Regioselective enzymatic acylation of pharmacologically interesting nucleosides in 2-methyltetrahydrofuran, a greener substitute for THF

Article abstract of DOI:10.1039/b818992g

1-β-Arabinofuranosyl uracil, 9-β-arabinofuranosyl adenosine, 2′-O-(2-methoxyethyl)-5-methyl uridine, adenosine and uridine were enzymatically acylated with hexanoic anhydride and vinyl esters by CALB lipase (lipase B from Candida antarctica) with excellen

Full text of DOI:10.1039/b818992g

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PAPER
www.rsc.org/greenchem | Green Chemistry
Regioselective enzymatic acylation of pharmacologically interesting
nucleosides in 2-methyltetrahydrofuran, a greener substitute for THF
Yolanda Simeo´,a Jose´ Vicente Sinisterraa,b and Andre´s R. Alca´ntara*a,b
Received 27th October 2008, Accepted 12th March 2009
First published as an Advance Article on the web 25th March 2009
DOI: 10.1039/b818992g
1-b-Arabinofuranosyl uracil, 9-b-arabinofuranosyl adenosine, 2¢-O-(2-methoxyethyl)-5-methyl
uridine, adenosine and uridine were enzymatically acylated with hexanoic anhydride and vinyl
esters by CALB lipase (lipase B from Candida antarctica) with excellent regioselectivity in many
cases and analytical reaction yields above 90%. The influence of the stereochemistry of the
hydroxyl group on C-2¢ was studied. Some of these esterifications were carried out in
2-methyltetrahydrofuran (MeTHF), which is described as an excellent substitute for THF
in biocatalysed processes in organic media. This application for this green solvent is a
proof-of-concept opening the use of MeTHF in biotransformations.
protecting groups and tedious separation processes, rendering
Introduction
low yields of the mono-O-acyl derivative.4 In turn, regioselective
Nucleosides and derivatives are relevant compounds in terms of
enzymatic acylation of nucleosides5 and hydrolysis6 of the
pharmacological properties.1 They have shown, among others,
corresponding esters are being intensively investigated due to
activity against some kinds of viruses (including the human
their feasibility and high efficiency. In general, biocatalysts used
immunodeficiency virus) and carcinogenic cells. For this reason,
in these processes (lipases) are relatively cheap and environmen-
enormous efforts have been undertaken in the last 20 years
tally friendly. Their use often avoids the need for protecting
to synthesize derivatives with potent antiviral and antitumor
groups, the reaction conditions are mild, they can show high
activities.2 In this sense, the goal of the present research work
regioselectivity (reducing the appearance of side reactions) that
was the regioselective synthesis of 1-b-arabinofuranosyl uracil
can be modulated by modification of the reaction parameters
(ara-U) and 9-b-arabinofuranosyl adenosine (ara-A) derivatives
and, in the case of immobilized enzymes, it is possible to reuse
to be tested as prodrugs with antitumor activity.
them,7 so that the economical sustainability can be increased.
Ara-U and ara-A are appealing compounds due to their
The benefits of using biotransformations can be further
cytotoxic activity. Nevertheless, they are extremely hydrophilic
improved with a rational selection of the solvent. In this
and, consequently, poorly absorbed enterically, so a different
sense, we present an esterification study of the above-mentioned
administration route would be desirable. For this reason, ap-
substrates with several vinyl esters as acylating agents and
propriate derivatives of the above-mentioned biologically active
using different lipases and solvents. Remarkable results were
products should be developed. Different chemical structures
obtained for the acylation of ara-U in 2-methyltetrahydrofuran
can be proposed to produce nucleoside prodrugs, but ester
(MeTHF), which is a versatile aprotic solvent that is being
formation is the most interesting reaction because of the high
used more often in industrial synthetic processes because of its
activity of human esterases, which can quickly liberate the drug.
favorable properties.8 In classical organic chemistry, MeTHF is
Besides, esters have higher lipophilicity, which facilitates the
increasingly being used as a THF substitute in the preparation
permeability through cell membranes. In fact, several esters from
of Grignard’s reagents,9a for low-temperature lithiation,9a,9b for
nucleoside analogues (such as acyclovir) have been prepared.3
lithium aluminium hydride reductions,9a for the Reformatsky
Among all possible derivatives, those with a small side chain
reaction,9c as well as for metal-catalyzed coupling reactions.9d–9f
in the OH-5¢ position could give rise to products with enhanced
In fact, MeTHF has a higher log P value (0.99)9g compared to
pharmacokinetic properties. Indeed, the acylation on C-5¢ would
THF (0.49),9g and it is partially miscible with water (solubility
lead to a prodrug that could be administered transdermally or
of water in MeTHF ranges from 4% to 5% upon heating from
as a solid pharmaceutical preparation.
0 to 70.6 ◦C; solubility of MeTHF in water ranges from 21%
Although chemical acylations of nucleosides have been re-
to 6% in the same temperature range);8 on the other hand,
ported in few cases, this methodology usually implies the use of
it has a boiling point of 89 ◦C, slightly higher than that of
THF and, therefore, solvent evaporation during reaction is
aOrganic and Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Universidad Complutense de Madrid, Plaza de Ramo´n y
Cajal s/n, 28040, Madrid, Spain. E-mail: andresr@farm.ucm.es;
Fax: +34 913941822; Tel: +34 913941820
reduced. Besides, this solvent forms an azeotrope with 10.6%
water, allowing the recycling of dry MeTHF; and gives clean
water phase separations, useful for two phase reactions and
product recovery. In many senses, MeTHF resembles toluene
in terms of physical properties.8 Most importantly, MeTHF
bIndustrial Biotransformations Service, Scientific Park of Madrid, calle
Santiago Grisol´ıa 2, 28760, Tres Cantos, Madrid, Spain
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is the only aprotic solvent similar to THF that derives from
renewable resources, i.e. it has low environmental impact, since
it is produced from furfural, which is a chemical isolated
from corn crops, sugar cane bagasse and oat hulls. Therefore,
substitution of THF or other solvents like dichloromethane or
dichloroethane for MeTHF will render greener processes, as the
3R desiderations—reduce, recycle and reuse—are all met by the
introduction of MeTHF. As far as we know, and excluding a
short statement in a old paper by Nakamura et al.9g (as one
of many solvents and, of course, without emphasizing its green
character at that time), this is the first description of MeTHF as
a solvent in biotransformations.
took place regioselectively at the hydroxyl group on C-5¢,
which is the preferred position for the lipase employed to
react as demonstrated by 2¢-deoxynucleosides.11 These deriva-
tives were characterized mainly by the 13C NMR data, since
the signal of the carbon supporting the acyl group (C-5¢)
moved downfield compared to the non-acylated. Thus, the
neighboring carbon atom (C-4¢) shifted upfield. The same
regioselectivity has been obtained in the hydrolysis of nucleoside
esters.6
Final conversion reached different values at the equilibrium
with the different solvents, probably because of their different
water miscibility and, therefore, also because of their different
water activity. In fact, when using MeTHF, after 6 h, 90%
of product 5¢-O-hexanoyl-1-b-arabinofuranosyl uracil (5) was
detected, but the yield decreased to 80% at 8 h, remaining
stable after this time. The reaction in THF proceeded slightly
slower but constantly increased up to a final value of 95% at
24 h. A similar behavior was observed for the esterification of
uridine (7) under the same reaction conditions, in MeTHF the
highest yield (95%) of 5¢-O-hexanoyl uridine (9) was obtained
after 4 h, decreasing then to 88% at 24 h; while in THF,
the acylation proceeded slower but the product formation
continuously increased up to 95% at 24 h. This fact may
be caused by the different water miscibility of the solvents:
thus, due to the liberation of hexanoic acid as the reaction
is proceeding, the micropH in the enzyme surroundings may
be altered, as described in ref. 12a, and causing a reversion
in the equilibrium by promoting the acidolysis of the acylated
product,12b this effect is more important when using MeTHF
vs. THF.
