 ppm), H-4 (∆δ=1.51) and H-6 (∆δ=0.39 ppm) in comparison with
the corresponding peaks in the spectrum of 4. Moreover, the coupling constant measured for the H-
1
resonance (4.99 ppm, d, J =3.5 Hz) also indicated in this case an inversion of the configuration
1,2
at C-1 and consequently 9 could be assigned the structure of 2-acetamido-3-O-acetyl-2-deoxy-α-D-
glucopyranoside.
From the data above it can be inferred that in the alcoholysis of 4, Novozym has a moderate
regioselectivity and two compounds 7 and 8 are initially formed. These, which bear ester functions on
carbons of suitable configuration, become substrates for the enzyme and are converted into 9. This was
the only product (yield 95%) when the reaction was protracted for 10 days.

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Table 1
Enzymatic alcoholysis with butanol of 3 and 4a
For the sake of an easy comparison, the pertinent data for the enzymatic alcoholysis of 3 and 4 are
summarised in Table 1.
2.3. Enzymatic esterification of 6 and 9
In order to prepare a partial ester of glucosamine with a single unprotected hydroxyl at C-4, not
accessible by alcoholysis, compound 6 was esterified in THF with vinyl acetate in the presence of
Novozym. The reaction was very fast, being completed in 15 min, to give the desired 2-acetamido-1,3,6-
tri-O-acetyl-2-deoxy-α-D-glucopyranoside 10, whose structure was confirmed by spectroscopic analysis.
Formation of peracetyl α-glucosamine was not observed, even after protracted incubation. This can
be explained by two concurrent factors: lower reactivity of the secondary hydroxyl and steric hindrance
after the esterification of the primary hydroxyl.
Under the conditions above, compound 9 undergoes a rapid (15 min) conversion, affording a single
product (yield 95%) which, on the basis of spectral data, was assigned the structure of 2-acetamido-3,6-
di-O-acetyl-2-deoxy-α-D-glucopyranoside 11. In this case the α-configuration of the C-1 hydroxyl does
not allow enzymatic recognition, and a derivative with both C-1 and C-4 free hydroxyl is obtained.

G. Nicolosi et al. / Tetrahedron: Asymmetry 10 (1999) 2891–2897
2895
Compounds 10 and 11 can undergo inversion of configuration at C-4 according to standard
5,8
procedures and are useful synthetic precursors of galactosamine, a compound more valuable than its
epimer, glucosamine.
3. Conclusions
The results obtained show that the commercially available lipase Candida antarctica (Novozym 435)
can be used profitably to catalyse alcoholysis with butanol of both peracetyl α-glucosamine and peracetyl
β-glucosamine. The reaction is regioselective and its course is related to the configuration of the C-1
carbon atom. The α-epimer 3, and the β-epimer 4, undergo preferential alcoholysis of the ester group at
C-6 and C-1, respectively. Under forcing conditions the reaction causes complete removal of the acetates
at C-4,6 for compound 3. The epimer 4, subjected to prolonged incubation, undergoes partial deprotection
at C-4,6 and at C-1,4,6, this latter product being the only one when the reaction is prolonged up to the
exhaustion of the substrate.
The primary hydroxyl in partial esters 6 and 9 can be easily acetylated with vinyl acetate in the
presence of Novozym to give compounds 10 and 11, useful as starting materials for the preparation
of galactosamine.
4. Experimental
4.1. Materials and methods
1H NMR spectra were recorded at 250 MHz on a Bruker AC 250 instrument. Mass spectra (FAB) were
recorded on a Kratos MS 50 instrument using dinitrobenzyl alcohol as matrix. Optical rotations were
measured at 25°C on a Jasco 135 instrument. Melting points were determined using a Kofler apparatus.
All reactions were monitored by thin-layer chromatography (TLC) carried out on Merck 60 F254 plates
9
and using anisaldehyde–sulfuric acid as chromogenic reagent. Column chromatography was on a Si gel
(
Merck, 0.063–0.200 mm, gradient of THF in Et O as the eluent).
2
Lipases from Aspergillus niger, Aspergillus orizae, Candida rugosa, Mucor javanicus, Rizophus
javanicus and porcine pancreas lipase were purchased from Sigma. Pseudomonas cepacia lipase was
a gift from Amano Co. Candida antarctica and Mucor miehei lipases were a gift from Novo Nordisk.
4.2. Preparation of peracetyl α-glucosamine 3 and peracetyl β-glucosamine 4
The epimeric peracetyl derivatives 3 and 4 were prepared according to standard procedures.10 The
5
products were recrystallised from ethanol to give pure peracetyl α-glucosamine 3, mp 135–136°C (lit.
11
1
36–137°C); [α] +89.4 (c 1, CHCl ) (lit. [α] +92.0), and peracetyl β-glucosamine 4, mp 185–188°C
D
3
D
10
10
(
lit. mp 187°C), [α] +1.2 (c 1, CHCl ) (lit. [α] +1.0).
D
3
D
4.3. Enzymatic alcoholysis of 3 and 4
Butanol (3 mL) and Novozym 435 (3 g) were added to a solution of the substrate (1 g of 3 or 4) in
THF (40 mL) and the mixture was shaken (300 rpm) at 45°C. At the end of the incubation (96 h) reaction
was quenched by filtering off the enzyme and the filtrate was evaporated in vacuo.

