Regioselective lipase acylation as a useful tool for separation and selective protection of β-D-Gal(1→4)-D-GlcNAc and β-D-Gal(1→3)-D-GlcNAc disaccharides

Article abstract of DOI:10.1016/S0957-4166(00)00342-6

Supported lipase from Candida antarctica (Chirazyme) was employed for a regioselective protection of the 2-azido derivatives 1 and 2, synthetic equivalents of β-D-Gal(1→3)-D-GlcNAc and β-D-Gal(1→4)-D-GlcNAc (N-acetyl lactosamine), respectively. The selectivity of the enzyme towards 1 and 2 was also exploited for an easy separation of the mixture of the two compounds obtained from a straightforward synthetic approach. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(00)00342-6

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 11 (2000) 3647–3651
Regioselective lipase acylation as a useful tool for
separation and selective protection of
b-
D
-Gal(1“4)-
D
-GlcNAc and b-
D
-Gal(1“3)- -GlcNAc
D
disaccharides†
Barbara La Ferla,a,* Luigi Lay,a Giovanni Russoa and Luigi Panzab
aUniversita` degli Studi di Milano, Dipartimento di Chimica Organica e Industriale,
Centro di Studio sulle Sostanze Organiche Naturali del CNR, via G. Venezian, 21–20133 Milano, Italy
bUniversita` degli Studi del Piemonte Orientale, Facolta` di Farmacia, Dipartimento di Scienze Chimiche, Alimentari,
Farmaceutiche e Farmacologiche (DISCAFF), Viale Ferrucci, 33–28100 Novara, Italy
Received 21 July 2000; accepted 28 August 2000
Abstract
Supported lipase from Candida antarctica (Chirazyme®) was employed for a regioselective protection of
the 2-azido derivatives 1 and 2, synthetic equivalents of b-
D
-Gal(1“3)-
D
-GlcNAc and b-
D
-Gal(1“4)-D-
GlcNAc (N-acetyl lactosamine), respectively. The selectivity of the enzyme towards 1 and 2 was also
exploited for an easy separation of the mixture of the two compounds obtained from a straightforward
synthetic approach. © 2000 Elsevier Science Ltd. All rights reserved.
A convenient approach to the preparation of complex carbohydrates is based on convergent
block synthesis. Therefore, it is particularly interesting to have access to selectively protected
building blocks to be used as donors or acceptors for assembly into more complex structures.
The disaccharides b-
D
-Gal(1“4)-
D
-GlcNAc (N-Acetyl lactosamine) and b-
D
-Gal(1“3)- -Glc-
D
NAc are among the most important components of glycoproteins and glycolipids, which play a
central role in many cellular recognition phenomena. Moreover, they are contained in human
milk oligosaccharides which exert a protective action on breast fed infants towards many
infectious diseases. The great biological relevance of these disaccharides, combined with their
scarce availability and extremely high cost, has stimulated many groups in the search of
convenient and easy strategies for their synthesis.1–8 Among other approaches, there has been a
growing interest in the use of lipases and proteases, in both hydrolysis and transesterification
reactions, as a tool for selective protection and deprotection of carbohydrates.9 We recently
* Corresponding author.
†
In memory of Prof. A. Verbert.
0957-4166/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00342-6

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B. La Ferla et al. / Tetrahedron: Asymmetry 11 (2000) 3647–3651
achieved the regioselective enzymatic acylation of lactose building blocks, which were used for
the synthesis of higher oligosaccharides.10
Our interest towards both human milk oligosaccharides and enzymatic acylation of carbo-
hydrates, induced us to exploit our chemo-enzymatic approach for the preparation of a variety
of differently protected building blocks related to the title disaccharides in order to have access
to a family of higher oligosaccharides for biological testing.
Previous observations revealed that 2-acetamido sugars are poor substrates for lipases.
Therefore the behavior of these two disaccharides towards enzymatic acylation was explored on
the corresponding 2-azido derivatives. The 2-azido derivatives 1 and 2, which are synthetic
equivalents of b-
D
-Gal(1“3)-
D
-GlcNAc and b-
D
-Gal(1“4)- -GlcNAc, respectively, were sepa-
D
rately submitted to selective enzymatic acetylation using lipase from Candida antarctica and
vinyl acetate as acylating agent in different solvents. Interesting and unexpected results are
reported in Table 1.
Table 1
Enzymatic selective acylation of disaccharides 1 and 2
Entry Compound Conditions
°C Time (h)
Products Acylation position
Yield %
1
2
1
1
THF, vinylacetate
CH3CN, vinylacetate
40 24
40 24
3
3
4
5
9
9
6
6%-OAc
6%-OAc
6,6%-OAc
6,2%,6%-OAc
6%-OAc
90
8
70
16
90
90
90
3
4
5
2
2
3
THF, vinylacetate
CH3CN, vinylacetate
CH3CN,
45 40
45 40
6%-OAc
6%-OAc, 6-OCOCH2Cl
28
4
vinylchloroacetate
CH3CN,
vinylchloroacetate
6
3
35
1
6
7
8
6%-OAc, 6-OCOCH2Cl
67
29
91
6%-OAc,
6,2%-OCOCH2Cl
6%,6-OAc,
7
4
CH3CN,
40 12
vinylchloroacetate
2%-OCOCH2Cl
Compound 2 was selectively acetylated at the 6%-position, both in THF and in acetonitrile, in
90% yield, as already observed for the benzyl lactoside,11 but only if the reaction was effected
at 40–45°C; at lower temperatures it did not react. Compound 1 behaved differently to
compound 2. In fact, the acetylation reaction of 1 carried out in THF at 40°C (entry 1) afforded
only the 6%-monoacetylated compound 3 in a shorter time than compound 2 (entry 3). Moreover,
on switching the solvent from THF to CH3CN (entry 2) the reaction did not stop after the
expected acetylation of the 6%-position, but a second selective acetylation at the 6-position
occurred, affording 4 as the main product. A small amount of the 6,6%,2%-tri-O-acetyl derivative
5 was also recovered. This surprising behavior has not previously been observed, as lipases
usually acylate only the primary hydroxyl on the non-reducing end on a disaccharide. This result
was exploited for a straightforward selective differential protection of the two primary hydroxyl
groups, which can be chemically achieved only in many steps. By effecting the first acetylation
in THF we obtained the 6%-O-acetyl derivative 3 (Scheme 1). Compound 3 was then allowed to
react with a different acylating agent (vinylchloroacetate) in acetonitrile affording 6 through a
second selective acylation at position 6. The temperature control was crucial for a successful