Results and discussion
1-b-Arabinofuranosyl uracil (ara-U, 1) and 9-b-arabino-
furanosyl adenosine (ara-A, 15), two cytotoxic compounds, were
regioselectively acylated with vinyl esters by several lipases in dif-
ferent solvents with the aim of producing nucleoside derivatives
with enhanced pharmacological properties (Scheme 1, Table 1).
Most of the published research on enzymatic esterification of
nucleosides is based on 2¢-deoxy derivatives.10 However, we
show herein the broad applicability of lipase B from Candida
antarctica (CALB) in such processes, since it is also capable of
distinguishing among three hydroxyl groups.
Scheme 1
With the aim of synthesizing derivatives of interesting cy-
totoxic agents ara-U and ara-A with short side chains in the
molecule, the acylation of 1-b-arabinofuranosyl uracil with
vinyl acetate (VA) by CALB in 2-methyltetrahydrofuran was
performed. After 6 h, 93% of the corresponding derivative in the
OH-5¢ position was obtained (product 2), along with 2% of start-
ing material and 5% of 3¢,5¢-O-diacetyl-1-b-arabinofuranosyl
uracil (6) (HPLC results, Table 2). This means that high
regioselectivity was achieved with the above-mentioned lipase
in only one step and short reaction time avoiding the use of
a protection/deprotection strategy. Therefore, this procedure
is atom economical and also environmentally friendly, since
MeTHF was used. It is noteworthy that the reaction without
enzyme did not work at all and only the initial substrate was
detected.
Acylation of 1-b-arabinofuranosyl uracil (ara-U)
Ara-U (1) was esterified at room temperature with hexanoic
anhydride by Novozyme435, an immobilised CALB, using
two solvents independently, THF and MeTHF. This process
Table 1 Structure of nucleosides and derivatives; U = uracil, 5-MU =
5-methyluracil, A = adenine
Base
R1
R2
R3
R4
Product
U
U
U
U
U
U
U
U
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
OH
OH
OH
O(CH2)2OCH3
O(CH2)2OCH3
O(CH2)2OCH3
O(CH2)2OCH3
O(CH2)2OCH3
H
H
H
H
H
H
H
H
1
COCH3
2
COCH2CH3
CO(CH2)2CH3
CO(CH2)4CH3
3
4
5
Results with other vinyl esters such as vinyl propionate (VP)
and butyrate (VB) largely paralleled those of vinyl acetate
(Table 2). In the case of VP and VB, once the maximum reaction
COCH3 COCH3
6
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
7
COCH3
CO(CH2)4CH3
H
COCH3
COCH2CH3
CO(CH2)2CH3 13
CO(CH2)4CH3 14
H
COCH3
COCH2CH3
CO(CH2)2CH3 18
CO(CH2)4CH3 19
H
8
U
9
5-MU
5-MU
5-MU
5-MU
5-MU
A
A
A
A
A
10
11
12
Table 2 Results obtained from the esterification of ara-U with CALB
and several vinyl esters in MeTHF
Ara-U (%)
5¢-Ester (%)
3¢,5¢-Diester (%)
15
16
17
Time/h VA VP VB VA VP VB VA
VP
VB
H
H
H
OH
OH
4
6
8
13
2
3
6
4
1
1
—
83
93
91
90
88
93
93
93
91
94
94
94
4
5
6
6
7
7
7
5
5
5
6
—
—
—
A
A
20
H
CO(CH2)4CH3 21
24
—
10
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Table 3 Comparison of results obtained from the esterification of ara-
U with vinyl esters in MeTHF by Pseudomonas fluorescens and CALB
acetyl groups came directly from the reagent (VA). The marked
preference for the hydroxyl group on C-3¢ as the second place
to acylate was attributed to the higher steric hindrance of the
2¢-OH, which is situated on the b-position like the nitrogenated
base. In fact, uridine was treated with vinyl acetate under the
same reaction conditions, giving rise after 192 h to 39% of the
derivative esterified in the 5¢-position (8) and 56% of a 70 : 30
mixture (ratio estimated from the NMR spectra) of the 2¢,5¢-
O- and 3¢,5¢-O-diacetyl compounds, which were impossible to
separate by traditional analytical methods. In this case, the OH
at the C-2¢ is not hindered by the base and, therefore, is also
accessible for acylation.
Ara-U (%)
5¢-Ester (%)
3¢,5¢-Diester (%)
Time/h Enzyme VA VP VB VA VP VB VA
VP
VB
4
4
6
6
8
CALB 13
6
4
83 88 91
56 37 50 40 60 49
93 93 94
30 33 45 64 62 51
91 93 94
20 29 34 74 63 60
4
4
5
6
6
6
6
3
7
5
7
5
1
5
4
5
PFL
CALB
PFL
2
—
1
CALB
PFL
3
—
1
8
8
6
24
24
CALB
PFL
—
2
—
2
—
1
90 93 94 10
85 72 70 13
7
26
6
19
High yields, but lower regioselectivity, was reported by
Uemura et al. for the acylation of 2¢-deoxyuridine with hexanoic
anhydride by PFL in several organic solvents (DMA, DMSO
and DMF) at room temperature after 24 h.16
yield was achieved (6 h) it did not decrease with time, while with
VA it was slightly lower.
To check the viability of using some other biocatalysts,
acylation of ara-U with vinyl esters was also tested with other
lipases, such as those from native lyophilized Mucor javanicus
(MJL), Candida rugosa (CRL), Pseudomonas cepacea (PCL) and
Pseudomonas fluorescens (PFL), but the yields were lower than
10%, probably due to aggregation of these native preparations
in the organic solvent and the concomitant diffusion problems.
This problem is overcome by the use of a lipase supported on
a carrier, such as in the CALB derivative used in this study, in
which all lipase molecules are more accessible to the substrate.13
The only exception was PFL, although enzymatic esterification
with this lipase led to lower yields than CALB and higher
proportion of the diacyl derivative in both 5¢ and 3¢ positions,
as shown in Table 3.
Acylation of 9-b-arabinofuranosyl adenosine (ara-A)
Likewise, a comparative study of the ara-A (15) esterification
with hexanoic anhydride by CALB in MeTHF and THF was
performed. In this case, THF turned to be a better solvent for
the substrate, giving rise to higher yields than MeTHF. In fact,
81% of 5¢-O-hexanoyl-9-b-arabinofuranosyl adenosine (19) was
rendered after 28 h.
A similar behavior was observed for the acylation of adenosine
(20) under the same reaction conditions, achieving the highest
yield after 30 h for the esterification in THF (21). In general,
longer reaction times are needed for nucleosides with a purine-
type base.17
Taking into account that for the esterification of ara-A with
hexanoic anhydride by CALB much higher yields were rendered
in THF, this was the solvent of choice for the reactions using
vinyl esters as acylating agents. As mentioned before, nucleosides
with a purine-type base reacted with a lower conversion rate
than those with a pyrimidine-type, i.e. longer reaction times
were needed for ara-A.
In Table 5, it can be noticed that the highest yield was
achieved after 24 h for VP and VB, and 28 h for VA (88%).
