Stereoselective adenylate deaminase (5′-adenylic acid deaminase, AMPDA)-catalyzed deamination of 5′-alkyl substituted adenosines: A comparison with the action of adenosine deaminase (ADA)

Article abstract of DOI:10.1016/j.tetasy.2003.11.007

The enzymes adenylate deaminase (AMPDA) and adenosine deaminase (ADA) are able to catalyze the stereoselective hydrolytic deamination of (5′R,S)-methyl-2′,3′-isopropylidene adenosine, but the 5′-butyl analog is a substrate only for AMPDA, which stereospec

Full text of DOI:10.1016/j.tetasy.2003.11.007

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 203–206
Stereoselective adenylate deaminase (50-adenylic acid deaminase,
AMPDA)-catalyzed deamination of 50-alkyl substituted adenosines:
a comparison with the action of adenosine deaminase (ADA)
Pierangela Ciuffreda, Angela Loseto and Enzo Santaniello*
Dipartimento di Scienze Precliniche, LITA Vialba, Universitꢀa degli Studi di Milano, Via G. B. Grassi, 74, 20157 Milano, Italy
Received 1 October 2003; accepted 11 November 2003
Abstract—The enzymes adenylate deaminase (AMPDA) and adenosine deaminase (ADA) are able to catalyze the stereoselective
hydrolytic deamination of (50R,S)-methyl-20,30-isopropylidene adenosine, but the 50-butyl analog is a substrate only for AMPDA,
which stereospecifically converts the (50S)-isomer to the corresponding inosine derivative.
ꢀ 2003 Elsevier Ltd. All rights reserved.
1. Introduction
NH2
N
OH
N
N
N
N
N
Adenosine deaminase [ADA, EC 3.5.4.4] and adenylate
deaminase (50-adenylic acid deaminase, AMPDA, EC
3.5.4.6) catalyze the deamination of adenosine 1a and
adenylic acid (adenosine 50-phosphate, AMP) 1b to the
corresponding inosines 2a,b (Fig. 1).
a. R=H
b. R=CH3
c. R=C4H9
N
N
R
O
R
O
HO
HO
O
O
O
O
3
4
NH2
N
OH
Figure 2.
N
N
N
N
N
N
N
was preferentially converted to the corresponding
inosine 4b2 (Fig. 3).
RO
RO
O
O
ADA / AMPDA
a. R=H
b. R=PO3H2
OH OH
OH OH
NH2
N
OH
1
2
N
N
N
N
N
Figure 1.
H
H
N
N
HO
HO
CH3
O
CH3
O
ADA
Recently, we have reported that both enzymes are able
to deaminate also 20,30-isopropylidene adenosine 3a
(Fig. 2), thus showing that some steric hindrance at the
20,30-positions is tolerated well by both enzymes.1
O
O
O
O
(S)-3b
(S)-4b
Figure 3.
Taking advantage of the above observation, we pre-
pared (50R,S)-50-methyl-20,30-isopropylidene adenosine
3b and found that the deamination catalyzed by ADA
proceeded stereoselectively and that the (50S)-isomer
We have now investigated the action of AMPDA on
(50R,S)-50-methyl-20,30-isopropylidene adenosine 3b and
extended the study to another 50-alkyl substituted
derivative, namely (50R,S)-50-butyl-adenosine 3c (Fig. 2).
The AMPDA-catalyzed deamination to the corre-
sponding inosine 4c was also compared to the same
reaction performed in the presence of ADA.
* Corresponding author. Tel.: +39-0250319691; fax: +39-0250319631;
e-mail: enzo.santaniello@unimi.it
0957-4166/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.11.007

