An expedient biocatalytic procedure for abasic site precursors useful in oligonucleotide synthesis

Article abstract of DOI:10.1039/c1ob05739a

Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable green method useful for industrial applications. Highly re

Full text of DOI:10.1039/c1ob05739a

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Organic &
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PAPER
An expedient biocatalytic procedure for abasic site precursors useful in
oligonucleotide synthesis†
Sau´l Mart´ınez-Montero,a Susana Ferna´ndez,a Yogesh S. Sanghvi,b Vicente Gotor*a and Miguel Ferrero*a
Received 11th May 2011, Accepted 23rd May 2011
DOI: 10.1039/c1ob05739a
Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been
accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable
green method useful for industrial applications. Highly regioselective acylation and deacylation
reactions with 1,2-dideoxy-D-ribose are described resulting in excellent yield. A fast, atom-efficient and
convenient synthesis of 3-, and 5-O-DMTr-1,2-dideoxyribose 17 and 19 has been achieved. These
compounds are useful precursors for the preparation of phosphoramidites required for the assembly of
oligonucleotides containing the tetrahydrofuran abasic lesions.
Introduction
Abasic sites are one of the most common cellular DNA damage in
the genome. They result from the cleavage of the glycosidic bond,
which can occur spontaneously under physiological conditions,
or during the intermediate steps of base excision repair when
DNA is exposed to various endogenous and exogenous damaging
agents.1 For example, antitumor antibiotic bleomycin is reported
to cause cleavage of thymine-deoxyribose glycosidic bond in
DNA.2 A variety of bifunctional agents, such as mitomycin C and
cisplatin can react with bases in DNA leading to the generation
of abasic sites.3 This process of cellular damage is also responsible
for interstrand cross-links in duplex DNA leading to cytotoxic
effects.4,5 Therefore, studies of repair of DNA is important for
maintaining the integrity of the genome.6 In vivo, the abasic sites
are believed to be repaired by the action of cellular endonucleases.7
Several oligonucleotide analogues containing abasic sites have
been synthesized to determine the structural basis of these
lesions that are responsible for their distinct biochemical effects.8
Noteworthy is the unoxidized abasic site from the hydrolysis of the
glycosidic bond (1, AP; Chart 1) and the oxidized abasic lesions
Chart 1
C4-AP (2, C4-oxidized abasic site), 2-deoxyribonolactone (3, L),
or 5-(2-phosphoryl-1,4-dioxobutane) (4, DOB), which inhibits
cleotides containing the tetrahydrofuran group (5) act as substrates
repair by DNA polymerase b.9 Other abasic lesions such as the
for AP endonucleases repair in Escherichia coli, and also serve as
tetrahydrofuran (5, F), the hydrocarbon derivatives CPA (6) and
effective templates for AuV reverse transcriptase and other DNA
CPE (7), the lactam (8, Lm), the ketone (9, K) and the methylene
cyclopentane (10, MCP) analogues have already been reported as
polymerases of eukaryotic and prokaryotic origin.8c,10 In addition,
abasic sugars were introduced into therapeutic siRNAs to inhibit
probes of replication in cells. It has been shown that oligodeoxynu-
degradation by nucleases.11
The widespread use of furanoid glycals as key intermediates for
the synthesis of interesting biological compounds12 has aroused
considerable interest in scalable and cost-efficient procedures for
their preparation. Most of the synthetic methods employed 2-
deoxy-D-ribose as starting material.8c,13 In 1984, Millican et al.10b
reported the synthesis of 1,2-dideoxy-D-ribose starting from 2¢-
deoxyadenosine via destructive protocol (loss of atoms) where
aDepartamento de Qu´ımica Orga´nica e Inorga´nica and Instituto Universi-
tario de Biotecnolog´ıa de Asturias, Universidad de Oviedo, 33006, Oviedo,
(Asturias), Spain. E-mail: vgs@uniovi.es, mferrero@uniovi.es; Fax: +34
985 103 448; Tel: +34 985 103 448
bRasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA, 92024-6615, USA
1
† Electronic supplementary information (ESI) available: H, 13C, DEPT,
19F NMR spectra data. See DOI: 10.1039/c1ob05739a
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Table 1 Enzymatic hydrolysis of di-O-toluoyl-1,2-dideoxyribose (11)
Entry Enzyme 11 : Enzymea T/◦C t (h) Conv. (%)b 12 (%)b,c
purine portion of the molecule was cleaved off to give the cyclic
enol ether. In 1994, Pedersen et al.14 and subsequently in 2002
Beigelman et al.15 employed thymidine as starting material in the
preparation of the 1,2-dideoxy-D-ribose. Clearly, these protocols
are not atom efficient where a good portion of the molecule is
sacrificed to obtain 1,2-dideoxy-D-ribose.
1
2
3
4
CAL-Bd 1 : 1
PSL-Ce
1 : 2
PSL-SDf 1 : 2
CVLg
1 : 0.25
55
50
50
50
48
2
15
1.5
>97
>97
>97
>97
>97
>97 (95)
>97
>97
Biocatalytic methods have been recognized as practical proce-
dures in the nucleoside area.16 For manipulation of protecting
groups, application of biocatalysts in organic synthesis has be-
come an attractive alternative to conventional chemical methods.
Thus, enzyme–catalyzed chemical transformations, which satisfy
increasingly stringent environmental constraints, are in great
demand by the pharmaceutical and chemical industries.
Herein we describe an efficient, scalable and atom economical
(non-destructive) synthesis of 1,2-dideoxy-D-ribose from simple
starting materials that are currently synthesized on metric ton scale
for the synthesis of 2-deoxynucleosides.17 Herein, we explored the
potential of the enzymatic catalysis for the manipulation of 1,2-
dideoxy-D-ribose with two orthogonal protecting groups at any
site that is desired. The orthogonally protected 1,2-dideoxy-D-
ribose could serve as very useful building-block in the synthesis of
carbohydrate analogues and in the generation of building-blocks
required for inserting abasic sites in oligonucleotides with greater
ease.
a Ratio 11 : enzyme (w/w). b Calculated by 1H NMR. c Percentage of
isolated yields are given in parentheses. d Novozym 435. e Lipase PS
“Amano” immobilized on ceramic. f Crude lipase PS “Amano” SD.
g Cromobacterium viscosum lipase.
