An expedient synthesis of flexible nucleosides: Via a regiocontrolled enzymatic glycosylation of functionalized imidazoles

Article abstract of DOI:10.1039/c7ob01850a

A versatile two-step synthesis of C4- and C5-arylated 2′-deoxyribosylimidazoles was elaborated using enzymatic N-transglycosylation followed by microwave-assisted Pd-catalysed arylation reactions. We report herein the reaction conditions that permit managing regioselectivity (N3 versus N1-isomers) in the enzymatic glycosylation of 4-iodoimidazole using the nucleoside N-deoxyribosyltransferase from L. leichmannii. Regiocontrolled glycosylation was also observed among several other imidazole derivatives studied, providing simple access to isomers not readily accessible by chemical routes. Finally, a series of flexible nucleosides was obtained in one step from 4- or 5-iodo-imidazole nucleosides by the Suzuki-Miyaura cross-coupling reaction with (hetero)aryl-boronic acids in aqueous media. Moreover, this chemoenzymatic approach is compatible with a one-pot two-step process affording a straightforward access to a broad array of potential anticancer and antiviral drugs as well as new DNA building blocks.

Full text of DOI:10.1039/c7ob01850a

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An expedient synthesis of flexible nucleosides via a regiocontrolled enzymatic
glycosylation of functionalized imidazoles
S. Vichier-Guerre, L. Dugué, F. Bonhomme and S. Pochet*
Unité de Chimie et Biocatalyse, Institut Pasteur, CNRS, UMR3523, 28 rue du Dr Roux, 75724 Paris
Cedex 15
*Corresponding author: sylvie.pochet@pasteur.fr
Abstract
A versatile two-step synthesis of C4 and C5-arylated 2’-deoxyribosylimidazoles was elaborated using
enzymatic N-transglycosylation followed by microwave-assisted Pd-catalysed arylation reactions. We
report herein the reaction conditions that permit to manage the regioselectivity (N3 versus N1-isomers) in
the enzymatic glycosylation of 4-iodoimidazole using the nucleoside N-deoxyribosyltransferase from L.
leichmannii. Regiocontrolled glycosylation was also observed among several other imidazole derivatives
studied, providing simple access to isomers not readily accessible by chemical routes. Finally, a series of
flexible nucleosides was obtained in one-step from 4 or 5-iodo-imidazole nucleoside by Suzuki-Miyaura
cross-coupling reaction with (hetero)aryl-boronic acids in aqueous media. Moreover, this
chemoenzymatic approach is compatible with a one-pot two-step process affording a straightforward
access to a broad array of potential anticancer and antiviral drugs as well as new DNA building blocks.
Keywords: Chemoenzymatic synthesis, Flexible Nucleoside, Microwave irradiation, Nucleoside 2’-
deoxyribosyltransferase, Regioselective glycosylation, Suzuki-Miyaura cross-coupling
1

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Introduction
Nucleoside analogues constitute a major class of biologically active compounds that are widely used as
anticancer and antiviral drugs.1, 2 Most of these nucleoside analogues are routinely produced by multi-
step and time-consuming chemical procedures, including the stereocontrolled formation of the glycosidic
linkage and several protection/deprotection steps of the sugar and nucleobase moieties. Over the past
few decades, biocatalysts have emerged as a powerful and attractive alternative to conventional chemical
synthesis. In particular, enzymes that catalyze the formation of the glycosidic bond have been successfully
exploited in the synthesis of natural and unnatural nucleosides.3-6 Two main classes of enzymes are
currently used for transferring pentosyl residue from a donor nucleoside to an acceptor base: nucleoside
phosphorylases (NP, EC 2.4.2.1) and nucleoside N-deoxyribosyltransferases (NDT, EC 2.4.2.6). Interestingly,
NDTs have been shown to tolerate a wide range of modified nucleobases from azole derivatives7-9 to
expanded-size purines.10-12
In the last decade, a class of shape-modified nucleosides called fleximers, where the purine base has been
split into its imidazole and pyrimidine subunits, has been introduced by Seley-Radke et al.13The imidazole
ring remains attached to the sugar moiety, while the pyrimidine ring is linked via a single C–C bond to the
C4 (proximal fleximer) or C5 (distal fleximer) of the imidazole (Fig. 1).14, 15 This connectivity confers
additional degrees of conformational freedom and torsional flexibility to the molecule, while still retaining
the requisite hydrogen bonding and aromatic features necessary for base pairing and molecular
recognition. These modified nucleobases have attracted significant attention as biochemical tools or
potential therapeutic agents.16-18
Fig. 1 Chemical structures of distal and proximal FlexA
The chemical synthesis of these nucleoside analogues remains challenging.15 The distal fleximers were
initially prepared from their tricyclic “expanded purine” counterparts through desulfurization with Raney
nickel.15, 19 The proximal fleximers were synthesized via cross-coupling reactions to connect the imidazole
and pyrimidine rings.15 We recently described the use of nucleoside transferases (NDT-II from L.
leichmannii and PNP from E. coli) to easily convert a chemically diverse set of 4-aryl and 4-heteroaryl
imidazole derivatives20 into the corresponding nucleoside derivatives (Scheme 1).12
2

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Ar
N
HO
Enzymatic
N-transglycosylation
N
Ar
N
O
NDT or PNP
N
H
OH
X = H or OH
X
4-(hetero)aryl imidazoles
imidazole based nucleosides
Scheme 1 Synthetic route to imidazole-based nucleosides
During the course of our study, we noticed that efficient 2’-deoxyribosylation of the pyrimidinone
derivative 1a at both N1 and N3 sites using NDT-II from L. leichmannii (Scheme 2). More precisely, the
transferase reaction between 1a and thymidine led to the initial formation of the N1 and N3 glycosylated
products (2a and 3a, respectively). When the conversion was allowed to proceed to completion, 2a was
isolated as the sole product suggesting that the N3 isomer 3a (kinetic product) was progressively
converted into the thermodynamically more stable N1 isomer 2a. However, in the case of the other
studied 4-arylated imidazole derivatives, the corresponding target N1 nucleosides were isolated as the
sole products.12
Scheme 2 NDT-catalysed transglycosylation of 1a into 2a and 3a.
While the nucleobase exchange reaction is highly regioselective for natural substrates, base analogues
may be glycosylated at multiple sites.11, 21, 22 There are only few examples of 2’-deoxyribosylimidazole
derivatives being synthesized by biocatalysts. Betbeder et al.7 have described the transglycosylation of
ethyl 5-aminoimidazole 4-carboxamide using NDT from L. leichmannii to produce 5-amino-1-(2-deoxy-ß-
D-ribosyl)imidazole 4-carboxamide as the sole product. Regioselective glycosylation at N1 position was
also reported starting from imidazole 4-carboxamide.8 However, using the whole cells of E. coli as a
biocatalyst, Ewing et al.23 have reported the 2-deoxy-D-ribosylation of ethyl 5-aminoimidazole 4-
carboxylate affording a mixture of ß-N1 and N3 isomers.
We thus surmised that it might be possible to control the regioselectivity in the glycosylation of 4-
iodoimidazole, thus providing a simple access to the N1 and N3 isomers in good yields. The resulting 4-
and 5-iodo-imidazole derivatives could be further engaged in cross-coupling reactions with boronic acid
derivatives, affording C-4 or C-5 aryl-imidazole nucleosides. We report here how this scheme was
validated experimentally.
3

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Results and Discussion
First, we studied the NDT-catalysed transfer reaction between 4-iodoimidazole (1b) and thymidine as a
function of enzyme concentration, incubation time and temperature. Reaction mixtures (1 mL) typically
contained 10 mM 4-iodoimidazole, 40 mM thymidine and variable amount of enzyme in 10 mM citrate
buffer (pH 6.0) containing 5% v/v DMSO. Conversion rate was monitored by reverse phase HPLC.
As shown in Table 1, for a given enzyme concentration (0.5 mg/mL), conversion of 4-iodoimidazole (1) at
50°C, which is reported to be the optimal temperature for NDT,24 led to the simultaneous formation of
two glycosylated products, which were isolated by reverse-phase HPLC and identified by NMR
1
spectroscopy as the N1 and N3 isomers (2b and 3b) (Table 1, entry 1). The H-13C HMBC spectra (see
Supporting Information) indicate a clear correlation between H-1’ and C(H)-5 for compound 2b and
between H-1’ and C(q)-5 for compound 3b.
