Promiscuous enzyme-catalyzed regioselective Michael addition of purine derivatives to α,β-unsaturated carbonyl compounds in organic solvent

Article abstract of DOI:10.1016/j.tet.2009.01.056

Regioselective Michael addition of purine derivatives to α,β-unsaturated carbonyl compounds could be catalyzed by d-aminoacylase amano (DA) in DMSO. The influence of reaction conditions on the Michael addition, including solvent, temperature, and enzyme c

Full text of DOI:10.1016/j.tet.2009.01.056

Tetrahedron 65 (2009) 2531–2536
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Promiscuous enzyme-catalyzed regioselective Michael addition of purine
derivatives to
a,
b-unsaturated carbonyl compounds in organic solvent
*
Jun-Liang Wang, Jian-Ming Xu, Qi Wu, De-Shui Lv, Xian-Fu Lin
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 March 2008
Received in revised form 10 January 2009
Accepted 13 January 2009
Available online 20 January 2009
Regioselective Michael addition of purine derivatives to a,b-unsaturated carbonyl compounds could be
catalyzed by
addition, including solvent, temperature, and enzyme concentration was systematically investigated.
Then we extended this methodology to six structurally diverse purine derivatives and a variety of
D-aminoacylase amano (DA) in DMSO. The influence of reaction conditions on the Michael
a,b-
unsaturated carbonyl compounds. 21 Michael adducts were selectively synthesized in moderate to high
yields. It is the first report on enzyme-catalyzed Michael addition for the preparation of purine
derivatives.
Keywords:
D
-Aminoacylase
Ó 2009 Elsevier Ltd. All rights reserved.
Michael addition
Purine derivatives
a,b-Unsaturated compounds
1. Introduction
utilization of natural materials for catalytic application is a very
charming strategy.
Synthesis of nucleoside derivatives has gained more attention
in the past decades because of their antiviral and antitumor abil-
ity.1 However, rare study has been focused on purine derivatives
due to their low reactivity and low solubility. Moreover, regiose-
lective reaction is difficult to undergo when purine derivatives are
used as the substrates since the alkylation of 2-aminopurines
usually gives rise to mixtures of N-9 and N-7 materials.2 Hence,
control of the regioselectivity is crucial during the synthetic pro-
cedure. Geen3 reported Michael addition with 2-aminopurines
catalyzed by potassium carbonate in N,N-dimethylformamide at
room temperature, by extending the reaction time and increasing
the quantity of Michael acceptors to obtain the exclusive N-9
alkylated products. In another way, Stephane Guillarme4 adopted
protected or deaza natural bases as the Michael donor for the
synthesis of several acyclic nucleosides. Recently, Microwave irra-
diation technique was introduced to the Michael addition of purine
derivatives, exclusive N-9 alkylated products could be obtained in
a short time.5 However, these methods always need special
equipment or unrecycling catalysts. Recently, the emphasis of sci-
ence and technology has diverted into environmentally friendly
and sustainable resources and processes. Therefore, direct
Enzymes are efficient catalysts in organic and bioorganic
synthesis. Besides the natural catalytic ability to catalyze
a primary reaction, many enzymes can also catalyze secondary
reaction at an active site, which is termed ‘catalytic promiscuity’.6
For example, lipase can catalyze the formation of C–C,7a C–N,7b
C–O,7c and C–S7d through Michael addition, arylmalonate decar-
boxylase can catalyze aldol additions,7e racemase can catalyze
PLP-dependent aldol additions.7f Recently, enzyme promiscuity
was also discovered at alternate-site besides the known natural
catalytically active site.8
In our former works, we have demonstrated the promiscuous
enzyme-catalyzed Michael addition of imidazole and pyr-
imidine.9a–e Taking the low reactivity of purine derivatives and
their antiviral and antitumor ability, and the highly regioselectivity
of enzyme as catalyst into account, it is very interesting and
meaningful for us to exploit the catalytic capability of enzyme on
the synthesis of purine derivatives .In this paper, we reported the
enzyme-catalyzed Michael addition of purine derivatives (Fig. 1) to
a,b-unsaturated carbonyl compound catalyzed by D-aminoacylase.
Then the reaction was optimized by investigating the influence of
reaction conditions on the Michael addition, including solvent,
temperature, and enzyme concentration. This methodology was
extended to a variety of structurally diverse purine derivatives and
a,b-unsaturated carbonyl compound. 21 products were obtained in
moderate to high yield under the catalysis of
DMSO at 50 ꢀC successfully.
D-aminoacylase in
* Corresponding author. Fax: þ86 571 87952618.