Enzymatic hydrolysis of 11 with PSL revealed that this lipase
showed the same 5-O-acyl group selectivity as CAL-B (entries 2
and 3, Table 1). The rate of hydrolysis with PSL was significantly
faster compared to CAL-B furnishing exclusively 12 in excellent
yield (95%). Interestingly, PSL displayed opposite selectivity
during hydrolysis of di-O-acyl-b-D-2¢-deoxynucleosides suggesting
that the presence of the nucleosidic base is pivotal in how the
substrate fits in the active site of the enzyme.19 Similar results
were also obtained with the enzyme CVL furnishing the 3-O-acyl
derivative in < 2 h (entry 4, Table 1). Nevertheless, PSL-C was the
enzyme of choice for the transformation of 11 into 12 on large-
scale. This is because PSL-C is immobilized and provides several
advantages such as enhanced stability, reusability, convenient
separation by filtration from the reaction mixture, preventing
protein contamination in the product, and consistent performance
of the commercial preparations. Additionally, easy isolation of 12
has been accomplished via extraction avoiding expensive column
chromatography. Furthermore, the toluoyl protecting group in 11
appears to be an ideal substrate for recognition by lipase. It also
offers easy detection by UV and more importantly, facilitates the
extraction of 12 in organic solvent.
Results and discussion
We have previously reported that Candida antarctica lipase B
(CAL-B) catalyzes the hydrolysis at the 5¢-O-acyl group of 2¢-
deoxynucleosides selectively, whereas Pseudomonas cepacia lipase
(PSL) shows unusual regioselectivity towards the acyl group at
3¢-position.18 As an extension of our earlier studies and further
exploration of enzyme substrate specificity and selectivity, we
elected di-O-toluoyl-1,2-dideoxy-D-ribose (11) as the starting
material.
The di-O-toluoyl-protected 11 was conveniently prepared
from the commercial 1-a-chloro-3,5-di-O-p-toluoyl-2-deoxy-
ribofuranose following literature procedure.17 The regioselective
hydrolysis of 11 is shown in Scheme 1 and the experimental data
are summarized in Table 1. For an initial screening of suitable
lipases, enzymatic hydrolysis of 11 was carried out with CAL-B,
PSL, and Cromobacterium viscosum lipase (CVL). Treatment of 11
with 0.15 M KH2PO4 (pH 7) containing 1,4-dioxane as cosolvent
in the presence of CAL-B at 55 ◦C afforded after 48 h (monitored
by TLC) the 5-hydroxyl derivative 12 as the only product (entry 1,
Table 1). As expected, CAL-B exhibited complete regioselectivity
in the hydrolysis of the acyl group at the 5-position of compound
11.
Next, we decided to prepare the 5-O-acyl derivative through
the enzymatic acylation of 1,2-dideoxy-D-ribose expecting that
the complementary behaviour of these lipases in hydrolysis and
acylation reactions may enable the synthesis of the desired
regioisomer. 1,2-Dideoxy-D-ribose was conveniently prepared by
the treatment of 11 with sodium methoxide in MeOH at reflux to
afford 13 in 89% yield (Scheme 2).
Scheme 2
Evaluation of the selective acylation of 13 in the presence
of immobilized CAL-B and PSL as catalysts was undertaken.
We employed commercially available vinyl benzoate (14a) as the
acylating agent for this study. Therefore, 1,2-dideoxy-D-ribose (13)
was dissolved in anhydrous THF under nitrogen and treated with
2 equiv of vinyl benzoate and CAL-B or PSL (Scheme 3). Re-
gioselective acylation was observed with both enzymes furnishing
Scheme 1
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Scheme 4
Although excellent selectivity was also achieved with p-methoxy
vinylbenzoate (14c), CAL-B-catalyzed acylation of 13 gave lower
conversion (entry 8, Table 2). The acylating agent 14c contains
an electron donating group lowering the formation of acylated
15c catalyzed by CAL-B compared to the vinylbenzoate and
p-methyl vinylbenzoate discussed above. Similar behaviour was
observed with PSL-C. However, this enzyme drives the reaction to
completion after 26 h at 50 ◦C (entry 9, Table 2). Interestingly, the
acylation proceeded faster with p-nitro vinylbenzoate (14d), which
possesses an electron withdrawing group. 1,2-Dideoxy-D-ribose
was converted selectively to the 5-O-p-nitrobenzoyl derivative 15d
with both CAL-B and PSL-C (entries 10 and 11, Table 2). The
later furnished 15d in 90% yield within 0.5 h at 50 ◦C. To further
enhance our understanding of the influence of p-substituent
during enzymatic acylation, the reaction was conducted with p-
fluoro vinylbenzoate (14e). The presence of a weakly deactivating
group in the para position of the benzoyl group provided longer
reaction times relative to the nitro derivative (entries 12 and 13
vs. entries 10 and 11, Table 2), but significantly shorter than
electron donating methyl or methoxy substituents. Overall, the
regioselective 5-O-acylation was observed with all of the enol
esters tested, providing shorter reaction times with PSL-C than
CAL-B.
Scheme 3
exclusively 5-O-benzoyl-1,2-dideoxyribose (15a). Shorter reaction
times were achieved with PSL-C, which affords 15a in almost
quantitative yield after 3 h at 50 ◦C (entries 1, 2, and 3, Table 2).
Importantly, pure 15a was isolated via precipitation without a need
for column chromatographic purification. It is noteworthy that
we are using less equiv of vinyl benzoate compared to an earlier
protocol reported by us for the preparation of 5¢-O-benzoyl-2¢-
deoxynucleoside derivatives.20 During the transformation of 13 to
15a, it is not necessary to use an excess of vinyl benzoate to drive
the reaction to completion. Compared to the earlier nucleoside
studies, in case of carbohydrate 13, the overall reaction time is
shorter without loss of selectivity.