Table 1 NDT-catalysed transglycosylation reactions of 4-iodoimidazole 1ba
O
NH
O
I
HO
N
O
N
N
N
N
NH
O
N
O
H
OH
HO
HO
I
I
N
O
O
+
Ll-NDT
N
H
OH
OH
Citrate buffer
pH 6.5
1b
2b
3b
Entry
Enzyme
(µg)
Incubation
temperature (°C)
Incubation
time (h)
% Productsb
1b
2b
3b
1
2
500
250
125
62.5
500
500
250
250
125
125
500
250
125
62.5
50
50
50
50
20
20
20
20
20
20
37
37
37
37
3
3
0
82
18
13
17
22
32
9
50
35
31
26
42
15
30
9
37
48
47
40
49
25
47
16
43
33
44
42
42
3
3
4
3
5
3
6
10
3
7
60
23
75
30
6
8
10
3
9
10
11
12
13
14
10
3
27
60
44
34
21
3
12
24
37
3
3
a
Reaction conditions: 10 mM acceptor in 5% v/v DMSO, 40 mM thymidine in 10 mM citrate buffer pH 6.5 (1 mL), variable amounts of enzyme
(from 12.5 to 100 µL at 5 mg/mL), incubation temperature (°C) and time (h) as indicated in Table 1.
b Conversions were determined by reverse-phase HPLC analysis of an aliquot of the incubation mixtures monitored at 230 nm.
As previously observed for the conversion of imidazole derivative 1a into 2’-deoxynucleosides 2a/3a, the
4

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rate of the glycosylated products changed as a function of time (Fig. 2A). When complete conversion of 1b
was observed, the amount of the N1 product 2b had reached 82%, while the early amount of N3 isomer
3b had decreased (down to 18% after 3 h). This interconversion indicates that 3b acts also as a donor for
the transfer reaction, 3b being a better substrate than 2b. Indeed, when the nucleoside 3b was incubated
in the presence of thymine and NDT in the above conditions, we observed the initial conversion of
thymine into thymidine, which in turn is slowly converted into 2b (see Fig. 3A). Under the same conditions,
the nucleoside 2b appeared quite stable toward the deoxyribosyl transfer reaction, as only a trace of
thymidine was detected after a long incubation time (see Fig. 3B).
A
B
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
1b
2b
3b
1b
2b
3b
0
60
120
180
240
300
360
0
60
120
180
240
300
360
Fig. 2 HPLC monitoring at 230 nm of the crude reaction mixture of 1b with thymidine in the presence of NDT (500
µg/mL panel A or 125 µg/mL panel B) at 50°C. Experiments were done in triplicate. See experimental section for
elution conditions.
A
B
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
1b
2b
3b
T
dT
1b
2b
3b
T
dT
0
180
360
540
720
900
1080
0
180
360
540
720
900
1080
Fig. 3 HPLC monitoring at 230 nm of the crude reaction mixture of 3b (panel A) or 2b (panel B) with thymine in the
presence of NDT (125 µg/mL) at 50°C. Experiments were done in duplicate. See experimental section for elution
conditions.
Reducing the enzyme concentration by a factor 4 (compared entries 1 and 3, Table 1) resulted in a nearly
complete conversion of 1b (17% of remaining 1b after 2h at 50°C) with the formation of the N3 derivative
3b as a major product (47%) compared to the N1 product 2b (36%). No change in the products ratio was
observed after 2 h, the reaction having reached equilibrium (Fig. 2B). When incubation was performed at
20°C, as anticipated the conversion rate was slow and long incubation time was required to achieve a
nearly complete conversion (9% of remaining 1b after 10h), 3b was found to be the major glycosylated
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product whatever the concentration enzyme is used (Table 1, entries 5-10). An incubation temperature of
37°C led to a similar products profile as compared to 20°C, conversion of 1b was only reached faster
(Table 1, entries 11-14). The highest enzyme concentration (entry 11) caused an increase in 2b/3b ratio
due to the reversibility of the reaction.
We also examined the influence of the 2’-deoxyribosyl donor, 2’-deoxycytidine (dC) or 2’-deoxyadenosine
(dA) in place of thymidine (dT), on the kinetic of the reaction and the products distribution, while
maintaining the incubation temperature at 37°C and the enzyme concentration at 125 µg/mL.
Table 2 Effect of donor-acceptor on the NDT productsa
Entry Acceptor Donor
% Conversion at 3 hb
% Conversion at 8 hb
1
2
3
4
5
1b
1b
1b
1c
1c
dT
dC
dA
dT
dC
1b 24
2b 34
2b 22
2b 18
2c 17
2c 15
3b 42
1b 11
2b 47
2b 29
2b 28
2c 37
2c 28
3b 42
1b 43
1b 63
1c 36
1c 48
3b 35
3b 19
3c 47
3c 37
1b 29
1b 48
1c 28
1c 36
3b 42
3b 24
3c
3c
35
36
a
Reaction conditions: 10 mM acceptor 1b or 1c in 5% v/v DMSO, 40 mM donor as indicated in the Table, 10 mM citrate buffer pH 6.5 in the
presence of 125 µg/mL NDT, incubation temperature of 37°C.
b Conversions were determined by reverse-phase HPLC analysis of an aliquot of the incubation mixtures monitored at 230 nm.
As shown in Table 2, thymidine was found to be the best donor (i.e. conversion of 1b was nearly complete
after 3 h as compared to dC and dA, entries 1-3). The N3 isomer 3b was early formed as the major
compound whatever the donor used and was further converted into N1 isomer 2b when the reaction has
gone almost to completion (or reached equilibrium). When 4-bromoimidazole (1c) was used as the
acceptor, comparable results were obtained with the concomitant formation of 2c and 3c, but the
conversion was a little slower than using 1b (Table 2, entries 4 & 5).
Based on these data, it appears possible to manage the regioselectivity of the NDT-catalysed reaction
using 4-iodoimidazole as substrate by varying enzyme concentration and incubation time for altering the
products distribution (i.e. the N3/N1 ratio).
We next studied the scope and limitations of the transfer reaction in terms of regioselectivity using a
small set of 4-substituted and 4,5-disubstituted imidazole derivatives (1d-1r). The efficiency and
selectivity profile were determined by HPLC analysis of the crude reaction mixtures. The glycosylated
6

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products were isolated and further characterized by NMR and mass spectrometry. Conversions after 3 h
of incubation are summarized in Table 3. Imidazole bearing a small functional group at position 4, such as
hydroxymethyl (1d), carboxamide (1e), methyl (1f) and ethyl ester (1g) carboxylic acid, carboxyaldehyde
(1h) and carbonitrile (1i), were found to be substrates with different reactivity patterns in terms of
conversion rate and N1/N3 ratio (Table 3, entries 3-8). Thus, 1d and 1e were rapidly converted under the
conditions used and the corresponding N1-glycosylated bases (2d and 2e, respectively) were isolated as
the sole products in nearly quantitative yield.8
Table 3 NDT-catalysed transglycosylation reactions of imidazole derivatives 1b-1qa
O
NH
O
R1
R2
R1
HO
N
O
N
N
N
N
NH
O
N
H
O
R2
R1
R2
OH
HO
HO
N
O
O
+
Ll-NDT
N
H
OH
OH
Citrate buffer
pH 6.5
1b-1q
2b-2q
3b-3q
Entry R1
R2
Remaining acceptorb
(%)
N1 isomerb (%)
N3 isomerb (%)
1
I
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1b
1c
1d
1e
1f
24
36
2b
2c
2d
2e
2f
34
17
3b
42
47
2
Br
3c
3d
3e
3f
3
CH2OH
CONH2
0
100
96
nd
4
4
nd
5
COOCH3
22
68
10
6
COOCH2CH3
CHO
1g
1hc
1ic
1j
10
2g
2h
2i
88
3g
3h
3i
2
7
31
41
28
8
CN
44
39
17
9
CH2COOCH3
CH2COOH
26
100d
2j
70
3j
4
10
11
12
13
14
15
1k
1l
2k
2l
nd
3k
3l
nd
CH=CHCOOH
CH2CH2NH2
CH2CH(NH2)COOH
CH2CH(NH2)CH2OH
CN
78
22
nd
1m
1n
1o
1p
1q
100d
100
100
13 (8e)
60 (44e)
2m
2n
2o
2p
2q
nd
3m
3n
3o
3p
3q
nd
nd
nd
nd
nd
NH2
CN
4 (39e)
7 (33e)
83 (53e)
33 (23e)
16
CONH2
a
Reaction conditions: 10 mM acceptor in 5% v/v DMSO, 40 mM thymidine in 10 mM citrate buffer pH 6.5, in the presence of 125 µg NDT,
incubation at 37°C for 3 h.
b % conversions after 3 h at 37°C determined by reverse-phase HPLC analysis of an aliquot of the incubation mixtures monitored at 230 nm.
c
The corresponding N1/N3-glycosylated products were not separable by HPLC under the analytical conditions used. The N1/N3 ratio was
determined by NMR analysis.
d 20% conversion in the presence of 500 µg NDT after 48 h at 37°C.
e % conversions after 16 h for compound 1p or 24 h for compound 1q at 50°C.
nd : not detected
Conversion of imidazole derivatives 1f-1j proceeded more slowly. HPLC analyses showed the formation of
both N1 and N3 products that further shifted toward glycosylation at N1 (Table 3, entries 5-8).
