Deamination of 5′-substituted-2′,3′-isopropylidene adenosine derivatives catalyzed by adenosine deaminase (ADA, EC 3.5.4.4) and complementary enzymatic biotransformations catalyzed by adenylate deaminase (AMPDA, EC 3.5.4.6): A viable route for the preparation of 5′-substituted inosine derivatives

Article abstract of DOI:10.1016/S0040-4020(02)00575-6

Adenosine deaminase (ADA) catalyzes the deamination of 2′,3′-isopropylidene adenosine and the corresponding 5′-amino derivative in a 3% dimethylsulfoxide aqueous solution. Whereas ADA is unable to convert other 5′-substituted derivatives (acetate, acetamido, azide), the enzyme adenylate deaminase (AMPDA) accepts all the above compounds as substrates for their biotransformation to the corresponding 5′-substituted inosine derivatives.

Full text of DOI:10.1016/S0040-4020(02)00575-6

TETRAHEDRON
Pergamon
Tetrahedron 58 (2002) 5767±5771
Deamination of 50-substituted-20,30-isopropylidene adenosine
derivatives catalyzed by adenosine deaminase (ADA, EC 3.5.4.4)
and complementary enzymatic biotransformations catalyzed by
adenylate deaminase (AMPDA, EC 3.5.4.6): a viable route for the
preparation of 50-substituted inosine derivatives
Pierangela Ciuffreda, Angela Loseto and Enzo Santaniellop
Á
Dipartimento di Scienze Precliniche, Universita degli Studi di Milano, LITA Vialba Via G. B. Grassi, 74-20157 Milano, Italy
Received 27 March 2002; revised 16May 2002; accepted 6June 2002
AbstractÐAdenosine deaminase (ADA) catalyzes the deamination of 20,30-isopropylidene adenosine and the corresponding 50-amino
derivative in a 3% dimethylsulfoxide aqueous solution. Whereas ADA is unable to convert other 50-substituted derivatives (acetate, aceta-
mido, azide), the enzyme adenylate deaminase (AMPDA) accepts all the above compounds as substrates for their biotransformation to the
corresponding 50-substituted inosine derivatives. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
2. Results and discussions
2.1. Synthesis of 50-derivatives of 20,30-isopropylidene
adenosine as possible substrates for ADA-catalyzed
deamination
Adenosine deaminase [ADA, EC 3.5.4.4] is an enzyme that
catalyzes the rapid and irreversible deamination of adeno-
sine 1a to inosine 2a and may be considered as a valuable
biocatalyst for the biotransformation of a wide range of
structurally modi®ed purine nucleosides.1 The recently
reported three-dimensional structures of the enzyme2,3
have added valuable information on the structural pre-
requisites that the purine and ribose moieties have to possess
in order to be accepted as substrates by the enzyme. A
considerable body of evidence has been gained to establish
that the 50-hydroxy group is necessarily required for the
activity of ADA, whereas some ¯exibility is possible at
the 20,30-positions.4,5
As expected by the crucial role played by the 50-hydroxy
group, the acetate 3b should not be enzymatically
In previous papers,6,7 we have shown that some steric
hindrance at the positions 20 and 30 is well tolerated
by the enzyme, since adenosine 20,30-diacetates 1b and
20,30-isopropylidene adenosine 1c may be converted by
means of ADA to the corresponding inosine derivatives
2b,c. We decided to extend the above observations pre-
paring a few 50-analogs of 20,30-isopropylidene adenosine
3a, namely compounds 3b±e, and submit them to the
ADA-catalyzed deamination (Figs. 1 and 2).
Figure 1. ADA-catalyzed deamination of adenosine derivatives 1a±c.
Keywords: biotransformations; adenosin deaminase; deamination.
Corresponding author. Tel.: 139-2-38210472; fax: 139-2-38210295;
e-mail: enzo.santaniello@unimi.it
p
Figure 2. Structure of compounds 3a±e.
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00575-6

5768
P. Ciuffreda et al. / Tetrahedron 58 (2002) 5767±5771
Scheme 1. Lipase-catalyzed acetylation of 20,30-isopropylidene adenosine
3a.