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P. Ciuffreda et al. / Tetrahedron: Asymmetry 15 (2004) 203–206
2. Results and discussion
NH2
N
NHBz
N
N
N
N
N
N
2.1. Synthesis of (50R,S)-50-methyl- and (50R,S)-50-butyl-
20,30-isopropylidene adenosine, 3b and 3c
N
HO
HO
O
O
For the synthesis of 50-C-alkyl-adenosines 3b,c, we
required the corresponding N7-protected 50-aldehyde
that can be obtained by oxidation of the N7-benzoyl
derivative 5, in turn prepared from 20,30-isopropylidene
adenosine 3a by an in situ benzoylation–debenzoylation
procedure.3 This method has been described nearly four
decades ago and we found that it is still the most reliable
procedure for the preparation of the N7-benzoyl deriv-
ative 54 since final yields are superior or comparable to
other methods.5;6 For the oxidation of compound 5 to
the aldehyde 8 the well established method described by
Moffatt is preferable.7 It should be briefly pointed out
that this oxidation procedure is effected with
dicyclohexylcarbodiimide–DMSO in the presence of
N,N0-diphenylethylenediammine. This allows the isola-
tion of the imidazoline 68 that is further hydrolyzed to
the corresponding aldehyde by treatment with a resin in
the acid form. The aldehyde is isolated in the hydrate
form 7 and only azeotropic distillation with benzene (or
toluene) may remove water and afford the carbonyl
form of the aldehyde 8. However, the water removal
generally causes extensive decomposition and yields of
this critical step never exceeded 30–40%. The reaction of
the aldehyde 8 with excess MeMgCl or BuMgBr allowed
the preparation of the required (50R,S)-50-methyl and
(50R,S)-50-butyl derivatives 9 and 10 (20% and 32% yield
from compound 5) (Scheme 1). Hydrolysis of the
N7-benzoyl group in compounds 9 and 10 to the
required (50R,S)-50-methyl and butyl derivatives 3b and
3c may be quantitatively achieved by treatment with
methanol solutions of aqueous ammonia.
1.DMSO/DCC/H+
2.(PhNHCH2)2
BzCl / Py
NaOH/Py
O
O
O
O
5
3a
NHBz
NHBz
N
N
N
N
N
N
Ph
O
N
OH
N
N
HO
N
benzene
reflux
O
Dowex 50
THF
Ph
O
O
O
O
6
7
NHBz
N
N
N
N
CH3
O
NH2
N
HO
N
N
MeMgCl / THF
O
O
N
O
H
O
9
NHBz
O
O
N
N
N
N
8
C4H9
O
HO
BuMgBr / THF
O
O
10
Scheme 1.
2.2. AMPDA-catalyzed deamination of (50R,S)-50-
methyl- and (50R,S)-50-butyl-20,30-isopropylidene adeno-
sine, 3b and 3c
dant isomer of the diasteromeric mixture (50R,S)-50-
butyl-adenosine 3c into the corresponding inosine 4c.11
The major isomer could be completely consumed after
4 h and the unreacted nucleoside was also used to
establish the stereochemical outcome of the enzymatic
The 3:1 diastereomeric mixture of synthetic (50R,S)-50-
methyl-20,30-isopropylidene adenosine 3b was deter-
1
mined by HPLC and 500 MHz H NMR analysis and
1
the (50R)-3b was the major isomer.2 The AMPDA-cat-
alyzed deamination of (50R,S)-3b was carried out in a
phosphate buffer at pH 6.5 and proceeded to stereo-
selectively transform in 10 min the minor isomer into the
(50S)-inosine derivative 4b.9 The unreacted (50R)-adeno-
sine 3b required an additional 3 h to be deaminated to
the corresponding (50R)-inosine. With the substrate 3b
AMPDA behaves similarly to ADA,2 taking into
account the difference in rate to reach complete trans-
formation of the (50S)-isomer (10 min for AMPDA vs
1 h for ADA). A fast reaction of AMPDA took place as
well with (50R,S)-butyl-adenosine 3c that had been
synthesized as a mixture of two diastereomers in a ratio
4:1 (as established by HPLC and NMR analysis).10
Interestingly, no reaction occurred with ADA, indicat-
ing that steric hindrance at position 50 is not tolerated by
the enzyme. On the contrary, AMPDA-catalyzed reac-
tion completely transformed in 25 min, the less abun-
reaction by the H NMR method adopted to determine
the absolute configurations of secondary alcohols.12 Our
results indicate that the unreacted diastereomer is (50R)-
50-butyl-adenosine (50R)-3c and that, consequently,
AMPDA is able to catalyze the preferential deamination
of (50S)-50-butyl-adenosine derivative (50S)-3c. We have
therefore established that AMPDA is able to catalyze
the preferential deamination of (50S)-50-methyl and
50-butyl-adenosine derivatives 3b,c to the corresponding
inosines 4b,c (Fig. 4). The results so far obtained for
AMPDA and ADA indicate that stereochemically pure
(50R)-50-methyl-20,30-isopropylidene adenosine 3b and
the corresponding (50S)-inosine 4b may be prepared by
the action of ADA and AMPDA. Whereas ADA is not
suitable for the transformation of (50R,S)-50-butyl-20,30-
isopropylidene adenosine 3c, AMPDA-catalyzed
deamination allows the preparation of stereochemically
pure (50R)-50-butyl-20,30-isopropylidene adenosine (50R)-