7

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Surprisingly, 4-acetic acid imidazole (1k) was found to be a poor substrate compared to the methyl ester
derivative (1j). A 4-fold higher enzyme concentration and long incubation time were required to observe a
20% conversion rate into N1/N3 products. When more sterically demanding substituents at position 4 of
the imidazole ring (1l-1o, entries 11-14) were used as substrates, no or poor conversion were detected
even in the presence of higher enzyme concentration, incubation time and temperature. We12 and
others10, 11 previously shown that the catalytic active site of NDT tolerates a variety of large heterocycles
as acceptors. The lack of transfer activity observed for some imidazole derivatives such as histidine, which
occurs in the lactobacteria cells hosting the NDT enzyme, might be explained by counterselection of the
active site during evolution.
Finally we examined the recognition of 4,5-disubstituted imidazoles 1p and 1q, precursors of purine
analogues.25, 26 Chemical glycosylation of 1p have been previously reported via the sodium salt procedure
affording both N1 and N3 glycosylated products in 66% yield with a 1:2 ratio, respectively.25 An additional
deprotection step is needed to isolate compounds 2p and 3p (62% and 70%, respectively). Both imidazole
derivatives 1p and 1q were found to be substrates of NDT and converted into the N1 and N3 isomers in
good overall yields with a ratio depending on the experimental conditions (Table 3, entries 15-16). The
achievable NDT-catalysed interconversion of 3p (or 3q) into 2p (or 2q) is of significant interest to facilitate
access to minor isomers compared to the classical synthetic approach.
The 4- and 5-halogeno-imidazole deoxynucleosides provide valuable starting points for further chemical
diversification. Thus, the palladium-catalysed Suzuki-Miyaura reaction27 represents one of the most
powerful methods for the C-modification of nucleobases and nucleosides.28 The C–C cross-coupling
reactions are usually performed in organic solvents using protected nucleobases and nucleosides. More
recently, alternative methodologies involving aqueous-phase coupling reactions from unprotected
halonucleobases and halonucleosides were developed.29-31
Applying our conditions optimized above to the transglycosylation of 1b on a 5.5-mmol scale, we
successfully isolated the N1/N3 isomers 2b/3b in large quantity and in good yields. We next studied
conditions for cross-coupling Suzuki-Miyaura reaction starting from 4(or 5)-iodo-imidazole derivatives 2b
(or 3b) with a set of aryl- and heteroarylboronic acids (Fig. 4).
N
F
N
B(OH)2
B(OH)2
Br
B(OH)2
4
5
6
H2N
N
MeO
N
OMe
tBuO
N
OtBu
N
N
N
B(OH)2
B(OH)2
B(OH)2
7
8
9
8

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Fig. 4 Aryl and heteroarylboronic acids (4-9) used in this study
We recently reported the efficient arylation of 4-iodoimidazole under microwave (MW) irradiation.20 First
we applied these conditions (Table 4, conditions A) to 4-iodo derivative 2b, which was reacted with 2
equiv. of phenylboronic acid (4) in the presence of Na2CO3 and Pd(Ph3)4 in a 1/1 mixture of DMF/water at
110°C for 1 h. While the starting material was nearly consumed, the targeted product 10.1 was isolated in
moderate yield (Table 4, entry 1). We next considered the optimized coupling conditions involving 5-iodo-
2’-deoxyuridine.32 Thus, performing the reaction in water without co-solvents in the presence of Pd(OAc)2
and PPh3 under MW irradiation at 110°C, slightly improved the yield of 10.1 (Table 4, entry 2), affording
10 .1 in 54% yield (data not shown).
Table 4 Suzuki cross-coupling reaction of 2b and 3b with arylboronic acids 4-9
Entry Nucleoside Ar-B(OH)2 Conditionsa Temperature/ Products Yield (%)
Time
1
2
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
3b
3b
3b
3b
3b
4
4
4
4
4
5
5
6
6
7
8
8
9
9
9
4
5
6
6
7
A
B
C
C
C
C
A
C
A
C
C
A
C
C
A
C
A
A
A
C
110°C/1 h
10.1
10.1
10.1
10.1
10.1
10.2
10.2
10.3
10.3
10.4
10.5
10.5
38
45
120°C/1 h
90°Cb/2 h
3
35
4
150°C/5 min
120°C/5 min
120°C/10 min
110°C/1 h
57
5
78
6
30
7
60
8
120°C/10 min
80°C/1 h
39
9
48
10
11
12
13
14
15
16
17
18
19
20
120°C/5 min
120°C/10 min
120°C/1 h
70
58
51
120°C/20 min 10.6/10.7
150°C/1 min 10.6/10.7
20/12
37/3
76
110°C/1 h
120°C/10 min
110°C/1 h
10.6
11.1
11.2
11.3
11.3
11.4
70
60
110°C/1 h
22
80°C/1 h
40
120°C/5 min
60
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21
22
23
3b
3b
3b
8
8
9
C
A
A
120°C/10 min
110°C/1 h
11.4
11.4
11.6
25
46
80
110°C/1 h
a Reagents and conditions: 2b or 3b (0.1 mmol), arylboronic acid (1.25 mmol), 1M Na2CO3 (3 mmol)
Method A: Pd(PPh3)4 (0.08 mmol) in DMF/H2O (1/1, 14 mL/mmol);
Method B: Pd(OAc)2 (0.08 mmol), PPh3 (0.08 mmol) in H2O (14 mL/mmol);
Method C: Pd(PPh3)4 (0.08 mmol), TPPS (0.08 mmol), in CH3CN/H2O (1/1, 14 mL/mmol);
b Conventional heating
Finally, the use of Pd(OAc)2/TPPS catalytic system29 in a mixture of acetonitrile/water in the presence of
Na2CO3 (conditions C) gave a complete conversion of 2b in 5 min under MW irradiation at 120°C, affording
10.1 in 78% yield (Table 4, entry 3). Lower or higher temperature resulted in rather low product yields
(entries 4 and 5). It is worth noting that bromide 2c was found to react slowly under these optimal
conditions.
Coupling reactions of 2b with the pyridinyl- and pyrimidinyl-boronic acid derivatives 5-9 (Fig. 3) were next
attempted under these conditions, resulting in the formation of compounds 10.2-10.6 with yield ranging
from 48 to 76% (Table 4, entries 6-15). The use of Cs2CO3 in place of Na2CO3 did not improve the coupling
yield of 5 (data not shown). In the case of the 3,5-pyrimidinone derivative 9, the partial deprotection of t-
butoxy group was observed using the Pd(OAc)2/TPPS system (conditions C) and two products 10.6 and
10.7 were isolated, even if the reaction temperature was increased and reaction time was shortened. The
use of Pd(Ph3)4 as catalyst (conditions A) gave the fully protected product 10.6 in high yield.
These optimal conditions were next applied to the coupling reaction of 5-iodo derivative 3b with the set
of boronic acid derivatives 5-9. Similar trends were observed and the C-5 aryl-imidazole derivatives 11.1-
11.6 were isolated in 40–80% yield (Table 4, entries 16-23).
Finally, we examined the feasibility of a one-pot two-step process, performing the Suzuki-Miyaura
arylation without work-up of the intermediate iodoimidazole derivative. As a proof of concept, we
carried out the enzymatic step under conditions that yielded a mixture of N1 and N3 isomers 2b
/3b
(80% conversion). After enzyme denaturation, the crude reaction mixture was treated with phenyl
boronic acid
purification.