Figure 3. AMPDA-catalyzed deamination of adenosine derivatives 3a±c.
transformed. We have prepared the substrate 3b by a lipase-
catalyzed acetylation in an organic solvent, since the direct
chemical acetylation of compound 3a was complicated by
the formation of mono- and bis-acetamido derivatives at the
N6-amino group. For the enzymatic preparation of 3b from
3a the lipase from Candida antarctica (CAL) was preferred,
due to the selectivity of this enzyme towards the 50-position
of adenosine or inosine derivatives.8 The reaction was
carried out in THF at room temperature under irreversible
transesteri®cation conditions using vinyl acetate as acyl
transfer agent. The acetate 3b was quantitatively formed
from 3a in 12 h, with no trace of N6-acetylation (Scheme 1).
action of the enzyme. We then prepared 50-deoxy-50-amino
adenosine 3d in order to verify if an amino group at the
position 50 could provide the hydrogen bonding network
that seems necessary to a nucleoside structure for an
ADA-catalyzed deamination. It was also expected that the
corresponding 50-acetamide 3e should not be a substrate of
the enzyme. For the preparation of the required 50-amino
and 50-acetamido compounds 3d and 3e, we started with the
N6-benzoylated compound 4, that was converted into the
corresponding tosylate (Scheme 2). Displacement by
sodium azide of the above intermediate in dimethyl forma-
mide at 808C afforded the debenzoylated azide 3c. Among
several methods described for the reduction of a nucleoside
azide,10,11 we were able to obtain a quantitative yield of the
required amino compound 3d by catalytic hydrogenation
(10% Pd±C)12 of the azide 3c. A selective enzymatic acetyl-
ation of the 50-amino group of 3d was attempted, but no
reaction occurred in the presence of CAL. Therefore, the
50-acetamido derivative 3e was prepared by chemical
acetylation of 3d, that afforded a mixture of 50- and
N6-benzoylated products. Subsequent treatment of this
mixture with ammonia solution in methanol afforded the
required acetamido derivative 3e.
Due to low solubility of the substrate, the ADA-catalyzed
deamination could be carried out either at 608C or in a 3%
DMSO aqueous solution. We have previously shown that at
the above temperature the enzymatic activity is still com-
patible with a quantitative transformation of a substrate6 and
it has been reported that the presence of polar solvents does
not inhibit the ADA activity on adenosine 1a.9 Preliminary
experiments were carried out on a DMSO solution of iso-
propylidene adenosine 3a in aqueous phosphate buffer (pH
7.4) so that a ®nal 3% concentration of DMSO was reached.
The substrate 3a was quantitatively deaminated in 15 min to
the inosine derivative 5a, this result con®rming that the
above mixed DMSO-aqueous solution did not deactivate
the enzyme. Therefore we decided to use this medium for
the ADA-catalyzed deamination of the acetate 3b and
substrates 3c±e. In these conditions, the acetate 3b was
not deaminated to any extent and this result again con®rmed
the essential role of the 50-hydroxy group for the catalytic
The 50-amino nucleoside 3d was deaminated rather slowly,
if compared to the corresponding adenosine 3a (6h versus
15 min), thus con®rming the essential role played by the
hydrogen bond network around the 50-position. In fact, we
have later observed that the 50-azido and the 50- acetamido
derivatives 3c and 3e were not substrates for the ADA-
catalyzed deamination.
AMPDA-catalyzed deamination of 50-derivatives of
20,30-isopropylidene adenosine (compounds 3a±e).
Compared with ADA, the properties and the possible use
of adenylate deaminase (50-adenylic acid deaminase,
AMPDA, EC 3.5.4.6) as biocatalyst are much less explored.
This enzyme catalyzes the deamination of adenylic acid
(adenosine 50-phosphate, AMP) to inosine 50-phosphate
(IMP), a reaction that is of considerable commercial
value, due to the use of IMP as a ¯avoring in the food
industry (Fig. 3).