E-mail address: llc123@zju.edu.cn (X.-F. Lin).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.01.056

2532
J.-L. Wang et al. / Tetrahedron 65 (2009) 2531–2536
Table 2
Screen of reaction conditions of DA-catalyzed Michael addition of 6-benzylamino-
purine 1a to methyl acrylate 2aa
NHCH2Ph
N
NH2
Cl
N
N
N
N
N
N
N
N
Entry
Amount of catalyst (mg)
Solvent
Temperature (ꢀC)
Yieldb (%)
N
N
N
H
N
H
1
2
3
4
5
6
7
8
5
5
5
5
5
DMSO
DMF
50
50
50
50
50
50
50
50
50
40
25
48
14
<1
4
<1
<1
44
44
42
23
8
N
H
1a
1c
1b
Dioxane
Pyridine
Chloroform
Cyclohexane
DMSO
DMSO
DMSO
DMSO
DMSO
NO2
N
5
N
S
H
N
O
OH
N
10
15
20
5
N
HN
N
9
10
11
N
N
H
N
H
N
N
5
1d
1e
1f
a
Experimental conditions: 0.5 M 6-benzylaminopurine, 2.5 M methyl acrylate,
5 mg enzyme, 1 ml solvent, 50 ꢀC, 24 h.
Figure 1. The structure of purine derivatives.
b
All yields were detected by HPLC.
Michael addition reaction could only be performed in some polar
solvents such as DMSO and DMF (entries 1 and 2, Table 2). In other
solvents including dioxane, pyridine, chloroform, and cyclohexane,
only trace product was detected, which may be ascribed to the low
solubility of 6-benzylaminopurine in those solvents (entries 3–6,
Table 2). Then the influence of enzyme concentration on the re-
action was examined. As shown in Table 2, when the enzyme
concentration increased from 5 mg/ml to 20 mg/ml (entries 1 and
7–9, Table 2), the yield decreased lightly. The optimal enzyme
concentration was 5 mg/ml. Next, the influence of reaction tem-
perature on the enzymatic Michael addition reaction was also
considered. It was found that the yield decreased with the decrease
of temperature (entries 1, 10, and 11, Table 2). The yield was only 8%
at 25 ꢀC. The highest yield was obtained at 50 ꢀC. All these studies
promoted us to use DMSO as the solvent in the presence of 5 mg/ml
enzyme concentration at 50 ꢀC to probe the generality of the con-
jugated addition processes.
Having the optimal conditions in hand, we applied this method
to other Michael acceptors and the results were shown in Table 3.
Examination of the results of the different acrylate reveals that the
chain length of the ester plays a minimal role in governing the
reactivity of the conjugate addition. As the carbon chain of alcohol
moiety increased, slight decrease in yield was observed (entries
1–3, Table 3). The reactions of 6-benzylaminopurine (1a) with
methyl vinyl ketone or acrylonitrile could also provide good yields
(entries 4 and 5, Table 3). Compared with acrylate and acrylonitrile,
methyl vinyl ketone presented the highest activity. Then a series of
2. Results and discussion
In order to demonstrate the specific catalytic effect of catalysts,
we performed some control experiments. The reaction of 6-benzyl-
aminopurine 1a with methyl acrylate 2a in the absence of enzyme
led to low yield adduct (8%) in 72 h. In contrast, the reaction in the
presence of DA and AA is up to 50.7-fold and 28-fold faster (entries 2
and 6, Table 1). Besides, the initial reaction rate is practically
proportional to the enzyme amount, also suggesting the catalytic
effect of the enzyme (entries 2 and 3, Table 1). When the reactants
were incubated with denatured D-aminoacylase (pre-treated with
urea at 100 ꢀC for 24 h) the rate is practically equal to the bovine
serum albumin (BSA), ruling out the possibility that the polymeric
support or the similar amino acid distribution on the protein surface
has promoted the process. All these results suggest that the specific
active sites and the tertiary structure of
D-aminoacylase are
responsible for the Michael addition reaction. Three widely used
hydrolases, Candida antarctica lipase B (CAL-B), Lipase from porcine
pancreas (PPL), and alkaline protease from Bacillus subtilis (PA), can
only accelerate the reaction by 2.1, 7.3, and 10.7-fold, respectively.
Among all the selected catalysts, D-aminoacylase (DA) was the best
catalyst for this Michael addition. Thus, DA was selected for the
further study.