To demonstrate the general application of acylation protocol
with other acyl derivatives and to study the influence of the elec-
tronic nature of the acyl moiety, we carried out the enzymatic acy-
lation with four p-substituted vinyl benzoates as acylating agents.
Thus, vinyl esters 14b–e were synthesized in good yields through
trans-vinylation reaction of the corresponding carboxylic acid in
the presence of palladium(II) acetate as catalyst (Scheme 4).21
When p-methyl vinylbenzoate (14b) was used for the acylation
of 13, total selectivity at the primary hydroxyl was observed with
both PSL-C and CAL-B. The rate of conversion was higher with
PSL-C, which afford 15b after 3 h in 97% yield (entry 4, Table 2).
To optimize conditions for CAL-B, longer reaction times were
examined. However, a maximum 85% conversion was reached
after 168 h at 55 ◦C (entries 5, 6, and 7, Table 2).
It is noteworthy that conventional acylation of 13 with an
acid chloride was non-selective furnishing mixtures of acylated
products. As an example, the reaction of 13 with 1.5 equiv
of 4-methylbenzoyl chloride in pyridine as solvent and DMAP
as catalyst offered 50% of the 5-O-esterified product, 2% of
the 3-O-acylated product, and 11% of the diacyled derivative
and 37% of starting material despite of extended reaction
(5 days).
Table 2 Enzymatic acylation of 1,2-dideoxy-D-ribose (13)
To make this enzymatic process attractive for industrial appli-
cations, we use the vinyl ester in 1 : 1.1 ratio with respect to the
starting material and tested the efficacy of enzyme recycle on a
5 mmol scale (Table 3). The reaction of 13 with 1.1 equiv of
vinyl benzoate catalyzed by PSL-C was completed in 7.5 h and
Entry R
Enzyme 13 : Enzymea T/◦C t (h) Conv. (%)b 15 (%)b,c
1
2
3
H
H
H
CAL-Bd 1 : 1
PSL-Ce 1 : 2
PSL-IMf 1 : 2
55
50
50
50
55
55
55
55
50
55
50
55
50
62
3
5
>97
>97
>97
>97
42
>97
>97 (98)
>97
>97 (97)
>97
4
5
6
7
8
9
10
11
12
13
Me PSL-Ce 1 : 2
Me CAL-Bd 1 : 1
Me CAL-Bd 1 : 1
Me CAL-Bd 1 : 1
OMe CAL-Bd 1 : 1
OMe PSL-Ce 1 : 2
NO2 CAL-Bd 1 : 1
NO2 PSL-Ce 1 : 2
3
65
96
168 85
59
>97
>97
Table 3 Recycling study of PSL-C in the enzymatic acylation of 13a
96
26
57
32
>97
>97
>97
>97 (86)
>97
>97 (90)
>97
>97 (86)
Run
t (h)
Conv. (%)b
15a (% isolated yield)
1
2
3
4
5
7.5
8.5
26
32
54
>97
>97
>97
91
96
96
94
86
86
0.5 >97
96 90
2.5 >97
F
F
CAL-Bd 1 : 1
PSL-Ce 1 : 2
a Ratio 13 : enzyme (w/w). b Calculated by 1H NMR. c Percentage of
isolated yields are given in parentheses. d Novozym 435. e Lipase PS
“Amano” immobilized on ceramic. f Lipase PS “Amano” SD immobilized
on diatomaceous earth.
89
a The reaction was carried out on 5 mmol scale at 50 ◦C, in 1 M
concentration with 1.1 equiv of vinyl benzoate. b Calculated by 1H NMR.
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work-up of the reaction provided crude 5-O-benzoyl-1,2-dideoxy-
D-ribose, which was easily isolated in pure state via extraction
and drying under vacuum (96%). Next, recycling experiments
using the recovered lipase were carried out. To improve the
activity, the immobilized enzyme was reactivated by hydration.
For that, the enzyme was poured into water, filtered, and dried
under vacuum. Five consecutive catalytic cycles were performed
and excellent yields have been achieved. With the exception of
longer reaction times in subsequent runs similar behaviour of the
PSL-C was observed. A conversion of ~90% was reached in the
fourth and fifth catalytic cycle. Despite lower efficiencies in the
4/5 cycles, the product is easily isolated following the standard
extraction work-up which removes the starting material in aq.
wash. Overall, the isolation of 15a was accomplished without
column chromatography.
Conclusions
We have developed a green protocol for the preparation of 3-,
and 5-O-DMTr-1,2-dideoxy-D-ribose, which are of interest due to
their incorporation into therapeutic oligonucleotides as abasic site
precursors. Candida antarctica lipase B and Pseudomonas cepacia
lipase catalyze the regioselective hydrolysis of di-O-toluoyl-1,2-
dideoxy-D-ribose furnishing the 3-O-toluoyl derivative in quanti-
tative yield. Meanwhile, both lipases exhibited complementary
behaviour in the acylation reaction of 1,2-dideoxy-D-ribose,
providing exclusively the opposite regioisomer. The presence of
electron donating substituents in the para-position of the benzoate
vinyl ester decreased the rate of enzymatic acylation. However,
the reaction with electron withdrawing group in the acylating
agent proceeded at a faster rate. The protocol described herein
offers a fast, reliable and scalable route to orthogonally protected
carbohydrate building-blocks in excellent overall yields. Specially,
the 3-O-DMTr precursor is very useful when reverse amidite
is required for therapeutic oligonucleotides synthesis. Moreover,
enzyme catalyzed reactions are less hazardous, less polluting,
and less energy-consuming than the conventional chemistry-base
methods. In addition, the enzyme has been reused to make
the process further economical for industrial applications. In
summary, we have developed atom efficient and low environmental
impact protocols without sacrificing the stereo- and regiocontrol
requirements of the modern day carbohydrate chemistry.