This way, esterification of ara-A with vinyl propionate by CALB
in THF gave rise to 98% of the 5¢ derivative (compound 17)
with total regioselectivity (2% of starting material remained
unreacted). In the case of vinyl butyrate, regioselectivity was
also excellent, since 97% of 5¢-O-butanoyl-9-b-arabinofuranosyl
adenosine (18) was detected together with 2% of substrate
and 1% of the corresponding 3¢,5¢-dibutyrate derivative. The
reaction with vinyl acetate underwent slightly slower and
rendered a lower yield (product 16), but the regioselectivity was
Nevertheless, it is difficult to draw a direct comparison
between the two catalysts, since they are used in a different state
(native lyophilized preparation (PFL) or immobilised derivative
(CALB)), and also because of their different intrinsic declared
activity (see Experimental). We would like to emphasise that
not many crude lipases are able to work efficiently in such a
polar solvent as THF, it is well known that more hydrophobic
solvents are generally preferred;14 in fact, only very stable
catalysts such as CALB (or the recently described use of crude
lipase from Pseudomonas stutzeri in THF)15 can be employed.
Anyhow, when the reaction time was extended to several days
the proportion of the diester (6) notably increased (Table 4).
Remarkably, 2¢,5¢-O-diacetyl-1-b-arabinofuranosyl uracil was
not detected at all.
In order to prove if the second acylation took place through
an acyl transfer process or if it was indeed an esterification, 5¢-O-
acetyl-1-b-arabinofuranosyl uracil (2) was dissolved in MeTHF
and maintained with stirring for 192 h at room temperature,
but no transformation occurred. This result confirms that both
Table 5 Results obtained from the esterification of ara-A with CALB
and several vinyl esters in THF
Table 4 Esterification of ara-U with vinyl acetate by Pseudomonas
fluorescens in MeTHF at longer reaction times
Ara-A (%)
5¢-Ester (%)
3¢,5¢-Diester (%)
Time/h
Ara-U (%)
5¢-Ester (%) (2)
3¢,5¢-Diester (%) (6)
Time/h VA VP VB VA VP
VB VA
VP
VB
24
48
72
96
192
3
82
76
72
69
59
14
24
28
31
41
4
6
8
24
28
47
43
38
19
10
46
41
31
2
41
40
29
2
53
57
62
79
88
54
59
69
98
95
59
60
71
97
92
—
—
—
2
—
—
—
—
5
—
—
—
1
—
—
—
—
—
2
2
6
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Table 6 Results obtained from the esterification of 2¢-O-methoxyethyl-
5-methyluridine with CALB and several vinyl esters in MeTHF
Experimental
General
2¢-O-methoxyethyl-5-
methyluridine (%)
5¢-Ester (%)
Ara-U and ara-A were kindly provided by Pro. Bio. Simt.
S.p.A., 2¢-O-methoxyethyl-5-methyl uridine by Ravi Chemi-
cals and MeTHF by Penn Specialty Chemicals Inc. CALB
(Novozyme435, immobilized lipase from Candida Antarctica
B) was a gentle donation of Novozymes, Spain. Lipases from
Mucor javanicus (MJL), Candida rugosa (CRL), Pseudomonas
cepacea (PCL) and Pseudomonas fluorescens (Amano Lipase
AK) were purchased from Sigma-Aldrich. All other chemicals
were obtained from commercial sources. Reaction progress was
followed by HPLC on an Agilent LC1200 using the C18 column
Mediterranea Sea from Teknokroma, and by TLC on Kieselgel
Plates 60 F254 (SDS). TLC plates were visualized under UV light
or revealed with 10% H2SO4 in methanol and heating. Column
chromatography was carried out, if necessary, on silica gel 60,
AC, 40–63 mm (purchased from SDS). NMR spectra of samples
in DMSO-d6 were recorded on a Bruker Avance 250 (250 MHz)
Time/h
VA
VP
VB
VA
VP
VB
2
4
6
8
24
2
2
2
90
93
94
94
94
95
91
92
89
73
89
85
91
89
62
—
—
—
—
—
—
—
—
—
—
—
—
similar to the values measured for vinyl butyrate. Esterification
of ara-A with the same vinyl esters but employing lipases
from other microorganisms (MJL, CRL, PCL and PFL) was
also tested. As occurred with the ara-U acylation, only PFL
produced the 5¢ ester derivative, but with rather irrelevant
yields.
1
spectrometer. Compound assignments were based on H, 13C,
Acylation of 2¢-O-(2-methoxyethyl)-5-methyl uridine
HMQC and HMBC NMR experiments.
2¢-O-Alkyl nucleosides are considered to be key building blocks
of several second generation antisense oligonucleotides in
clinical development. As promising therapeutic agents, much
effort is being made towards the development of novel nuclease
resistant oligonucleotides, which are capable of hybridizing
with appropriate specificity and affinity to complementary
sequences thus acting as effective inhibitors of gene expression.18
However, these nucleotides should be modified to avoid rapid
degradation by cellular nucleases. Therefore, we have esterified
2¢-O-(2-methoxyethyl)-5-methyl uridine following the same
strategy as for ara-A and ara-U.
Acylation of the latter with hexanoic anhydride in MeTHF
gave rise directly to the diester in 3¢ and 5¢ position, achieving
the highest yield after 6 h (89% of 2¢-O-(2-methoxyethyl)-3¢,5¢-
dihexanoyl-5-methyl uridine, 14). However, the fact that the
reaction was not regioselective and that 8% of compound 14 was
formed in the sample without enzyme, prompted us to focus our
attention again to vinyl esters. Reaction of this starting material
with vinyl acetate rendered 94% of the 5¢ acetyl derivative
(compound 11) after 6 h (Table 6). Slightly lower results were
obtained with vinyl propionate (92%, product 12) and vinyl
butyrate (91%, compound 13). This way, the regioselective
synthesis of nucleoside derivatives with only the hydroxyl group
at C-3¢ free was accomplished.
Experimental procedures
Esterification of ara-U with hexanoic anhydride
Ara-U (120 mg, 0.5 mmol) was dissolved in 50 mL of an-
hydrous MeTHF and 0.5 g of molecular sieves, 0.5 g of
CALB (Novozyme435, 20% w/w loading,19 10 000 PLU g-1)20
and 0.35 mL of hexanoic anhydride (1.5 mmol) were added,
maintaining the reaction with orbital shaking for 24 h at room
temperature. After that, MeOH was added, the reaction mixture
was filtered, neutralized with saturated NaHCO3 solution and
dried with anhydrous Na2SO4. Then, silica gel was added,
the solvent was evaporated at reduced pressure and the crude
subsequently purified by column chromatography to give 75 mg
(45%) of 5¢-O-hexanoyl-1-b-arabinofuranosyl uracil (5): white
solid; 1H NMR (DMSO-d6, 250 MHz): d 0.85 (3H, t, J = 6.3 Hz,
CH3CH2CH2), 1.26 (4H, m, CH3CH2CH2), 1.54 (2H, t, J =
6.7 Hz, CH2CH2CO), 2.33 (2H, t, J = 7.2 Hz, CH2CH2CO),
3.95 (2H, m, H-3¢ and H-4¢), 4.01 (1H, br s, H-2¢), 4.21 (1H, dd,
J1 = 3.4 Hz, J2 = 11.8 Hz, 1H-5¢), 4.30 (1H, dd, J1 = 7.2 Hz,
J2 = 11.8 Hz, 1H-5¢), 5.58 (1H, d, J = 8.1 Hz, H-5), 5.67 (1H,
br s, OH-3¢), 5.76 (1H, d, J = 3.9 Hz, OH-2¢), 6.04 (1H, d,
J = 3.4 Hz, H-1¢), 7.51 (1H, d, J = 8.1 Hz, H-6), 11.34 (1H,
br s, NH); 13C NMR (DMSO-d6, 63 MHz): d 14.2 (CH3), 22.2
(CH3CH2CH2), 24.5 (CH2CH2CO), 31.0 (CH3CH2CH2), 33.7
(CH2CH2CO), 63.8 (C-5¢), 75.0 and 76.4 (C-2¢ and C-3¢), 82.1
(C-4¢), 85.7 (C-1¢), 100.5 (C-5), 142.6 (C-6), 150.8 (C-2), 163.6
(C-4), 173.2 (CO). Anal. Calcd. for C15H22N2O7: C, 52.63; H,
6.48; N, 8.18%. Found: C, 52.36; H, 6.44; N, 7.76%.