4. The coupling products 2b and 3b were isolated in 56% overall yield after HPLC
Conclusion
We have developed a straightforward and highly efficient two-step chemoenzymatic procedure for the
synthesis of C-4 and C-5 aryl-imidazole 2’-deoxynucleosides. We found experimental conditions that
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affect the regioselectivity in the glycosylation of 4-iodoimidazole using NDT-II from L. leichmannii, making
possible the preparation of the kinetic product of the reaction, i.e. the N3 glycosylated isomer, in good
yield. Regiocontrolled enzymatic glycosylation was also observed among several other imidazole
derivatives studied, providing simple access to regioisomers not readily accessible by chemical routes.
These results illustrate that NDT can accommodate a variety of nucleobases, because of its flexibility in
both accepting and donating hydrogen bonds.11 To our knowledge, regioselectivity control in the transfer
reactions catalysed by NDT has never been reported so far. Once in hand, the 4 and 5-iodo-imidazole
nucleosides have been successfully engaged in Suzuki-Miyaura cross-coupling reactions with a set of
(hetero)aryl-boronic acid derivatives. We also demonstrated that our synthetic approach is compatible
with a one-pot two-step process, i.e. enzymatic glycosylation of 4-iodoimidazole followed by Suzuki-
Miyaura arylation without work-up of the iodoimidazole nucleoside. Compared to the chemical routes
described so far, the chemoenzymatic strategy reported here is easier and less labor consuming,
providing a straightforward access to original proximal or distal fleximers. These results will be beneficial
to the further development of these artificial nucleobases as drug candidates or DNA building blocks.
Studies are currently underway to explore their potential as polymerase substrates.
Experimental Section
General information
All chemicals were obtained from commercial sources and used without further purification unless
otherwise noted. All solvents were analytical grade purity. Microwave reactions were performed on a
microwave Biotage initiator 2 oven under an inert atmosphere of argon. Reactions were monitored by
thin layer chromatography (TLC) on pre-coated Merck silica gel plates (60 F254/0.2 mm), by LC-MS (Agilent
1100 series) or by HPLC (Agilent 1100 series) using a C18 reverse phase column. Analytical HPLC was
carried out on an Agilent system (1100 series) equipped with a diode-array detector using a C18 reverse
phase column (Kromasil, 5 μm, 100 Å; column 1: 4.6 x 150 mm or column 2: 4.6 x 150 mm) at a flow rate
of 1mL/min and a linear gradient of acetonitrile in 10 mM triethylammonium acetate buffer (TEAA) over
20 min (Gradients: G1, 5 to 40%; G2, 0 to 7%; G3, 0 to 20%; G4, 5 to 25%; G5, 0 to 10%; G6, 5 to 60%).
Purification by HPLC was carried out on Agilent 1100 Series system on a C18 reverse phase column
(Kromasil, 5 μm, 100 Å, 10 x 250 mm) using a flow rate of 4.0 mL/min and an isocratic or linear gradient of
acetonitrile in 10 mM TEAA over 20 min. Yields refer to chromatographically and spectroscopically pure (>
1
95 %) compounds. H and 13C NMR spectra were recorded on a Bruker Advance 400 operating at 400.13
MHz and 100.62 MHz, respectively. Chemical shifts are given in ppm (
δ) relative to residual solvent peak,
1
coupling constants (J) are reported in Hertz and standard abbreviations are used. Assignment of H and
1
13C signals was performed by analysis of the correlated homonuclear H,1H-COSY and heteronuclear
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1H,13C-HMBC, 1H,13C-HSQC spectra. High-resolution mass spectra were recorded on a Waters Q-TOF micro
MS instrument under electrospray ionization in positive ionization mode using a mobile phase of
acetonitrile/water with 0.1 % formic acid.
General procedure for the enzymatic glycosylation of imidazole derivatives
Nucleoside 2-deoxyribosyltransferases class II from L. leichmannii (LlNDT-II or NDT) was produced and
purified as described.33
Analytical scale synthesis: Typically, enzymatic reaction mixtures (1 mL) contained imidazole derivative
(0.01 mmol) as acceptor and thymidine (0.04 mmol) as donor. A solution of acceptor and donor in DMSO
(5% v/v) was added slowly to 10 mM citrate buffer (pH 6.0) to reach a final volume of 1 mL. The reaction
was started by adding NDT (variable amounts at 5 µg/µL) and run at 20°C, 37°C and 50°C (see Tables 1 -3).
Conversion was monitored by analytical reverse phase HPLC.
Preparative scale synthesis: Experimental conditions described above were adapted by increasing the
volume and mass of reactants. The amount of NDT at 5 µg/µL, the reaction time and temperature were
adjusted in order to obtain in a single experiment both regioisomers in a significant amount, while
recovering relatively little amounts of starting material. The products were isolated after purification by
reverse phase HPLC.
1-(2-Deoxy-β-D-ribofuranosyl)-4-iodo-1H-imidazole (2b) and 1-(2-deoxy-β-D-ribofuranosyl)-5-iodo-1H-
imidazole (3b): Starting from 1b (1.0 g, 5.20 mmol) and thymidine (5.0 g, 20.0 mmol) in the presence of
NDT (62.5 mg) at 20°C for 20 h (78% conversion). 2b: 709 mg (45% yield); tR = 11.35 min (column 1, G1);
Analytical and spectral data are in accordance with previously published data;34 HRMS (ESI-TOF) m/z calcd
1
for [C8H11IN2O3+H]: 310.9893, found: 310.9885. 3b: 445 mg (28% yield); tR = 11.68 min (column 1, G1); H
NMR (DMSO-d6):
(m, 2H, H-5’, H-5”), 3.83 (m, 1H, H-4’), 4.31 (m, 1H, H-3’), 4.90 (t, J = 5.7 Hz, 1H, OH-5’), 5.30 (d, J = 4.0 Hz,
1H, OH-3’), 5.91 (dd, J = 6.1, 7.5 Hz, 1H, H-1’), 7.02 (d, J = 1.0 Hz, 1H, H-4), 8.11 (d, J = 1.0 Hz, 1H, H-2); 13
NMR (DMSO-d6): 40.9, 62.0, 71.0, 87.2, 88.3, 136.2, 138.4, 137.2; HRMS (ESI-TOF) m/z calcd for
[C8H11IN2O3+H]: 310.9893, found: 310.9892.
δ 2.27 (ddd, J = 3.3, 6.0, 13.3 Hz, 1H, H-2’), 2.41 (ddd, J = 6.0, 7.3, 13.3 Hz, 1H, H-2”), 3.51
C
δ
4-Bromo-1-(2-Deoxy-β-D-ribofuranosyl)-1H-imidazole (2c) and 5-bromo-1-(2-deoxy-β-D-ribofuranosyl)-
1H-imidazole (3c): Starting from 1c (340 mg, 2.35 mmol) and thymidine (2.3 g, 9.50 mmol) in the
presence of NDT (30 mg) at 37°C for 2.5 h (86% conversion). 2c: 133 mg (21% yield); tR = 10.36 min
(column 1, G1); 1H NMR (DMSO-d6):
δ 2.22 (ddd, J = 3.3, 6.0, 13.3 Hz, 1H, H-2’), 2.33 (ddd, J = 6.0, 7.3, 13.3
Hz, 1H, H-2”), 3.50 (m, 2H, H-5’, H-5”), 3.81 (m, 1H, H-4’), 4.28 (m, 1H, H-3’), 4.89 (t, J = 5.7 Hz, 1H, OH-5’),
5.24 (d, J = 4.0 Hz 1H, OH-3’), 6.01 (t, J = 6.5 Hz, 1H, H-1’), 7.51 (d, J = 1.6 Hz, 1H, H-5), 7.86 (d, J = 1.6 Hz,
1H, H-2); 13C NMR (DMSO-d6):
δ 41.2, 62.1, 71.1, 86.4, 88.3, 114.8, 117.0, 137.2; HRMS (ESI-TOF) m/z calcd
for [C8H1179BrN2O3+H]: 263.0031, found: 263.0031. [C8H1181BrN2O3+H]: 265.0012, found: 265.0025. 3c: 200
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mg (32% yield); tR = 11.43 min (column 1, G1); 1H NMR (DMSO-d6):
δ 2.28 (ddd, J = 3.4, 6.3, 13.4 Hz, 1H, H-
2’), 2.44 (ddd, J = 5.9, 7.3, 13.4 Hz, 1H, H-2”), 3.50 (m, 2H, H-5’, H-5”), 3.83 (m, 1H, H-4’), 4.32 (m, 1H, H-
3’), 4.95 (br s, 1H, OH-5’), 5.32 (br s, 1H, OH-3’), 5.96 (dd, J = 6.3, 7.1 Hz, 1H, H-1’), 7.01 (d, J = 1.1 Hz, 1H,
H-4), 8.01 (d, J = 1.1 Hz, 1H, H-2); 13C NMR (DMSO-d6):
δ 40.6, 61.9, 70.9, 85.3, 88.3, 102.3, 129.5, 137.2;
HRMS (ESI-TOF) m/z calcd for [C8H1179BrN2O3+H]: 263.0031, found: 263.0031, for [C8H1181BrN2O3+H]:
265.0012, found: 265.0021.
