Large-scale manufacturing of all four 2′-deoxynucleosides via novel strategies including a chemo-enzymatic process

Article abstract of DOI:10.1081/NCN-100002539

A chemical synthesis of 2-deoxyribose-1-phosphate 2 and its enzymatic conversion into purine 2′-deoxynucleosides (dNus) are shown. Besides the chemoenzymatic process for purine dNus, a modified process for practical dC preparation is also established. Consequently, a series of practical manufacturing processes of all four dNus have been realized via novel strategies.

Full text of DOI:10.1081/NCN-100002539

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LARGE-SCALE MANUFACTURING OF ALL FOUR
2′-DEOXYNUCLEOSIDES VIA NOVEL STRATEGIES
INCLUDING A CHEMO-ENZYMATIC PROCESS
Hironori Komatsu a , Hirokazu Awano a , Hiroharu Tanikawa a , Kiyoshi Itou b & Ichirou
Ikeda b
a Chemical Synthesis Laboratory, Mitsui Chemicals Inc. , 1144 Togo, Mobara-shi, Chiba,
297-0017, Japan
b Life Science Laboratory, Mitsui Chemicals Inc. , 1144 Togo, Mobara-shi, Chiba,
297-0017, Japan
Published online: 07 Feb 2007.
To cite this article: Hironori Komatsu , Hirokazu Awano , Hiroharu Tanikawa , Kiyoshi Itou & Ichirou Ikeda (2001) LARGE-
SCALE MANUFACTURING OF ALL FOUR 2′-DEOXYNUCLEOSIDES VIA NOVEL STRATEGIES INCLUDING A CHEMO-ENZYMATIC
PROCESS, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1291-1293
To link to this article: http://dx.doi.org/10.1081/NCN-100002539
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1291–1293 (2001)
LARGE-SCALE MANUFACTURING OF ALL
FOUR 2ꢀ-DEOXYNUCLEOSIDES VIA NOVEL
STRATEGIES INCLUDING A
CHEMO-ENZYMATIC PROCESS
Hironori Komatsu,1,∗ Hirokazu Awano,1 Hiroharu Tanikawa,1
Kiyoshi Itou,2 and Ichirou Ikeda2
1Chemical Synthesis Laboratory
2Life Science Laboratory, Mitsui Chemicals Inc.,
1144 Togo, Mobara-shi, Chiba 297-0017, Japan
ABSTRACT
A chemical synthesis of 2-deoxyribose-1-phosphate 2 and its enzymatic con-
version into purine 2ꢀ-deoxynucleosides (dNus) are shown. Besides the chemo-
enzymatic process for purine dNus, a modified process for practical dC prepa-
ration is also established. Consequently, a series of practical manufacturing
processes of all four dNus have been realized via novel strategies.
Due to the recent success with clinical development of antisense drugs, de-
mand for 2ꢀ-deoxynucleosides (dNus) is increasing rapidly. The dNus are key
raw materials for the preparation of antisense drugs. Since current supply com-
pletely depends on salmon milt, development of an effective large-scale production
method that avoids the dependence of such natural resources has been an im-
portant objective for us. Although, thymidine is presently synthesized in a large
scale all over the world including our plant, synthesis of 2ꢀ-deoxycytidine (dC)
7 requires alternative chemistry for its scale-up. Furthermore, the synthesis of
purine dNus still remains a practical challenge using strategies reported in the
literature (1).
1291
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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KOMATSU ET AL.
Figure 1. a, 1) phosphate; 2) hydrolysis, 63%; b, adenine, PNPase, 82%; c, guanine, PNPase, 72%.
Chemo-Enzymatic Process for dA and dG (Fig. 1)
Preparation of purine dNus, dA 3 and dG 4 is a practical challenge since the
reaction of purine base and activated 2-deoxyribose produces both anomeric and
regio-isomers (1). A combination of chemical synthesis of enzymic substrate 2 and
itsenzymaticconversion(2)hasrealizedapracticalwayforthemanufacturingofdA
and dG. Thus, a phosphate addition to 3, 5-O-bis(4-chlorobenzoyl)-2-deoxyribosyl-
1-chloride 1 followed by hydrolysis afforded 2 in 63% isolated yield. Following
enzymatic conversion of the synthetic 2-deoxyribose-1-phosphate 2 using purine
nucleoside phosphorylase (PNPase) gave dA 3 or dG 4 as a single product in
82% and 72% isolated yields, respectively. Optimization of the reaction conditions
and the reaction mechanism are further investigated. Their details will be reported
elsewhere.
A Practical Method for dC Preparation (Fig. 2)
Many experimental applications have been reported for preparation of vari-
ous dC derivatives (3). However, they weren’t suitable to manufacture of dC 7 for
practical reasons like the requirement of excess reagents, slow reaction, or require-
ment of high-dilution conditions. An alteration of conventional reactive amines into
N-methylpiperidine has accelerated the reaction rate enough to establish a manu-
facturing method for dC 7. Thus, dC 7 was synthesized through an ammonolysis
step (5 → 6) in 53% isolated total yield starting from 1.
In summary, a series of practical manufacturing processes of dNus are demon-
strated. For purine dNus, a chemo-enzymatic strategy using a practically applicable

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MANUFACTURING OF ALL FOUR 2ꢀ-DEOXYNUCLEOSIDES
1293
Figure 2. a, persililated uracil; b, 1) TsCl, TEA, N-methylpiperidine; 2) NH4OH, 55%; c, hydrolysis
then HCl, 96%.
process resulted in an anomer-specific conversion into dA 3 or dG 4. By a modified
synthetic method, dC 7 was prepared in a practical way.
REFERENCES
1. Kawakami, H. et al., Chem. Lett., 1989, 235–238.
2. Friedkin, M. et al., J. Biol. Chem., 1950, 184, 449–459; Friedkin, M. et al., J. Biol.
Chem., 1950, 184, 461–464.
3. Reese, C. B. et al., Tetrahedron Lett., 1980, 21, 2265–2268.

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