Conclusion
The described enzymatic processes represent an excellent
methodology to obtain valuable nucleoside derivatives. Reac-
tions took place in many cases in short reaction times (6 h) and
with total regioselectivity, which is arduous to achieve by pure
chemical means due to the presence of various hydroxyl groups.
A comparison between THF and environmentally friendly
MeTHF was established, both solvents behaving in a similar way,
while for the acylation of ara-U and uridine, MeTHF rendered
better conversions. This green solvent can be really valuable in
the biotransformations field.
Acylation of uridine (7) with hexanoic anhydride
0.5 g of molecular sieves, 0.5 g of CALB and 0.35 mL of
hexanoic anhydride were added to a solution of 120 mg
uridine in 50 mL anhydrous MeTHF. After 24 h with orbital
shaking at room temperature, MeOH was added, the reaction
mixture filtered, neutralized with saturated NaHCO3 solution
and dried with anhydrous Na2SO4. Silica gel was added, the
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solvent was evaporated at reduced pressure and the residue was
chromatographed on a silica gel column to give 71 mg (42%)
of 5¢-O-hexanoyl uridine (9): white solid; 1H NMR (DMSO-d6,
250 MHz): d 0.87 (3H, m, CH3), 1.27 (4H, m, CH3CH2CH2),
1.52 (2H, m, CH2CH2CO), 2.34 (2H, t, J = 7.3, CH2CH2CO),
3.97 (2H, m, H-3¢ and H-4¢), 4.08 (1H, t, J = 5.0 Hz, H-2¢),
4.18 (1H, dd, J1 = 5.3 Hz, J2 = 12.1 Hz, 1H-5¢), 4.26 (1H, dd,
J1 = 3.6 Hz, J2 = 12.1 Hz, 1H-5¢), 5.33 (1H, br s, OH-3¢), 5.52
(1H, br s, OH-2¢), 5.67 (1H, d, J = 8.1 Hz, H-5), 5.76 (1H, d,
J = 5.8 Hz, H-1¢), 7.64 (1H, d, J = 8.1 Hz, H-6), 11.41 (1H,
br s, NH); 13C NMR (DMSO-d6, 63 MHz): d 14.2 (CH3), 22.2
(CH3CH2CH2), 24.5 (CH2CH2CO), 31.0 (CH3CH2CH2), 33.6
(CH2CH2CO), 64.0 (C-5¢), 70.1 (C-3¢), 73.0 (C-2¢), 81.4 (C-4¢),
89.0 (C-1¢), 102.3 (C-5), 141.1 (C-6), 151.0 (C-2), 163.4 (C-4),
173.1 (CO). Anal. Calcd. for C15H22N2O7: C, 52.63; H, 6.48; N,
8.18%. Found: C, 52.83; H, 6.56; N, 7.62%.
(2H, t, J = 7.2 Hz, CH3CH2CH2CO), 3.95 (2H, m, H-3¢ and H-
4¢), 4.03 (1H, m, H-2¢), 4.23 (1H, dd, J1 = 3.8 Hz, J2 = 11.8 Hz,
1H-5¢), 4.32 (1H, dd, J1 = 7.1 Hz, J2 = 11.8 Hz, 1H-5¢), 5.60
(1H, d, J = 8.1 Hz, H-5), 5.72 (1H, br s, OH-3¢), 5.80 (1H, d,
J = 4.2 Hz, OH-2¢), 6.05 (1H, d, J = 4.2 Hz, H-1¢), 7.52 (1H,
d, J = 8.1 Hz, H-6), 11.36 (1H, br s, NH); 13C NMR (DMSO-
d6, 75 MHz): d 13.7 (CH3CH2CH2CO), 18.3 CH3CH2CH2CO),
35.6 (CH3CH2CH2CO), 63.7 (C-5¢), 75.0 and 76.4 (C-2¢ and C-
3¢), 82.1 (C-4¢), 85.8 (C-1¢), 100.5 (C-5), 142.6 (C-6), 150.8 (C-2),
163.7 (C-4), 173.1 (CO). Anal. Calcd. for C13H18N2O7: C, 49.68;
H, 5.77; N, 8.91%. Found: C, 49.67; H, 5.77; N, 8.70%.
Synthesis of 3¢,5¢-O-acetyl-1-b-arabinofuranosyl uracil (6).
1 g of molecular sieves, 1 g◦of Pseudomonas fluorescens (activity
≥20 000 U g-1, pH 8.0, 55 C using triolein as substrate)21 and
276 mL of vinyl acetate were added to a solution of 240 mg
of ara-U in 100 mL of anhydrous MeTHF. After 8 days with
orbital shaking at room temperature, MeOH was added, the
reaction mixture filtered, neutralized with saturated NaHCO3
solution and dried with anhydrous Na2SO4. Then, silica gel
was added, the solvent evaporated at reduced pressure and the
residue chromatographed on a silica gel column to give 158 mg
(55%) of compound 2 and 139 mg (42%) of 3¢,5¢-O-acetyl-1-b-
Esterification of ara-U with vinyl esters
Ara-U (120 mg, 0.5 mmol) was dissolved in 50 mL of anhydrous
MeTHF and 0.5 g of molecular sieves, 0.5 g of CALB and
1.5 mmol of vinyl ester (138 mL vinyl acetate, 163 mL vinyl
propionate or 190 mL vinyl butyrate) were added, maintaining
the reaction with orbital shaking for 7 h at room temperature.
After that, MeOH was added, the reaction mixture filtered,
neutralized with saturated NaHCO3 solution and dried with
anhydrous Na2SO4. Then, silica gel was added and the solvent
evaporated to dryness. Finally, the residue was purified by
column chromatography over silica gel.
1
arabinofuranosyl uracil (6): white solid; H NMR (DMSO-d6,
250 MHz): d 2.06 (3H, s, CH3CO), 2.10 (3H, s, CH3CO), 4.15
(1H, ddd, J1 = J2 = 3.8 Hz, J3 = 7.0 Hz, H-4¢), 4.22 (1H, m,
H-2¢), 4.32 (2H, m, 2H-5¢), 4.96 (1H, br s, H-3¢), 5.63 (1H, d,
J = 8.1 Hz, H-5), 6.02 (1H, d, J = 3.9 Hz, H-1¢), 6.16 (1H, d,
J = 4.3 Hz, OH-2¢), 7.57 (1H, d, J = 8.1 Hz, H-6), 11.41 (1H,
br s, NH); 13C NMR (DMSO-d6, 63 MHz): d 21.0 (CH3CO),
21.1 (CH3CO), 63.5 (C-5¢), 72.5 (C-2¢), 78.4 (C-3¢), 79.8 (C-4¢),
85.8 (C-1¢), 100.7 (C-5), 142.4 (C-6), 150.7 (C-2), 163.6 (C-4),
170.1 (CO), 170.6 (CO). Anal. Calcd. for C13H16N2O8: C, 47.56;
H, 4.91; N, 8.53%. Found: C, 47.83; H, 5.62; N, 7.09%.