4-Hydroxymethyl-1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazole (2d) and 5-hydroxymethyl-1-(2-deoxy-β-
D-ribofuranosyl)-1H-imidazole (3d): Starting from 1d as chlorhydrate salt (14 mg, 0.10 mmol) and
thymidine (100 mg, 0.4 mmol) in the presence of NDT (1 mg) at 50°C for 2 h (80% conversion). A mixture
of 2d and 3d (10/1): 10.4 mg (47% yield); tR = 6.93 min (column 2, G2); 1H NMR (DMSO-d6):
δ 2.18 (ddd, J =
3.1, 5.9, 13.2 Hz, 1H, H-2’ of 2d), 2.25 (ddd, J = 2.9, 6.1, 13.2 Hz, 0.1H, H-2’ of 3d), 2.35 (ddd, J = 5.9, 7.3,
13.2 Hz, 1H, H-2” of 2d), 2.35 (ddd, J = 5.9, 7.3, 13.2 Hz, 0.1H, H-2” of 3d), 3.48 (m, 2.2H, H-5’ and H-5” of
2d, H-5’ and H-5” of 3d), 3.78 (m, 1.1H, H-4’ of 2d and 3d), 4.28 (m, 1.1H, H-3’ of 2d and 3d), 4.21 (d, J =
5.5 Hz, 2H, CH2 of 2d), 4.45 (d, J = 4.5 Hz, 0.2H, CH2 of 3d), 4.81 (t, J = 5.6 Hz, 1H, CH2OH of 2d), 4.86 (m,
1.1H, OH-5’ of 2d and 3d), 5.08 (t, J = 5.0 Hz, 0.1H, CH2OH of 3d), 5.22 (m, 1.1H, OH-3’ of 2d and 3d), 5.98
(dd, J = 6.0, 6.7 Hz, 1H, H-1’ of 2d), 6.02 (dd, J = 6.0, 7.3 Hz, 0.1H, H-1’ of 3d), 6.78 (s, 0.1H, H-4 of 3d), 7.16
(s, 1H, H-5 of 2d), 7.74 (d, J = 1.3 Hz, 1H, H-2 of 2d), 7.88 (br s, 0.1H, H-2 of 3d); 13C NMR (DMSO-d6):
δ
41.2(2d), 41.3(3d), 51.2 (C5), 53.1 (3d), 58.2 (2d), 62.1 (3d), 62.4 (2d), 71.0 (3d), 71.3 (2d), 84.6 (3d), 85.8
(2d), 87.9 (3d), 88.0 (2d), 114.4 (2d), 127.8 (3d), 131.8 (3d), 136.3 (2d), 143.6 (2d); HRMS (ESI-TOF) m/z
calcd for [C9H14N2O4+H]: 215.1032, found: 215.1031.
1-(2-Deoxy-β-D-ribofuranosyl)-1H-imidazole-4-carboxamide (2e): Starting from 1e (7.9 mg, 0.07 mmol)
and thymidine (70 mg, 0.29 mmol) in the presence of NDT (350 µg) at 50°C for 2 h (97% conversion). 2e:
14.2 mg (85% yield); tR = 8.04 min (column 2, G3); Spectral data in accordance with previously published
data8; HRMS (ESI-TOF) m/z calcd for [C9H13N3O4+Na]: 250.0804, found: 250.0814.
Methyl-1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazole-4-carboxylate (2f) and methyl-1-(2-deoxy-β-D-
ribofuranosyl)-1H-imidazole-5-carboxylate (3f): Starting from 1f (20 mg, 0.16 mmol) and thymidine (150
mg, 0.62 mmol) in the presence of NDT (250 µg) at 37°C for 2 h (51% conversion). 2f: 12.3 mg (29% yield);
tR = 12.28 min (column 2, G3); 1H NMR (DMSO-d6):
δ 2.26 (ddd, J = 3.3, 6.1, 13.4 Hz, 1H, H-2’), 2.37 (ddd, J
= 5.8, 7.1, 13.4 Hz, 1H, H-2”), 3.53 (m, 2H, H-5’, H-5”), 3.74 (s, 3H, CH3), 3.84 (m, 1H, H-4’), 4.31 (m, 1H, H-
3’), 4.94 (t, J = 5.4 Hz, 1H, OH-5’), 5.27 (d, J = 3.8 Hz, 1H, OH-3’), 6.08 (t, J = 6.7 Hz, 1H, H-1’), 7.99 (d, J =
1.4 Hz, 1H, H-5), 8.08 (d, J = 1.4 Hz, 1H, H-2); 13C NMR (DMSO-d6):
δ 41.6, 51.5, 62.0, 71.1, 86.5, 88.4,
124.5, 133.0, 138.0, 163.1; HRMS (ESI-TOF) m/z calcd for [C10H14N2O5+Na]: 265.0800, found: 265.0802. 3f:
1
8.1 mg (19% yield); tR = 16.65 min (column 2, G3); H NMR (DMSO-d6):
δ 2.23 (dt, J = 6.1, 13.2 Hz, 1H, H-
2’), 2.34 (ddd, J = 4.5, 6.2, 13.2 Hz, 1H, H-2”), 3.58 (m, 2H, H-5’, H-5”), 3.78 (s, 3H, CH3), 3.84 (m, 1H, H-4’),
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4.27 (m, 1H, H-3’), 4.98 (br s, 1H, OH-5’), 5.27 (br s, 1H, OH-3’), 6.56 (t, J = 6.0 Hz, 1H, H-1’), 7.66 (d, J = 1.0
Hz, 1H, H-4), 8.34 (d, J = 1.0 Hz, 1H, H-2); 13C NMR (DMSO-d6):
42.4, 52.0, 61.4, 70.0, 86.4, 88.2, 121.8,
δ
137.9, 140.5, 160.4; HRMS (ESI-TOF) m/z calcd for [C10H14N2O5+Na]: 265.0800, found: 265.0806.
Ethyl-1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazole-4-carboxylate (2g) and ethyl-1-(2-deoxy-β-D-
ribofuranosyl)-1H-imidazole-5-carboxylate (3g): Starting from 1g (100 mg, 0.8 mmol) and thymidine (650
mg, 2.6 mmol) in the presence of NDT (750 µg) at 37°C for 2 h (38% conversion). 2g: 33.8 mg (18% yield);
tR = 9.25 min (column 1, G4); Spectral data in accordance with previously published data8; HRMS (ESI-TOF)
m/z calcd for [C11H16N2O5+Na]: 279.0957, found: 279.0949. 3g: 3.6 mg (4% yield); tR = 14.27 min (column 1,
G4); 1H NMR (DMSO-d6):
δ 1.29 (t, J = 7.4, 3H, CH3), 2.21 (dt, J = 6.0, 13.2 Hz, 1H, H-2’), 2.36 (ddd, J = 4,5,
6.0, 13.2 Hz, 1H, H-2”), 3.59 (m, 2H, H-5’, H-5”), 3.87 (m, 1H, H-4’), 4.26 (m, 3H, CH2, H-3’), 4.99 (m, 1H,
OH-5’), 5.25 (br s, 1H, OH-3’), 6.56 (t, J = 6.1 Hz, 1H, H-1’), 7.65 (d, J = 0.9 Hz, 1H, H-4), 8.33 (br s, 1H, H-2);
13C NMR (DMSO-d6):
δ 14.6, 42.6, 60.8, 61.4 70.0, 86.4, 88.1, 122.1, 137.8, 140.4, 160.0; HRMS (ESI-TOF)
m/z calcd for [C11H16N2O5+Na]: 279.0957, found: 279.0955.