Then the reaction conditions were optimized by using the Mi-
chael addition of 6-benzylaminopurine 1a to methyl acrylate 2a as
model reaction. As indicated in Table 2, the enzyme-catalyzed
sterically hindered Michael acceptors with either
a-methyl or
Table 1
b-methyl group were studied under the same conditions (entries
Initial rates (Vo) of the Michael addition of 6-benzylaminopurine 1a to methyl ac-
rylate 2a in the presence of different catalysts
Table 3
NHCH2Ph
NHCH2Ph
Michael addition of 6-benzylaminopurine to various Michael acceptors
N
N
N
10mg catalyst
N
O
NHCH2Ph
NHCH2Ph
O
N
2ml DMSO, 50 °C
N
N
H
R2
N
N
O
2a
O
N
N
5mg DA
N
R1
3a
1a
R3
N
1ml DMSO, 50 °C ,
72 h
N
N
H
R3
R2
Yielda (%)
N
Yielda (%)
V0 (mM hꢁ1
)
Vr
b
Entry
Catalyst
Amount (mg)
R
1
1a
2a-i
3a-i
1
2
3
4
5
6
7
8
9
d
d
10
5
10
10
10
10
10
10
8
92
88
10
11
81
15
37
58
0.15
7.6
3.9
0.22
0.25
4.2
0.31
1.1
1.0
50.7
26.0
1.5
1.7
28.0
2.1
D
-Aminoacylase
-Aminoacylase
Entry
Time (h)
R1
R2
R3
D
DA denaturedc
1
2
3
4
5
6
7
8
9
72
72
72
48
72
72
72
48
48
H
H
H
H
H
H
CH3
CH3
H
H
H
H
H
H
CH3
H
H
CH3
COOCH3
(3a) 92
(3b) 81
(3c) 78
(3d) 90
(3e) 84
(3f) 42
(3g) 39
(3h) 67
(3i) 62
BSA
COOCH2CH3
COO(CH2)3CH3
COCH3
CN
COOCH3
COOCH3
COOCH]CH2
COOCH]CH2
Acylase ‘amano’
CAL-B
PPL
7.3
10.7
PA
1.6
a
Experimental conditions: 0.2 M 6-benzylaminopurine, 0.4 M methyl acrylate,
10 mg enzyme, 2 ml DMSO, 50 ꢀC, 72 h. All yields were detected by HPLC.
b
Relative initial rate to the reaction in absence of enzyme.
Enzyme predenatured with urea at 100 ꢀC for 24 h.
c
a
All yields were detected by HPLC.

J.-L. Wang et al. / Tetrahedron 65 (2009) 2531–2536
2533
Table 4
Michael addition of other purine derivatives to
a
,
b
-unsaturated carbonyl compound promoted by DAa
Entry
Nucleophile
Acceptor
Time (h)
Product
NH2
Yieldb (%)
O
N
N
N
1
1b
72
(3j) 59
O
O
O
N
N
O
O
Cl
O
N
N
2
3
1c
1c
72
48
(3k) 80
(3l) 94
O
N
Cl
N
N
O
O
N
N
NO2
N
O
N
S
4
1d
48
(3m) 95
N
O
N
O
N
N
O
NO2
N
N
S
5
1d
48
(3n) 91
N
O
N
O
N
N
NO2
N
N
S
N
6
1d
72
(3o) 81
CN
O
N
N
N
CN
O
O
O
O
N
7
8
1e
1e
48
48
(3p) 97
(3q) 98
N
O
O
O
N
N
O
O
N
N
N
O
N
N
N
O
NC
O
N
N
N
9
1e
1f
1f
48
48
48
(3r) 77
(3s) 98
(3t) 98
CN
O
N
O
CN
N
N
O
10
11
O
O
O
N
O
O
O
N
N
O
N
O
NC
N
N
N
12
1f
48
(3u) 70
CN
N
CN
a
Reaction conditions: purine derivatives (0.2 mmol),
All yields were detected by HPLC.
a,b
-carbonyl compound (1 mmol) in 1 mL DMSO at 50 ꢀC.
b

2534
J.-L. Wang et al. / Tetrahedron 65 (2009) 2531–2536
6–9, Table 3). All the reactions reached the equilibrium after 48 or
72 h and only provide moderate yield. Compared with substituted
methyl acrylates, the substituted vinyl acrylates showed good re-
activity and higher yields were observed (entries 6 and 9; 7 and 8,
Table 3). It is worthwhile to mention that all of the products were
alkylated at the N-9 position according to the 1H NMR and 13C NMR
spectral analysis. HMBC spectra also ascertained that the site of
alkylation was N-9 rather than N-7 or another position.
analytical grade and were dried by storing over activated 3 Å
molecular sieves for 24 h prior to use. All other reagents were used
as-received. 1H and 13C NMR spectra were recorded on Bruker
AVANCE DMX-500 spectrometer at 500 MHz and 125 MHz in CDCl3
or DMSO-d6, respectively. Chemical shifts are reported in parts per
million (d), relative to the internal standard of tetramethylsilane
(TMS). IR spectra were measured with a Nicolet Nexus FTIR 670
spectrophotometer. Mass spectrometry data were obtained on
Brucker Esquire-LC for electro-spray (MS-ES) measurements (sol-
vents: methanol; positive mode). Melting points were determined
using XT-4 apparatus and were not corrected.