5¢-O-acetyl-1-b-arabinofuranosyl uracil (2). 61 mg (43%):
1
white solid; H NMR (DMSO-d6, 250 MHz): d 2.06 (3H, s,
CH3CO), 3.93 (2H, m, H-3¢ and H-4¢), 4.02 (1H, ddd, J1 = J2 =
4.5 Hz, J3 = 7.0 Hz, H-2¢), 4.21 (1H, dd, J1 = 4.0 Hz, J2 =
11.8 Hz, 1H-5¢), 4.30 (1H, dd, J1 = 7.1 Hz, J2 = 11.8 Hz, 1H-5¢),
5.60 (1H, d, J = 8.1 Hz, H-5), 5.71 (1H, d, J = 3.9 Hz, OH-
3¢), 5.78 (1H, d, J = 4.5 Hz, OH-2¢), 6.05 (1H, d, J = 4.5 Hz,
Acylation of uridine with vinyl acetate by Pseudomonas
fluorescens
H-1¢), 7.52 (1H, d, J = 8.1 Hz, H-6), 11.35 (1H, br s, NH); 13
C
NMR (DMSO-d6, 63 MHz): d 21.5 (CH3), 64.5 (C-5¢), 75.5 and
77.0 (C-2¢ and C-3¢), 82.5 (C-4¢), 86.2 (C-1¢), 101.0 (C-5), 143.1
(C-6), 151.3 (C-2), 164.1 (C-4), 171.1 (CO). Anal. Calcd. for
C11H14N2O7: C, 46.16; H, 4.93; N, 9.79%. Found: C, 46.17; H,
5.00; N, 9.45%.
Uridine (240 mg, 1 mmol) was dissolved in 100 mL of anhydrous
MeTHF and 1 g of molecular sieves, 1 g of Pseudomonas
fluorescens and 276 mL of vinyl acetate (3 mmol) were added,
maintaining the reaction with orbital shaking for 8 days at room
temperature. After that, MeOH was added, the reaction mixture
filtered, neutralized with saturated NaHCO3 solution and dried
with anhydrous Na2SO4. Then, silica gel was added and the
solvent evaporated to dryness. Finally, the residue was purified
by column chromatography over silica gel yielding 148 mg (53%)
of a mixture consisting of 3¢,5¢-O-diacetyl uridine and 2¢,5¢-O-
diacetyl uridine, and 106 mg (38%) of 5¢-O-acetyl uridine (8):
5¢-O-propanoyl-1-b-arabinofuranosyl uracil (3). 87 mg
1
(58%): white solid; H NMR (DMSO-d6, 300 MHz): d 1.03
(3H, t, J = 7.5 Hz, CH3CH2CO), 2.34 (2H, dd, J1 = 7.5 Hz,
J2 = 15.6 Hz, CH3CH2CO), 3.92 (2H, m, H-3¢ and H-4¢), 4.01
(1H, m, H-2¢), 4.23 (2H, m, 2H-5¢), 5.58 (1H, d, J = 8.1 Hz,
H-5), 5.67 (1H, d, J = 3.4 Hz, OH-3¢), 5.74 (1H, d, J = 4.5 Hz,
OH-2¢), 6.02 (1H, d, J = 4.2 Hz, H-1¢), 7.49 (1H, d, J = 8.1 Hz,
H-6), 11.30 (1H, br s, NH); 13C NMR (DMSO-d6, 75 MHz):
d 9.3 (CH3CH2CO), 27.1 (CH3CH2CO), 63.8 (C-5¢), 75.0 and
76.4 (C-2¢ and C-3¢), 82.0 (C-4¢), 85.7 (C-1¢), 100.5 (C-5), 142.6
(C-6), 150.8 (C-2), 163.7 (C-4), 174.0 (CO). Anal. Calcd. for
C12H16N2O7: C, 48.00; H, 5.37; N, 9.33%. Found: C, 47.86; H,
5.38; N, 9.04%.
1
white solid; H NMR (DMSO-d6, 250 MHz): d 2.09 (3H, s,
CH3CO), 3.99 (2H, m, H-3¢ and H-4¢), 4.19 (3H, m, 2H-5¢ and
H-2¢), 5.37 (1H, d, J = 4.5 Hz, OH-3¢), 5.55 (1H, d, J = 4.9 Hz,
OH-2¢), 5.71 (1H, d, J = 8.0 Hz, H-5), 5.77 (1H, d, J = 4.7 Hz,
H-1¢), 7.67 (1H, d, J = 8.0 Hz, H-6), 11.42 (1H, br s, NH);
13C NMR (DMSO-d6, 63 MHz): d 21.0 (CH3), 64.1 (C-5¢), 70.1
(C-3¢), 73.0 (C-2¢), 81.4 (C-4¢), 89.0 (C-1¢), 102.4 (C-5), 141.2
(C-6), 151.0 (C-2), 163.5 (C-4), 170.6 (CO). Elemental analysis
(Found: C, 46.53; H, 5.19; N, 8.89%. Calc. for C11H14N2O7: C,
46.16; H, 4.93; N, 9.79%).
5¢-O-butanoyl-1-b-arabinofuranosyl uracil (4). 84 mg (54%):
white solid; 1H NMR (DMSO-d6, 300 MHz): d 0.90 (3H, t, J =
7.4 Hz, CH3CH2CH2CO), 1.57 (2H, m, CH3CH2CH2CO), 2.33
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Esterification of ara-A with hexanoic anhydride
evaporated to dryness. Finally, the residue was purified by
column chromatography over silica gel.
0.5 g of molecular sieves, 0.5 g of CALB and 0.35 mL of
hexanoic anhydride were added to a solution of 135 mg of
ara-A in 50 mL of anhydrous THF. After 24 h with orbital
shaking at room temperature, MeOH was added, the reaction
mixture filtered, neutralized with saturated NaHCO3 solution
and dried with anhydrous Na2SO4. Silica gel was added, the
solvent was evaporated at reduced pressure and the residue was
chromatographed on a silica gel column to give 58 mg (31%)
of 5¢-O-hexanoyl-9-b-arabinofuranosyl adenosine (19): yellow
solid; 1H NMR (DMSO-d6, 250 MHz): d 0.67 (3H, t, J = 6.9 Hz,
CH3), 1.05 (4H, m, CH3CH2CH2), 1.31 (2H, m, CH2CH2CO),
2.12 (2H, t, J = 7.2 Hz, CH2CH2CO), 3.78 (1H, ddd, J1 = J2 =
3.6 Hz, J3 = 7.5 Hz, H-4¢), 3.97 (2H, d, J = 3.6 Hz, H-2¢ and
H-3¢), 4.08 (1H, dd, J1 = 3.6 Hz, J2 = 11.7 Hz, 1H-5¢), 4.19
(1H, dd, J1 = 7.5 Hz, J2 = 11.7 Hz, 1H-5¢), 5.62 (2H, br s, OH-
2¢ and OH-3¢), 6.11 (1H, d, J = 4.2 Hz, H-1¢), 7.10 (2H, br s,
NH2), 7.93 and 7.95 (2H, s, H-2 and H-8); 13C NMR (DMSO-d6,
63 MHz): d 14.2 (CH3), 22.2 (CH3CH2CH2), 24.5 (CH2CH2CO),
31.0 (CH3CH2CH2), 33.6 (CH2CH2CO), 64.1 (C-5¢), 75.4 and
76.0 (C-2¢ and C-3¢), 81.3 (C-4¢), 83.9 (C-1¢), 118.5 (C-5), 140.7
(C-8), 149.7 (C-4), 152.9 (C-2), 156.3 (C-6), 173.2 (CO). Anal.