P. Ciuffreda et al. / Tetrahedron: Asymmetry 15 (2004) 203–206
205
NH2
N
NH2
N
OH
N
N
N
N
N
N
N
H
H
N
N
N
R
HO
HO
OH
O
R
R
O
O
AMPDA
O
O
O
O
O
O
R= CH3, C3H9
(R,S)-3b,c
(S)-4b,c
(R)-3b,c
Figure 4.
3c and the corresponding (50S)-50-butyl-20,30-isopropyl-
idene inosine (50S)-4c.
2. Ciuffreda, P.; Loseto, A.; Santaniello, E. Tetrahedron:
Asymmetry 2002, 13, 239–241.
ꢁ
3. Chladek, S.; Smrt, J. Collect. Czech. Chem. Commun.
1964, 29, 214–232.
4. Compound 5: 1H NMR (CDCl3) d 9.29 (1H, s, NH), 8.74
(1H, s, H-8), 8.09 (1H, s, H-2), 8.00 (2H, d, J ¼ 7:7,
o-PhH), 7.58 (1H, dd, J ¼ 7:7, 7.7 Hz, m-PhH), 7.42 (2H,
t, J ¼ 7:7 Hz, p-PhH), 5.95 (1H, d, J ¼ 5:6 Hz, H-10), 5.21
(1H, dd, J ¼ 5:6, 5.6 Hz, H-20), 5.09 (1H, dd, J ¼ 1:4,
5.6 Hz, H-30), 4.55 (1H, ddd, J ¼ 1:4, 1.4, 2.1 Hz, H-40),
3.98 (1H, dd, J ¼ 1:4, 12.6 Hz, H-50a), 3.80 (1H, dd,
J ¼ 2:1, 12.6 Hz, H-50b), 1.63 (3H, s, CCH3), 1.36 (3H, s,
CCH3).
5. Vrudhula, V. M.; Kappler, F.; Afshar, C.; Ginell, S. L.;
Lessinger, L.; Hampton, A. J. Med. Chem. 1989, 32, 885–
890.
6. Shuto, S.; Obara, T.; Toriya, M.; Hosoya, M.; Snoeck, R.;
Andrei, G.; Balzarini, J.; De Clercq, E. J. Med. Chem.
1992, 35, 324–331.
3. Conclusions
Our results show that ADA and AMPDA are able to
catalyze the deamination of a 20,30-isopropylidene
adenosine in a stereoselective fashion when a methyl
group is present at the 50-position. For both enzymes,
the (50S)-isomer is preferentially deaminated to the
corresponding inosine derivative and the unreacted
(50R)-adenosine may be recovered. This result may
constitute an additional example of the application of
two important biocatalysts in the nucleoside area for
chemo-enzymatic preparation of modified purine
nucleosides. In contrast to AMPDA, ADA seems more
sensitive to the steric hindrance at the 50 position, since
this enzyme is unable to catalyze the deamination of
(50R,S)-50-butyl derivative 3c. However, the high reac-
tion rate observed for AMPDA-catalyzed conversion of
50-butyl derivative 3c raises another interesting question.
In fact, at the pH required for the enzymatic transfor-
mation of the physiological substrate of AMPDA, that
is AMP, the phosphate at the position 50 should be in the
anionic form and AMPDA should accept ionic or polar
substituents at that position. This is in contrast with the
results obtained with the 50-butyl derivative 3c and other
nucleosides bearing apolar substituents at the
50-position.1 The results obtained so far highlight also a
main difference in the action of the AMPDA, compared
to the other deaminating enzyme ADA.
7. Ranganathan, R. S.; Jones, G. H.; Moffatt, J. G. J. Org.
Chem. 1974, 39, 290–297.
8. The most significant signals for compound 6 are: 8.70 (1H,
s, H-8), 7.86 (1H, s, H-2), 6.16 (1H, d, J ¼ 2:1 Hz, H-10),
5.72 (1H, d, J ¼ 2:8 Hz, H-50), 5.20 (1H, dd, J ¼ 6:3,
6.3 Hz, H-30), 5.16 (1H, dd, J ¼ 2:1, 6.3 Hz, H-20), 4.62
(1H, dd, J ¼ 2:8, 6.3 Hz, H-40) 1.47 (3H, s, CCH3), 1.31
(3H, s, CCH3).
9. Compounds 3b,c (0.02 g) in phosphate buffer (50 mM,
6 mL, pH 6.5) with 3% DMSO were treated with AMPDA
(from Aspergillus species, Sigma, 0.107 units/mg solid,
20 mg) for the time indicated in the text. The progress of
reactions was monitored by HPLC (3b: phosphate buffer
pH 6.0/CH3 CN, 8:2; 3c: phosphate buffer pH 6.0/CH3
CN, 7:3). When the reaction was complete, the solution
was lyophilized to afford inosines 4b,c.
10. 3c, major diastereomer: HPLC tR ¼ 18:5 min (phosphate
buffer pH 6.0/CH3CN, 7:3); 1H NMR (CD3OD) d 8.28
(1H, s, H-8), 8.19 (1H, s, H-2), 6.11 (1H, d, J ¼ 3:4 Hz, H-
10), 5.25 (1H, dd, J ¼ 3:4, 6.0 Hz, H-20), 5.07 (1H, dd,
J ¼ 2:7, 6.0 Hz, H-30), 4.15 (1H, dd, J ¼ 2:7, 3.4 Hz,
H-40), 3.74 (1H, dt, J ¼ 3:4, 4.0 Hz, H-50), 1.60 (3H, s,
CCH3), 1.54–1.41 (m, 2H, CH2), 1.35–1.23 (m, 4H,
CH2 · 2), 1.38 (3H, s, CCH3), 0.87 (3H, t, J ¼ 6:0 Hz,
CH3); minor diastereomer: HPLC tR ¼ 17:2 min (phos-
Acknowledgements
ꢀ
This work has been financially supported by Universita
degli Studi di Milano (Fondi FIRST).
1
phate buffer pH 6.0/CH3 CN, 7: 3); H NMR (CD3OD) d
8.36 (1H, s, H-8), 8.18 (1H, s, H-2), 6.13 (1H, d,
J ¼ 3:4 Hz, H-10), 5.15 (1H, dd, J ¼ 3:4, 6.0 Hz, H-20),
5.00 (1H, dd, J ¼ 2:7, 6.0 Hz, H-30), 4.26 (1H, dd, J ¼ 2:7,
3.4 Hz, H-40), 3.74 (1H, dt, J ¼ 3:4, 4.0 Hz, H-50), 1.61
(3H, s, CCH3), 1.54–1.41 (m, 2H, CH2), 1.35–1.23 (m, 4H,
CH2 · 2), 1.37 (3H, s, CCH3), 0.90 (3H, t, J ¼ 6:0 Hz,
CH3).
References and notes
1. Ciuffreda, P.; Loseto, A.; Santaniello, E. Tetrahedron
2002, 58, 5767–5771.
11. Compound (S)-4c HPLC tR ¼ 10:1 min (phosphate buffer
pH 6.0/CH3CN, 7:3); 1H NMR (CD3OD) d 8.21 (1H, s,

