Improvement on lipase catalysed regioselective O-acylation of lactose: A convenient route to 2′-O-fucosyllactose

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IMPROVEMENT ON LIPASE CATALYSED
REGIOSELECTIVE O-ACYLATION OF LACTOSE:A
CONVENIENT RUOTE TO 2′-O- FUCOSYLLACTOSE
Anna Rencurosi a , Laura Poletti a , Luigi Panza b & Luigi Lay c
a Dipartimento di Chimica Organicae Industriale , Centro di Studio sulle Sostanze
Organiche Naturali del CNR , Università degli Studi di Milano , via G. Venezian, 21–20133,
Milano, Italy
b Facoltà di Farmacia, Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e
Farmacologiche (DISCAFF),Viale Ferrucci, 33–28100 , Università degli Studi del Piemonte
Orientale , Novara, Italy
c Facoltà di Farmacia, Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e
Farmacologiche (DISCAFF),Viale Ferrucci, 33–28100 , Università degli Studi del Piemonte
Orientale , Novara, Italy
Published online: 16 Aug 2006.
To cite this article: Anna Rencurosi , Laura Poletti , Luigi Panza & Luigi Lay (2001) IMPROVEMENT ON LIPASE CATALYSED
REGIOSELECTIVE O-ACYLATION OF LACTOSE:A CONVENIENT RUOTE TO 2′-O- FUCOSYLLACTOSE , Journal of Carbohydrate
Chemistry, 20:7-8, 761-765
To link to this article: http://dx.doi.org/10.1081/CAR-100108289
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J. CARBOHYDRATE CHEMISTRY, 20(7&8), 761–765 (2001)
COMMUNICATION
IMPROVEMENT ON LIPASE CATALYSED
REGIOSELECTIVE O-ACYLATION OF LACTOSE:
A CONVENIENT ROUTE TO 2ꢀ-O-
FUCOSYLLACTOSE1
Anna Rencurosi,1 Laura Poletti,1 Luigi Panza,2 and Luigi Lay,2,*
1Università 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
2Università degli Studi del Piemonte Orientale, Facoltà di Farmacia,
Dipartimento di Scienze Chimiche, Alimentari,
Farmaceutiche e Farmacologiche (DISCAFF),
Viale Ferrucci, 33–28100 Novara, Italy
Although enormous progress has been made in the past ten years, the synthesis of
complex carbohydrates still remains a severe problem. Conventional syntheses of
these molecules often dictate multiple protections and deprotections of various hy-
droxyl groups, dramatically increasing the number of steps required. On the other
hand, enzymes offer the opportunity to carry out cheap, highly chemo and regios-
elective transformations, providing new approaches to tackle many synthetic prob-
lems encountered in carbohydrate synthesis. In particular, lipases allow highly re-
gioselective O-acylations. Therefore, a growing interest in enzymatic manipulation
of protecting groups for the synthesis of oligosaccharides has been developed dur-
ing recent years.2
Recently, we reported on the use of lipase catalysed acylation for designing
useful building blocks in oligosaccharide synthesis.3,4 Excellent selectivity for 6ꢀ-
O-acylation was observed on various disaccharidic substrates (such as lactose,
maltose and cellobiose) using different acylating agents,3 Candida antarctica li-
pase being the best biocatalyst. However, our procedure contained a main draw-
back: the high polarity of the sugar substrates required the use of tert-amyl alcohol
as a solvent, whose high boiling point made the work up of the reaction mixture
quite laborious, thus rendering the method impractical. An important improvement
761
Copyright © 2001 by Marcel Dekker, Inc.
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Table 1. Experimental Conditions and Yields of Enzymatic Acylation Reactions
Yield
(%)
Entry
Substrate
Acylating Agent
Vinyl acetate
Conditions
Product
1
2
3
4
5
6
1
1
1
1
2a
2a
THF, 40°C, 3 days
CH3CN, 40°C, 4 days
THF, 40°C, 4 days
THF, 40°C, 2 days
CH3CN, 45°C, 4 days
CH3CN, 45°C, 5 days
2a
2a
2b
2c
3a
3b
73
64
83
78
65
62
Vinyl acetate
Trifluoroethyl levulinate
Vinyl chloroacetate
Trifluoroethyl levulinate
Vinyl chloroacetate
was achieved when we observed that when using relatively low boiling solvents
such as THF or CH3CN, the enzymatic acylations proceeded with equal efficiency,
despite the poor solubility of the substrates. For example, employing THF as a sol-
vent, we could introduce a chloroacetyl group at the 6ꢀ-OH of lactose with very
high regioselectivity and chemical yield (78%) and with a much easier product re-
covery.4 This finding prompted us to explore in more detail the behaviour of the
Candida antarctica lipase in different solvents and on various biologically relevant
substrates.5 In the present communication we show further achievements obtained
in the functionalization of lactose and a new straightforward synthesis of 2ꢀ-O-fu-
cosyllactose 7.6
In a preliminary screening of different organic solvents, THF and CH3CN
gave the best results. In THF, 6ꢀ-O-acylated lactosides were obtained regioselec-
tively from 17 at 40 °C in 73–83 % yields from different acylating agents with Can-
dida antarctica lipase8 (Table 1). The same reaction performed in CH3CN (Table
1, entry 2) showed less selectivity, giving, besides the 6ꢀ-O-monoacylated deriva-
tive as the main product, fair amounts (16%) of diacylated disaccharides. However,
when compound 2a (Scheme 1) was submitted to a second acylation with lipase
from Candida antarctica in CH3CN at 45 °C, an excellent regioselectivity for the
2ꢀ-OH was observed with both the acylating agents employed (Table 1, entries 5
and 6). All the reactions were very clean, without formation of by-products, al-
lowing full recovery of the unreacted substrate. Thus, a double sequential acyla-
tion, changing the solvent and using orthogonal protecting groups, provides an
easy access to a family of lactose building blocks.
We took advantage from our chemo-enzymatic approach to design a new
convenient route to the synthesis of 2ꢀ-O-fucosyllactose 7, which, after lactose,
was found to be the most abundant component of the human milk oligosaccharides
Scheme 1.