Information available from the literature seems to indicate
that, compared to ADA, AMPDA is able to accept a wider
range of substrates, but is less stereoselective.13 AMPDA
from Aspergillus sp. is commercially available and we
decided to use this enzyme on compounds 3a±e, previously
used as substrates for ADA-catalyzed deamination. For this
enzyme we again used a 3% DMSO aqueous solution as the
Scheme 2. Synthesis of compounds 3c±e, substrates of the deaminating
enzymes ADA and AMPDA: (i) TsCl/py, 08C, 2 h, 93%; (ii) NaN3/DMF,
808C, 1 h, 80%; (iii) H2/Pt(C), rt, 5 h, 90%; (iv) Ac2O/py, 08C, 2 h; NH3/
MeOH, rt, 4 h, 80%.

P. Ciuffreda et al. / Tetrahedron 58 (2002) 5767±5771
Table 1. ADA- and AMPDA-catalyzed deamination of adenosine derivatives 3a±e
5769
Substrate
AMPDA reaction time (min)a
ADA reaction time (min)a
Vo(AMPDA)/Vo(ADA)b
Product (yield, %)c
3a
3b
3c
3d
3e
45
20
30
180
10
15
0.2
5a (98)
5b (92)
5c (94)
5d (98)
5e (95)
No reaction
No reaction
360
.1000
.1000
9.3
No reaction
.1000
a
Time for complete reaction.
b
Initial rates of the enzyme-catalyzed reactions were measured by HPLC (see Section 4 for details). Vo(AMPDA)/Vo(ADA).1000 means that at high ADA
concentration no reaction was detected after several days of incubation.
In all case enzyme-catalyzed reactions proceeded quantitatively without formation of by-products (HPLC); yields refer to isolated products.
c
medium, once it was established that isopropylidene adeno-
sine 3a was readily deaminated by AMPDA in 45 min
(Table 1).
column cromatography were monitored by TLC and
HPLC. HPLC analyses were carried out on a Jasco HPLC
instrument with an Uvidec 100 II UV detector operating
at 260 nm using an Alltech Hypersil BDS C18
(4.6mm £250 mm). The eluant was phosphate buffer at
pH 6.0 containing CH3CN in the following ratio: 80/20
for 3a and 3b, 70/30 for 3c, 85/15 for 3d and 3e at a ¯ow
rate of 1 mL/min. Thin-layer chromatography (TLC) was
performed using Merck silica gel 60 F254 precoated plates
with a ¯uorescent indicator. Flash chromatography14 was
performed using Merck silica gel 60 (230±400 mesh) with
appropriate mixtures of CH2Cl2 and MeOH as eluant. All
reagents were obtained from commercial sources and used
without further puri®cation. The enzymes were obtained as
follows: immobilized lipase from C. antarctica (Novozym
435w, Novo Nordisk), adenosine deaminase from calf
intestinal mucosa (Sigma, type II, 2.2 units/mg protein),
50-adenylic acid deaminase from Aspergillus sp. (Sigma,
0.107 units/mg protein). The starting nucleoside 1a was
purchased from Aldrich, compounds 1c and 4 were prepared
according to literature procedures.15,16
It was gratifying to us to ®nd that all compounds were
substrates for the catalytic action of AMPDA and that this
enzymatic deamination allows a viable preparation of the
inosine derivatives 5b±e from the 50-substituted adenosine
derivatives 3b±e.
3. Conclusion
We have shown that the two deaminating enzymes, ADA
and AMPDA, show a difference in their substrate selec-
tivity. Both enzymes can tolerate substitution at the
20,30-positions, but, as a salient difference, the presence
of a hydroxy group at the 50-position is necessary for
ADA-catalyzed reactions. This can be substituted by
another polar group such as amino, although the reaction
is much slower. AMPDA seems a more versatile biocatalyst
in the nucleoside ®eld, since it is able to convert all the
compounds 3a±e to the corresponding inosine derivative
5a±e in a time suitable for preparative purposes. The results
obtained may ®nd explanation through knowledge of
the three-dimensional structure of the enzymes. However,
this information is not available for AMPDA. Further
research is needed in order to de®ne the structural features
that a nucleoside derivative must possess in order to become
a substrate for AMPDA. We have shown that AMPDA is
a more versatile biocatalyst than ADA for the preparation
of a great variety of 6-oxopurine ribosides.
4.1.1. 50-Acetyl 20,30-O-isopropylidene adenosine (3b).