Calcd. for C16H23N5O5: C, 52.59; H, 6.34; N, 19.17%. Found: C,
51.63; H, 6.01; N, 19.33%.
5¢-O-acetyl-9-b-Arabinofuranosyl adenosine (16). 102 mg
(65%): white solid; H NMR (DMSO-d6, 250 MHz): d 2.04
1
(3H, s, CH3CO), 3.98 (1H, m, H-4¢), 4.20 (2H, br s, H-2¢ and
H-3¢), 4.33 (2H, m, H-5¢), 5.80 (1H, d, J = 4.1 Hz, OH-3¢), 5.85
(1H, d, J = 4.5 Hz, OH-2¢), 6.32 (1H, d, J = 4.2 Hz, H-1¢), 7.32
(2H, br s, NH2), 8.16 and 8.17 (2H, s, H-2 and H-8); 13C NMR
(DMSO-d6, 63 MHz): d 21.0 (CH3), 64.3 (C-5¢), 75.4 and 76.0
(C-2¢ and C-3¢), 81.2 (C-4¢), 83.9 (C-1¢), 118.5 (C-5), 140.8 (C-8),
149.7 (C-4), 152.9 (C-2), 156.2 (C-6), 170.7 (CO). Anal. Calcd.
for C12H15N5O5: C, 46.60; H, 4.89; N, 22.64%. Found: C, 45.96;
H, 4.63; N, 22.51%.
5¢-O-propanoyl-9-b-Arabinofuranosyl
adenosine
(17).
111 mg (68%): white solid; 1H NMR (DMSO-d6, 250 MHz): d
1.03 (3H, t, J = 7.5 Hz, CH3), 2.35 (2H, dd, J1 = 7.5 Hz, J2 =
14.9 Hz, CH3CH2), 3.99 (1H, m, H-4¢), 4.20 (2H, br s, H-2¢ and
H-3¢), 4.34 (2H, m, H-5¢), 5.79 (1H, d, J = 3.7 Hz, OH-3¢), 5.85
(1H, d, J = 4.1 Hz, OH-2¢), 6.31 (1H, d, J = 4.0 Hz, H-1¢),
7.32 (2H, br s, NH2), 8.15 and 8.16 (2H, s, H-2 and H-8); 13C
NMR (DMSO-d6, 63 MHz): d 9.3 (CH3), 27.1 (CH3CH2), 64.1
(C-5¢), 75.4 and 75.9 (C-2¢ and C-3¢), 81.2 (C-4¢), 83.8 (C-1¢),
118.5 (C-5), 140.7 (C-8), 149.7 (C-4), 152.9 (C-2), 156.2 (C-6),
174.0 (CO). Anal. Calcd. for C13H17N5O5: C, 48.29; H, 5.30; N,
21.66%. Found: C, 47.97; H, 4.90; N, 21.68%.
Acylation of adenosine with hexanoic anhydride
5¢-O-Butanoyl-9-b-arabinofuranosyl adenosine (18). 107 mg
1
0.5 g of molecular sieves, 0.5 g of CALB and 0.35 mL of
hexanoic anhydride were added to a solution of 135 mg of
adenosine in 50 mL of anhydrous THF. After 24 h with orbital
shaking at room temperature, MeOH was added, the reaction
mixture filtered, neutralized with saturated NaHCO3 solution
and dried with anhydrous Na2SO4. Silica gel was added, the
solvent was evaporated at reduced pressure and the residue was
chromatographed on a silica gel column to give 62 mg (33%) of
5¢-O-hexanoyl adenosine (21): white solid; 1H NMR (DMSO-d6,
250 MHz): d 0.82 (3H, t, J1 = J2 = 6.8 Hz, CH3), 1.21 and 1.48
(6H, m, CH2), 2.28 (2H, t, J1 = J2 = 7.3 Hz, CH2CO), 4.08 (1H,
m, H-4¢), 4.26 (3H, m, H-3¢ and 2H-5¢), 4.68 (1H, m, H-2¢), 5.41
(1H, d, J = 4.6 Hz, OH-3¢), 5.62 (1H, d, J = 5.8 Hz, OH-2¢), 5.91
(1H, d, J = 4.9 Hz, H-1¢), 7.32 (2H, br s, NH2), 8.15 (1H, s, H-8),
8.31 (1H, s, H-2); 13C NMR (DMSO-d6, 63 MHz): d 14.1 (CH3),
22.1 (CH3CH2), 24.4 (CH2CH2CO), 30.9 (CH3CH2CH2), 33.6
(CH2CO), 64.0 (C-5¢), 70.6 (C-3¢), 73.2 (C-2¢), 81.8 (C-4¢),
88.1 (C-1¢), 119.5 (C-5), 140.1 (C-8), 149.7 (C-4), 153.0 (C-
2), 156.4 (C-6), 173.1 (CO). Anal. Calcd. for C16H23N5O5:
C, 52.59; H, 6.34; N, 19.17%. Found: C, 52.17; H, 6.20;
N, 18.65%.
(63%): white solid; H NMR (DMSO-d6, 250 MHz): d 0.88
(3H, t, J = 7.3 Hz, CH3), 1.55 (2H, ddd, J1 = J2 = 7.3 Hz, J3 =
14.7 Hz, CH3CH2), 2.32 (2H, t, J1 = J2 = 7.1 Hz, CH2CO), 3.98
(1H, m, H-4¢), 4.27 (2H, m, H-2¢ and H-3¢), 4.37 (2H, m, H-5¢),
5.76 (1H, d, J = 3.7 Hz, OH-3¢), 5.82 (1H, d, J = 4.2 Hz, OH-2¢),
6.31 (1H, d, J = 4.0 Hz, H-1¢), 7.31 (2H, br s, NH2), 8.13 and
8.16 (2H, s, H-2 and H-8); 13C NMR (DMSO-d6, 63 MHz): d
13.8 (CH3), 18.3 (CH3CH2), 35.6 (CH2CO), 64.1 (C-5¢), 75.4 and
76.0 (C-2¢ and C-3¢), 81.3 (C-4¢), 83.9 (C-1¢), 118.5 (C-5), 140.7
(C-8), 149.7 (C-4), 152.9 (C-2), 156.3 (C-6), 173.1 (CO). Anal.
Calcd. for C14H19N5O5: C, 49.85; H, 5.68; N, 20.76%. Found: C,
49.82; H, 5.57; N, 20.97%.
Acylation of 2¢-O-(2-methoxyethyl)-5-methyl uridine with
hexanoic anhydride
0.5 g of molecular sieves, 0.5 g of CALB and 0.35 mL of
hexanoic anhydride were added to a solution of 160 mg
2¢-O-(2-methoxyethyl)-5-methyl uridine in 50 mL of anhydrous
MeTHF. After 24 h with orbital shaking at room temperature,
MeOH was added, the reaction mixture filtered, neutralized
with saturated NaHCO3 solution and dried with anhydrous
Na2SO4. Silica gel was added, the solvent was evaporated at
reduced pressure and the residue was chromatographed on a
silica gel column to give 86 mg (43%) of 2¢-O-(2-methoxyethyl)-
Esterification of ara-A with vinyl esters
Ara-A (135 mg, 0.5 mmol) was dissolved in 50 mL of anhydrous
MeTHF and 0.5 g of molecular sieves, 0.5 g of CALB and
1.5 mmol of vinyl ester (138 mL vinyl acetate, 163 mL vinyl
propionate or 190 mL vinyl butyrate) were added, maintaining
the reaction with orbital shaking for 24 h at room temperature.