Ionic liquids have proven to be environmentally green
and attractive reaction media for biocatalysis.22 Therefore,
we
tested
[BMIM]-[PF6]
(1-butyl-3-methylimidazolium)
hexafluorophosphate for lipase catalyzed acylation of 1,2-
dideoxy-D-ribose and compared it to the THF as solvent. The
acylation of 13 with vinyl benzoate (1.1 equiv) in [BMIM]-[PF6] at
50 ◦C catalyzed by PSL-C proceeded with high regioselectivity in
10 h, only trace amount of diacyl derivative (<2%) was detected
compared to the reaction in THF.
The enzymatic catalysis provides useful synthetic intermediates
that can be employed in the assembly of other complex molecules.
For example, dimethoxytrityl (DMTr) protected 17 and 19 are
key precursors for the synthesis of corresponding amidites that
are used for incorporation into therapeutic oligonucleotides.11
Therefore, synthesis of 17 and 19 was envisioned from a common
starting material 11 in a straightforward manner (Scheme 5). The
tritylation of 12 and 15 with dimethoxytrityl chloride furnished 16
and 18 in 87% and 83% yield, respectively. The synthetic ease and
accessibility of these orthogonally protected 1,2-dideoxy-D-ribose
16 and 18 may further enhance the value of these molecules in
carbohydrate chemistry. Next, the toluoyl or benzoyl groups are
selectively cleaved with sodium methoxide in MeOH, leading to
the 5- and 3-O-DMTr monomers 17 and 19, useful building blocks
in carbohydrate and oligonucleotide synthesis.
Experimental section
Enzyme activities
Pseudomonas cepacia lipase (PSL-C, Amano PS-C Type II, 1950
U/g) was purchased from Aldrich. Candida antarctica lipase B
(CAL-B, Novozym 435, 7300 PLU/g) was a gift from Novo
Nordisk Co. Chromobacterium viscosum lipase (CVL, 4100 U/mg)
was a gift from Genzyme Co. Pseudomonas cepacia lipase powder
immobilized in diatomaceous earth (PSL-IM, 943 U/g) and
Pseudomonas cepacia powder as a crude enzyme preparation con-
taining dextrin as diluents (PSL-SD, 24700 U/g) were generously
provided by Amano Europe.
Scheme 5
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Enzymatic hydrolysis of di-O-toluoyl-1,2-dideoxyribose. Synthesis
of 3-O-toluoyl-1,2-dideoxy-D-ribose (12)
MHz): d 22.3 (CH3), 98.5 (C-2), 126.8 (Ci), 129.8 (Cm), 130.6 (Co),
142.0 (C-1), 145.0 (Cp), 164.3 (C O).
To a solution of 11 (400 mg, 1.13 mmol) in 1,4-dioxane
(3.7 mL) was added 0.15 M phosphate buffer pH 7 (7.4 mL) and
the corresponding lipase [ratio of 11:CAL-B was 1 : 1 (w/w); ratio
of 11:PSL was 1 : 2 (w/w); ratio of 11:CVL was 1 : 0.25 (w/w)]. The
mixture was allowed to react at 250 rpm for the time and at the
temperature indicated in Table 1. The reactions were monitored
by TLC (10% MeOH/CH2Cl2). The enzyme was filtered off and
washed with CH2Cl2, the solvents were distilled under vacuum,
and the residue was taken up in CH2Cl2 and washed with NaHCO3
(aq). The combined organic layers were dried over Na2SO4 and
evapor◦ated to give pure 12. Rf (10% MeOH/CH2Cl2): 0.52. mp:
88–90 C. [a]2D0 = +26 (c 1.0, CH2Cl2). IR (KBr): u 3460, 3055,
Vinyl 4-methoxybenzoate (14c)
White solid. Rf (20% EtOAc/hexane): 0.57. mp: 52–54 ◦C. IR
(KBr): u 3055, 2971, 2842, 1726, 1645, 1607 cm-1. 1H NMR
(CDCl3, 300.13 MHz): d 3.86 (s, 3H, OCH3), 4.67 (d, 1H, H-
2, 3JHH 6.3 Hz), 5.04 (d, 1H, H-2, 3JHH 14.2 Hz), 6.95 (d, 2H, Hm,
3JHH 9.0 Hz), 7.52 (dd, 1H, H1 3JHH 6.3 Hz, 3JHH 14.2 Hz), 8.06 (d,
1H, Ho, 3JHH 8.4 Hz). 13C NMR (CDCl3, 75.5 MHz): d 55.4 (CH3),
97.6 (C-2), 113.8 (Cm), 121.2 (Ci), 132.1 (Co), 141.5 (C-1), 163.3-
163.9 (C O + Cp). HRMS (ESI+) calc for C10H10NaO3 [M+Na]+:
201.0522, found: 201.0510.
1
2987, 1713, 1612 cm-1. H NMR (CDCl3, 300.13 MHz): d 2.23
Vinyl 4-nitrobenzoate (14d)
(m, 2H, H-2), 2.40 (s, 3H, CH3), 4.15 (m, 3H, 2H1+H4), 5.33 (dt,
◦
Pale yellow solid. Rf (20% EtOAc/hexane): 0.57. mp: 74–76 C.
3
3
3
1H, JHH 2.4, JHH 6.4 Hz), 7.23 (d, 2H, Hm, JHH 8.3 Hz), 7.91
IR (KBr): u 3056, 2987, 1737, 1647, 1609, 1530 cm-1. H NMR
1
3
(d, 2H, Ho, JHH 8.3 Hz). 13C NMR (CDCl3, 75.5 MHz): d 22.3
(CDCl3, 300.13 MHz): d 4.80 (dd, 1H, H-2, 2JHH 2.0 Hz, 3JHH 6.0
(CH3), 33.8 (C2), 63.6 (C5), 68.3 (C1), 77.2 (C3), 85.6 (C4), 127.7
(Ci), 129.8 (Cm), 130,3 (Co), 144.7 (Cp), 167.2 (C O). MS (ESI+,
m/z): 237 [(M+H)+, 10%], 259 [(M+Na)+, 100]. HRMS (ESI+)
calc for C13H16NaO4 [M+Na]+: 259.0941, found: 259.0938.