The structure of the purine derivatives also affected the results
of the Michael addition reaction. Six kinds of structurally diverse
purine derivatives underwent Michael addition with methyl acry-
late, methyl vinyl ketone or acrylonitrile favorably and the results
were summarized in Table 4. As shown in Table 4, the Michael
addition of adenine with methyl acrylate only provided the corre-
sponding adduct in 59% yield after 72 h (entry 1, Table 4). The re-
activity of adenine was relatively lower than that of
6-benzylaminopurine, probably due to the poor solubility of ade-
nine in DMSO. Then the Michael additions of 6-chloropurine, aza-
thiopurine with methyl acrylate, methyl vinyl ketone, and
acrylonitrile were examined (entries 2–6, Table 4). It was found that
all the reactions could generate the corresponding mono Michael
adducts highly selectively at the N-9 position in good yields after
48–72 h. When allopurinol and 6-hydroxypurine were tested in
Michael addition with methyl acrylate, methyl vinyl ketone, and
acrylonitrile, bis-adducts were favored in good yields without
mono adduct products unexpectedly (entries 7–12, Table 4).
According to the 1H NMR and 13C NMR spectral analysis, both the
N-2 and N-9 positions of allopurinol or 6-hydroxypurine were
alkylated. HMBC spectra also ascertained that the sites of alkylation
were N-2 and N-9 rather than N-2 and N-7 or another position. The
Michael addition of allopurinol formed bis-adducts because allo-
purinol has two active NH functionalities. A similar explanation
could be elucidated for 6-hydroxypurine due to that 6-hydroxy-
purine can be converted to the similar structure of allopurinol
in equilibrium.
4.2. Typical procedure
Purine derivatives (0.5 mmol) and
a,b-unsaturated carbonyl
compounds (2.5 mmol) were added to a 10 mL conical flask
containing 1 ml DMSO and the mixture was shaken at ambient
temperature for a period of time. The reaction was terminated by
filtering off the enzyme and the DMSO was evaporated in reduced
pressure. The product was isolated by silica gel column chromato-
graphy with an eluent consisting of petrol ether/ethyl acetate
(1/1 v/v).
4.2.1. 3-(6-Benzylamino-purin-1-yl)-propionic acid methyl
ester (3a)
Yellow solid, mp 124–125 ꢀC; IR (KBr): 3366, 3097, 1724,
1613 cmꢁ1
;
1H NMR (CDCl3,
d
, ppm): 8.52 (s, 1H), 7.87 (s, 1H),
7.41–7.27 (m, 5H), 6.45 (s,1H), 4.85 (s, 2H), 4.59 (t, 2H, J 6.0 Hz), 3.63
(s, 3H), 2.92 (t, 2H, J 6.0 Hz); 13C NMR (CDCl3,
, ppm): 171.6, 155.0,
d
153.4, 140.7, 138.8, 128.9, 128.0, 127.7, 127.4, 121.0, 52.3, 42.0, 39.6,
34.2; ESI-MS (m/z): 312 (MþH).
4.2.2. 3-(6-Benzylamino-purin-9-yl)-propionic acid ethyl ester (3b)
White solid, mp 67–68 ꢀC; IR (KBr): 3264, 3059, 1729,
1614 cmꢁ1 1H NMR (CDCl3,
; d, ppm): 8.39 (s, 1H), 7.74 (s, 1H),
7.35–7.25 (m, 5H), 6.54 (s, 1H), 4.86 (s, 2H), 4.45 (t, 2H, J 6.0 Hz),
3. Conclusion
4.13–4.08 (m, 2H), 2.89 (t, 2H, J 6.0 Hz), 1.20 (t, 3H, J 6.8 Hz); 13C
NMR (CDCl3, d, ppm): 170.8, 154.6, 153.0, 140.5, 138.5, 128.6, 128.5,
An efficient and highly regioselective enzymatic Michael addi-
tion for the preparation of purine derivatives was developed. The
influence of reaction conditions on the Michael addition, including
solvent, temperature, and enzyme concentration was investigated.
Moderate to high yields were obtained when 5 mg/ml DA was used
to catalyze the Michael addition in DMSO at 50 ꢀC. This strategy
works with a broad range of structurally diverse purine derivatives.
Further study on the promiscuity of DA was under way.
127.6, 127.4, 119.6, 61.0, 39.3, 34.2, 14.0; ESI-MS (m/z): 326 (MþH).
4.2.3. 3-(6-Benzylamino-purin-9-yl)-propionic acid butyl esterꢁ(13c)
;
1H
Yellow solid, mp 65–66 ꢀC; IR (KBr): 3265, 1733, 1616 cm
NMR (CDCl3, d, ppm): 8.39 (s, 1H), 7.75 (s, 1H), 7.37–7.25 (m, 5H),
6.45 (s, 1H), 4.86 (s, 2H), 4.45 (t, 2H, J 6.4 Hz), 4.13 (t, 2H, J 6.8 Hz),
2.90 (t, 2H, J 6.4 Hz), 1.58–1.51 (m, 2H), 1.32–1.24 (m, 2H), 0.88
(t, 3H, J 7.2 Hz); 13C NMR (CDCl3,
d, ppm): 170.9, 154.6, 153.0, 140.5,
138.4, 130.4, 128.6, 127.7,127.4, 119.6, 65.0, 39.4, 34.1, 30.6, 30.4, 19.1,
4. Experimental
13.6; ESI-MS (m/z): 354 (MþH).
