A chemoenzymatic synthesis of deoxy sugar esters involving stereoselective acetylation of hemiacetals catalyzed by CALB

Article abstract of DOI:10.1016/j.molcatb.2010.09.008

An extension of the scope of the chemoenzymatic strategy for the synthesis of stereochemically pure pyranose deoxy sugar esters of different carboxylic acids has been achieved. The objective of the work was to extend the strategy to the synthesis of furanose deoxy sugar derivatives and additionally, to N-Boc-protected amino acid esters. With all used carboxylic acids (deoxycholic acid, α-methoxyphenylacetic acid, N-Boc-l-phenylalanine and N-Boc-l-tyrosine) the lipase-catalyzed stereoselective acetylation of furanose or pyranose hemiacetal moiety as a key step afforded one desired stereochemically pure acetylated hemiacetal deoxy sugar ester in high de.

Full text of DOI:10.1016/j.molcatb.2010.09.008

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
A chemoenzymatic synthesis of deoxy sugar esters involving stereoselective
acetylation of hemiacetals catalyzed by CALB
Ly Villoa,∗, Malle Kreena, Marina Kudryashovaa, Andrus Metsalaa, Sven Tampa, Ülo Lillea,
Tõnis Pehkb, Omar Parvea
a Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia
b Department of Chemical Physics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
a r t i c l e i n f o
a b s t r a c t
Article history:
An extension of the scope of the chemoenzymatic strategy for the synthesis of stereochemically pure
pyranose deoxy sugar esters of different carboxylic acids has been achieved. The objective of the work
was to extend the strategy to the synthesis of furanose deoxy sugar derivatives and additionally, to N-Boc-
protected amino acid esters. With all used carboxylic acids (deoxycholic acid, ˛-methoxyphenylacetic
acid, N-Boc-l-phenylalanine and N-Boc-l-tyrosine) the lipase-catalyzed stereoselective acetylation of
furanose or pyranose hemiacetal moiety as a key step afforded one desired stereochemically pure acety-
lated hemiacetal deoxy sugar ester in high de.
Received 19 August 2010
Received in revised form
15 September 2010
Accepted 22 September 2010
Available online 29 September 2010
Keywords:
Deoxy sugar ester
© 2010 Elsevier B.V. All rights reserved.
Chemoenzymatic synthesis
Lipase-catalyzed acetylation
Furanose and pyranose hemiacetal
N-Boc-amino acid ester
1. Introduction
pounds have been prepared predominantly by the enzymatic
acylation [6,10]. A lot of work has been done with hydrolases,
The synthesis of (deoxy) sugars and their derivatives of both,
pyranose and furanose form continues to be one of the focus
points of organic synthesis as well as drug development [1]. Also,
a number of functionalized tetrahydrofurans and tetrahyropy-
rans, which represent parent ring systems for deoxy furanosides
and pyranosides, respectively, are embedded in many biologically
important natural products [2,3]. Several carbohydrates and their
derivatives have proven to be efficient and versatile tools in stere-
oselective synthesis. The compounds of this type are also used as
organocatalysts, chiral auxiliaries, ligands, etc. [4]. The carbohy-
drate esters of amino acids have found application as sweetening
agents and surfactants, as well as in the delivery of physiologically
active agents [5]. Recently, some of the esters have shown anti-
tumor, plant growth inhibiting and antibiotic activities [6,7]. The
sulfur-containing glucosamine esters are used for the synthesis of
glycopeptides [8]. Therefore, different approaches are needed to
achieve variable structures of carbohydrate derivatives.
among which mainly lipases can be found. Lipases, called the
“workhorses” of biocatalysis, are widely used to catalyze stereose-
lective reactions in both laboratory scale as well as in an industrial
scale fine organic synthesis [11]. These enzymes are applicable in
the acyl transfer reactions, for example, in the reactions involv-
ing esters, carboxylic acids, alcohols, etc., playing an important
role in the kinetic resolution of racemic mixtures. Lipases have
been used for separation of ˛- and ˇ-glucopyranosides [12], but
also for derivatization of carbohydrates in synthesis of oligosac-
charides [13] as well as in a chemoenzymatic approach for the
synthesis of several regioprotected fructose derivatives [14], etc.
Amino acid esters of carbohydrates have been prepared by Lohith
and Divakar who used lipases to obtain mixtures of mono- and
diesters [15].
The sugars as well as deoxy sugars form two types of thermody-
namically favored cyclic hemiacetals, i.e. furanoses and pyranoses.
These rings of carbohydrates, five- and six-membered, respectively,
greatly differ in their molecular geometry and, therefore, in the
accessibility of the corresponding hydroxyl groups to the enzyme.
From this has arisen the main purpose of the current study – to
investigate whether the chemoenzymatic synthetic strategy devel-
oped initially for the synthesis of pyranose deoxy sugar esters [16]
is applicable for the synthesis of their furanose counterparts. The
basic concept of the approach is as follows (Scheme 1):
For the synthesis of deoxy sugars many methods have been
developed [9]. Several chemical methods are available for the
synthesis of sugar esters [7]. However, in last decades these com-
∗
Corresponding author. Tel.: +372 620 2816; fax: +372 620 2828.
E-mail address: lee@chemnet.ee (L. Villo).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.09.008

L. Villo et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
45
H
H
nose) esters. A comparative study of the stereoselectivity and yield
of the products of CALB-catalyzed acetylation was performed.
O
H
O
O
O
O
Br
O
R
O
R
lipase-catalyzed
deacetylation
O-alkylation of
R-COOH
2. Experimental section
AcO
AcO
HO
2.1. General methods and materials
1
spontaneous
cyclization
1H and 13C NMR spectra were recorded in CDCl3 solutions on a
Bruker 400, 500 or 800 MHz spectrometers. All signals were refer-
enced relatively to solvent signal (7.27 ppm for 1H and 77.00 ppm
for 13C). 2D FT methods were used for the full assignment of NMR
spectra. TLC was performed using DC-Alufolien Kieselgel 60 F254
(Merck) silica gel plates and the compounds were visualized by
staining upon heating with anisaldehyde solution. Column chro-
matography was performed on Merck silica gel 60 (230–400 mesh).
Commercial reagents were used without purification. The Can-
dida antarctica lipase B (CALB) preparation (Novozym® 435) was
obtained from Novozymes A/S.
The O-alkylation of carboxylic acids as well as the lipase-
catalyzed acetylation and deacetylation reactions (General Pro-
cedures A, B and C) were performed according to the standard
procedures (only incubation times were varied in accordance with
reaction velocity) [16].
lipase-catalyzed
diastereoselective
acetylation
AcO
O
HO
O
O
O
O
O
R
R
Scheme 1. A chemoenzymatic approach for the synthesis of deoxy sugar esters.
1. A carboxylic acid is O-alkylated by a deoxy sugar precursor (˛-
bromo-ω-acetoxy aldehyde (1)).
2. The following lipase-catalyzed hydrolysis cleaves the acetate
with high regioselectivity.
3. A spontaneous cyclization takes place affording a mixture of
hemiacetal deoxy sugar esters.
4. Only one of the hemiacetal deoxy sugar ester diastereomers is
acetylated by the lipase. A kinetic dynamic resolution of hemi-
acetal stereoisomers takes place. The stereopreference of the
CALB was in accordance with the Kazlauskas’ rule for secondary
alcohols.