20,30-O-Isopropylidene adenosine 1c (154 mg, 0.5 mL),
vinyl acetate (1.5 mmol) and CAL (200 mg) were sus-
pended in THF (10 mL). The mixture was allowed to
react at 608C for 12 h, the progress of the reaction being
monitored by TLC (CH2Cl2/MeOH, 9:1). The enzyme was
®ltered off, washed with MeOH, and the solvents were
removed under reduced pressure. The residue was puri®ed
by ¯ash chromatography (CH2Cl2/MeOH, 95:5) to give
50-acetyl 20,30-O-isopropylidene adenosine 3b (160 mg,
92%) as a white powder: mp 164±1658C (lit.17 mp
25
1
1678C); [a]D ˆ229.8 (c 1, MeOH); H NMR (CD3OD)
d 8.22 (1H, s, H-2), 8.21 (1H, s, H-8), 6.19 (1H, d, Jˆ
2.7 Hz, H-10), 5.51 (1H, dd, Jˆ2.7, 6.7 Hz, H-20), 5.07
(1H, dd, Jˆ3.4, 6.7 Hz, H-30), 4.43 (1H, ddd, Jˆ3.4, 4.7,
6.0 Hz, H-40), 4.26(1H, dd, Jˆ4.7, 12.0 Hz, H-50a), 4.22
(1H, dd, Jˆ6.0, 12.0 Hz, H-50b), 1.95 (3H, s, OCOCH3),
1.59 (3H, s, CCH3), 1.36(3H, s, CC H3).
4. Experimental
4.1. General
Melting points were recorded on Stuart Scienti®c SMP3
instrument and are uncorrected. IR spectra were recorded
on a Nicolet 510 Fourier transform spectrophotometer.
1H NMR spectra were recorded on Bruker AM-500 spectro-
4.1.2. 50-Deoxy-50-azido-20,30-O-isopropylidene adeno-
sine (3c). N6-Monobenzoyl 20,30-O-isopropylidene adeno-
sine 4 (1 g, 2.4 mmol) was evaporated three times with
pyridine, then dissolved in pyridine (15 mL) and the solu-
tion cooled with an external ice bath. Tosyl chloride (1.1 g,
6.0 mmol) was added and the solution stirred for 2 h at 08C.
After addition of cold water, the mixture was extracted with
ethyl acetate. The resulting solution was sequentially
washed with HCl, NaHCO3, brine, dried over anhydrous
1
meter operating at 500.13 MHz. The H NMR chemical
shifts are reported in parts per million, using as reference
the signal for residual solvent protons (7.24 for CDCl3, 3.30
for CD3OD). Coupling constants (J) are given in Hz and the
1
NMR signals were assigned by H-homodecoupling and
COSY experiments. Optical rotations were measured on a
Perkin±Elmer 241 polarimeter (sodium D line at 258C) for
solutions in MeOH. The progress of all reactions and

5770
P. Ciuffreda et al. / Tetrahedron 58 (2002) 5767±5771
Na2SO4 and the solvent evaporated under reduced pressure.
The residue (1.26g, 93%) was suf®ciently pure by TLC
(CH2Cl2/MeOH, 95:5) to be used in the next step without
Jˆ6.0, 14.0 Hz, H-50a), 3.45 (1H, dd, Jˆ5.4, 14.0 Hz,
H-50b), 1.95 (3H, s, NHCOCH3), 1.58 (3H, s, CCH3), 1.36
(3H, s, CCH3). Anal. calcd for C15H20N6O4: C, 51.72; H,
5.79; N, 24.12. Found: C, 51.60; H, 5.73; N, 24.09.