4.1. General remarks
4.2.4. 4-(6-Benzylamino-purin-9-yl)-butan-2-one (3d)
White solid, mp 106–107 ꢀC; IR (KBr): 3267, 1717, 1626 cmꢁ1; 1H
Alkaline protease from Bacillus subtilis (10 U/mg, 1 U corre-
sponds to the amount of enzyme, which liberates 1 mmol folin-
NMR (DMSO-d6,
7.35–7.20 (m, 5H), 4.70 (s, 2H), 4.31 (t, 2H, J 6.4 Hz), 3.10 (t, 2H,
J 6.8 Hz), 2.10 (s, 3H); 13C NMR (DMSO-d6,
, ppm): 206.7, 154.7,
152.6, 141.3, 140.6, 128.5, 127.5, 126.9, 122.3, 42.5, 38.4, 30.1; ESI-MS
(m/z): 296 (MþH); HRMS (ESI) m/z calcd for [MþH] C16H18N5O
296.1433, found 296.1441.
d, ppm): 8.31 (s, 1H), 8.25 (s, 1H), 8.19 (s, 1H),
positive amino acids and peptides per minute at pH 7.5 and 37 ꢀC)
was obtained from Wuxi Enzyme Co. Ltd. (Wuxi, PR China). Lipase
immobilized on acrylic resin from Candida antarctica (ꢂ10,000 U/g,
recombinant, expressed in Aspergillus oryzae) and Lipase from
porcin pancreas (PPL) (30–90 U/mg protein, one unit will hydrolyze
d
1.0
from Sigma (Steinheim, Germany).
ichia coli (10,000 U/mg, 1 U is defined as enzyme quantity, which
produces 1 mol of -amino acid per 30 min) and acylase ‘amano’
(AA) from A. oryzae (ꢂ30,000 U/g, 1 U is defined as enzyme
quantity, which produces 1 mol of -amino acid per 30 min) were
purchased from Amano Enzyme Inc (Japan). All solvents were
m
equiv of triacetin in 1 h at pH 7.7 at 37 ꢀC) were purchased
-Aminoacylase from Escher-
4.2.5. 3-(6-Benzylamino-purin-9-yl)-propionitrile (3e)
D
White solid, mp 160–161 ꢀC; IR (KBr): 3279, 3099, 1620 cmꢁ1
;
1H NMR (DMSO-d6,
5H), 6.67 (s, 1H), 4.87 (s, 2H), 4.42 (t, 2H, J 6.8 Hz), 2.99 (t, 2H,
J 6.8 Hz); 13C NMR (DMSO-d6,
, ppm): 154.8, 153.4, 139.2, 138.3,
d, ppm): 8.39 (s, 1H), 7.64 (s, 1H), 7.38–7.34 (m,
m
D
d
m
L
128.6, 127.6, 127.4, 119.8, 116.6, 39.7, 29.7, 18.8; ESI-MS (m/z): 279
(MþH).

J.-L. Wang et al. / Tetrahedron 65 (2009) 2531–2536
2535
4.2.6. 3-(6-Benzylamino-purin-9-yl)-2-methyl-propionic acid
(t, 2H, J 6.8 Hz), 3.75 (s, 3H), 3.66 (s, 3H), 2.96 (t, 2H, J 6.8 Hz); 13C
NMR (CDCl3, , ppm): 171.0, 156.1, 151.7, 150.6, 149.7, 145.0, 137.9,
131.0, 116.7, 52.0, 40.0, 33.4, 33.1; ESI-MS (m/z): 364 (MþH);
HRMS (ESI) m/z calcd for [MþH] C13H14N7O4S 364.0750, found
364.0824.
methyl ester (3f)
d
Colorless oil; IR (KBr): 3259, 3068, 1736, 1622 cmꢁ1
;
1H NMR
(CDCl3,
1H), 4.86 (s, 2H), 4.46–4.40 (m, 1H), 4.28–4.24 (m, 1H), 3.65 (s, 3H),
3.22–3.13 (m, 1H), 0.86 (d, 3H, J 7.2 Hz); 13C NMR (CDCl3,
, ppm):
d, ppm): 8.41 (s, 1H), 7.75 (s, 1H), 7.39–7.26 (m, 5H), 6.29 (s,
d
171.1, 154.6, 153.1, 140.7, 140.3, 138.3, 128.5, 127.5, 127.3, 119.4, 45.5,
39.6, 29.5, 14.8; ESI-MS (m/z): 325 (MþH); HRMS (ESI) m/z calcd for
[MþH] C17H20N5O2 326.1539, found 326.1545.