2.1.1. General Procedure A: the O-alkylation of a carboxylic acid
followed by the lipase-catalyzed deacetylation
Carboxylic acid (1 mmol) was dissolved in acetonitrile (8 ml),
4 eq. of DIPEA was added, followed by 0.7 mmol of bromoaldehyde
(1 or 2) dissolved in 2 ml of acetonitrile. The reaction was moni-
tored by TLC. After disappearance of the starting material (1 or 2)
Novozym® 435 (600 mg) and H2O (5 mmol) were added, the mix-
ture was shaken at rt and the reaction was monitored by TLC. The
solution was then diluted with Et2O, the enzyme was filtered off,
and the solution was washed with water, 1 M NaHSO4, water and
brine, and dried with Na2SO4. The product was evaporated and
purified by column chromatography over silica.
˛-Bromo-ω-acetoxy pentanal (1) was used as a precursor of the
deoxy sugar (3,4-dideoxy-d-ribose and 3,4-dideoxy-d-arabinose)
moiety in the synthesis of pyranose deoxy sugar esters [16]. For
the synthesis of furanose deoxy sugar esters racemic ˛-bromo-ω-
acetoxy butanal (2), as a deoxy sugar precursor (affording 3-deoxy
d-erythrose and 3-deoxy d-threose), has to be used.1 Thus, one of
the goals of the current work was to investigate the applicability
of the above approach to the synthesis and especially resolving of
the stereoisomers of furanose deoxy sugar esters. An additional
important step to be made in this study in the development of the
synthetic strategy was the synthesis of deoxy sugar esters of N-Boc-
protected ˛-amino acids (involving deoxy sugar in both, furanose
as well as pyranose form)
In this work all starting compounds and targets are mere exam-
ples representing corresponding type of organic compounds.
In order to obtain furanose deoxy sugar esters the reaction
sequence in the Scheme 1 was followed using ˛-bromobutanal
2 for the O-alkylation of carboxylic acids. Four carboxylic acids
were chosen for the deoxy sugar ester synthesis. First, deoxycholic
acid (DCA2; structures in Table 1) as a sterically demanding dihy-
droxy carboxylic acid bearing unprotected hydroxyl groups was
chosen. (The glycoconjugates of bile acids itself have long been
a subject of interest to researchers from different fields [18].) ˛-
Methoxyphenylacetic acid (MPA) was involved because of its role
of the chiral derivatizing agent allowing to determine the absolute
stereochemistry of the ester diastereomers by measuring of differ-
ential shielding effects in the NMR spectra of diastereomers [19,20].
As an extension of the synthetic approach two N-Boc-protected
˛-amino acids, viz. N-Boc-l-phenylalanine and N-Boc-l-tyrosine
were introduced in the synthesis of stereochemically pure 3-deoxy
d-erythrose (furanose) esters as well as 3,4-dideoxy d-ribose (pyra-
2.1.2. General Procedure B: the lipase-catalyzed acetylation of
hemiacetal compounds (9–14)
To the solution of 0.2 mmol of hemiacetal in 4 ml of chloroform,
1 ml of vinyl acetate and 200 mg of Novozym® 435 were added. The
reaction mixture was shaken at rt and the reaction was monitored
by TLC. After the process had been completed, the enzyme was fil-
tered off and the solution evaporated. The products were separated
by column chromatography over silica.
2.1.3. General Procedure C: the lipase catalyzed deacetylation of
compounds 15–20
To the solution of 0.5 mmol of an acetylated compound in 8 ml
of acetonitrile (containing 2% of water) 350 mg of Novozym® 435
was added. The reaction mixture was shaken at rt and the process
was monitored by TLC. The enzyme was filtered off, the reaction
mixture was evaporated and the products were purified by column
chromatography over silica.
2.2. Compound characterization
2.2.1. Deoxycholic acid 4ꢀ-acetoxy-1ꢀ-formyl-propyl ester (3)
TLC: Rf = 0.4 (EtOAc). Reaction time 28 h.
1
The synthesis and characterization of ω-acetylated aldehydes 1 and 2 (Scheme 2)
has been described in detail [17].
2.2.2. (S)-(+)-˛-Methoxyphenylacetic acid
4ꢀ-acetoxy-1ꢀ-formyl-propyl ester (4)
2
Abbreviations: DCA – deoxycholic acid, MPA – ˛-methoxyphenylacetic acid,
N-Boc-Phe – N-Boc-l-phenylalanine, N-Boc-Tyr – N-Boc-l-tyrosine, Boc – tert-
butoxycarbonyl group, CALB – Candida antarctica lipase B.
TLC: Rf = 0.14 (30% EtOAc/hexane). Reaction time 3 h.

46
L. Villo et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
Table 1
The yield and stereoisomeric purity of the products.
Structure of acid residueR-COOH
Abbr.
Comp. no
Yield (%)
de
DCA
3-Ac-DCA
DCA
9
15
9a
35a
27b
54c
–
22
OH
>98
12
>98d
1
9
3
HO
H
MPA
10
16
10a
44a
33b
42
–
>98
9
O
2
>98d
4
6
11
11
13
17
19
11a
13a
62a
72a
41b
44b
68
–
–
>98
>98
>98d
>98d
N-Boc-Phe
O
10
O
9
8NH
7
6
2
89
4
3
N-Boc-Tyr
12
14
18
20
12a
32a
53a
27b
24b
80
–
–
>98
>98
>98d
11
10
O
O
9
8NH
HO
7
2
6
4
3
a
b
c
Overall yield for the “one-pot synthesis”.
Theoretically, maximum yield of the reaction can be 50%.
Conversion rate was 80%.
d
Two anomers are accounted as one diastereomer.
2.2.3. N-(tert-Butoxycarbonyl)-l-phenylalanine
4ꢀ-acetoxy-1ꢀ-formyl-propyl ester (5)
47.15 < 47.11 (C17), 46.45 < 46.44 (C13), 42.0 (C5), 36.2 (C4), 36.0
(C20), 35.2 (C1), 35.14 < 35.11 (C8), 34.1 (C10), 33.5 (C9), 31.3 < 31.2
(C23), 30.73 < 30.72 (C22), 30.3 (C2), 28.5 (C11), 29.27 > 29.25 (C4ꢀ),
27.50 < 27.49 (C16), 27.1 (C6), 26.1 (C7), 23.7 (C15), 23.1 (C19),
17.22 < 17.21 (C21), 12.7 (C18) ppm; cis: 173.85 < 173.83 (C24),
95.1 < 95.0 (C2ꢀ), 73.16 < 73.15 (C12), 71.7 (C3), 78.26 < 78.23 (C3ꢀ),
64.4 < 64.3 (C5ꢀ), 48.14 < 48.13 (C14), 47.1 < 47.0 (C17), 46.5 < 46.4
(C13), 42.0 (C5), 36.3 (C4), 36.0 (C20), 35.2 (C1), 35.1 (C8), 34.1
(C10), 33.5 (C9), 31.01 < 30.97 (C23), 30.69 < 30.68 (C22), 30.3 (C2),
28.51 < 28.46 (C11), 28.37 (C4ꢀ), 27.50 < 27.46 (C16), 27.1 (C6), 26.1
(C7), 23.7 (C15), 23.1 (C19), 17.3 < 17.2 (C21), 12.7 (C18) ppm. MS
(m/z): 460.15, 391.10, 373.15, 355.15, 273.10, 255.10. IR (neat,
cm−1): 1045, 1244, 1374, 1740, 2866–2938, 3426. Anal. Calcd for
C28H46O6 (478.74): C, 70.24; H, 9.71. Found: C, 70.17; H, 9.68.