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P. Ciuffreda et al. / Tetrahedron: Asymmetry 15 (2004) 203–206
H-8), 7.92 (1H, s, H-2), 5.87 (1H, d, J ¼ 3:4 Hz, H-10), 5.10
preparing the corresponding (S)- and (R)-MTPA esters,
that were analyzed by 1H NMR according to the MosherÕs
modified method (Ohtani, I.; Kusumi, T.; Kashman, Y.;
Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092–
4096). The DdðdS ꢀ dRÞ values of the protons of the
ribose moiety ðDd < 0Þ and the 50-butyl chain protons
ðDd > 0Þ were in agreement with the proposed R-config-
uration.
(1H, dd, J ¼ 3:4, 6.0 Hz, H-20), 5.02 (1H, dd, J ¼ 2:7,
6.0 Hz, H-30), 4.37 (1H, dd, J ¼ 2:7, 3.4 Hz, H-40), 3.75
(1H, dt, J ¼ 3:4, 4.0 Hz, H-50), 1.61 (3H, s, CCH3), 1.59–
1.48 (m, 2H, CH2), 1.39–1.24 (m, 4H, CH2 · 2), 1.36 (3H,
s, CCH3), 0.86 (3H, t, J ¼ 6:0 Hz, CH3).
12. The configuration of the unreacted (R)-3c was assigned
converting the compound into the N6-benzoate 10 and