1
puri®cation; H NMR (CDCl3) d 8.69 (1H, s, H-2), 8.12
(1H, s, H-8), 8.03±8.00 (4H, m, 2£benzoyl o and 2£tosyl
o), 7.58 (1H, dd, Jˆ7.0, 7.0 Hz, benzoyl p), 7.50 (2H, dd,
Jˆ7.0, 7.0 Hz, benzoyl m), 7.50 (2H, d, Jˆ8.4 Hz, tosyl m),
6.13 (1H, d, Jˆ2.8 Hz, H-10), 5.34 (1H, dd, Jˆ2.8, 6.3 Hz,
H-20), 5.02 (1H, dd, Jˆ2.8, 6.3 Hz, H-30), 4.49 (1H, ddd,
4.2. Enzymatic deamination of adenosine derivatives
3a±e to inosine derivatives 5a±e
0
Jˆ2.8, 4.2, 5.6Hz, H-4 ), 4.25 (1H, dd, Jˆ4.2, 10.5 Hz,
Compounds 3a±e (20 mg) in phosphate buffer (50 mM,
10 mL, pH 7.4 for ADA and pH 6.5 for AMPDA) containing
3% DMSO were treated with ADA (2 mg) or AMPDA
(20 mg) for the time indicated in Table 1. The progress of
reactions was monitored by HPLC (3a and 3b: phosphate
buffer pH 6.0/CH3CN, 80:20; 3c: phosphate buffer pH 6.0/
CH3CN, 70:30; 3d and 3e: phosphate buffer pH 6.0/CH3CN,
85:15). The solution was lyophilized and the residue crystal-
lized from methanol±water as white solids (compounds
5b,e). Compounds 5c and 5d resisted to several attempts
of crystallization. 20,30-isopropylidene inosine (5a) showed
physical characteristics in agreement with published data.7
H-50a), 4.22 (1H, dd, Jˆ5.6, 10.5 Hz, H-50b), 2.36(3H, s,
tosyl CH3), 1.58 (3H, s, CCH3), 1.35 (3H, s, CCH3). To a
solution of tosylate (1.25 g, 2.2 mmol) in dry DMF (25 mL),
NaN3 was added (572 mg, 8.8 mmol) and the reaction
heated at 808C for 1 h. Excess NaN3 was removed by ®ltra-
tion and the solution was diluted with ethyl acetate then
washed with water. The organic solution was dried over
anhydrous Na2SO4 and the solvent evaporated at reduced
pressure. The residue was puri®ed by ¯ash chromatography
(CH2Cl2/MeOH, 95:5) to afford 50-deoxy-50-azido-20,30-O-
isopropylidene adenosine 3c as an amorphous solid
(585 mg, 80%): IR (nmax, cm21, KBr) 3434, 2924, 2109,
1
1637, 1384, 1249, 1092; H NMR (CDCl3) d 8.34 (1H, s,
4.2.1. 50-Acetyl 20,30-O-isopropylidene inosine (5b). White
25
1
H-2), 7.94 (1H, s, H-8), 6.09 (1H, d, Jˆ2.1 Hz, H-10), 5.42
(1H, dd, Jˆ2.1, 6.3 Hz, H-20), 5.03 (1H, dd, Jˆ3.5, 6.3 Hz,
H-30), 4.37 (1H, ddd, Jˆ3.5, 5.6, 5.6 Hz, H-40), 3.60±3.53
(2H, m, part AB of system ABX, H-50a and H-50b), 1.60
(3H, s, CCH3), 1.37 (3H, s, CCH3). Anal. calcd for
C13H16N8O3: C, 46.98; H, 4.85; N, 33.72. Found: C,
46.74; H, 4.62; N, 33.68.
powder, mp 229±2308C, [a]D ˆ222.6( c 1, MeOH); H
NMR (CD3OD) d 8.18 (1H, s, H-2), 8.06(1H, s, H-8), 6.21
(1H, d, Jˆ2.7 Hz, H-10), 5.42 (1H, dd, Jˆ2.7, 6.7 Hz, H-20),
5.04 (1H, dd, Jˆ3.4, 6.7 Hz, H-30), 4.45 (1H, ddd, Jˆ3.4,
4.7, 6.0 Hz, H-40), 4.27 (1H, dd, Jˆ4.7, 12.0 Hz, H-50a),
4.24 (1H, dd, Jˆ6.0, 12.0 Hz, H-50b), 1.97 (3H, s,
OCOCH3), 1.59 (3H, s, CCH3), 1.38 (3H, s, CCH3). Anal.
calcd for C15H18N4O6: C, 51.43; H, 5.18; N, 15.99. Found:
C, 51.34; H, 5.06; N, 14.83.