TLC: Rf = 0.2 (EtOAc). Flash chromatography eluent: 30% → 100%
EtOAc/hexane.
TLC: Rf = 0.15 (30% EtOAc/hexane). Reaction time 6 h.
2.2.4. N-(tert-Butoxycarbonyl)-l-tyrosine
4ꢀ-acetoxy-1ꢀ-formyl-propyl ester (6)
TLC: Rf = 0.25 (50% EtOAc/hexane). Reaction time 6 h.
2.2.5. N-(tert-Butoxycarbonyl)-l-phenylalanine
4ꢀ-acetoxy-1ꢀ-formyl-butyl ester (7)
TLC: Rf = 0.2 (30% EtOAc/hexane). Reaction time 24 h.
2.2.6. N-(tert-Butoxycarbonyl)-l-tyrosine
4ꢀ-acetoxy-1ꢀ-formyl-butyl ester (8)
TLC: Rf = 0.25 (40% EtOAc/hexane). Reaction time 24 h.
2.2.7. Deoxycholic acid 2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl ester
(9)
The reaction was carried out following the General Proce-
dure A starting with 1 mmol of DCA to yield 172 mg (35%,
overall yield for “one-pot synthesis”) of 9 in 120 h. 1H NMR
2.2.8. (S)-(+)-˛-Methoxyphenylacetic acid
2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl ester (10)
The reaction was carried out following the General Procedure
A starting with 1 mmol of MPA to yield 111 mg (44%, overall yield
for “one-pot synthesis”) of 10 in 25 h. 1H NMR (500 MHz, CDCl3)
ı 7.42, 7.36, 7.34, 5.50, 5.39, 5.35, 5.18, 5.12, 4.86, 4.84, 4.77,
4.76, 4.10, 3.83, 2.41, 2.28, 1.95, 1.68 ppm. 13C NMR (125 MHz,
CDCl3) ı trans: 170.14 < 170.09 (C1), 135.71 < 135.68 (C3), 128.89
(C6), 128.7 (C7), 128.7 (C5), 127.19 > 127.17 (C4), 127.19 (C8),
100.01 < 99.98 (C2ꢀ), 82.3 < 82.2 (C2), 78.8 > 78.7 (C3ꢀ), 66.71 > 66.67
(C5ꢀ), 57.33 > 57.29 (C9), 29.4 > 29.0 (C4ꢀ) ppm; cis: 170.01 < 169.94
(C1),135.6 < 135.5 (C3), 128.85 (C6), 128.6 (C7), 128.7 < 128.64(C5),
127.1 > 127.0 (C4), 127.1 (C8), 95.4 < 95.1 (C2ꢀ), 82.1 < 81.9 (C2),
74.0 > 73.8 (C3ꢀ), 64.4 > 64.3 (C5ꢀ), 57.25 > 57.24 (C9), 28.7 > 28.3
(C4ꢀ) ppm. MS (m/z):251.95, 164.95, 121.05, 105.00, 91.05, 77.00. IR
(neat, cm−1): 699, 1032–1105, 1178–1247, 1494, 1739, 2831–2942,
(500 MHz, CDCl3)
ı trans: 5.36, 5.06, 4.13/4.04, 3.97, 3.59,
2.39/1.93, 2.37/2.23, 1.84/1.25, 1.82/1.25, 1.79, 1.78/1.51, 1.77/1.33,
1.72/0.96, 1.69, 1.66/1.36, 1.59/1.06, 1.53, 1.50, 1.41/1.12, 1.40,
1.38, 0.95, 0.89, 0.66 ppm; cis: 5.47, 5.00, 4.10/3.85, 3.97, 3.59,
2.27/2.08, 2.42/2.32, 1.84/1.25, 1.82/1.25, 1.79, 1.78/1.51, 1.77/1.33,
1.72/0.96, 1.69, 1.66/1.36, 1.59/1.06, 1.53, 1.50, 1.41/1.12, 1.40,
1.38, 0.95, 0.89, 0.66 ppm. 13C NMR3 (125 MHz, CDCl3) ı trans:
173.87 < 173.85 (C24), 100.31 > 100.28 (C2ꢀ), 73.16 < 73.14 (C12),
71.7 (C3), 78.24 < 78.23 (C3ꢀ), 66.73 < 66.72 (C5ꢀ), 48.2 (C14),
3
Carbon atoms of deoxy sugar moiety are indicated with prime symbol.

L. Villo et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
47
4.67, 4.64, 4.51, 4.43, 3.51, 2.89, 2.08, 2.02, 1.75, 1.67, 1.54 ppm. 13
C
NMR (125 MHz, CDCl3) ı 172.0, 171.8, 171.72, 171.66 (C1), 155.7,
155.5 (C9), 155.46, 155.43, 155.38, 155.32 (C7), 130.5, 130.3 (C5),
126.93, 126.87, 126.80 (C4), 115.6, 115.52, 115.45 (C6), 91.5, 91.2
(C2ꢀ), 80.4, 80.3, 80.2, 80.1 (C10), 72.0, 71.8, 71.3, 71.1 (C3ꢀ), 62.7,
62.6, 61.9, 61.3 (C6ꢀ), 55.1, 54.82, 54.79, 54.56 (C2), 37.3, 37.2, 36.9,
36.6 (C3), 28.23, 28.20 (C11), 24.6, 24.5, 24.1 (C4ꢀ), 22.6, 22.4, 22.3
(C5ꢀ) ppm. MS (m/z): 306.90, 263.90, 245.90, 200.85, 178.90, 146.95,
136.00, 107.00, 84.00, 57.05. IR (neat, cm−1): 734, 1046, 1169–1247,
1370–1517, 1739, 2978, 3374. Anal. Calcd for C19H27NO7 (381.47):
C, 59.82; H, 7.15; N, 3.67. Found: C, 59.96; H, 7.16; N, 3.66. TLC:
Rf = 0.5 (20% isopropanol/hexane). Flash chromatography eluent:
8% → 20% isopropanol/hexane.
3441. Anal. Calcd for C13H16O5 (252.29): C, 61.89; H, 6.41. Found: C,
61.81; H, 6.38. TLC: Rf = 0.35 (50% EtOAc/hexane). Flash chromatog-
raphy eluent: 30% → 50% EtOAc/hexane.
2.2.9. N-(tert-Butoxycarbonyl)-l-phenylalanine
2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl ester (11)4
The reaction was carried out following the General Procedure
A starting with 1 mmol of Boc-Phe to yield 217 mg (62%, overall
yield for “one-pot synthesis”) of 11 in 24 h. 1H NMR (500 MHz,
CDCl3) ı 7.22, 7.21, 7.08, 7.07, 5.25, 5.15, 5.00, 4.47, 3.95, 2.99,
2.98, 2.45, 2.28, 1.99, 1.82, 1.34 ppm. 13C NMR (125 MHz, CDCl3)
ı 171.5 (C1), 155.2 (C9), 135.89/135.83 (C4), 129.5/129.4 (C5),
128.6/128.5 (C6), 127.1/126.9 (C7), 99.9 (C2ꢀ), 80.2 (C10), 79.3/79.1
(C3ꢀ), 64.5 (C5ꢀ), 54.5 (C2), 38.4/38.3 (C3), 28.3 (C4ꢀ), 25.2 (C11)
ppm. MS (m/z): 332.90, 259.90, 247.95, 233.90, 85.95, 70.00, 57.00.