After that, MeOH was added, the reaction mixture filtered,
neutralized with saturated NaHCO3 solution and dried with
anhydrous Na2SO4. Then, silica gel was added and the solvent
1
3¢,5¢-dihexanoyl-5-methyl uridine (14): yellow solid; H NMR
(DMSO-d6, 250 MHz): d 0.88 (6H, m, 2 ¥ CH3), 1.28 (8H, m,
2 ¥ CH3CH2CH2), 1.52 (4H, m, 2 ¥ CH2CH2CO), 1.81 (3H, s,
CH3), 2.19 (2H, t, J = 7.3 Hz, CH2CH2CO), 2.35 (2H, t, J =
7.3 Hz, CH2CH2CO), 3.23 (3H, s, OCH3), 3.45 (2H, t, J =
4.6 Hz, OCH2CH2OCH3), 3.67 (2H, m, OCH2CH2OCH3), 4.04
(3H, m, H-2¢, H-3¢ and H-4¢), 4.24 (1H, m, 2H-5¢), 5.85 (1H,
860 | Green Chem., 2009, 11, 855–862
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d, J = 4.9 Hz, H-1¢), 7.46 (1H, s, H-6), 11.43 (1H, br s, NH);
13C NMR (DMSO-d6, 63 MHz): d 12.5 (CH3), 14.1 and 14.2
(2 ¥ CH3CH2CH2), 22.1 and 22.2 (2 ¥ CH3CH2CH2), 24.4 and
24.6 (CH2CH2CO), 31.0 and 31.1 (2 ¥ CH3CH2CH2), 33.7 and
34.1 (CH2CH2CO), 58.4 (OCH3), 63.8 (C-5¢), 69.0 (C-3¢), 69.4
(OCH2CH2OCH3), 71.6 (OCH2CH2OCH3), 80.6 and 81.6 (C-2¢
and C-4¢), 87.0 (C-1¢), 110.1 (C-5), 136.4 (C-6), 150.9 (C-2), 164.0
(C-4), 173.1 and 175.0 (2 ¥ CO). Anal. Calcd. for C25H40N2O9:
C, 58.58; H, 7.87; N, 5.47%. Found: C, 56.40; H, 7.52; N, 5.87%.
11.44 (1H, br s, NH); 13C NMR (DMSO-d6, 63 MHz): d 13.0
and 14.2 (CH3 and CH3CH2CH2CO), 18.7 CH3CH2CH2CO),
36.1 (CH3CH2CH2CO), 59.0 (OCH3), 64.2 (C-5¢), 69.5 and 69.9
(C-3¢ and OCH2CH2OCH3), 72.1 (OCH2CH2OCH3), 81.1 and
82.1 (C-2¢ and C-4¢), 87.6 (C-1¢), 110.7 (C-5), 136.9 (C-6), 151.4
(C-2), 164.6 (C-4), 173.5 (CO). Anal. Calcd. for C17H26N2O8: C,
52.84; H, 6.78; N, 7.25%. Found: C, 52.61; H, 6.80; N, 6.95%.
Acknowledgements
Esterification of 2¢-O-(2-methoxyethyl)-5-methyl uridine with
vinyl esters
The authors thank Penn Specialty Chemicals Inc. for kindly
providing us 2-methyltetrahydrofuran, Pro. Bio. Simt. S.p.A.
for ara-U and ara-A, Ravi Chemicals for 2¢-O-methoxyethyl-5-
methyl uridine and Novozymes for CALB. The financial support
by the SOLFSAVE project funded by the European Union, the
project S-0505/PPQ/0344 by the Comunidad Auto´noma de
Madrid, and projects CTQ2006-15692-C02-01 and CTQ2006-
09052 from MEC (Spanish Ministry of Education and Science)
is gratefully acknowledged.
2¢-O-(2-methoxyethyl)-5-methyl uridine (120 mg, 0.5 mmol) was
dissolved in 50 mL of anhydrous MeTHF and 0.5 g of molecular
sieves, 0.5 g of CALB and 1.5 mmol of vinyl ester (138 mL vinyl
acetate, 163 mL vinyl propionate or 190 mL vinyl butyrate) were
added, maintaining the reaction with orbital shaking for 5 h at
room temperature. After that, MeOH was added, the reaction
mixture filtered, neutralized with saturated NaHCO3 solution
and dried with anhydrous Na2SO4. Then, silica gel was added
and the solvent evaporated to dryness. Finally, the residue was
purified by column chromatography over silica gel.
Notes and references
1 (a) C. K. Chu, and D. C. Baker, Nucleosides and Nucleotides as
Antitumor and Antiviral Agents, Plenum Press, New York, 1993;
(b) L. B. Townsend, Chemistry of Nucleosides and Nucleotides,
Plenum Press, New York, 1988.
2 (a) S. D. Chamberlain, A. R. Moorman, L. A. Jones, P. de Miranda,
D. J. Reynolds, G. W. Kozalka and T. A. Krenitsky, Antiviral Chem.
Chemother., 1992, 3, 371; (b) E. K. Hamamura, M. Prystasz, J. P. H.
Verheyden, J. G. Moffatt, K. Yamaguchi, N. Uchida, K. Sato, A.
Nomura, O. Shiratori, S. Takase and K. Katagiri, J. Med. Chem.,
1976, 19, 654.
3 (a) L. Colla, E. de Clercq, R. Busson and H. Vanderhaeghe, J. Med.
Chem., 1983, 26, 602; (b) L. M. Beauchamp, G. F. Orr, P. de Miranda,
T. Burnette and T. A. Krenitsky, Antiviral Chem. Chemother., 1992,
3, 157; (c) X. Li, Q. Wu, D.-S. Lv and X.-F. Lin, Bioorg. Med. Chem.,
2006, 14, 3377.
4 (a) S. Shimokawa, J. Kimura and O. Mitsunobu, Bull. Chem. Soc.
Jpn., 1976, 49, 3357; (b) D. C. Baker, T. H. Haskell, S. R. Putt and
B. J. Sloan, J. Med. Chem., 1979, 22, 273.
2¢-O-(2-methoxyethyl)-5¢-acetyl-5-methyl
uridine
(11).
128 mg, 70%: yellow solid; 1H NMR (DMSO-d6, 250 MHz): d
1.81 (3H, s, CH3), 2.08 (3H, s, CH3CO), 3.23 (3H, s, OCH3), 3.48
(2H, m, OCH2CH2OCH3), 3.68 (2H, m, OCH2CH2OCH3), 4.05
(3H, m, H-2¢, H-3¢ and H-4¢), 4.22 (2H, m, H-5¢), 5.32 (1H, d,
J = 5.8 Hz, OH-3¢), 5.84 (1H, d, J = 4.8 Hz, H-1¢), 7.47 (1H, s,
H-6), 11.44 (1H, br s, NH); 13C NMR (DMSO-d6, 63 MHz):
d 12.5 (CH3), 21.0 (CH3CO), 58.5 (OCH3), 63.9 (C-5¢), 69.0
(C-3¢), 69.4 (OCH2CH2OCH3), 71.6 (OCH2CH2OCH3), 80.6
and 81.6 (C-2¢ and C-4¢), 87.1 (C-1¢), 110.2 (C-5), 136.4 (C-6),
150.9 (C-2), 164.1 (C-4) and 170.6 (CO). Anal. Calcd. for
C15H22N2O8: C, 50.28; H, 6.19; N, 7.82%. Found: C, 50.50; H,
6.26; N, 7.28%.