B. La Ferla et al. / Tetrahedron: Asymmetry 11 (2000) 3647–3651
3649
reaction. In fact, when the temperature is kept around 28–29°C only compound 6 (entry 5) was
formed, while at higher temperature (35°C) (entry 6) a partial third acylation at position 2%
provides compound 6 along with compound 7. Taking advantage of these observations, we were
able to introduce the chloroacetyl group selectively at position 2% of compound 4 to obtain 8 in
high yield (entry 7).
Scheme 1. Selective enzymatic acylation. Reagents and conditions: (a) THF, vinyl acetate, 40°C, 24 h; (b) CH3CN,
vinyl acetate, 40°C, 24 h; (c) CH3CN, vinyl chloroacetate, 28°C, 4 h; (d) CH3CN, vinyl chloroacetate, 25°C, 1 h; (e)
CH3CN, vinyl chloroacetate, 40°C, 12 h; (f) THF, vinyl acetate, 45°C, 40 h; (g) CH3CN, vinyl acetate, 45°C, 40 h
We also exploited the different reactivity of lipase from Candida antarctica to develop a
practical route to the 2-azido derivatives 1 and 2. A quick approach to the synthesis of
compounds differentially protected at the anomeric position, described by Schmidt et al.,12 was
based on the glycosylation of diol 10 with the galactosyl trichloroacetamidate 12. The reaction
(Scheme 2) afforded a 4/5 mixture of compounds 13 and 14, which were separated only by
tedious medium pressure chromatography. Applying a similar synthetic strategy with acceptor
11, in which we put an acetyl group on position 6 in place of the benzyl group used by Schmidt,
we obtained compounds 15 and 16 in a ratio of 85/15, and a 76% yield (based on the recovered
acceptor). This mixture was submitted to a first crude chromatographic separation, which
afforded 32% of pure 15 and 44% of a 2.8/1 mixture of 15 and 16 as estimated by NMR. The
mixture was deacetylated and submitted to enzymatic acetylation using vinyl acetate as acylating
agent in THF at 35°C for 5 days. Using the above conditions, the more reactive compound 1
was acetylated, whereas compound 2 remained unreacted.13 The reaction mixture was now easily
separated using standard flash chromatography: from compound 1 the 6%-monoacetylated 314
was isolated in 72% yield together with a little amount of 6,6%-diacetylated 4 (25%), whereas pure
compound 2 was recovered unreacted (Table 2).
Scheme 2. Synthesis of compounds 1 and 2

