Efficient chemo-enzymatic syntheses of pharmaceutically useful unnatural 2′-deoxynucleosides

Article abstract of DOI:10.1081/NCN-200060154

Our chemo-enzymatic method was successfully applied to the synthesis of 2-chloro-2′-deoxyadenosine (CdA, cladribine) in two ways: 1) direct conversion of chemically synthesized 2-deoxy-α-D-ribose 1-phosphate (dRP) to CdA; 2) a two-step route via 9-(2-deoxy-β-D-ribos-1-yl)-2,6- dichloropurine (Cl₂Pu-dR, 5). Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200060154

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EFFICIENT CHEMO-ENZYMATIC SYNTHESES OF
PHARMACEUTICALLY USEFUL UNNATURAL 2′-
DEOXYNUCLEOSIDES
Hironori Komatsu a & Tadashi Araki a
a Catalysis Science Laboratory, Mitsui Chemicals, Inc., Chiba, Japan
Version of record first published: 15 Nov 2011.
To cite this article: Hironori Komatsu & Tadashi Araki (2005): EFFICIENT CHEMO-ENZYMATIC SYNTHESES OF
PHARMACEUTICALLY USEFUL UNNATURAL 2′-DEOXYNUCLEOSIDES, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7,
1127-1130
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):1127–1130, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060154
EFFICIENT CHEMO-ENZYMATIC SYNTHESES OF
PHARMACEUTICALLY USEFUL UNNATURAL
2’-DEOXYNUCLEOSIDES
5
Hironori Komatsu and Tadashi Araki
Catalysis Science Laboratory, Mitsui
Chemicals, Inc., Chiba, Japan
5
Our chemo-enzymatic method was successfully applied to the synthesis of 2-chloro-2’-deoxyadenosine
(CdA, cladribine) in two ways: 1) direct conversion of chemically synthesized 2-deoxy-a-D-ribose 1-
phosphate (dRP) to CdA; 2) a two-step route via 9-(2-deoxy-b-D-ribos-1-yl)-2,6-dichloropurine (Cl2Pu-
dR, 5).
Keywords 2-Deoxy-a-D-Ribose 1-Phosphate, dRP, 2-Chloro-2’-Deoxyadenosine, Cladribine,
Purine Nucleoside Phosphorylase, Glycosylation
INTRODUCTION
Efficient synthetic methods for 2’-deoxynucleosides (dNus) has been under
development over the past few decades. We previously reported a chemo-
enzymatic method and its application to the syntheses of natural dNus.[1,2] The
method consists of three distinctive technologies: 1) stereoselective synthesis of 2-
deoxyribose 1-a-phosphate (dRP) by crystallization-induced asymmetric transfor-
mation;[3] 2) an efficient method to expedite an enzymatic conversion by adding
Mg(OH)2; 3) development of a new enzyme for 2’-deoxycytidine. To expand the
application of this method,[4] syntheses of various pharmaceutically useful unnatural
*
dNus such as CdA have been examined (Scheme 1).
RESULTS AND DISCUSSIONS
Phosphate (1) was stereoselectively synthesized as described.[3] Preparation of
dRP, however, was slightly modified, since its cyclohexylammonium salt was highly
soluble in MeOH and tedious isolation steps were required. Deprotection of 1 by
*
For previous syntheses of CdA, see: Ref. [5].
Address correspondence to Hironori Komatsu, Catalysis Science Laboratory, Mitsui Chemicals, Inc.,
Sodegaura-shi, Chiba, Japan.
1127
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1128
H. Komatsu and T. Araki
SCHEME 1
NH3/MeOH gave an ammonium salt of dRP (2) as crystals directly from the
reaction solution (Scheme 2).
A direct enzymatic glycosylation pathway was first examined using 2
(Scheme 3). Glycosylation of 2-chloroadenine (4) with 2 would be the simplest
synthetic route to CdA. Difficulties in the preparation of dRP prevented its
application. Our chemo-enzymatic method accomplished this synthetic strategy. As
previously reported,[6] 4 was prepared by amination of 3 in NH3/MeOH. The
reaction required high temperature (160°C) in a sealed tube and long reaction time
(24 h). Unlike the reported method, simple filtration was sufficient to obtain 4 in
pure form. Enzymatic glycosylation of 4 with 2 was performed in H2O at 45°C in
the presence of purine nucleoside phosphorylase (PNPase, self-cloned in E. coli)y
and Mg(OH)2 in 98% HPLC yield. Recrystallization from MeOH afforded pure
CdA in 88% isolated yield.
A two-step synthetic route via Cl2Pu-dR (5) was next investigated (Scheme 4).
The same glycosylation condition as described above was carried out first in the
presence of Mg(OH)2. The slightly alkaline condition partially hydrolyzed or
ammonolyzed the 6-Cl group of 3. Without Mg(OH)2, sparingly soluble 5
crystallized directly from the reaction solution, which facilitated the enzymatic
conversion. Thus, enzymatic glycosylation of 3 with 2 in H2O at 45°C in the
presence of PNPase gave 5 in high isolated yield (91%). Ammonolysis of 5 was
performed in NH4OH/CH3CN. In contract to the ammonolysis of 3 or acetylated
*
5, the reaction proceeded at moderate temperature to give 98% HPLC yields. The
relative hydrophilic property of 5, compared to 3 or acetylated 5, increased its
reactivity in a polar reaction medium (NH4OH/CH3CN). This made two-step route
advantageous. Finally, treatment with anion exchange resin [IER (ꢀOH)] followed
by recrystallization from EtOH gave CdA in 79% isolated yield.
In summary, synthesis of CdA was successfully performed using our novel
chemo-enzymatic strategy. Two synthetic routes were demonstrated. One is a direct
yFor a preparation of PNPase, see: Refs. [7,8].