4.2.14. 4-[6-(3-Methyl-5-nitro-3H-imidazol-4-ylsulfanyl)-purin-
9-yl]-butan-2-one (3n)
Yellow solid, mp 37–38 ꢀC; IR (KBr): 3451, 3115, 3076, 1713,1627,
1567, 1536 cmꢁ1; 1H NMR (CDCl3,
d
, ppm): 8.53 (s, 1H), 8.15 (s, 1H),
7.75 (s, 1H), 4.50 (t, 2H, J 6.0 Hz), 3.75 (s, 3H), 3.09 (t, 2H, J 6.0 Hz),
2.10 (s, 3H); 13C NMR (CDCl3,
, ppm): 205.4, 156.0, 151.6, 150.5,
4.2.7. 3-(6-Benzylamino-purin-9-yl)-butyric acid methyl ester (3g)
Colorless oil; IR (KBr): 3270, 3031, 1736, 1620 cmꢁ1 1H NMR
;
d
(CDCl3,
1H), 5.05–4.96 (m, 1H), 4.87 (s, 2H), 3.62 (s, 3H), 3.23–3.17 (m, 1H),
2.91–2.85 (m, 1H), 1.68 (d, 3H, J 6.8 Hz); 13C NMR (CDCl3,
, ppm):
d, ppm): 8.39 (s, 1H), 7.63 (s, 1H), 7.39–7.25 (m, 5H), 6.70 (s,
149.7, 145.4, 137.9, 131.0, 116.8, 42.1, 38.4, 33.1, 29.9; ESI-MS (m/z):
370 (MþNa); HRMS (ESI) m/z calcd for [MþH] C13H14N7O3S
348.0801, found 348.0811.
d
170.6, 154.6, 152.7, 138.7, 138.5, 128.5, 127.6, 127.3, 120.1, 51.8, 48.6,
39.3, 20.0; ESI-MS (m/z): 326 (MþH); HRMS (ESI) m/z calcd for
[MþH] C17H20N5O2 326.1539, found 326.1531.
4.2.15. 4-[6-(3-Methyl-5-nitro-3H-imidazol-4-ylsulfanyl)-purin-
9-yl]-butyronitrile (3o)
Yellow solid, mp 187–188 ꢀC; IR (KBr): 3447, 3123,
4.2.8. 3-(6-Benzylamino-purin-9-yl)-butyric acid vinyl ester (3h)
1569,1533 cmꢁ1
;
1H NMR (CDCl3,
d
, ppm): 8.48 (s, 1H), 8.17 (s,
1H), 7.75 (s, 1H), 4.55 (t, 2H, J 6.8 Hz), 3.70 (s, 3H), 3.03 (t, 2H,
J 6.8 Hz); 13C NMR (CDCl3,
, ppm): 156.5, 151.9, 150.4, 149.5,
Yellow oil; IR (KBr): 3273, 3031, 1753, 1619 cmꢁ1 1H NMR
;
(DMSO-d6, d, ppm): 8.31 (s, 1H), 8.25 (s, 1H), 8.19 (s, 1H), 7.35–7.21
d
(m, 5H), 7.13–7.08 (m, 1H), 5.06–4.97 (m, 1H), 4.84–4.80 (m, 1H),
4.68 (s, 1H), 4.63–4.61 (m, 1H), 3.20–3.14 (m, 2H), 1.57 (d, 3H,
144.1, 138.1, 130.9, 116.5, 40.7, 39.8, 33.1, 18.5; ESI-MS (m/z): 331
(MþH); HRMS (ESI) m/z calcd for [MþH] C12H11N8O2S 331.0647,
found 331.0719.
J 6.8 Hz); 13C NMR (DMSO-d6,
d, ppm): 168.1, 152.5, 141.4, 140.6,
140.0, 128.5, 127.5, 126.9, 121.2, 99.0, 47.9, 20.6; ESI-MS (m/z): 338
(MþH); HRMS (ESI) m/z calcd for [MþH] C18H20N5O2 338.1539,
found 338.1564.