IR (neat, cm−1):702, 1048, 1168–1248, 1369, 1605, 1738–1739,
2980, 3365. Anal. Calcd for C18H25NO6 (351.44): C, 61.51; H,
7.18; N, 3.99. Found: C, 61.69; H, 7.20; N, 4.01. TLC: Rf = 0.6
(20% isopropanol/hexane). Flash chromatography eluent: 10% iso-
propanol/hexane.
2.2.13. 3-Acetyl deoxycholic acid
(2ꢀS,3ꢀR)-2ꢀ-acetoxy-tetrahydrofuran-3ꢀ-yl ester (15)
The reaction was carried out following the General Procedure B
to yield 210 mg (27%) of 15 in 240 h. 1H NMR (500 MHz, CDCl3) ı
6.15, 5.20, 4.71, 4.14, 3.99, 2.40/2.27, 2.09, 2.01, 2.06/1.78, 1.87/1.27,
1.85/1.53, 1.83/1.24, 1.81/1.37, 1.81, 1.77, 1.76/1.03, 1.69, 1.61/1.05,
1.56, 1.51, 1.44, 1.43, 1.41/1.10, 1.40, 0.98, 0.92, 0.68 ppm. 13C NMR
(125 MHz, CDCl3) ı 173.3 (C24), 170.8 (C25), 169.7 (C27), 99.5 (C2ꢀ),
76.8 (C3ꢀ), 74.2 (C3), 73.1 (C12), 68.2 (C5ꢀ), 48.2 (C14), 47.2 (C17),
46.4 (C13), 41.7 (C5), 35.9 (C20), 35.0 (C8), 34.8 (C1), 34.1 (C10),
33.5 (C9), 32.1 (C4), 31.0 (C23), 30.6 (C22), 29.4 (C4ꢀ), 28.6 (C11),
27.4 (C16), 26.9 (C6), 26.4 (C2), 25.9 (C7), 23.5 (C15), 23.1 (C19),
21.5 (C26), 21.2 (C28), 17.2 (C21), 12.7 (C18) ppm. [␣]25546 = 23.1
(c 2.96, MeOH). MS (m/z): 523, 495, 484, 410, 368, 341, 256, 129. IR
(neat, cm−1): 1044, 1244, 1374, 1448, 1739, 2940, 3535. Anal. Calcd
for C32H50O8 (562.82): C, 68.28; H, 8.97. Found: C, 68.09; H, 8.95.
TLC: Rf = 0.25 (30% EtOAc/hexane). Flash chromatography eluent:
25% EtOAc/hexane.
2.2.10. N-(tert-Butoxycarbonyl)-l-tyrosine
2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl ester (12)
The reaction was carried out following the General Procedure
A starting with 1 mmol of Boc-Tyr to yield 117 mg (32%, overall
yield for “one-pot synthesis”) of 12 in 24 h. 1H NMR (500 MHz,
CDCl3) ı 6.88, 6.86, 6.7, 6.6, 5.2, 5.0, 4.95, 4.4, 4.04, 4.02, 2.9, 2.8,
2.3, 2.0, 1.3 ppm. 13C NMR (125 MHz, CDCl3) ı 172.0, 171.8, 171.7,
155.8, 155.5, 155.4, 130.5, 127.3, 127.0, 115.7, 100.1, 100.0, 80.7,
80.6, 79.3, 79.1, 66.7, 66.6, 60.7, 37.6, 37.1, 29.2, 29.1, 28.4, 25.1,
21.1 ppm. MS (m/z): 366.90, 348.90, 263.95, 249.90, 219.90, 146.90,
107.00, 87.00, 57.05. IR (neat, cm−1): 830, 1047, 1170–1247, 1517,
1616, 1739, 2937–2981, 3366. Anal. Calcd for C18H25NO7 (367.44):
C, 58.83; H, 6.87; N, 3.81. Found: C, 58.95; H, 6.88; N, 3.80. TLC:
Rf = 0.4 (10% isopropanol/toluene). Flash chromatography eluent:
10% isopropanol/toluene.
2.2.14. (S)-(+)-˛-Methoxyphenylacetic acid
(2ꢀS,3ꢀR)-2ꢀ-acetoxy-tetrahydrofuran-3ꢀ-yl ester (16)
The reaction was carried out following the General Procedure B
to yield 49 mg (33%) of 16 in 160 h. 1H NMR (500 MHz, CDCl3) ı 7.42,
7.37, 7.34, 6.18, 5.22, 4.78, 4.03/3.68, 3.42, 2.05, 1.73/2.25 ppm. 13
C
2.2.11. N-(tert-Butoxycarbonyl)-l-phenylalanine
2ꢀ-hydroxy-tetrahydropyran-3ꢀ-yl ester (13)
NMR (125 MHz, CDCl3) 169.7 (C1), 169.5 (C10), 135.7 (C3), 128.9
(C6), 128.7 (C5,7), 127.1 (C4,8), 99.3 (C2ꢀ), 82.1 (C2), 77.6 (C3ꢀ), 68.0
(C5ꢀ), 57.4 (C9), 29.3 (C4ꢀ), 21.1 (C11) ppm. [␣]25546 = +0.4 (c 1.38,
MeOH). MS (m/z): 293.90, 234.90, 164.90, 129.00, 121.00, 105.00,
91.00, 77.00. IR (neat, cm−1): 699, 1014, 1116, 1173–1231, 1367,
1494, 1748, 2907–2939. Anal. Calcd for C15H18O6 (294.33): C, 61.21;
H, 6.18. Found: C, 61.16; H, 6.19. TLC: Rf = 0.6 (50% EtOAc/hexane).
Flash chromatography eluent: 20% EtOAc/hexane
The reaction was carried out following the General Procedure
A starting with 1.3 mmol of Boc-Phe to yield 339 mg (72%, over-
all yield for “one-pot synthesis”) of 13 in 24 h. 1H NMR (500 MHz,
CDCl3) ı 7.32, 7.30, 7.27, 7.25, 7.21, 4.98, 4.88, 4.71, 4.76, 4.60, 4.55,
4.01, 3.93, 3.53, 3.12, 2.09, 1.97, 1.76, 1.67, 1.64, 1.58, 1.54, 1.43 ppm.
13C NMR (125 MHz, CDCl3) ı 171.6 > 171.4 (C1), 155.5 < 155.2
(C9), 135.94 > 135.86 (C4), 129.5 > 129.3 (C5), 128.7 > 128.5 (C6),
127.2 > 127.0 (C7), 94.4 > 91.7 (C2ꢀ), 80.3 < 80.0 (C10), 72.2 > 71.3
(C3ꢀ), 63.2 > 62.0 (C6ꢀ), 54.8 < 54.4 (C2), 38.1 (C3), 28.31 < 28.28
(C11), 25.7 > 24.6 (C4ꢀ), 22.7 > 22.3 (C5ꢀ) ppm. MS (m/z): 308.95,
290.95, 274.00, 248.05, 164.00, 147.90, 120.05, 100.00, 90.90,
84.00, 57.05. IR (neat, cm−1): 702, 1047, 1168–1245, 1371, 1498,
1718–1740, 2937–2979, 3365. Anal. Calcd for C19H27NO6 (365.47):
C, 62.44; H, 7.46; N, 3.83. Found: C, 62.32; H, 7.44; N, 3.82. TLC:
Rf = 0.3 (5% isopropanol/toluene). Flash chromatography eluent:
10% → 25% isopropanol/hexane.