4.1.3. 50-Deoxy-50-amino-20,30-O-isopropylidene adeno-
sine (3d). Compound 3c (200 mg, 0.60 mmol) was
dissolved in ethanol (10 mL) and 10% Pd/C (10 mg) was
added. The solution was left under of a hydrogen atmos-
phere (5 h) and then the catalyst removed by ®ltration. The
solvent was evaporated at reduced pressure and the residue
puri®ed by ¯ash chromatography (CH2Cl2/MeOH, 80:20) to
afford 50-deoxy-50-amino-20,30-O-isopropylidene adenosine
3d (166 mg, 90%) as a white powder: mp 206±2088C (dec),
4.2.2. 50-Deoxy-50-azido-20,30-O-isopropylidene inosine
1
(5c). Amorphous solid, H NMR (CD3OD) d 8.36(1H, s,
H-2), 8.12 (1H, s, H-8), 6.23 (1H, d, Jˆ2.7 Hz, H-10), 5.43
(1H, dd, Jˆ2.7, 6.3 Hz, H-20), 5.02 (1H, dd, Jˆ3.4, 6.3 Hz,
H-30), 4.37 (1H, ddd, Jˆ3.4, 5.6, 5.6 Hz, H-40), 3.61±3.54
(2H, m, part AB of system ABX, H-50a and H-50b), 1.59
(3H, s, CCH3), 1.37 (3H, s, CCH3). Anal. calcd for
C13H15N7O4: C, 46.85; H, 4.54; N, 29.42. Found: C,
46.75; H, 4.38; N, 29.26.
1
25
(lit.18 mp 204±2058C); [a]D ˆ235.8 (c 1, MeOH); H
NMR (CD3OD) d 8.26(1H, s, H-2), 8.20 (1H, s, H-8),
6.13 (1H, d, Jˆ3.4 Hz, H-10), 5.46(1H, dd, Jˆ3.4,
6.0 Hz, H-20), 5.00 (1H, dd, Jˆ3.4, 6.0 Hz, H-30), 4.22
(1H, ddd, Jˆ3.4, 6.0, 6.0 Hz, H-40), 2.92±2.84 (2H, m,
part AB of system ABX, H-50a and H-50b), 1.58 (3H, s,
CCH3), 1.36(3H, s, CC H3).
4.2.3. 50-Deoxy-50-amino-20,30-O-isopropylidene inosine
25
1
(5d). [a]D ˆ239.4 (c 1, MeOH); H NMR (CD3OD) d
8.21 (1H, s, H-2), 8.06(1H, s, H-8), .614 (1H, d,
Jˆ
3.4 Hz, H-10), 5.39 (1H, dd, Jˆ3.4, 6.0 Hz, H-20), 4.98
(1H, dd, Jˆ3.4, 6.0 Hz, H-30), 4.23 (1H, ddd, Jˆ3.4, 6.0,
6.0 Hz, H-40), 2.96±2.88 (2H, m, part AB of system ABX,
H-50a and H-50b), 1.58 (3H, s, CCH3), 1.36(3H, s, CC H3).
Anal. calcd for C13H17N5O4: C, 50.81; H, 5.58; N, 22.79.
Found: C, 50.63; H, 5.46; N, 22.62.
4.1.4.
50-Deoxy-50-acetamido-20,30-O-isopropylidene
adenosine (3e). To a solution of compound 3d (100 mg,
0.33 mmol) in dry pyridine (0.5 mL), acetic anhydride
(67 mg, 0.66 mmol) was added and the solution stirred for
2 h at 08C. After usual work-up, the resulting mixture of
acetates was dissolved in methanol saturated with ammonia
(10 mL). After stirring for 4 h at room temperature, the
solvent was evaporated to leave a yellow oil. The residue
was puri®ed by ¯ash chromatography (CH2Cl2/MeOH,
95:5) to afford title compound 3e as a white solid
4.2.4. 50-Deoxy-50-acetamido-20,30-O-isopropylidene ino-
1
25
sine (5e). Mp 178±1798C, [a]D ˆ249.6( c 1, MeOH); H
NMR (CD3OD) d 8.19 (1H, s, H-2), 8.09 (1H, s, H-8), 6.14
(1H, d, Jˆ2.7 Hz, H-10), 5.39 (1H, dd, Jˆ2.7, 6.7 Hz, H-20),
4.96(1H, dd, Jˆ3.4, 6.7 Hz, H-30), 4.27 (1H, ddd, Jˆ3.4,
6.0, 6.7 Hz, H-40), 3.50 (1H, dd, Jˆ6.0, 14.0 Hz, H-50a),
3.45 (1H, dd, Jˆ6.7, 14.0 Hz, H-50b), 1.93 (3H, s,
NHCOCH3), 1.57 (3H, s, CCH3), 1.35 (3H, s, CCH3).