4.2.16. 3-[5-(2-Methoxycarbonyl-ethyl)-4-oxo-4,5-dihydro-
pyrazolo[3,4-d]pyrimidin-1-yl]-propionic acid methyl ester (3p)
White solid, mp 117–118 ꢀC; IR (KBr): 3173, 3068, 1724,
4.2.9. 3-(6-Benzylamino-purin-9-yl)-2-methyl-propionic acid vinyl
ester (3i)
1613 cmꢁ1
;
1H NMR (CDCl3,
d
, ppm): 8.15 (s, 1H), 8.08 (s, 1H), 4.56
(t, 2H, J 6.4 Hz), 4.18 (t, 2H, J 6.4 Hz), 3.66 (s, 6H), 3.01 (t, 2H,
J 6.4 Hz), 2.82 (t, 2H, J 6.4 Hz); 13C NMR (CDCl3,
, ppm): 171.5, 170.9,
White solid, mp 62–63 ꢀC; IR (KBr): 3273, 3032, 1748,
1619 cmꢁ1
;
1H NMR (CDCl3,
d
, ppm): 8.37 (s, 1H), 7.65 (s, 1H),
d
7.35–7.25 (m, 5H), 7.20–7.15 (m, 1H), 4.84 (s, 2H), 4.86–4.82 (m, 1H),
4.58–4.56 (m, 1H), 4.44–4.39 (m, 1H), 4.27–4.22 (m, 1H), 3.26–3.17
158.9, 158.3, 149.5, 128.9, 106.7, 52.0, 51.9, 48.9, 42.6, 33.8, 33.0; ESI-
MS (m/z): 309 (MþH); HRMS (ESI) m/z calcd for [MþH] C13H17N4O5
309.1121, found 309.1141.
(m, 1H), 1.28 (d, 3H, J 7.6 Hz); 13C NMR (CDCl3,
d, ppm): 171.1, 154.6,
153.1, 140.7, 140.3, 138.4, 128.5, 127.5, 127.3, 119.3, 98.5, 45.5, 39.5,
29.5, 14.8; ESI-MS (m/z): 338 (MþH); HRMS (ESI) m/z calcd for
[MþH] C18H20N5O2 338.1539, found 338.1564.
4.2.17. 1,5-Bis-(3-oxo-butyl)-1,5-dihydro-pyrazolo[3,4-d]-
pyrimidin-4-one (3q)
White solid, mp 111–112 ꢀC; IR (KBr): 3086, 1710,1676 cmꢁ1; 1H
4.2.10. 3-(6-Amino-purin-9-yl)-propionic acid methyl ester (3j)
NMR (DMSO-d6,
J 6.8 Hz), 4.10 (t, 2H, J 6.4 Hz), 3.06 (t, 2H, J 6.8 Hz), 2.94 (t, 2H,
J 6.4 Hz), 2.12 (s, 3H), 2.09 (s, 3H); 13C NMR (DMSO-d6,
, ppm):
206.7, 152.6, 141.3, 128.5, 127.5, 126.9, 42.5, 38.4, 30.1; ESI-MS (m/z):
277 (MþH); HRMS (ESI) m/z calcd for [MþH] C13H17N4O3 277.1222,
found 277.1223.
d, ppm): 8.39 (s, 1H), 8.05 (s, 1H), 4.43 (t, 2H,
White solid, IR (KBr): 3291, 3130, 1727, 1607 cmꢁ1 1H NMR
;
(DMSO-d6,
d
, ppm): 8.12 (s, 1H), 8.08 (s, 1H), 7.20 (s, 2H), 4.36 (t, 2H,
, ppm):
d
J 6.4 Hz), 3.56 (s, 3H), 2.94 (t, 2H, J 6.4 Hz); 13C NMR (CDCl3,
d
171.4, 156.3, 152.8, 149.8, 141.3, 119.1, 52.0, 33.8; ESI-MS (m/z): 222
(MþH).
4.2.11. 3-(6-Chloro-purin-9-yl)-propionic acid methyl ester (3k)
4.2.18. 3-[1-(2-Cyano-ethyl)-4-oxo-1,4-dihydro-pyrazolo[3,4-
d]pyrimidin-5-yl]-propionitrile (3r)
White solid, mp 99–100 ꢀC; IR (KBr): 3126, 1725, 1509 cmꢁ1; 1H
NMR (DMSO-d6,
d
, ppm): 8.77 (s, 1H), 8.68 (s, 1H), 4.53 (t, 2H,
White solid, mp 161–162 ꢀC; IR (KBr): 3114, 1704 cmꢁ1; 1H NMR
J 6.8 Hz), 3.57 (s, 3H), 3.03 (t, 2H, J 6.8 Hz); 13C NMR (DMSO-d6,
d
,
(DMSO-d6,
4.27 (t, 2H, J 6.8 Hz), 3.14 (t, 2H, J 6.8 Hz), 3.02 (t, 2H, J 6.8 Hz); 13C
NMR (DMSO-d6, , ppm): 156.5, 151.9, 151.2, 135.6, 118.6, 105.4, 42.8,
d, ppm): 8.51 (s, 1H), 8.22 (s, 1H), 4.57 (t, 2H, J 6.8 Hz),
ppm): 171.3, 152.3, 151.8, 149.3, 148.1, 131.2, 52.0, 33.3; ESI-MS
(m/z): 241 (MþH).
d
41.6, 18.2, 17.7; ESI-MS (m/z): 243 (MþH); HRMS (ESI) m/z calcd for
4.2.12. 4-(6-Chloro-purin-9-yl)-butan-2-one (3l)
[MþH] C11H11N6O 243.0916, found 243.0951.