2.2.15. N-(tert-Butoxycarbonyl)-l-phenylalanine
(2ꢀS,3ꢀR)-2ꢀ-acetoxy-tetrahydrofuran-3ꢀ-yl ester (17)
The reaction was carried out following the General Procedure
B to yield 99 mg (41%) of 17 in 260 h. 1H NMR (500 MHz, CDCl3)
ı 7.31, 7.28, 7.17, 5.18, 4.99, 4.58, 4.08, 3.98, 3.09, 2.30, 2.07,
1.44 ppm. 13C NMR (125 MHz, CDCl3) ı 171.2 (C1), 169.5 (C12),
155.1 (C9), 135.8 (C4), 129.3 (C5), 128.7 (C6), 127.2 (C7), 99.4 (C2ꢀ),
80.2 (C10), 77.9 (C3ꢀ), 68.1 (C5ꢀ), 54.5 (C2), 38.5 (C3), 29.4 (C4ꢀ), 28.3
(C11), 21.1 (C13) ppm. MS (m/z): 333.00, 278.00, 276.95, 259.95,
164.00, 120.00, 70.00, 57.05. IR (neat, cm−1):702, 756, 1167–1233,
1368, 1498, 1605, 1715–1747, 2932–2979, 3373. [␣]25546 = −58.6
(c 1.41, MeOH). Anal. Calcd for C20H27NO7 (393.48): C, 61.05; H,
6.93; N, 3.56. Found: C, 61.12; H, 6.95; N, 3.55. TLC: Rf = 0.2 (20%
EtOAc/hexane). Flash chromatography eluent: 15% EtOAc/hexane.
2.2.12. N-(tert-Butoxycarbonyl)-l-tyrosine
2ꢀ-hydroxy-tetrahydropyran-3ꢀ-yl ester (14)
The reaction was carried out following the General Procedure
A starting with 1 mmol of Boc-Tyr to yield 240 mg (53%, overall
yield for “one-pot synthesis”) of 14 in 24 h. 1H NMR (500 MHz,
CDCl3) ı 7.01, 6.98, 6.75, 5.24, 5.19, 5.14, 5.12, 4.91, 4.88, 4.83,
2.2.16. N-(tert-Butoxycarbonyl)-l-tyrosine
(2ꢀS,3ꢀR)-2ꢀ-acetoxy-tetrahydrofuran-3ꢀ-yl ester (18)
The reaction was carried out following the General Proce-
dure B to yield 22 mg (27%) of 18 in 182 h. 1H NMR (500 MHz,
4
In the case of N-Boc-protected amino acids E- and Z-isomers (caused by urethane
bond in Boc-group) complicate the spectra by exchange broadened peaks.

48
L. Villo et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
CDCl3) ı 6.99, 6.76, 6, 32, 6.09, 5.16, 5.03, 4.51, 4.07/3.98, 2.98,
2.29/1.85, 2.11 ppm. 13C NMR (125 MHz, CDCl3) ı 171.3 (C1), 169.7
(C12), 155.22 (C7), 155.16 (C9), 130.3 (C5), 127.1 (C4), 115.5
(C6), 99.3 (C2ꢀ), 80.3 (C10), 77.7 (C3ꢀ), 68.0 (C5ꢀ), 54.6 (C2), 37.5
(C3), 29.3 (C4ꢀ), 28.2 (C11), 21.1 (C13) ppm. MS (m/z): 365.95,
306.90, 200.90, 178.90, 107.00, 84.00, 57.05. IR (neat, cm−1):
757, 1012–1108, 1165–1236, 1369–1517, 1615, 1744, 2935–3020,
3377. [␣]25546 = −45,4 (c 0.57, MeOH). Anal. Calcd for C20H27NO8
(409.48): C, 58.66; H, 6.66; N, 3.42. Found: C, 58.73; H, 6.64; N,
3.40. TLC: Rf = 0.3 (40% EtOAc/hexane). Flash chromatography elu-
ent: 30% EtOAc/hexane.
2.2.20. (S)-(+)-˛-Methoxyphenylacetic acid
(3ꢀR)-2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl ester (10a)
The reaction was carried out following the General Procedure
C to yield 11 mg (42%) of 10a in 72 h. 1H NMR (500 MHz, CDCl3) ı
trans: 7.3–7.5, 5.41, 5.13, 4.78, 4.08/3.87, 3.41, 2.31/1.71 ppm; cis:
7.3–7.5, 5.51, 5.04, 4.86, 4.01/3.76, 3.42, 2.21/1.96 ppm. 13C NMR
(125 MHz, CDCl3) ı trans: 170.0 (C1), 135.9 (C3), 128.83 (C6), 128.63
(C5,7), 127.07 (C4,8), 100.0 (C2ꢀ), 82.0 (C2), 78.8 (C3ꢀ), 66.8 (C5ꢀ),
57.2 (C9), 29.0 (C4ꢀ) ppm; cis: 170.1 (C1), 135.9 (C3), 128.89 (C6),
128.69 (C5,7), 127.14 (C4,8), 95.0 (C2ꢀ), 82.0 (C2), 73.9 (C3ꢀ), 64.2
(C5ꢀ), 57.4 (C9), 29.0 (C4ꢀ) ppm. [␣]25546 = 59.5 (c 0.3, MeOH). Flash
chromatography eluent: 20% EtOAc/hexane.
2.2.17. N-(tert-Butoxycarbonyl)-l-phenylalanine
2.2.21. N-(tert-Butoxycarbonyl)-l-phenylalanine
(2ꢀS,3ꢀR)-2ꢀ-acetoxy-tetrahydropyran-3ꢀ-yl ester (19)
(3ꢀR)-2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl ester (11a)
The reaction was carried out following the General Procedure B
to yield 121 mg (44%) of 19 in 200 h. 1H NMR (500 MHz, CDCl3) ı
7.30, 7.25, 7.16, 5.76, 4.99, 4.74, 4.60, 3.87/3.66, 3.09, 2.11, 2.00/1.65,
1.78/1.47 ppm. 13C NMR (125 MHz, CDCl3) ı 171.0 (C1), 169.2 (C12),
154.9 (C9), 135.7 (C4), 129.2 (C5), 128.5 (C6), 127.0 (C7), 90.9 (C2ꢀ),
79.9 (C10), 68.4 (C3ꢀ), 62.7 (C6ꢀ), 54.4 (C2), 38.3 (C3), 28.2 (C11),
24.1 (C4ꢀ), 20.6 (C5ꢀ), 21.0 (C13) ppm. MS (m/z): 347.00, 291.00,
274.00, 164.00, 128.05, 120.10, 100.05, 84.00, 57.05, 43.05. IR (neat,
cm−1): 702, 1012–1075, 1166–1233, 1369, 1498, 1605, 1716–1748,
2935–2976, 3374. [␣]25546 = −34.9 (c 1.69, MeOH). Anal. Calcd for
The reaction was carried out following the General Procedure
C to yield 24 mg (68%) of 11a in 260 h. 1H NMR (125 MHz, CDCl3)
ı 7.23, 7.21, 7.19, 7.09, 7.07, 5.25, 5.01, 4.99, 4.96, 4.94, 4.5, 4.48,
4.04, 3.99, 3.98, 3.85, 3.83, 3.10, 2.99, 2.98, 2.27, 2.25, 1.97, 1.67,
1.34 ppm. 13C NMR (500 MHz, CDCl3) ı 171.5, 155.1, 135.9, 135.9,
129.3, 128.7, 128.6, 127.2, 127.2, 100.2, 80.1, 79.1, 74.2, 66.8, 54.5,
38.5, 29.2, 28.3, 23.9, 14.2 ppm. [␣]25546 = −20.9 (c 0.77, MeOH).