3
3
Hz), 5.14 (dd, 1H, H-2, JHH 2.0 Hz, JHH 14.0 Hz), 6.95 (d, 2H,
Hm, JHH 9.0 Hz), 7.52 (dd, 1H, H-1 3JHH 6.3 Hz, JHH 13.9 Hz),
8.28 (m, 4H, Ho+Hm). 13C NMR (CDCl3, 75.5 MHz): d 99.5 (C-
2), 123.7 (Cm), 131.1 (Co), 134.3 (Ci), 141.1 (C-1), 161.8 (CP and
3
3
C
O).
Synthesis of 1,2-dideoxy-D-ribose (13)
Vinyl 4-fluorobenzoate (14e)
NaOMe (1.9 g, 35.25 mmol) was added to a solution of 11 (5 g,
14.1 mmol) in anhydrous MeOH (28 mL) at room temperature.
Reaction was stirred at reflux and monitored by TLC (10%
MeOH/CH2Cl2) indicating the total disappearance of the starting
material in 2 h. Once the reaction was concluded, solvents were
evaporated under vacuum and the residue was purified by flash
chromatography (gradient elution with 2–10% MeOH/CH2Cl2)
to obtain 13 as a colorless oil (1.48 g, 89% yield). Rf (10%
Colorless liquid. Rf (20% EtOAc/hexane): 0.57. mp: 52–54 ◦C.
IR: u 3093, 2958, 1737, 1646, 1604 cm-1. 1H NMR (CDCl3, 300.13
2
3
MHz): d 4.70 (dd, 1H, H-2, JHH 2.0 Hz, JHH 6.3 Hz), 5.06 (d,
1H, H-2, 3JHH 13.9 Hz), 7.14 (m, 2H, Hm), 7.48 (dd, 1H, H1, 3JHH
6.3 Hz, 3JHH 13.9 Hz), 8.14 (m, 2H, Ho). 13C NMR (CDCl3, 75.5
MHz): d 98.3 (C-2), 115.7 (d, Cm, 2JCF 22.1 Hz), 125.2 (Ci), 132.6
(d, Co, 3JCF 9.3 Hz), 141.3 (C-1), 162.6 (C O), 254.9 (d, Cp, 1JCF
64.1 Hz). 19F NMR (CDCl3, 282 MHz): d 104.4
1
MeOH/CH2Cl2): 0.20. IR: u 3370, 2945, 2882, 1441 cm-1. H
NMR (MeOH-d4, 300.13 MHz): d 1.95 (m. 1H, H-2), 2.09 (m,
1H, H-2), 3.51 (m, 2H, H-5), 3.75 (m, 1H, H4), 3.91 (m, 2H, H-1),
4.21 (dt, 1H, 3JHH 3.1 Hz, 3JHH 6.1 Hz). 13C NMR (MeOH-d4, 75.5
MHz): d 36.2 (C-2), 63.9 (C-5), 68.2 (C-1), 74.0 (C-3), 88.4 (C-
4). HRMS (ESI+) calc for C5H10NaO3 [M+Na]+: 141.0522, found:
141.0524.
Enzymatic acylation of 1,2-dideoxy-D-ribose
General procedure for the synthesis of 15a–e. A suspension
of 13 (59 mg, 0.5 mmol), the corresponding vinyl ester 14
(1 mmol), and the lipase [ratio of 13:CAL-B was 1 : 1 (w/w); ratio
of 13:PSL was 1 : 2 (w/w)] in anhydrous THF (2.5 mL) under
nitrogen was stirred at 250 rpm for the time and at the temperature
indicated in Table 2. The reactions were monitored by TLC (10%
MeOH/CH2Cl2). The enzyme was filtered off and washed with
CH2Cl2. The combined organic layers were washed with NaHCO3
(aq),dried over Na2SO4 and evaporated. The volatile acylation
age◦nts (14a, 14b, 14e) were removed by drying under vacuum at
60 C to give pure 15a, 15b and 15e. With acylating agents 14c–
d, the residue obtained after extraction was subjected to flash
chromatography (2% MeOH/CH2Cl2) to afford 15c–d as pure
white solids.
Synthesis of vinyl esters 14b–e
General Procedure. A mixture of carboxylic acid (16b–e)
(5 mmol), vinyl acetate (50 mL), Pd(OAc)2 (0.8 mmol; 1.6
mmol for 16d), and KOH (55 mg, 1 mmol) under nitrogen was
stirred overnight at room temperature. After filtration over Celite,
solvent was evaporated and the residue was purified by flash
chromatography on silica gel (20% EtOAc/hexane) to afford 14
(81% yield for 14b; 72% yield for 14c; 72% yield for 14d; 71% yield
for 14e).
Vinyl 4-methylbenzoate (14b)
5-O-Benzoyl-1,2-dideoxy-D-ribose (15a)
◦
Colorless liquid. Rf (20% EtOAc/hexane): 0.57. IR: u 3055, 2987,
1730, 1645, 1613 cm-1. 1H NMR (CDCl3, 300.13 MHz): d 2.40 (s,
3H, CH3), 4.67 (d, 1H, H-2, 3JHH 6.4 Hz), 5.06 (d, 1H, H-2, 3JHH
14.0 Hz), 7.24 (d, 2H, Ho, 3JHH 8.3 Hz), 7.51 (dd, 1H, 3JHH 6.4 Hz,
3JHH 14.0 Hz), 7.98 (d, 1H, 3JHH 8.4 Hz). 13C NMR (CDCl3, 75.5
98% yield. Rf (10% MeOH/CH2Cl2): 0.52. mp: 57–59 C. [a]D20
= +29 (c 1.0, CH2Cl2). IR (KBr): u 3445, 2986, 2953, 1721, 1602
cm-1. 1H NMR (CDCl3, 300.13 MHz): d 1.95 (m, 1H, H-2), 2.18
(m, 1H, H-2), 2.78 (br s, 1H, OH), 4.05 (m, 3H, H-1 + H-4), 4.36
(m, 3H, H-3 + H-5), 7.42 (t, 2H, Hm 3JHH 7.6 Hz), 7.55 (tt, 2H, Hp
5964 | Org. Biomol. Chem., 2011, 9, 5960–5966
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3
3JHH 1.3 Hz, JHH 6.7 Hz), 8.03 (m, 2H, Ho). 13C NMR (CDCl3,
Synthesis of 5-O-dimethoxytrityl-3-O-toluoyl-1,2-
75.5 MHz): d 34.9 (C-2), 64.9 (C-5), 67.1 (C-1), 73.5 (C-3), 83.8
(C-4), 128.4–129.6 (5C, Ci+Co+Cm), 133.2 (Cp), 166.6 (C O). MS
(ESI+, m/z): 259 [(M+Na)+, 100]. HRMS (ESI+) calc for C12H15O4
[M+H]+: 223.0965, found: 223.0959.