1-(2-Deoxy-β-D-ribofuranosyl)-1H-imidazole-4-carbaldehyde (2h) and 1-(2-deoxy-β-D-ribofuranosyl)-1H-
imidazole-5-carbaldehyde (3h): Starting from 1h (9.6 mg, 0.1 mmol) and thymidine (116 mg, 0.48 mmol)
in the presence of NDT (350 µg) at 50°C for 2 h (65% conversion). 2h: 7.2 mg (31% yield); tR = 8.85 min
(column 2, G2); 1H NMR (DMSO-d6):
δ 2.30 (ddd, J = 3.5, 6.0, 13.2 Hz, 1H, H-2’), 2.38 (ddd, J = 5.8, 7.2, 13.2
Hz, 1H, H-2”), 3.53 (m, 2H, H-5’, H-5”), 3.85 (m, 1H, H-4’), 4.32 (m, 1H, H-3’), 4.95 (br s, 1H, OH-5’), 5.30
(br s, 1H, OH-3’), 6.12 (t, J = 6.8 Hz, 1H, H-1’), 8.09 (d, 1H, H-5), 8.24 (s, 1H, H-2), 9.73 (s, 1H, CHO); 13C
NMR (DMSO-d6):
δ
41.6, 62.0, 71.1, 86.7, 88.5, 126.0, 139.0, 142.2, 185.8; HRMS (ESI-TOF) m/z calcd for
1
[C9H12N2O4+Na]: 235.0695, found: 235.0693. 3h: 6.0 mg (26% yield); tR = 13.37 min (column 2, G2); H
NMR (DMSO-d6):
(m, 1H, H-3’), 5.00 (br s, 1H, OH-5’), 5.27 (br s, 1H, OH-3’), 6.53 (t, J = 6.2 Hz, 1H, H-1’), 7.93 (s, 1H, H-4),
8.44 (s, 1H, H-2), 9.73 (s, 1H, CHO); 13C NMR (DMSO-d6):
42.3, 61.4, 70.1, 86.6, 88.4, 130.9, 141.8, 144.4,
δ 2.22 (m, 1H, H-2’), 2.37 (m, 1H, H-2”), 3.57 (m, 2H, H-5’, H-5”), 3.86 (m, 1H, H-4’), 4.28
δ
180.5; HRMS (ESI-TOF) m/z calcd for [C9H12N2O4+Na]: 235.0695, found: 235.0693.
1-(2-Deoxy-β-D-ribofuranosyl)-1H-imidazole-4-carbonitrile (2i) and 1-(2-deoxy-β-D-ribofuranosyl)-1H-
imidazole-5-carbonitrile (3i): Starting from 1i (11 mg, 0.12 mmol) and thymidine (115 mg, 0.47 mmol) in
the presence of NDT (350 µg) at 50°C for 2 h (67% conversion). 2i: 13.7 mg (56% yield); tR = 14.95 min
(column 2, G5); 1H NMR (DMSO-d6):
H-4’), 4.31 (m, 1H, H-3’), 6.10 (t, J = 6.3 Hz, 1H, H-1’), 8.12 (d, J = 1.1 Hz, 1H, H-5), 8.31 (d, J = 1.1 Hz, 1H, H-
2); 13C NMR (DMSO-d6):
41.5, 61.9, 70.9, 86.8, 88.6, 112.9, 115.9, 127.9, 139.0; HRMS (ESI-TOF) m/z
calcd for [ C9H11 N3O3+H]: 210.0879, found: 210.0885. 3i: 0.9 mg (4% yield); tR = 16.48 min (column 2, G5);
1H NMR (DMSO-d6):
2.36 (ddd, J = 3.8, 6.3, 13.5 Hz, 1H, H-2’), 2.51 (m, 1H, H-2”), 3.52 (m, 2H, H-5’, H-
δ 2.26-2.41 (m, 2H, H-2’, H-2”), 3.52 (m, 2H, H-5’, H-5”), 3.84 (m, 1H,
δ
δ
5”), 3.84 (m, 1H, H-4’), 4.31 (m, 1H, H-3’), 4.89 (br s, 1H, OH-5’), 5.37 (br s, 1H, OH-3’), 6.12 (t, J = 6.3 Hz,
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1H, H-1’), 7.89 (d, J = 1.1 Hz, 1H, H-4), 8.31 (d, J = 1.1 Hz, 1H, H-2); 13C NMR (DMSO-d6):
δ
40.5, 61.7, 70.8,
86.2, 88.7, 103.3, 111.9, 140.6, 141.2; HRMS (ESI-TOF) m/z calcd for [C9H11 N3O3+H]: 210.0879, found:
210.0885.
Methyl 2-[1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazol-4-yl]acetate (2j) and methyl 2-[1-(2-deoxy-D-
ribofuranosyl)-1H-imidazol-5-yl]acetate (3j): Starting from 1j (7 mg, 0.05 mmol) and thymidine (48 mg,
0.22 mmol) in the presence of NDT (315 µg) at 50°C for 4 h (84% conversion). 2j: 5.9 mg (42% yield); tR =
1
12.95 min (column 2, G3); H NMR (DMSO-d6):
δ 2.20 (ddd, J = 3.2, 6.3, 13.4 Hz, 1H, H-2’), 2.31 (ddd, J =
5.7, 7.5, 13.4 Hz, 1H, H-2”), 3.49 (m, 2H, H-5’, H-5”), 3.53 (s, 2H, CH2), 3.60 (s, 3H, CH3), 3.79 (m, 1H, H-4’),
4.28 (m, 1H, H-3’), 4.88 (br s, 1H, OH-5’), 5.23 (br s, 1H, OH-3’), 5.98 (dd, J = 6.1, 7.4 Hz, 1H, H-1’), 7.21 (s,
1H, H-5), 7.80 (d, J = 1.3 Hz, 1H, H-2); 13C NMR (DMSO-d6):
134.9, 136.4, 171.6; HRMS (ESI-TOF) m/z calcd for [C11H16N2O5+Na]: 279.0957, found: 279.0955. 3j: 5.9 mg
(42% yield); tR = 13.82 min (column 2, G3); 1H NMR (DMSO-d6):
2.21 (ddd, J = 3.3, 6.0, 13.4 Hz, 1H, H-2’-
δ 34.3, 41.2, 52.0, 62.3, 71.2, 86.0, 88.0, 115.5,
δ
C4), 2.31 (ddd, J = 6.0, 7.4, 13.4 Hz, 1H, H-2”), 3.47 (m, 2H, H-5’, H-5”), 3.63 (s, 3H, CH3), 3.78 (m, 3H, CH2,
H-4’), 4.28 (m, 1H, H-3’), 4.84 (t, J = 5.5 Hz, 1H, OH-5’), 5.23 (d, J = 4.1 Hz, 1H, OH-3’), 5.87 (dd, J = 6.2, 7.4
Hz, 1H, H-1’), 6.77 (br s, 1H, H-4), 7.87 (br s, 1H, H-2); 13C NMR (DMSO-d6): 13C NMR (DMSO-d6):
δ 29.8,
41.1, 52.4, 62.1, 71.0, 84.5, 88.0, 124.6, 128.6, 136.0, 170.7; HRMS (ESI-TOF) m/z calcd for
[C11H16N2O5+Na]: 279.0957, found: 279.0951.
1-(2-Deoxy-β-D-ribofuranosyl)-1H-imidazole-4-acrylic acid (2l) and 1-(2-deoxy-β-D-ribofuranosyl)-1H-
imidazole-5-acrylic acid (3l): Starting from 1l (16 mg, 0.11 mmol) and thymidine (112 mg, 0.46 mmol) in
the presence of NDT (100 µg) at 50°C overnight (89% conversion). A mixture of 2l and 3l (10/1.5): 25.1 mg
(85% yield); tR = 8.21 min (column 2, G5); 1H NMR (DMSO-d6):
δ 2.25 (ddd, J = 3.4, 6.2, 13.1 Hz, 1.15H, H-2’
2l and 3l), 2.34 (ddd, J = 5.9, 7.1, 13.1 Hz, 1.15H, H-2” 2l and 3l), 3.50 (m, 2.3H, H-5’ and H-5” 2l and 3l),
3.81 (m, 1H, H-4’ 2l), 3.85 (m, 0.15H, H-4’ 3l), 4.30 (m, 1.15H, H-3’ 2l and 3l), 6.03 (t, J = 6.0 Hz, 1H, H-1’
2l), 6.11 (t, J = 6.2 Hz, 0.15H, H-1’ 3l), 6.30 (d, J = 15.7 Hz, 1H, =CH 2l), 6.31 (d, J = 16 Hz, 0.15H, =CH 3l),
7.40 (d, J = 15.7 Hz, 1H, =CH 2l), 7.50 (d, J = 16 Hz, 0.15H, =CH 3l), 7.57 (s, 0.15H, H4 3l), 7.74 (d, J = 0.6 Hz,
1H, H5 2l), 7.94 (br s, 1H, H2 2l), 8.08 (s, 0.15H, H2 3l); 13C NMR (DMSO-d6):
δ 40.3 (3l), 41.2 (2l), 62.0 (3l),
62.2(2l), 71.0 (3l), 71.1(2l), 84.4 (3l), 86.1 (2l), 88.2 (C4), 88.3 (3l), 116.2 (2l), 118.2 (3l), 121.0 (2l), 128.4
(3l), 130.0 (3l), 132.2 (3l), 137.0 (2l), 138.0 (2l), 138.6 (2l and 3l), 168.1 (3l), 168.4 (2l); HRMS (ESI-TOF)
m/z calcd for [C11H14N2O5+H]: 255.0981, found: 255.0985.