TLC: Rf = 0.43 (10% isopropanol/toluene). Flash chromatography
eluent: 25% EtOAc/hexane.
C
21H29NO7 (407.51): C, 61.89; H, 7.19; N, 3.44. Found: C, 61.74; H,
2.2.22. N-(tert-Butoxycarbonyl)-l-tyrosine
7.17; N, 3.45. TLC: Rf = 0.5 (10% isopropanol/toluene). Flash chro-
matography eluent: 15% EtOAc/hexane.
(3ꢀR)-2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl ester (12a)
The reaction was carried out following the General Proce-
dure C to yield 12 mg (80%) of 12a in 216 h. 1H NMR (500 MHz,
2.2.18. N-(tert-Butoxycarbonyl)-l-tyrosine
CDCl3)
ı 6.99, 6.76, 5.38, 5.23, 5.14, 5.06, 4.53, 4.37, 4.13,
(2ꢀS,3ꢀR)-2ꢀ-acetoxy-tetrahydropyran-3ꢀ-yl ester (20)
4.07, 3.99, 3.85, 3.03, 2.98, 2.33, 2.19, 1.93, 1.75, 1.43 ppm. 13C
NMR (125 MHz, CDCl3) ı 171.6 (C1), 155.4 (C7), 155.2 (C9),
130.4 (C5), 127.1 (C4), 115.5 (C6), 99.9 > 95.5 (C2ꢀ), 80.5 (C10),
78.9 > 74.0 (C3ꢀ), 66.6 > 64.4 (C5ꢀ), 54.6 (C2), 37.6 (C3), 29.6 (C4ꢀ),
28.3 (C11) ppm. [␣]25546 = −18.9 (c 0.4, MeOH). TLC: Rf = 0.3
(10% isopropanol/toluene). Flash chromatography eluent: 40%
EtOAc/hexane.
The reaction was carried out following the General Proce-
dure B to yield 56 mg (24%) of 20 in 214 h. 1H NMR (500 MHz,
CDCl3) ı 6.98, 6.96, 6.75, 6.73, 5.77, 5.10, 4.75, 4.53, 4.39, 3.86,
3.66, 2.99, 2.88, 2.16, 2.01, 1.79, 1.68, 1.48, 1.41 ppm. 13C NMR
(125 MHz, CDCl3) ı 171.2 (C1), 169.4 (C12), 155.3 (C7), 155.2 (C9),
130.2 (C5), 126.9 (C4), 115.4 (C6), 90.9 (C2ꢀ), 80.2 (C10), 68.4
(C3ꢀ), 62.6 (C6ꢀ), 54.6 (C2), 37.4 (C3), 28.2 (C11), 24.0 (C4ꢀ), 21.0
(C13), 20.5 (C5ꢀ) ppm. MS (m/z): 423.00, 363.05, 348.95, 307.00,
201.00, 179.05, 106.90, 107.00, 84.00, 57.05. IR (neat, cm−1):
757, 1012–1108, 1165–1236, 1369–1517, 1615, 1744, 2935–3020,
3377. [␣]25546 = −38.8 (c 1.07, MeOH). Anal. Calcd for C12H29NO8
(423.51): C, 59.55; H, 6.92; N, 3.31. Found: C, 59.38; H, 6.90; N,
3.29. TLC: Rf = 0.4 (10% isopropanol/toluene). Flash chromatogra-
phy eluent: 30% EtOAc/hexane
2.2.23. N-(tert-Butoxycarbonyl)-l-phenylalanine
(3ꢀR)-2ꢀ-hydroxy-tetrahydropyran-3ꢀ-yl ester (13a)
The reaction was carried out following the General Procedure
C to yield 39 mg (89%) of 13a in 72 h. 1H NMR (500 MHz, CDCl3) ı
7.23, 7.20, 7.10, 5.02, 4.85, 4.66, 4.59, 3.90, 3.67, 3.45, 3.02, 2.04,
2.01, 1.62, 1.46, 1.34 ppm. 13C NMR (125 MHz, CDCl3) 171.8, 171.7
(C1), 155.5, 155.2 (C9), 136.0, 135.9 (C4), 129.4, 129.3 (C5), 128.6,
128.6 (C6), 127.2, 127.1 (C7), 94.2, 91.8 (C2ꢀ), 80.5, 80.1 (C10), 71.9,
71.1 (C3ꢀ), 62.8, 62.4 (C6ꢀ), 55.1, 54.7 (C2), 38.4, 37.8 (C3), 28.3
(C11), 25.4, 24.8 (C4ꢀ), 22.5, 22.2 (C5ꢀ) ppm. [␣]25546 = −11.1 (c 1.56,
MeOH). TLC: Rf = 0.45 (10% isopropanol/toluene). Flash chromatog-
raphy eluent: 30% EtOAc/hexane.
2.2.19. Deoxycholic acid (3ꢀR)-2ꢀ-hydroxy-tetrahydrofuran-3ꢀ-yl
ester (9a)
The reaction was carried out following the General Proce-
dure C to yield 32 mg (54%, conversion 80% in 21 days) of 9a.
1H NMR (800 MHz, CDCl3) 5.35 s > 5.47 d 4.2 Hz (H2ꢀ), 5.06 dd
5.6 and 0.9 Hz > 4.99 ddd 7.8, 7.2 and 4.2 Hz (H3ꢀ), 4.14–4.02 m
(H5ꢀ), 3.98 t 2 × 1.9 Hz (H12), 3.58 tt 2 × 4.8 and 2 × 10.6 Hz (H3),
2.42–2.21 m (H23, H4ꢀ), 1.94–0.95 m from ring and side chain pro-
tons of isomers, 0.95 d 6.4 Hz (H21), 0.89 s (H19), 0.661 < 0.659 s
(H18). ı 13C NMR (200 MHz, CDCl3) ı 173.92 < 173.91 (C24),
100.18 > 94.86 (C2ꢀ), 78.21 < 73.19 (C3ꢀ), 73.15 < 73.14 (C12), 71.64
(C3), 66.65 > 65.32 (C5ꢀ), 48.06 > 48.03 (C14), 47.06 > 46.99 (C17),
46.40 < 46.39 (C13), 41.95 < 41.94 (C5), 36.15 (C4), 35.89 < 35.88
(C8), 35.2 (C1), 35.17 (C1 and C20), 34.05 < 34.04 (C10),
33.43 (C9), 31.22 > 30.94 (C23), 30.66 > 30.60 (C22), 30.22 < 30.20
(C2), 29.17 > 28.27 (C4ꢀ), 28.42 > 28.40 (C11), 27.50 < 27.49 (C16),
27.04 > 27.03 (C6), 26.08 < 26.07 (C7), 23.64 < 23.62 (C15), 23.05
(C19), 17.15 < 17.13 (C21), 12.64 (C18). [␣]25546 = 36.3 (c 1.2,
MeOH). TLC: Rf = 0.2 (10% isopropanol/toluene). Flash chromatog-
raphy eluent: 70% EtOAc/hexane.