dideoxy-D-ribose (16)
To a solution of 12 (80 mg, 0.34 mmol), in anhydrous pyridine
(3.4 mL) was added 4,4¢-dimethoxytrityl chloride (127 mg,
0.37 mmol), and the mixture was stirred at room temperature.
The progress of the reaction was monitored by TLC (30%
hexane/CH2Cl2), which indicates the total disappearance of
starting material in 3 h. The mixture was concentrated under
reduced pressure and the crude product was purified by column
5-O-Toluoyl-1,2-dideoxy-D-ribose (15b)
Rf (10% MeOH/CH2Cl2): 0.52. mp: 45–47 ◦C. [a]D20 = +26 (c 1.0,
CH2Cl2). IR (KBr): u 3429. 3037, 2949, 1717, 1612 cm-1. 1H NMR
(CDCl3, 300.13 MHz), d 1.92 (m, 1H, H-2), 2.14 (m, 1H, H-2),
2.37 (s, 3H, CH3), 3.03 (br s, 1H, OH), 4.01 (m, 3H, H-1 + H-4),
4.31 (m, 3H, H-3 + H-5), 7.19 (d, 2H, Hm, 3JHH 8.1 Hz), 7.89 (d,
2H, Ho, 3JHH 8.1 Hz). 13C NMR (CDCl3, 75.5 MHz) d 21.5 (CH3),
34.9 (C-2), 64.7 (C-5), 67.1 (C-1), 73.3 (C-3), 83.8 (C-4), 126.8 (Ci),
129.0 (Cm), 129.6 (Co), 143.8 (Cp), 166.7 (C O). MS (ESI+, m/z):
237 [(M+H)+, 20%], 259 [(M+Na)+, 100]. HRMS (ESI+) calc for
C13H16NaO4 [M+Na]+: 259.0941, found: 259.922.
˚
chromatography using neutral silica gel 60 A (32–63 mm) pH 7
(20% hexane/CH2Cl2) to obtain 16 (158 mg, 87% yield). mp: 43–
45 ◦C. Rf (30% hexane/CH2Cl2): 0.37. [a]2D0 = 39 (c 1.0, CH2Cl2).
IR (KBr): u 3055, 2966, 1714, 1609 cm-1. H NMR (MeOH-d4,
1
300.13 MHz), d 2.09 (m, 1H, H-2), 2.33 (m, 1H, H-2), 2.39 (s, 3H,
CH3), 3.25 (m, 2H, 2H-5), 3.73 (s, 6H, OCH3), 4.09 (m, 3H, H1
and H4), 5.39 (dt, 1H, H3, 3JHH 2.2 Hz, 3JHH 6.4 Hz), 6.82 (d, 4H,
3
3
DMT, JHH 8.8 Hz), 7.25 (m, 9H, DMT), 7.43 (d, 2H, Hm, JHH
8.3 Hz), 7.88 (d, 2H, Ho JHH 8.3 Hz). 13C NMR (MeOH-d4, 75.5
3
MHz): d 21.7 (CH3), 33.7 (C-2), 55.6 (2C, OCH3), 65.3 (C-5), 68.6
(C-1), 78.3 (C-3), 84.7 (C-4), 87.5 (C-DMT), 114.1 (4C), 127.8–
131.3 (14C), 137.3 (2C, DMT), 145.5–146.4 (2C), 160.1 (2C), 167.6
(C O). MS (ESI+, m/z): 561 [(M+Na)+, 100]. HRMS (EI+) calc
for C34H34O6 [M+]: 538.2355, found: 538.2351.
5-O-p-Methoxybenzoyl-1,2-dideoxy-D-ribose (15c)
Rf (10% MeOH/CH2Cl2): 0.52. mp: 67–69 ◦C. [a]D20 = +21 (c 0.72,
CH2Cl2). IR (KBr): 3455, 3054, 2985, 1712, 1606 cm-1. 1H NMR
(CDCl3, 400.13 MHz): d 1.96 (m, 1H, H2), 2.19 (m, 1H, H2), 3.85
(s, 3H, OCH3), 4.02 (m, 2H, H-1), 4.08 (c, 1H, H-4, 3JHH 4.9 Hz),
Synthesis of 5-O-dimethoxytrityl-1,2-dideoxy-D-ribose (17)
3
4.35 (m, 3H, H-5 and H-3), 6.90 (d, 2H, Hm, JHH 8.9 Hz), 7.89
Compound 16 (100 mg, 0.19 mmol) was dissolved in anhydrous
MeOH (2.0 mL) and NaOMe (21 mg, 0.38 mmol) was added
at room temperature under continuous stirring. The reaction
is stirred at reflux for 2 h. The solvent was then evaporated
and the residue subjected to flash chromatography on silica gel
(40% EtOAc/hexane) to give 17 (64 mg, 83% yield). Rf (40%
EtOAc/hexane): 0.26. mp: 42–44 ◦C. [a]D20 = +14 (c 1.0, CH2Cl2).
3
(d, 2H, JHH 8.9 Hz). 13C NMR (CDCl3, 100.61 MHz): d 35.0
(C-2), 55.4 (OCH3), 64.7 (C-5), 67.2 (C-1), 73.5 (C-3), 84.0 (C-4),
113.7 (Cm), 122.1 (Ci), 131.8 (Co), 163.7 and 166.4 (Cp and C O).