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B. La Ferla et al. / Tetrahedron: Asymmetry 11 (2000) 3647–3651
Table 2
Separation of a mixture of disaccharides 1 and 2 by selective acylation
Compound
Conditions
Product
Yield %a
1+2
(2.8:1)
THF, vinylacetate,
Candida antartica,
35°C, 5 days
6,6%-OAc 4
6%-OAc 3
Unreacted 2
25
72
90
a The yield is calculated considering only the relevant starting compound.
In conclusion the above technique allows the regioselective introduction of different acyl
protecting groups on the title disaccharides. In particular compound 1 showed to be highly
versatile allowing the regioselective protection at position 6%, 6,6% or 6,6%,2% by tuning the reaction
conditions through the reactivity of the acylating agent and the proper choice of the solvent.
Moreover, exploiting the intrinsic different reactivity of the disaccharides 1 and 2 it was possible
to selectively acylate only compound 1, thus allowing their otherwise difficult separation. Such
disaccharidic building blocks, regioselectively protected in positions 6%, 6,6% or 6,6%,2% by lipase
catalyzed acylation, will be further elaborated and used for complex oligosaccharide synthesis.
Acknowledgements
This work was supported by EU-NOFA project (grant FAIR CT973142).
References
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2. Toepfer, A.; Schmidt, R. R. Carbohydr. Res. 1993, 247, 159–164.
3. Zhu, X. X.; Ding, P. Y.; Cai, M. S. Tetrahedron Lett. 1996, 37, 8549–8552.
4. Wrodnigg, T. M.; Stutz, A. E. Angew. Chem., Int. Ed. 2000, 38, 827–828.
5. Kimura, T.; Takayama, S.; Huang, H.; Wong, C. H. Angew. Chem., Int. Ed. Engl. 1996, 35, 2348–2350.
6. Li, J.; Wang, P. G. Tetrahedron Lett. 1997, 38, 7967–7970.
7. Vic, G.; Tran, C. H.; Scigelova, M.; Crout, D. H. G. Chem. Commun. 1997, 169–170.
8. Fang, J. W.; Xie, W. H.; Li, J.; Wang, P. G. Tetrahedron Lett. 1998, 39, 919–922.
9. Waldmann, H.; Sebastian, D. Chem. Rev. 1994, 94, 911.
10. La Ferla, B.; Lay, L.; Poletti, L.; Panza, L.; Russo, G. J. Carb. Chem. 2000, 19, 331–343.
11. Unpublished results.
12. Toepfer, A.; Kinzy, W.; Schmidt, R. R. Liebigs Ann. Chem. 1994, 449–464.
13. Only traces of the 6%-monoacetylated compound 9, derived from 2, were detected by TLC.
14. 1H NMR diagnostic data: 3 (MeOD, 300 MHz): l (ppm) 4.58 (d, 1H, J1,2=7.5 Hz, H-1), 4.44 (d, 1H, J1%,2%=7.5
Hz, H-1%), 4.31 (dd, 1H, J6%a,6%b=11.6, J5%,6%b=8.3 Hz, H-6%b), 4.19 (dd, 1H, J6%a,6%b=11.6, J5%,6%a=4.5 Hz,
H-6%a), all other protons have a l<3.90 ppm. 4 (MeOD, 300 MHz): l (ppm) 4.59 (d, 1H, J1,2=7.1 Hz, H-1), 4.44
(d, 1H, J1%,2%=7.1 Hz, H-1%), 4.40 (dd, 1H, J6%a,6%b=11.4, J5%,6%b=1.4 Hz, H-6%b), 4.31 (dd, 1H, J6%a,6%b=11.4,
J5%,6%a=8.4 Hz, H-6%a), 4.23 (dd, 1H, J6a,6b=11.7, J5,6b=4.2 Hz, H-6b), 4.17 (dd, 1H, J6a,6b=11.7,
J5,6a=7.2 Hz, H-6a), all other protons have a l<3.90 ppm. 6 (MeOD, 300 MHz): l (ppm) 4.61 (d, 1H, J1,2=7.1
Hz, H-1), 4.53 (dd, 1H, J6%a,6%b=11.5, J5%,6%b=1.6 Hz, H-6%b), 4.44 (d, 1H, J1%,2%=7.0 Hz, H-1%), 4.32 (dd, 1H,
J6%a,6%b=11.5, J5%,6%a=7.6 Hz, H-6%a), 4.27 (dd, 1H, J6a,6b=11.7, J5,6b=5.3 Hz, H-6b), 4.19 (dd, 1H,
J6a,6b=11.7, J5,6a=4.4 Hz, H-6a), 4.18 (s, 2H, CH2Cl), all other protons have a l<3.90 ppm. 7 (MeOD, 300

B. La Ferla et al. / Tetrahedron: Asymmetry 11 (2000) 3647–3651
3651
MHz): l (ppm) 5.12 (dd, 1H, J2%,3%=9.4, J1%,2%=8.3 Hz, H-2%), 4.70–4.15 (m, 10H, H-1,1%,6a,6b,6%a,6%b, 2CH2Cl),
all other protons have a l<3.90 ppm. 8 (CDCl3, 300 MHz): l (ppm) 5.12 (dd, 1H, J2%,3%=9.3, J1%,2%=8.2 Hz,
H-2%), 4.53 (d, 1H, J1,2=8.0 Hz, H-1), 4.49 (d, 1H, J1%,2%=7.1 Hz, H-1%), 4.44–4.27 (m, 3H, H-6%b,6%a,6b),
4.22–4.14 (m, 3H, H-6a, CH2Cl), all other protons have a l<4.0 ppm. 9 (MeOD, 300 MHz): l (ppm) 4.56 (d, 1H,
J1,2=7.7 Hz, H-1), 4.37 (d, 1H, J1%,2%=7.0 Hz, H-1%), 4.33 (dd, 1H, J6%a,6%b=11.7, J5%,6%b=8.6 Hz, H-6%b), 4.23
(dd, 1H, J6%a,6%b=11.7, J5%,6%a=4.0 Hz, H-6%a), all other protons have a l<3.90 ppm.
.