5-Amino-1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazole-4-carbonitrile (2p) and 4-amino-1-(2-deoxy-β-D-
ribofuranosyl)-1H-imidazole-5-carbonitrile (3p): Starting from 1p (13 mg, 0.12 mmol) and thymidine (116
mg, 0.48 mmol) in the presence of NDT (1.25 mg) at 50°C for 20 h (100% conversion). 2p: 5.4 mg (20%
yield); tR = 13.31 min (column 1, G1); Spectral data in accordance with previously published data;25 HRMS
(ESI-TOF) m/z calcd for [C9H11N4O3+H]: 225.0988, found: 225.0978. 3p: 16.5 mg (61% yield); tR = 11.24 min
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(column 1, G5); Spectral data in accordance with previously published data;25 HRMS (ESI-TOF) m/z calcd
for [C9H11N4O3+H]: 225.0988, found: 225.0985.
5-Cyano-1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazole-4-carboxamide (2q) and 4-cyano-1-(2-deoxy-β-D-
ribofuranosyl)-1H-imidazole-5-carboxamide (3q): Starting from 1q (17 mg, 0.12 mmol) and thymidine
(118 mg, 0.48 mmol) in the presence of NDT (625 µg) at 50°C for 2 h (51% conversion). 2q: 3.22 mg (9.7%
1
yield); tR = 8.56 min (column 1, G5); H NMR (DMSO-d6):
δ 2.39 (ddd, J = 4.0, 6.3, 10.2 Hz, 1H, H-2’), 2.56
(m, 1H, H-2”), 3.52 (m, 2H, H-5’, H-5”), 3.88 (m, 1H, H-4’), 4.34 (m, 1H, H-3’), 4.92 (br s, 1H, OH-5’), 5.38
(br s, 1H, OH-3’), 6.14 (t, J = 6.3 Hz, 1H, H-1’), 7.59 (br s, 1H, CONH2), 7.71 (br s, 1H, CONH2), 8.34 (s, 1H, H-
2). 13C NMR (DMSO-d6):
δ 40.5, 61.5, 70.6, 86.7, 88.9, 104.7, 111.1, 139.4, 145.8, 161.9. HRMS (ESI-TOF)
m/z calcd for [C10H12N4O4+Na]: 275.0756, found: 275.0753. 3q: 4.25 mg (13% yield); tR = 9.56 min (column
1
1, G1); H NMR (DMSO-d6): 2.35 (m, 2H, H-2’ and H-2”), 3.56 (m, 2H, H-5’, H-5”), 3.85 (m, 1H, H-4’), 4.29
(m, 1H, H-3’), 5.00 (br s, 1H, OH-5’), 5.30 (br s, 1H, OH-3’), 6.29 (t, J = 6.1 Hz, 1H, H-1’), 8.1 (br s, 1H,
CONH2), 8.2 (br s, 1H, CONH2), 8.34 (s, 1H, H-2); 13C NMR (DMSO-d6):
δ 42.1, 61.4, 70.4, 86.7, 88.6, 114.0,
114.9, 134.6, 139.0, 159.7; HRMS (ESI-TOF) m/z calcd for [C10H12N4O4+Na]: 275.0756, found: 275.0750.
General procedure for the Suzuki-Miyaura cross coupling reactions
Procedure A: A 1M Na2CO3 aqueous solution (0.5 mL) purged with argon were introduced into a mixture
purged with argon of 2b or 3b (0.1 mmol), boronic acid (0.16 mmol) and Pd(PPh3)4 (80 mg, 0.005 mmol) in
DMF (1.5 mL). The mixture was heated under microwave irradiation (for temperature and reaction time
see Table 4). When the reaction was complete, the reaction mixture was filtered and products were
isolated from the crude reaction mixture by HPLC on a C18 column followed by lyophilisation.
Procedure B: Under argon, 2b (0.1 mmol), PPh3 (1.46 mg, 0.006 mmol), Na2CO3 (15.9 mg, 0.15 mmol), and
boronic acid 4 (0.15 mmol) were dissolved in water (1 mL). Then Pd(OAc)2 (0.9 mg, 0.004 mmol) was
added to the mixture. The mixture was heated under microwave irradiation for 1 h at 120°C. The product
10.1 was isolated from the crude reaction mixture by HPLC on a C18 column followed by lyophilisation.
Procedure C: A mixture of water–acetonitrile (2:1, 1.2 ml) was added through a septum to an argon-
purged vial containing 2b or 3b (0.1 mmol), boronic acid (0.125 mmol), Pd(OAc)2 (1.12 mg, 0.005 mmol),
TPPS (14.2 mg, 0.025 mmol), Na2CO3 (32 mg, 0.3 mmol). The mixture was stirred under heating or under
microwave irradiation (for temperature and reaction time see Table 4). Products were isolated from the
crude reaction mixture by HPLC on a C18 column followed by lyophilisation.
Analytical and spectral data for compounds 10.1 to 10.5, 10.7 and 11.5 were in accordance with
previously published data.12
1-(2-Deoxy-β-D-ribofuranosyl)-4-phenyl-1H-imidazole (10.1): Following procedure C, 20.4 mg (78%
yield).
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2-Fluoro-3-[1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazol-4-yl]-pyridine (10.2): Following procedure A, 14.8
mg (60% yield).
5-Bromo-3-[1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazol-4-yl]-pyridine (10.3): Following procedure A,
16.3 mg (48% yield).
2-Amino-4-[1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazol-5-yl]-pyrimidine (10.4): Following procedure C,
19.4 mg (70% yield).
2,4-Di-methyl-4-[1-(2-deoxy-D-ribofuranosyl)-1H-imidazol-4-yl]-pyrimidine (10.5): Following procedure
C, 18.4 mg (58% yield).
2,4-Di-tert-butoxy-5-[1-(2-deoxy-D-ribofuranosyl)-1H-imidazol-4-yl]-pyrimidine
(10.6):
Following
procedure A, 31.0 mg (76% yield). Purification by silica gel column, gradient 0% to 12% MeOH in CH2Cl2; tR
1
= 18.79 min (column 1, G6); H NMR (DMSO-d6):
δ 1.57 (s, 9H, tBu), 1.67 (s, 9H, tBu), 2.26 (m, 1H, H-2’),
2.35 (m, 1H, H-2”), 3.51 (m, 2H, H-5’, H-5”), 3.83 (m, 1H, H-4’), 4.33 (m, 1H, H-3’), 4.89 (t, J = 5.1 Hz, 1H,
OH-5’), 5.28 (d, J = 3.6 Hz, 1H, OH-3’), 6.10 (t, J = 6.8 Hz, 1H, H-1’), 7.54 (s, 1H, H-5 Im), 7.93 (s, 1H, H-2 Im),
8.81 (s, 1H, H Pyr); 13C NMR: 28.6, 41,3, 62.3, 71.3, 80.0, 82.6, 86.1, 88.1, 110.6, 116.1, 133.9, 137.0,
155.0, 162.0, 165.5; HRMS (ESI-TOF) m/z calcd for [C20H30N4O5+H]: 407.2294, found: 407.2276.
5-[1-(2-Deoxy-β-D-ribofuranosyl)-1H-imidazol-4-yl]-pyrimidine-2,4-(1H,3H)-dione (10.7): Following
procedure C, 3.5 mg (12% yield).