3. Results and discussion
The detailed synthetic scheme of deoxy sugar esters consists of
following steps (Scheme 2):
Step 1. The first reaction of this step is the O-alkylation of car-
boxylic acid with bromoaldehyde (1 or 2). The O-alkylation is
followed by the lipase-catalyzed deacetylation of the ω-hydroxyl
group, the hydroxy aldehyde formed undergoes a spontaneous
cyclization affording a mixture of diastereomers of deoxy sugar
ester in hemiacetal form. These two reactions were carried out
as a “one-pot synthesis”. This increased the yield of the products
as the separation of the aldehyde intermediates 3–8 over silica
appeared to be quite complicated and the base used for alkylation
did not interfere with the enzymatic reaction. The diastereomeric

L. Villo et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
49
Step 1, "one-pot synthesis"
H
Step 3
H
Step 2
O
O
O
lipase, H2O,
CH3CN
AcO
O
HO
O
2'
2. lipase, H2O,
CH3CN
lipase, vinyl
acetate, CHCl3
HO
O
1. R-COOH,
DIPEA, CH3CN
O
O
O
Br
O
O
O
R
( )n
OAc
( )n
OAc
( )n
( )n
( )n
3'
R
O
R
R
R = DCA, MPA,
BOC-Phe
or BOC-Tyr
9a: R=DCA, n=1
10a: R=MPA, n=1
11a: R=BOC-Phe, n=1
12a: R=BOC-Tyr, n=1
13a: R=BOC-Phe, n=2
14a: R=BOC-Tyr, n=2
15: R=3-Ac DCA, n=1
16: R=MPA, n=1
17: R=BOC-Phe, n=1
18: R=BOC-Tyr, n=1
19: R=BOC-Phe, n=2
20: R=BOC-Tyr, n=2
9: R=DCA, n=1
10: R=MPA, n=1
11: R=BOC-Phe, n=1
12: R=BOC-Tyr, n=1
13: R=BOC-Phe, n=2
14: R=BOC-Tyr, n=2
3: R=DCA, n=1
4: R=MPA, n=1
5: R=BOC-Phe, n=1
6: R=BOC-Tyr, n=1
7: R=BOC-Phe, n=2
8: R=BOC-Tyr, n=2
1: n=2
2: n=1
Scheme 2. The chemoenzymatic synthesis of deoxy sugar esters of different carboxylic acids.
esters of hemiacetal deoxy sugar formed (9–14) were inseparable
by column chromatography and were only purified as a mixture
from side products.
Step 2. The diastereomeric mixture of each of the hemiacetal esters
(9–14) was further enzymatically acetylated in stereoselective
manner affording one diastereomer of trans-hemiacetal acetate
(15–20), which was easily separated by column chromatography
over silica.
Step 3. The deacetylation of individual hemiacetal acetates by
lipase gave desired stereochemically pure deoxy sugar esters,
albeit as a mixture of two anomers under the conditions used
(9a–14a).
was a mixture of stereoisomers of the open-chain (aldehyde) deoxy
sugar ester. In the lipase-catalyzed acetylation also the hydroxyl
group attached to the C3 atom of the steroid skeleton was reac-
tive, but the subsequent lipase-catalyzed deacetylation cleaved
both of the acetates. The latter reaction appeared to be a rather
time-consuming process (conditions were not optimized). The
deacetylation resulted during incubation with lipase for 21 days at
room temperature in the formation of the stereochemically pure
furanose deoxy sugar (3-deoxy d-erythrose) ester of DCA (9a),
which exists as a mixture of two anomers in solution.
3.2. The synthesis of furanose deoxy sugar esters of (S)-MPA
It should be emphasized that the lipase-catalyzed acetylation
(Step 2) is diastereoselective for the compounds investigated in
the current work; in this synthetic step the configuration of the
stereogenic center at C2 of deoxy sugar skeleton controls the stere-
ochemical result of the enzymatic reaction. The hemiacetal acetate
that is formed is of trans geometry and at the same time the stereo-
chemistry of this acetate is in accordance with the Kazlauskas’ rule
for secondary alcohols [16].
The “one-pot synthesis” of MPA hemiacetal deoxy sugar esters
(10) gave this stereoisomeric mixture with reasonable yield and
time. The resolution of diastereomers by lipase-catalyzed acetyla-
tion gave two products of which the more polar one was separated
over silica and identified by NMR to be the desired acetylated fura-
nose hemiacetal as an individual diastereomer (16) while the less
polar product decomposed in contact with silica gel during column
chromatography and thus remaining unidentified. The deacetyla-
tion of ester 16 proceeded smoothly (during 72 h) and selectively
– no byproducts were detected. The 3-deoxy d-erythrose ester 10a
was obtained in the form of a mixture of two anomers.
3.1. The synthesis of furanose deoxy sugar esters of DCA
An important feature of the synthesis of MPA esters regard-
ing the whole strategy towards the stereochemically pure furanose
deoxy sugar esters was the successful experimental determination
of the absolute configuration of deoxy sugar ester 10a, which is
preferably acetylated by CALB. This was easy to achieve by deter-
mination of the differential shielding effect for the Cꢀ4 atom of the
furanose ring from the 13C NMR spectrum of the diastereomeric
mixture 10. The corresponding value of chemical shift difference
(differential shielding), ꢀı, was found to be as large as 0.350 ppm
(29.35–29.00) for the pair of the ester diastereomers 10 (deter-
mined for the trans anomers). The individual deoxy sugar ester 10a
was identified by observed significant shielding of Cꢀ4 of furan ring
by the phenyl group. As a result, it was proven that CALB catalyzes
acetylation of furanose deoxy sugar ester hemiacetals preferring
the stereoisomer in accordance with the Kazlauskas’ rule for sec-
ondary alcohols. The same has been shown for pyranose deoxy
sugar hemiacetals [16].
The O-alkylation of the DCA carboxyl group with bromobutanal
(2) followed by the lipase-catalyzed deacetylation of the terminal
hydroxyl group of deoxy sugar provided a mixture of hemiacetal
deoxy sugar ester diastereomers (9) with 35% yield (Table 1). This
could be considered as a satisfactory result taking into account two
issues: 1) that DCA as an acyl group on C2-OH of furanose deoxy
sugar ester is accessible to the lipase and 2) adding methanol to the
reaction medium was necessary. The first point has been proven
by identification of some (modest) amount of parent deoxy sugar
(tetrahydrofuran-2,3-diol) in the crude product of the deacetyla-
tion. The solubility of DCA in acetonitrile medium is low; therefore,
methanol was added to improve the homogenization of the reac-
tion mixture of O-alkylation. (Unfortunately, methanol is not inert
towards an ˛-bromo-aldehyde under basic conditions used. As a
result – the yield of O-alkylation was somewhat diminished.) Due
to the presence of methanol in the reaction mixture, the deacety-
lation step necessitated no additional nucleophile (water has been
added in other cases) but only enzyme preparation. Along with the
target compound 9 also two byproducts were separated and iden-
tified as DCA methyl ester (7%) and parent deoxy sugar (10%). The
mixture of diastereomers purified over column chromatography
was subjected to the enzymatic acetylation, which appeared to be
slower as compared to corresponding pyranose deoxy sugar esters.