H. Komatsu and T. Araki
1129
SCHEME 2
SCHEME 3
SCHEME 4
enzymatic glycosylation pathway, and the other, a glycosylation-amination pathway.
This strategy will be useful as an efficient alternative method for the syntheses of
various unnatural 2’-deoxynucleosides.
REFERENCES
1. Komatsu, H.; Awano, H.; Tanikawa, H.; Itou, K.; Ikeda, I. Large-scale manufacturing of all four 2’-
deoxynucleosides via novel strategies including a chemo-enzymatic process. Nucleosides Nucleotides Nucleic
Acids 2001, 20, 1291.
2. Komatsu, H.; Awano, H.; Ishibashi, H.; Oikawa, T.; Ikeda, I.; Araki, T. Chemo-enzymatic syntheses of natural
and unnatural 2’-deoxynucleosides. Nucleic Acids Res. Suppl. 2003, 3, 101.
3. Komatsu, H.; Awano, H. First stereoselective synthesis of 2-deoxy-alpha-D-ribosyl-1-phosphate: novel
application of crystallization-induced asymmetric transformation. J. Org. Chem. 2002, 67, 5419.
4. Komatsu, H.; Araki, T. Chemo-enzymatic synthesis of 2’,3’-dideoxy-3’-fluoro-b-D-guanosine via 2,3-dideoxy-3-
fluoro-a-D-ribose 1-phosphate. Tetrahedron Lett. 2003, 44, 2899.
5. Janeba, Z.; Francom, P.; Robins, M.J. Efficient syntheses of 2-chloro-2’-deoxyadenosine (cladribine) from 2’-
deoxyguanosine(1). J. Org. Chem. 2003, 68, 989, and references therein.

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H. Komatsu and T. Araki
6. Brown, G.B.; Weliky, V.S. 2-Chloroadenine and 2-chloroadenosine. J. Org. Chem. 1958, 23, 125.
7. Jensen, K.F.; Nygaard, P. Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium.
Purification and some properties. Eur. J. Biochem. 1975, 51, 253.
8. Krenitsky, T.A.; Koszalka, G.W.; Tuttle, J.V. Purine nucleoside synthesis, an efficient method employing
nucleoside phosphorylases. Biochemistry 1981, 20, 3615.