1-(2-Deoxy-β-D-ribofuranosyl)-5-phenyl-1H-imidazole (11.1): Following procedure C, 18.3 mg (70%
yield); tR = 14.07 min (column 1, G6); 1H NMR (DMSO-d6):
δ 2.22 (ddd, J = 2.8, 5.8, 13.2 Hz, 1H, H-2’), 2.55
(ddd, J = 6.0, 8.1, 13.2 Hz, 1H,H-2”), 3.53 (m, 2H, H-5’, H-5”), 3.81 (m, 1H, H-4’), 4.31 (m, 1H, H-3’), 4.92 (br
s, 1H, OH-5’), 5.23 (br s, 1H, OH-3’), 5.09 (dd, J = 6.0, 7.8 Hz, 1H, H-1’), 7.01 (s, 1H, H-4 Im), 7.41 (m, 1H, H-
4 Ph), 7.48 (m, 4H, H-2, H-3, H-5, H-6 Ph), 8.11 (s, 1H, H-2 Im); 13C NMR (DMSO-d6):
δ 40.2, 54.9, 61.7,
70.8, 83.7, 87.7, 127.3, 127.9, 128.5, 128.8, 129.4, 132.7, 136.3; HRMS (ESI-TOF) m/z calcd for
[C14H16N2O3+H]: 261.1239, found: 261.1247.
2-Fluoro-3-[1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazol-5-yl]-pyridine (11.2): Following procedure A, 16.3
mg (60% yield); tR = 11.56 min (column 1, G6); 1H NMR (DMSO-d6): δ 2.22 (ddd, J = 3.0, 6.0, 13.3 Hz, 1H, H-
2′), 2.45 (m, 1H, H-2″), 3.48 (m, 2H, H-5′, H-5″), 3.76 (m, 1H, H-4′), 4.28 (m, 1H, H-3′), 4.90 (br s, 1H, OH-
5′), 5.23 (br s, 1H, H-3′), 5.73 (dd, J = 6.1, 7.7 Hz, 1H, H-1′), 7.11 (br s, 1H, H-4 Im), 7.51 (ddd, J = 2.0, 4.8,
7.1 Hz, 1H, H-5 Pyr), 8.06 (ddd, J = 2.0, 7.4, 9.7 Hz, 1H, H-4 Pyr) 8.18 (d, J = 0.9 Hz, 1H, H-2 Im), 8.32 (ddd, J
= 1.1, 1.9, 4.8 Hz, 1H, H-6 Pyr); 13C NMR (DMSO-d6): δ 41.1, 62.1, 71.2, 84.7, 88.2, 112.7 (d, J = 31 Hz),
122.9 (d, J = 4 Hz), 124.5 (d, J = 4 Hz), 130.3, 137.6, 143.1 (d, J = 3 Hz), 148.0 (d, J = 14 Hz), 160.2 (d, J = 236
Hz); HRMS (ESI-TOF) m/z calcd for [C13H14FN3O3+H]: 280.1097, found: 280.1094.
5-Bromo-3-[1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazol-5-yl]-pyridine (11.3): Following procedure A,
14.1 mg (40% yield); tR = 11.72 min (column 1, G6); 1H NMR (DMSO-d6): δ 2.26 (ddd, J = 3.4, 6.2, 13.4 Hz,
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1H, H-2′), 2.61 (ddd, J = 6.2, 7.6, 13.4 Hz, 1H, H-2″), 3.49 (m, 2H, H-5′, H-5″), 3.82 (m, 1H, H-4′), 4.30 (m,
1H, H-3′), 4.89 (t, J = 5.5 Hz, 1H, OH-5′), 5.27 (d, J = 4.5 Hz, 1H, OH-3′), 5.89 (dd, J = 6.2, 7.4 Hz, 1H, H-1′),
7.23 (d, J = 0.9 Hz, 1H, H-4 Im), 8.18 (br s, 1H, H-2 Im), 8.19 (t, J = 2.0 Hz, 1H, H-6 Pyr), 8.70 (d, J = 1.9 Hz,
1H, H-4 Pyr), 8.73 (d, J = 1.9 Hz, 1H, H-2 Pyr); 13C NMR (DMSO-d6): δ 40.2, 62.1, 71.2, 84.3, 88.4, 120.7,
128.0, 128.5, 130.2, 138.0, 147.7, 149.8; HRMS (ESI-TOF) m/z calcd for [C13H1479BrN3O3+H]: 340.0297,
found: 340.0298, for [C13H1481BrN3O3+H]: 342.0278, found: 342.0284.
2-Amino-5-[1-(2-deoxy-β-D-ribofuranosyl)-1H-imidazol-5-yl]-pyrimidine (11.4): Following procedure C,
1
16.8 mg (60% yield); tR = 7.20 min (column 1, G1); H NMR (DMSO-d6):
δ 2.22 (ddd, J = 3.1, 6.1, 13.3 Hz,
1H, H-2’), 2.54 (ddd, J = 6.1, 8.0, 13.4 Hz, 1H, H-2”), 3.49 (m, 2H, H-5’, H-5”), 3.79 (m, 1H, H-4’), 4.28 (m,
1H, H-3’), 4.89 (br s, 1H, OH-5’), 5.25 (d, J = 3.7 Hz, 1H, OH-3’), 5.77 (dd, J = 6.1, 8.0 Hz, 1H, H-1’), 6.89 (s,
2H, NH2), 6.99 (d, J = 0.9 Hz, 1H, H-4 Im), 8.09 (d, J = 0.9 Hz, 1H, H-2 Im), 8.29 (s, 2H, H-4, H-6 Pyr); 13C
NMR (DMSO-d6):
δ 40.3, 62.2, 71.2, 84.1, 88.2, 112.6, 127.8, 128.2, 136.6, 158.2, 163.5; HRMS (ESI-TOF)
m/z calcd for [C12H15N5O3+H]: 278.1253, found: 278.1257.
2,4-Di-methyl-5-[1-(2-deoxy-D-ribofuranosyl)-1H-imidazol-4-yl]-pyrimidine (11.5): Following procedure
A, 14.8 mg (46% yield).
2,4-Di-tert-butoxy-5-[1-(2-deoxy-D-ribofuranosyl)-1H-imidazol-5-yl]-pyrimidine
(11.6):
Following
procedure A from 0.05 mmol of 3b, 16.2 mg (80% yield). Purification by silica gel column, gradient 0% to
1
12% MeOH in CH2Cl2; tR = 17.28 min (column 1, G6); H NMR (DMSO-d6):
δ 1.55 (s, 9H, tBu), 1.60 (s, 9H,
tBu), 2.18 (ddd, J = 2.6, 5.7, 13.1 Hz, 1H, H-2’), 2.43 (ddd, J = 5.8, 8.1, 13.4 Hz, 1H, H-2”), 3.47 (m, 2H, H-5’,
H-5”), 3.74 (m, 1H, H-4’), 4.28 (m, 1H, H-3’), 4.89 (t, J = 5.0 Hz, 1H, OH-5’), 5.22 (d, J = 4 Hz, 1H, OH-3’),
5.66 (dd, J = 5.8, 8.1 Hz, 1H, H-1’), 6.91 (s, 1H, H-4 Im), 8.08 (s, 1H, H-2 Im), 8.16 (s, 1H, H Pyr); 13C NMR
(DMSO-d6):
δ 28.4, 28.5, 41.1, 62.2, 71.3, 80.8, 83.0, 84.4, 87.9, 105.9, 125.3, 128.9, 136.8, 159.5, 164.1,
167.7; HRMS (ESI-TOF) m/z calcd for [C20H30N4O5+H]: 407.2294, found: 407.2309.
On-pot two-step procedure
A mixture of 4-iodoimidazole (20 mg, 0. 1 mmol), thymidine (100 mg, 0.04 mmol) and NDT (2 mg) in 10
mM citrate buffer (pH 6.0, 8 mL) was heated at 50°C over 2 h 30 (80% conversion by LC-MS monitoring).
Enzyme denaturation was performed by heating for 1 min at 100 °C under MW irradiation. The crude
reaction mixture was then added to through a septum to an argon-purged vial containing boronic acid
(0.125 mmol), Pd(OAc)2 (1.12 mg, 0.005 mmol), TPPS (14.2 mg, 0.025 mmol) and Na2CO3 (32 mg, 0.3
mmol) in acetonitrile (4 mL). The mixture was heated for 5 min at 120°C under MW irradiation. Products
were isolated from the crude reaction mixture by reverse phase HPLC followed by lyophilisation, affording
10.1 (8.4 mg, 32%) and 11.1 (6.2 mg, 24%).
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Acknowledgements
The authors gratefully acknowledge financial support from the Institut Pasteur and CNRS.
Notes and references
# NMR spectra of new compounds are given as supplementary information.
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