The acetylation gave target compound as individual diastereomer
– acetylated cyclic trans hemiacetal deoxy sugar ester (15), which
was separated by flash chromatography. Another product obtained
The absolute configuration of 10a and of other resolved deoxy
sugar ester stereoisomers correspond to what has been depicted in
the Scheme 2.
3.3. The synthesis of furanose and pyranose deoxy sugar esters of
N-Boc-phenylalanine
The first two steps of the synthetic sequence integrated into a
“one-pot synthesis” of the esterification of N-Boc-l-phenylalanine

50
L. Villo et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
H
using either aldehyde 2 or 1 as a deoxy sugar precursor pro-
ceeded smoothly and gave hemiacetal deoxy sugar esters 11
and 13, respectively, both of them in good yield. Again, the
parent deoxy sugars – tetrahydrofuran-2,3-diol for the former
and tetrahydropyran-2,3-diol for the latter, were determined as
byproducts, evidently due to the breaking of the ester group formed
by the carboxylic acid N-Boc-Phe and 2-hydroxyl group of deoxy
sugar moiety. The latter evidence observed indicates not very
high functional group selectivity of the process in some cases.
Nevertheless, in both cases, the desired deoxy sugar esters were
obtained in good yield and the reaction time all together did not
exceed 48 h under standard conditions. On the contrary, the lipase-
catalyzed acetylation used for the separation of diastereomers
occurred slowly. The incubation of deoxy furanose ester (11) with
CALB during 260 h afforded only acetylated hemiacetal 17 that was
successfully separated and identified. In the case of the pyranose
deoxy sugar ester (13), again, only an acetylated desired product
19 was obtained, while the other starting diastereomer afforded
only traces of several byproducts that were discarded. The yields
(41% and 44%) of the hemiacetal acetates (17) and (19), respec-
tively, were high considering the fact that only one half of the
substance should be converted into the acetylated product. Finally,
the CALB-catalyzed deacetylation of the hemiacetal acetates (17)
and (19) resulted in the formation of the desired stereochemically
pure deoxy sugar esters (11a) and (13a) with good (68%) to excel-
lent (89%) yields, respectively. The reaction proceeded more easily
for the pyranose hemiacetal while the furanose hemiacetal acetate
required 3.5 times longer incubation time under the same condi-
tions.
O
O
OAc
OH
O
O
OH
CALB
CALB
Br
vinyl
acetate
deacetylation
Br
Br
Br
OAc
21
22
2
21
O
O
OAc
O
OH
OH
CALB
CALB
deacetylation
vinyl
acetate
O
R
O
R
O
R
O
O
O
9a-13a
9-14
15-20
Scheme 3. The chemoselectivity (and feasibility) of the reactions depends on the
substituent at the 3rd position of the furan cycle.
However, this does not apply for N-Boc-l-amino acid esters as,
apparently, the amino group has crucial influence on the decycliza-
tion of hemiacetal in the active site of the enzyme or causes (makes
possible) the occurrence of further uncontrolled reactions [21].
It is important to note that the CALB-catalyzed acetylation of the
˛-bromo-ω-hydroxy butanal (21, Scheme 3), which mainly exists
in a furanose hemiacetal form, results almost solely in the forma-
tion of an extended-chain ˛-bromo-ω-acetoxy butanal (2) [17]. The
current work has shown that the lipase-catalyzed acetylation of
furanose hemiacetal deoxy sugar esters 9–14 (the analogues of 21)
affords, prevalently, acetylated cyclic hemiacetals 15–20 in high
de. There is still no convincing rationalization available for this
important difference.
It has also been observed that the CALB-catalyzed deacetyla-
tion of the acetylated [17] stereoisomeric bromotetrahydrofuranols
22 (Scheme 3) failed in our hands. In this work, on the contrary,
the deacetylation of the related compounds 15–20 catalyzed by
the same enzyme was successful with most of the esters under
investigation producing hemiacetals 9a–13a.
In summary, we have to conclude that the substituents at the
3rd position of the 2-furanols greatly influence the selectivities of
the lipase-catalyzed acetylation; they may affect the feasibility of
the acetylation and deacetylation of this type of compounds in gen-
eral, independently of the stereochemistry of the substrate in some
cases.
3.4. The synthesis of furanose and pyranose deoxy sugar esters of
N-Boc-tyrosine
In the case of N-Boc-l-tyrosine, the O-alkylation was smooth
for both starting bromoaldehydes and also occurred similarly to
the above synthesis of analogous N-Boc-Phe derivatives. The O-
alkylation and the subsequent enzymatic hydrolysis step produced,
together with the desired deoxy sugar esters (12) and (14), respec-
tively, also the aforementioned parent deoxy sugars, as a major
byproduct. For the “one-pot synthesis” the overall yield of the pyra-
nose deoxy sugar ester of N-Boc-Tyr (14) was higher than that of
corresponding furanose deoxy sugar ester (12) but notably lower
than that of corresponding N-Boc-Phe esters (11) and (13). In the
lipase-catalyzed kinetic resolution of diastereomers the yield of
N-Boc-Tyr esters (18) and (20) was lower than that observed for N-
Boc-Phe esters (17) and (19). At the same time, there was no great
difference between the yield of acetylated furanose and pyranose
deoxy sugar esters in case of both of the N-Boc protected amino
acid esters.
4. Conclusions
The synthetic strategy developed previously for the synthesis
and stereochemical resolution of the pyranose dideoxy sugar esters
is also applicable to the most of the furanose deoxy sugar esters
investigated.
In the case of all four carboxylic acids tested – deoxycholic
acid, ˛-methoxyphenylacetic acid, N-Boc-phenylalanine and N-
Boc-tyrosine – the corresponding stereochemically pure 3-deoxy
d-erythrose ester was obtained in satisfactory to good yield.
In addition, 3,4-dideoxy d-ribose esters of N-Boc-phenylalanine
and N-Boc-tyrosine were synthesized in a diastereoselective man-
ner.
By evaluating yields of the products and incubation times
needed for the enzymatic resolution of stereoisomers, the overall
tendency is that pyranose deoxy sugar derivatives are, in gen-
eral (still, with one exception – 3,4-dideoxy d-ribose of N-Boc-Tyr
ester), slightly more proper target compounds for the current syn-
thetic strategy than furanose deoxy sugar derivatives.
Finally, it should be emphasized that for the synthesis of deoxy
sugar esters within the frames of the current synthetic approach –
the remote hydroxyl groups of carboxylic acids (for instance, DCA
and N-Boc-Tyr) need no protection.
The deacetylation of N-Boc-Tyr esters (18) and (20) were per-
formed by CALB in accordance with standard procedure. The
deacetylation of 18 led to the formation of the desired deoxy fura-
nose ester (12a) in good yield. On the other hand, lipase-catalyzed
deacetylation of pyranose deoxy sugar ester (20) remained, even
after several attempts, unsuccessful. Currently, it is hard to explain
this dramatic difference in the behavior of the pyranose vs. fura-
nose deoxy sugar esters. Further experimental investigation of this
synthesis supported by molecular modeling studies is needed to
clarify the reasons for such different results compared to the other
pyranose deoxy sugar esters.
In summary, the main difference between furanose and pyra-
nose deoxy sugar esters is that in most cases the lipase-catalyzed
acetylation of furanose hemiacetals produces only acetylated cyclic
form. On the contrary, when starting from pyranose deoxy sugar
esters, also an open-chain aldehyde form has often been gained
besides the stereochemically pure cyclic hemiacetal acetate [16].

L. Villo et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 44–51
51
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