5 (a) L. A. Condezo, J. Ferna´ndez-Lucas, C. A. Garc´ıa-Burgos, A. R.
Alca´ntara, and J. V. Sinisterra, Biocatalysis in the Pharmaceutical
and Biotechnology Industries, ed. R. N. Patel, Taylor & Francis, CRC
press, New York, 2006; (b) M. Ferrero and V. Gotor, Chem. Rev.,
2000, 100, 4319.
6 (a) J. Garc´ıa, S. Ferna´ndez, M. Ferrero, Y. S. Sanghvi and V. Gotor,
J. Org. Chem., 2002, 67, 4513; (b) M. A. Zinni, L. E. Iglesias and
A. M. Iribarren, J. Mol. Catal. B: Enzym., 2007, 47, 86.
7 K. Faber, Biotransformations in Organic Chemistry, Springer-Verlag,
Berlin-Heidelberg, 2000.
8 Information provided by Penn Specialty Chemicals Inc., and also
available from D. Aul and B. Comanita, Manufacturing Chemist,
May 2007, 33.
9 (a) D. F. Aycock, Org. Process Res. Dev., 2007, 11, 156; (b) S. Mitra,
S. R. Gurrala and R. S. Coleman, J. Org. Chem, 2007, 72, 8724; (c) S.
Nuwa, S. Handa, S. Miki, U.S. Patent, 20050043544; (d) J. Miller, J.
Penney, U.S. Patent, 20050137402; (e) S. Krishnan and S. Schreiber,
Org. Lett., 2004, 6, 4021; (f) D. Spring, S. Krishnan and S. Schreiber,
J. Am. Chem. Soc., 2000, 122, 5656; (g) K. Nakamura, M. Kinoshita
and A. Ohno, Tetrahedron, 1994, 50, 44681.
10 (a) F. Mor´ıs and V. Gotor, J. Org. Chem., 1993, 58, 653; (b) J. Garc´ıa,
S. Ferna´ndez, M. Ferrero, Y. S. Sanghvi and V. Gotor, Tetrahedron
Lett., 2004, 45, 1709.
11 (a) F. Mor´ıs and V. Gotor, Tetrahedron, 1994, 50, 6927; (b) X.-F. Sun,
N. Wang, Q. Wu and X.-F. Lin, Biotechnol. Lett., 2004, 26, 1019.
12 (a) M. D. Romero, L. Calvo, C. Alba, A. Daneshfar and H. S.
Ghaziaskar, Enzyme Microb. Technol., 2005, 37, 42; (b) J. Xu, J.
Zhu, T. Kawamoto, T. Atsuo and Y. Hu, Chin. J. Biotechnol., 1997,
13, 263.
2¢-O-(2-methoxyethyl)-5¢-propanoyl-5-methyl uridine (12).
130 mg, 68%: white solid; H NMR (DMSO-d6, 250 MHz): d
1
1.06 (3H, t, J = 7.4, CH3CH2CO), 1.81 (3H, s, CH3), 2.37 (2H,
dd, J1 = 7.5 Hz, J2 = 14.9 Hz, CH3CH2CO), 3.23 (3H, s, OCH3),
3.48 (2H, m, OCH2CH2OCH3), 3.68 (2H, m, OCH2CH2OCH3),
4.05 and 4.11 (3H, m, H-2¢, H-3¢ and H-4¢), 4.25 (2H, m, H-5¢),
5.33 (1H, d, J = 5.8 Hz, OH-3¢), 5.85 (1H, d, J = 4.8 Hz, H-1¢),
7.46 (1H, s, H-6), 11.45 (1H, br s, NH); 13C NMR (DMSO-d6,
63 MHz): d 9.3 (CH3CH2CO), 12.5 (CH3), 27.0 (CH3CH2CO),
58.5 (OCH3), 63.8 (C-5¢), 69.0 (C-3¢), 69.4 (OCH2CH2OCH3),
71.6 (OCH2CH2OCH3), 80.6 and 81.6 (C-2¢ and C-4¢), 87.1 (C-
1¢), 110.2 (C-5), 136.4 (C-6), 150.9 (C-2), 164.1 (C-4) and 173.9
(CO). Anal. Calcd. for C16H24N2O8: C, 51.61; H, 6.50; N, 7.52%.
Found: C, 51.56; H, 6.48; N, 7.28%.
2¢-O-(2-methoxyethyl)-5¢-butanoyl-5-methyl uridine (13).
1
128 mg, 65%: white solid; H NMR (DMSO-d6, 250 MHz): d
0.89 (3H, t, J = 7.3 Hz, CH3CH2CH2CO), 1.57 (2H, ddd, J1 =
J2 = J3 = 7.3 Hz, CH3CH2CH2CO), 1.81 (3H, s, CH3), 2.35 (2H,
t, J = 7.2 Hz, CH3CH2CH2CO), 3.23 (3H, s, OCH3), 3.48 (2H,
m, OCH2CH2OCH3), 3.67 (2H, m, OCH2CH2OCH3), 4.05 (3H,
m, H-2¢, H-3¢ and H-4¢), 4.24 (1H, m, 2H-5¢), 5.31 (1H, d, J =
5.7 Hz, OH-3¢), 5.84 (1H, d, J = 4.8 Hz, H-1¢), 7.46 (1H, s, H-6),
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13 Y. Mei, L. Miller, W. Gao and R. A. Gross, Biomacromolecules, 2003,
4, 70.
14 (a) P. Hudson, R. K. Eppler and D. S. Clark, Curr. Opin. Biotechnol.,
2005, 16, 637; (b) R. Chenevert, N. Pelchat and F. Jacques, Curr. Org.
Chem., 2006, 10, 1067.
T. Raku, R. Kurane and Y. Tokiwa, Biotechnol. Lett., 2000, 22,
883.
18 (a) M. Grotli, M. Douglas, R. Eritja and B. S. Sproat, Tetrahedron,
1998, 54, 5899; (b) K.-H. Altmann, P. Martin, N. M. Dean and B. P.
Monia, Nucleosides Nucleotides, 1997, 16, 917.
15 P. Hoyos, M. Fernandez, J. V. Sinisterra and A. R. Alca´ntara, J. Org.
Chem., 2006, 71, 7632.
16 A. Uemura, K. Nozaki, J.-I. Yamashita and M. Yasumoto, Tetrahe-
dron Lett., 1989, 30, 3817.
17 (a) Y. Tokiwa, H. Fan, T. Raku, and M. Kitagawa, Biocatalysis in
Polymer Science, ed. R. A. Gross, and H. N. Cheng, ACS Symp.
Ser. Nr. 840, Washington DC, 2003; (b) M. Kitagawa, H. Fan,
19 T. Nakaoki, Y. Mei, L. M. Miller, A. Kumar, B. Kalra, M. E. Miller,
O. Kirk, M. Christensen and R. A. Gross, Ind. Biotechnol., 2005, 1,
126.
20 PLU = propyl laurate units, defined as the amount of catalyst
producing 1 mmol of propyl laurate per minute, at 60 ◦C, Novozymes
analytical method F-9600369.
21 Information from www.Sigma-Aldrich.com.
862 | Green Chem., 2009, 11, 855–862
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