MS (ESI+, m/z): 275.0 [(M+Na)+, 100%]. HRMS (ESI+) calc for
C13H16NaO5 [M+Na]+: 275.0890, found: 275.0880.
1
IR (KBr): u 3433, 3055, 2987, 1609 cm-1. H NMR (MeOH-d4,
5-O-p-Nitrobenzoyl-1,2-dideoxy-D-ribose (15d)
300.13 MHz): d 1.86 (m, 1H, H2), 2.06 (m, 1H, H-2), 3.08 (m, 2H,
H-5), 3.75 (s, 6H, OCH3), 3.94 (m, 3H, H-1+H-4), 4.22 (dt,1H,
H-3, 3JHH 2.6 Hz, 3JHH 5.7 Hz), 6.83 (d, 4H, DMT, 3JHH 8.8 Hz),
7.26 (m, 7H, DMT), 7.44 (d, 2H, DMT, 3JHH 7.7 Hz). 13C NMR
(MeOH-d4, 75.5 MHz): d 36.1 (C-2), 56.0 (2C, OMe), 65.9 (C-5),
68.4 (C-1), 74.6 (C-3), 87.2 (C-4) and 87.7 (C-DMT), 114.3 (4C),
128.0–131.6 (9C), 137.6 (2C), 146.8 (1C), 160.4 (2C). MS (ESI+,
m/z): 443 [(M+Na)+, 10]. HRMS (EI+) calc for C26H28O5 [M+]:
420.1937, found: 420.1936.
Rf (10% MeOH/CH2Cl2): 0.52. mp: 90–92 ◦C. [a]D20 = +26 (c 1.0,
CH2Cl2). IR (KBr): 3352, 3074, 1716, 1606, 1527 cm-1. 1H NMR
(CDCl3, 300.13 MHz): d 1.96 (m, 1H, H-2), 2.21 (m, 1H, H-2),
4.00–4.12 (m, 3H, H-1 and H-4), 4.32–4.50 (m, 3H, H-3, and H-
5), 8.26 (m, 4H, Ho and Hm). 13C NMR (CDCl3, 75.5 MHz): d
35.0 (C-2), 65.7 (C-5), 67.2 (C-1), 73.6 (C-3), 83.6 (C-4), 123.6
(Cm), 130.8 (Co), 135.2 (Ci), 150.7 (Cp), 164.7 (C O). MS (ESI+,
m/z): 290.0 [(M+Na)+, 100%]. HRMS (ESI+) calc for C12H13NaO6
[M+Na]+: 290.0635, found: 290.0639.
Synthesis of 5-benzoyl-3-dimethoxytrityl-1,2-dideoxy-D-ribose
(18a) or 3-dimethoxytrityl-5-toluoyl-1,2-dideoxy-D-ribose (18b)
5-O-p-Fluorobenzoyl-1,2-dideoxy-D-ribose (15e)
To
a solution of 15a or 15b(0.34 mmol) in anhydrous
Rf (10% MeOH/CH2Cl2): 0.52. mp: 59–61 ◦C. [a]D20 = +41 (c 1.0,
CH2Cl2). IR (KBr): 3372, 3070, 1712, 1507, 1512 cm-1. 1H NMR
(CDCl3, 400.13 MHz): d 1.96 (m, 1H, H-2), 2.20 (h, 1H, H-2,
3JHH 8.0 Hz), 3.99–4.10 (m, 3H, H-1 and H-4), 4.32–4.50 (m, 3H,
H-3, and H-5), 7.11 (m, 2H, Hm), 8.06 (m, 2H, Ho). 13C NMR
(CDCl3, 100.61 MHz): d 35.1 (C-2), 65.0 (C-5), 67.2 (C-1), 73.6
(C-3), 83.8 (C-4), 115.6 (d, Cm, 2JCF 22.0 Hz), 130.8 (Co), 126.0 (Ci),
132.3 (Co), 165.7 (C O), 165.9 (d, Cp, 2JCF 254.5 Hz). 19F NMR
(CDCl3, 282 MHz): d 105.2. MS (ESI+, m/z): 263.0 [(M+Na)+,
100%]. HRMS (ESI+) calc for C12H13FNaO6 [M+Na]+: 263.0690,
found: 263.0690.
pyridine (3.4 mL) was added 4,4¢-dimethoxytrityl chloride
(127 mg, 0.37 mmol), and the mixture was stirred at 70 ◦C.
The progress of the reaction was monitored by TLC (30%
hexane/CH2Cl2) which indicates the total disappearance of the
starting material in 3 h. The mixture was concentrated under
reduced pressure and the crude product was purified by column
˚
chromatography using neutral silica gel 60 A (32–63 mm) pH 7
(20% hexane/CH2Cl2) to obtain 18.
18a. 83% yield. mp: 47–49 ◦C. Rf (30% hexane/CH2Cl2): 0.27.
IR (KBr): u 3055, 2986, 2957, 1720, 1608 cm-1. 1H NMR (MeOH-
d4, 300.13 MHz): d 1.58 (m, 1H, H-2), 1.74 (m, 1H, H-2), 3.74
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(s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.84–4.05 (m, 5H, H-1, H-4,
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3
3
H-5), 4.23 (dt, 1H, H-3, JHH 2.2 Hz, JHH 5.9 Hz), 6.85 (m, 4H,
DMT), 7.10–7.69 (m, 11H), 7.61 (t, 1H, Hp, 3JHH 1.3 Hz, 3JHH 6.8
Hz), 7.88 (m, 2H, Ho). 13C NMR (MeOH-d4, 75.5 MHz): d 35.0
(C-2), 55.6 (2C, OCH3), 65.7 (C-5), 68.5 (C-1), 76.8 (C-4), 84.4
and 88.3 (C-3+C-DMT), 114.1 (4C), 127.8–134.2 (15C), 137.6 and
137.8 (2C), 147.0 (1C), 160.2 (2C), 167.5 (C O). MS (ESI+, m/z):
547 [(M+Na)+, 100%], 1099 [(2 M+Na)+, 40]. HRMS (EI+) calc
for C33H32O6 [M+]: 524.2199, found: 524.2204.