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G. Nicolosi et al. / Tetrahedron: Asymmetry 10 (1999) 2891–2897
4.3.1. Chromatographic purification of the residues from 3 gave 5 and 6
(
+)-2-Acetamido-1,3,4-tri-O-acetyl-2-deoxy-α-D-glucopyranoside 5: oil (320 mg, yield 36%); [α]
D
5
1
+
61.7 (c 1, CHCl ) (lit. [α] +57.0). FABMS and H NMR data are in agreement with those reported in
3
D
5
the literature. (+)-2-Acetamido-1,3-di-O-acetyl-2-deoxy-α-D-glucopyranoside 6: white solid (186 mg,
1
yield 24%), mp 167–170°C, [α] +80.1 (c 1, CHCl ). FABMS and H NMR data are in agreement with
D
3
5
those reported in the literature.
4.3.2. Chromatographic purification of the residues from 4 gave 7, 8 and 9
(
+)-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranoside 7: oil (276 mg, yield 31%), [α]
D
4
1
+
52.2 (c 1, CHCl ). FABMS and H NMR data are in agreement with those reported in the literature.
3
(
−)-2-Acetamido-1,3-di-O-acetyl-2-deoxy-β-D-glucopyranoside 8: oil (80 mg, yield 10%) [α] −8.8 (c
D
1
1
1
5
, CHCl ). H NMR (250 MHz, CDCl ): 1.92 (s, 3H, NHAc), 2.09 (s, 6H, 3-Ac and 4-Ac), 2.11 (s, 3H,
3
3
-Ac), 3.58 (m, 1H, H-5), 3.77–3.88 (m, 3H, H-4 and H-6), 4.35 (ddd, J=10.0, 9.6, 8.7 Hz, 1H, H-2),
.10 (dd, J=10.0, 9.5 Hz, 1H, H-3), 5.69 (d, J=8.7 Hz, 1H, H-1), 6.52 (d, J=9.6 Hz, 1H, NH). FABMS:
+
[M+H] at m/z 306. Anal. calcd for C H NO : C, 47.21; H, 6.27; N, 4.59; found: C, 47.10; H, 6.22;
12
19
8
N, 4.60. (+)-2-Acetamido-3-O-acetyl-2-deoxy-α-D-glucopyranoside 9: solid (70 mg, yield 10%), mp
1
1
3
55–157°C, [α] +22.4 (c 1, EtOH). H NMR (250 MHz, CD OD): 1.93 (s, 3H, NHAc), 2.01 (s, 6H,
D
3
-Ac and 4-Ac), 3.58 (dd, J=9.7, 9.6 Hz, 1H, H-4), 3.78 (m, 2H, H-6), 3.86 (m, 1H, H-5), 4.06 (dd,
J=10.7, 3.5 Hz, 1H, H-2), 5.08 (d, J=3.5 Hz, 1H, H-1), 5.20 (dd, J=10.7, 9.7 Hz, 1H, H-3). FABMS:
+
[
M+H] at m/z 264. Anal. calcd for C H NO : C, 45.63; H, 6.51; N, 5.32; found: C, 44.50; H, 6.45; N,
10 17 7
5.36.
4.4. Enzymatic esterification of 6 and 9
Novozym 435 (700 mg) was added to a solution of the substrate (200 mg, 0.07 mmol of 6; or 0.07
mmol of 9) in THF (20 mL) containing vinyl acetate (0.4 mL, 0.5 mmol) and the suspension was kept at
5°C under stirring (300 rpm). After 15 min the reaction was quenched, the enzyme filtered off, and the
filtrate was taken to dryness.
4
4.4.1. Chromatographic purification of the residues from 6 gave 10
(
+)-2-Acetamido-1,3,6-tri-O-acetyl-2-deoxy-α-D-glucopyranoside 10: oil, (215 mg, yield 96%), [α]
D
5
1
+
60.3 (c 1, CHCl ) (lit. [α] +62.0). FABMS and H NMR data are in agreement with those reported
in the literature.
3
D
5
4.4.2. Chromatographic purification of the residues from 9 gave 11
(
+)-2-acetamido-3,6-di-O-acetyl-2-deoxy-α-D-glucopyranoside 11: oil (220 mg, yield 95%), [α]
D
1
+
33.5 (c 1, EtOH). H NMR (250 MHz, CD OD): 1.92 (s, 3H, NHAc), 2.03 (s, 6H, 3-Ac and 4-Ac),
3
2.05 (s, 3H, 1-Ac), 3.53 (dd, J=9.8, 9.3 Hz, 1H, H-4), 4.02–4.09 (m, 2H, H-2 and H-5), 4.22 (dd, J=11.8,
5
.2 Hz, 1H, H-6), 4.35 (dd, J=11.8, 2.2 Hz, 1H, H-6), 5.03 (d, J=3.5 Hz, 1H, H-1), 5.18 (dd, J=10.7,
+
9.3 Hz, 1H, H-3). FABMS: [M+H] at m/z 306. Anal. calcd for C H NO : C, 47.21; H, 6.27; N, 4.59;
12
19
8
found: C, 47.08; H, 6.24; N, 4.63.

G. Nicolosi et al. / Tetrahedron: Asymmetry 10 (1999) 2891–2897
2897
Acknowledgements
The authors gratefully acknowledge Dr. Alfio Guglielmino for his assistance during the preparation
of his degree thesis. This work was financially supported by the CNR ‘Target Project on Biotechnology’
(Roma, Italy) and by the MURST (PRIN, Roma, Italy) as well as by a grant from the Università degli
Studi di Catania (Progetti di Ricerca di Ateneo, Catania, Italy).
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