Anal. calcd for C15H19N5O5: C, 51.57; H, 5.48; N, 20.05.
Found: C, 51.39; H, 5.29; N, 19.98.
25
(100 mg, 80%): mp 169±1708C, [a]D ˆ216.9 (c 1,
1
MeOH); H NMR (CD3OD) d 8.25 (1H, s, H-2), 8.24
(1H, s, H-8), 6.13 (1H, d, Jˆ3.3 Hz, H-10), 5.43 (1H, dd,
Jˆ3.3, 6.7 Hz, H-20), 4.97 (1H, dd, Jˆ3.0, 6.7 Hz, H-30),
4.30 (1H, ddd, Jˆ3.0, 5.4, 6.0 Hz, H-40), 3.55 (1H, dd,

P. Ciuffreda et al. / Tetrahedron 58 (2002) 5767±5771
5771
7. Ciuffreda, P.; Loseto, A.; Santaniello, E. Tetrahedron: Asym-
metry 2002, 13, 239±241.
Acknowledgements
8. Prasad, A. K.; Wengel, J. Nucleosides Nucleotides 1996, 15,
1347±1359.
Á
This work has been ®nancially supported by Universita
degli Studi di Milano (Fondi ex 60%) and the Italian
National Council for Research (CNR, Target Project in
Biotechnology).
9. Bolen, D. W.; Fisher, J. R. Biochemistry 1969, 11, 4239±
4246.
10. Scriven, E.; Turnbull, K. Chem. Rev. 1988, 88, 297±368.
11. Samano, M. C.; Robbins, M. J. Tetrahedron Lett. 1991, 32,
6293±6296.
References
12. Ceulemans, G.; Vandendriessche, F.; Rozensky, J.;
Herdewijn, P. Nucleosides Nucleotides 1995, 14, 117±127.
13. Margolin, A. L.; Borcherding, D. R.; Wolf-Kugel, D.;
Margolin, N. A. J. Org. Chem. 1996, 59, 7214±7218.
14. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,
2923±2925.
1. Ferrero, M.; Gotor, V. Chem. Rev. 2000, 100, 4319±4347.
2. Marrone, T. J.; Straatsma, T. P.; Briggs, J. M.; Wilson, D. K.;
Quiocho, F. A.; McCammon, J. A. J. Med. Chem. 1996, 39,
277±284.
3. Wilson, D. K.; Quiocho, F. A. Biochemistry 1993, 32, 1689±
1694.
15. Hampton, A. J. Am. Chem. Soc. 1961, 83, 3640±3645.
Â
16. Chladek, S.; Smrt, J. Collect. Czech. Chem. Commun. 1964,
4. Bloch, A.; Robins, M. J.; McCarthy, Jr., J. R. J. Med. Chem.
1967, 10, 908±912.
Â
5. Maury, G.; Daiboun, T.; Elalaoui, A.; Genu-Dellac, C.;
29, 214±232.
17. Brown, D. M.; Haynes, L. J.; Todd, A. R. J. Chem. Soc. 1950,
Â
Perigaud, C.; Bergogne, C.; Gosselin, G.; Imbach, J.-L.
3299±3304.
18. Reeve, A. M.; Townsend, C. A. Tetrahedron 1998, 54,
15959±15975.
Nucleosides Nucleotides 1991, 10, 1677±1692.
6. Ciuffreda, P.; Casati, S.; Santaniello, E. Tetrahedron 2000, 56,
3239±3243.