18b. 83% yield. mp: 48–50 ◦C. Rf (30% hexane/CH2Cl2): 0.27.
[a]2D0 = +13 (c 1.0, CH2Cl2). IR (KBr): u 3055, 2966, 1716, 1609
1
cm-1. H NMR (MeOH-d4, 300.13 MHz): d 1.53 (m, 1H, H-2),
1.70 (m, 1H, H-2), 2.38 (s, 3H, CH3), 3.70 (s, 3H, OCH3), 3.73 (s,
3H, OCH3), 3.80–4.10 (m, 5H, H-1, H-4, H-5), 4.23 (dt, 1H, H-3,
3JHH 2.2 Hz, 3JHH 5.9 Hz), 6.82 (m, 4H, DMT), 7.09–7.39 (m, 9H),
7.46 (d, 2H, Hm, 3JHH 8.3 Hz), 7.88 (d, 2H, Ho, 3JHH 8.1 Hz). 13
C
NMR (MeOH-d4, 75.5 MHz): d 21.9 (CH3), 35.4 (C2), 56.0 (2C, O-
Me), 65.9 (C-5), 68.9 (C-1), 77.1 (C-4), 84.8–84.6 (C-3+C-DMT),
114.1 (4C) 128.2–131.9 (14C), 137.9–138.2 (2C), 145.6 and 147.4
(2C), 160.6 (2C), 168.0 (C O). MS (ESI+, m/z): 561 [(M+Na)+,
100%], 1099 [(2M+Na)+, 60]. HRMS (EI+) calc for C34H34O6 [M+]:
538.2355, found: 538.2347.
9 L. Guan and M. M. Greenberg, J. Am. Chem. Soc., 2010, 132, 5004–
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Synthesis of 3-dimethoxytrityl-1,2-dideoxy-D-ribose (19)
Same procedure as described above for the synthesis of 17. 86%
yield. Rf (40% EtOAc/hexane): 0.24. mp: 54–56 ◦C. [a]D20 = +36 (c
1.0, CH2Cl2). IR (KBr): u 3406, 3055, 2986, 1609 cm-1. 1H NMR
(MeOH-d4, 300.13 MHz): d 1.34 (m, 1H, H-2), 1.56 (m, 1H, H-
3
2
2), 3.15 (dd, 1H, H-5, JHH 5.3 Hz, JHH 11.9 Hz), 3.30 (dd, 1H,
3
2
H-5, JHH 3.5 Hz, JHH 12.7 Hz), 3.75 (s, 6H, 2OMe), 3.83 (m,
3
3
3H, 2H1+H4), 4.14 (dt, 1H, H3, JHH 1.7 Hz, JHH 5.7 Hz), 6.84
(m, 4H, DMT), 7.27 (m, 7H, DMT), 7.46 (m, 2H, DMT). 13C
NMR (MeOH-d4, 75.5 MHz): d 35.8 (C2), 56.0 (2C, OMe), 63.7
(C5), 68.8 (C1), 77.2 (C3), 88.0–88.4 (Ct+C4), 87.7 (C4), 114.4 (4C),
128.2–131.9 (9C), 138.4 (2C), 147.4 (1C), 160.5 (2C). MS (ESI+,
m/z): 443 [(M+Na)+, 100%], 863 [(2M+Na)+, 20]. HMRS (EI+)
calc for C26H28O5 [M+]: 420.1937, found: 420.1942.
Procedure for the recycling of PSL-C
A stirred suspension of 13 (590 mg, 5 mmol), vinyl benzoate
(814 mg, 5.5 mmol), and PSL-C (1.18 g) in anhydrous THF (5 mL)
under nitrogen was heated at 50 ◦C and 250 rpm for 7.5 h. Once
the reaction is finished, the enzyme was filtered off and washed
with CH2Cl2. The combined filtrate were washed with NaHCO3
(aq), dried over Na2SO4 and evaporated. The small excess of vinyl
benzoate was removed under vacuum at 60 ◦C to afford 15a in 96%
yield. This process was repeated four times employing the same
PSL-C (see Table 3). To reactivate the enzyme, PSL-C was poured
into water, filtered, and dried under vacuum after each cycle.
16 (a) M. Ferrero and V. Gotor, Chem. Rev., 2000, 100, 4319–4347; (b) M.
Ferrero and V. Gotor, Monatsh. Chem., 2000, 131, 585–616.
17 The synthesis of 3,5-di-O-toluoyl-1,2-dideoxyribose was accomplished
by the procedure described in reference 13c starting with 1a-chloro-3,5-
di-O-p-toluoyl-2-deoxyribofuranose available in commercial quantity
from RI Chemical Inc. (Orange, CA, USA).
18 J. Garc´ıa, S. Ferna´ndez, M. Ferrero, Y. S. Sanghvi and V. Gotor, J. Org.
Chem., 2002, 67, 4513–4519.
19 I. Lavandera, S. Ferna´ndez, J. Magdalena, M. Ferrero, H. Grewal,
C. K. Savile, R. J. Kazlauskas and V. Gotor, ChemBioChem, 2006, 7,
693–698.
Notes and references
20 J. Garc´ıa, S. Ferna´ndez, M. Ferrero, Y. S. Sanghvi and V. Gotor,
Tetrahedron Lett., 2004, 45, 1709–1712.
21 M. Lobell and M. P. Schneider, Synthesis, 1994, 375–377.
22 F. van Rantwijk and R. A. Sheldon, Chem. Rev., 2007, 107, 2757–2785.
1 E. C. Friedberg, G. C. Walker, W. Siede, R. D. Wood, R. A. Schultz
and T. Ellenberger, in DNA Repair and Mutagenesis, Second Ed., ASM
Press, Washington, D. C., USA, 2006.
5966 | Org. Biomol. Chem., 2011, 9, 5960–5966
This journal is
The Royal Society of Chemistry 2011
©