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763
in 77 % of Caucasian women classified as secretors.9 It seems reasonable that 2ꢀ-
O-fucosyllactose may play an important role in determining the antiadhesive prop-
erties of human milk oligosaccharides, which have been demonstrated to represent
a protection against infections for breast-fed infants during the lactation period.10
To the best of our knowledge, only three chemical syntheses of 2ꢀ-O-fuco-
syllactose have been reported so far, all of them relying on a tri-O-isopropylidene
lactose derivative as a key intermediate.11 However, our procedure offers some ad-
vantages, such as better overall yield and easier removal of the protective groups.
Our synthesis of the 2ꢀ-O-fucosyllactose was accomplished as follows. An acetyl
and a levulinoyl group were sequentially introduced on the benzyl lactoside 17 to
give compound 3a in 47% overall yield (95% conversion). After conventional
acetylation of the remaining hydroxyl groups and selective removal of levulinoyl
ester, acceptor 4 was glycosylated with the ꢁ-trichloroacetimidate of the 3,4 di-O-
acetyl-2-O-benzyl-L-fucopyranose 512 to give the protected 2ꢀ-O-fucosyllactose
6
13 in 70 % yield (Scheme 2).
The deprotection to 2ꢀ-O-fucosyllactose was very cleanly and easily accom-
plished by standard Zemplèn deacetylation and hydrogenolysis (Pd/C, H2,
MeOH/H2O, 24 h) of the remaining benzyls leading to 2ꢀ-O- fucosyllactose 7 in
quantitative yield (Scheme 2). The optical rotation ([ꢁ]D ꢂ52.3, c 0.6, H2O, after
three days) as well as 1H and 13C NMR data from compound 7 are in agreement
with those reported in the literature;6,14 1H NMR (300 MHz, D2O): ꢃ 5.51, H-1ꢄ
Scheme 2.

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RENCUROSI ET AL.
(ꢁ/ꢅ), J ꢆ 2.7 Hz, 5.43, H-1ꢁ, J ꢆ 3.7 Hz, 4.72, H-1ꢀ (ꢁ/ꢅ), J ꢆ 7.7 Hz, (the sig-
nal of H-1ꢅ is overlapped with the solvent peak at ꢃ 4.80–4.85); 13C NMR (75.44
MHz, D2O): ꢃ 103.11 (C-1ꢀ), 102.15 (C-1ꢄ), 98.72 (C-1ꢅ), 94.64 (C-1ꢁ).
In conclusion, we described a new and easy chemo-enzymatic synthesis of
the biologically relevant 2ꢀ-O-fucosyllactose exploiting a versatile lactose building
block, obtained by a double sequential acylation at 6ꢀ-OH and 2ꢀ-OH with two or-
thogonal protecting groups catalysed by lipase from Candida antarctica.
ACKNOWLEDGMENTS
This work was supported by EU-NOFA project (grant FAIR CT973142).
REFERENCES
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5.26 (d, 1H, J1ꢄ,2ꢄ ꢆ 3.9 Hz, H-1ꢄ), 5.25 (t, 1H, J3ꢄ,4ꢄ ꢆ J4ꢄ,5ꢄ ꢆ 4.6 Hz, H-4ꢄ), 5.15 (dd,
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ꢆ 10.5 Hz, H-3ꢀ), 5.00 (t, 1H, H-2), 4.86 (d, 1H, J ꢆ 12.4 Hz, CHPh), 4.59 (d, 1H, J
ꢆ 12.4 Hz, CHPh), 4.59 (unresolved, 1H, H-6a), 4.58 (d, 1H, CHPh), 4.53 (d, 1H,
CHPh), 4.51 (d, 1H, J1,2 ꢆ 7.6 Hz, H-1), 4.43 (d, 1H, J1ꢀ,2ꢀ ꢆ 7.6 Hz, H-1ꢀ), 4.37 (qd,
1H, H-5ꢄ), 4.30 (dd, 1H, J6a,5 ꢆ 4.7 Hz, J6a,6b ꢆ 12.1 Hz, H-6b), 4.15ꢂ4.02 (m, 2H,
H-6ꢀa, H-6ꢀb), 3.88 (t, 1H, J3,4 ꢆ J 4,5 ꢆ 9.0 Hz, H-4), 3.85ꢂ3.79 (m, 3H, H-2ꢀ, H-
5ꢀ, H-2ꢄ), 3.62 (ddd, H-5), 2.12ꢂ1.86 (6s, 24 H, CH3CO), 1.13 (d, 3H, J5ꢄ,6ꢄ ꢆ 6.6
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C-3ꢄ, C-4ꢄ, C-5ꢄ), 73.18, 70.69 (2 CH2Ph), 20.74ꢂ20.46 (CH3CO), 15.65 (C-6ꢄ)
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Received March 19, 2001
Accepted September 5, 2001

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