White solid, mp 183–184 ꢀC; IR (KBr): 3103, 1604 cmꢁ1; 1H NMR
(DMSO-d6,
d
, ppm): 8.77 (s, 1H), 8.73 (s, 1H), 4.63 (t, 2H, J 6.8 Hz),
4.2.19. 3-[1-(2-Methoxycarbonyl-ethyl)-6-oxo-1,6-dihydro-purin-
9-yl]-propionic acid methyl ester (3s)
3.17 (t, 2H, J 6.8 Hz), 2.10 (s, 3H); 13C NMR (DMSO-d6,
d
, ppm): 206.5,
161.9, 151.9, 151.8, 151.7, 142.5, 122.5, 43.6, 41.6, 30.2; ESI-MS (m/z):
225 (MþH); HRMS (ESI) m/z calcd for [MþH] C9H10ClN4O 225.0465,
found 326.0455.
White solid, mp 116–117 ꢀC; IR (KBr): 3100, 1736, 1687 cmꢁ1; 1H
NMR (DMSO-d6, d, ppm): 8.39 (s, 0.5H), 8.30 (s, 0.5H), 8.22 (s, 0.5H),
8.09 (s, 0.5H), 4.54 (t, 1H, J 6.4 Hz), 4.39 (t, 1H, J 6.4 Hz), 4.22 (t, 2H,
J 5.6 Hz), 3.60 (s, 3H), 3.59 (s, 3H), 2.99 (m, 2H), 2.82 (t, 2H, J 5.6 Hz);
4.2.13. 3-[6-(3-Methyl-5-nitro-3H-imidazol-4-ylsulfanyl)-purin-
13C NMR (DMSO-d6,
d, ppm): 171.7, 171.3, 157.1, 156.4, 149.0, 123.6,
9-yl]-propionic acid methyl ester (3m)
114.5, 52.0, 42.7, 42.4, 35.1, 34.0, 33.1; ESI-MS (m/z): 309 (MþH);
HRMS (ESI) m/z calcd for [MþH] C13H17N4O5 309.1121, found
309.1130.
Yellow solid, IR (KBr): 3447, 3115, 1735, 1566, 1534 cmꢁ1 1H
;
NMR (CDCl3, d, ppm): 8.55 (s, 1H), 8.15 (s, 1H), 7.76 (s, 1H), 4.55

2536
J.-L. Wang et al. / Tetrahedron 65 (2009) 2531–2536
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4.2.20. 1,9-Bis-(3-oxo-butyl)-1,9-dihydro-purin-6-one (3t)
White solid, IR (KBr): 3005,1712,1685 cmꢁ1; 1H NMR (DMSO-d6,
d
, ppm): 8.27 (s, 1H), 8.16 (s, 1H), 4.45 (t, 2H, J 6.4 Hz), 4.13 (t, 2H,
J 6.8 Hz), 3.09 (t, 2H, J 6.8 Hz), 2.95 (t, 2H, J 6.4 Hz), 2.10 (s, 3H), 2.09
(s, 3H); 13C NMR (DMSO-d6,
, ppm): 207.1, 206.6,158.9,154.0,148.0,
d
144.9, 114.5, 72.6, 60.6, 43.8, 30.2; ESI-MS (m/z): 277 (MþH); HRMS
4. Guillarme, S.; Legoupy, S.; Aubertin, A.-M.; Olicard, C.; Bourgougnon, N.; Huet, F.
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4.2.21. 3-[9-(2-Cyano-ethyl)-6-oxo-6,9-dihydro-purin-1-yl]-
propionitrile (3u)
White solid, mp 218–219 ꢀC; IR (KBr): 1712, 1697 cmꢁ1; 1H NMR
(DMSO-d6,
4.28 (t, 2H, J 6.4 Hz), 3.18 (t, 2H, J 6.4 Hz), 3.02 (t, 2H, J 6.0 Hz); 13C
NMR (DMSO-d6, , ppm): 157.2, 153.8, 147.9, 145.1, 118.7, 114.4, 72.6,
d, ppm): 8.39 (s, 1H), 8.36 (s, 1H), 4.60 (t, 2H, J 6.0 Hz),
d
60.6, 19.9, 17.6; ESI-MS (m/z): 243 (MþH); HRMS (ESI) m/z calcd for
[MþH] C11H11N6O 243.0916, found 243.0930.
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Acknowledgements
This investigation has enjoyed financial support from the Nat-
ural Science Foundation of China (No. 20572099), Ph.D. Programs
Foundation of Ministry of Education of China (20060335031), and
the Zhejiang Provincial Natural Science Foundation (Project No.
2008-Y407086).
References and notes
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