U.S. patent application number 11/287212 was filed with the patent office on 2006-05-04 for selective process for producing an anomer of a 1-phosphorylated saccharide derivative and process for producing a nucleoside.
This patent application is currently assigned to MITSUI CHEMICALS, INC.. Invention is credited to Tomoyuki Ando, Tadashi Araki, Tamotsu Asano, Hirokazu Awano, Junya Fujiwara, Nobuyuki Fukazawa, Ichirou Ikeda, Kiyoshi Ito, Hironori Komatsu, Kyoko Maruyama, Hitoki Miyake, Takeshi Nakamura, Katsutoshi Tsuchiya, Hideki Umetani, Takahiro Yamauchi.
Application Number | 20060094869 11/287212 |
Document ID | / |
Family ID | 27342316 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060094869 |
Kind Code |
A1 |
Komatsu; Hironori ; et
al. |
May 4, 2006 |
Selective process for producing an anomer of a 1-phosphorylated
saccharide derivative and process for producing a nucleoside
Abstract
A desired isomer is selectively prepared by phosphorolyzing and
isomerizing an anomer mixture of a 1-phosphorylated saccharide
derivative while crystallizing one of the isomers to displace the
equilibrium. Furthermore, using the action of a nucleoside
phosphorylase, a nucleoside is prepared from the 1-phosphorylated
saccharide derivative obtained and a base with improved
stereoselectivity and a higher yield. This process is an
anomer-selective process for preparing a 1-phosphorylated
saccharide derivative and a nucleoside.
Inventors: |
Komatsu; Hironori;
(Mobara-shi, JP) ; Awano; Hirokazu; (Mobara-shi,
JP) ; Fukazawa; Nobuyuki; (Mobara-shi, JP) ;
Ito; Kiyoshi; (Mobara-shi, JP) ; Ikeda; Ichirou;
(Mobara-shi, JP) ; Araki; Tadashi; (Mobara-shi,
JP) ; Nakamura; Takeshi; (Mobara-shi, JP) ;
Asano; Tamotsu; (Mobara-shi, JP) ; Fujiwara;
Junya; (Mobara-shi, JP) ; Ando; Tomoyuki;
(Mobara-shi, JP) ; Tsuchiya; Katsutoshi;
(Mobara-shi, JP) ; Maruyama; Kyoko; (Mobara-shi,
JP) ; Umetani; Hideki; (Mobara-shi, JP) ;
Yamauchi; Takahiro; (Mobara-shi, JP) ; Miyake;
Hitoki; (Mobara-shi, JP) |
Correspondence
Address: |
BUCHANAN INGERSOLL PC;(INCLUDING BURNS, DOANE, SWECKER & MATHIS)
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
MITSUI CHEMICALS, INC.
Chiyoda-ku
JP
|
Family ID: |
27342316 |
Appl. No.: |
11/287212 |
Filed: |
November 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09958305 |
May 7, 2002 |
|
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PCT/JP01/00968 |
Feb 13, 2001 |
|
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11287212 |
Nov 28, 2005 |
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Current U.S.
Class: |
536/26.1 |
Current CPC
Class: |
C07H 19/10 20130101;
C07H 19/00 20130101; C07H 19/20 20130101 |
Class at
Publication: |
536/026.1 |
International
Class: |
C07H 19/04 20060101
C07H019/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2000 |
JP |
2000-033212 |
Mar 10, 2000 |
JP |
2000-067333 |
Nov 9, 2000 |
JP |
2000-341960 |
Claims
1. A process for selectively preparing either .alpha. or .beta.
isomer of a 1-phosphorylated saccharide derivative monomer
comprising the steps of phosphorolyzing and isomerizing an anomer
mixture of a 1-phosphorylated saccharide derivative to give .alpha.
and .beta. isomers of the 1-phosphorylated saccharide derivative
monomer and selectively crystallizing one of these isomers to
displace the equilibrium between these anomers.
2. A process for selectively preparing either .alpha. or .beta.
isomer of a 1-phosphorylated saccharide derivative monomer
comprising the steps of phosphorolyzing and isomerizing an anomer
mixture of a 1-phosphorylated saccharide derivative represented by
formula (1): ##STR24## where R.sup.1 and R.sup.2 independently
represents hydrogen, methyl, protected hydroxymethyl or protected
carboxyl; R.sup.3 represents acyl; R.sup.4 represents a protective
group for hydroxy; X represents halogen, alkoxy or alkylthio; W
represents oxygen or sulfur; Z represents oxygen, sulfur or
optionally substituted carbon; m represents an integer of 1 to 3; n
represents 0 or 1; p and q represents an integer of 0 to 4; and r
represents 0 or 1; provided that p, q, r and n meet the conditions
of p+r.ltoreq.n+1 and q.ltoreq.2x(n+1)-2x(p+r) when Z is oxygen or
sulfur and of p+r.ltoreq.n+2 and q.ltoreq.2x(n+2)-2x(p+r) when Z is
carbon, to give .alpha. and .beta. isomers of the 1-phosphorylated
saccharide derivative monomer and selectively crystallizing one of
these isomers to displace the equilibrium between these
anomers:
3. A process for preparing a 1-phosphorylated saccharide derivative
monomer represented by formula (3): ##STR25## wherein R.sup.1 and
R.sup.2 independently represents hydrogen, methyl, hydroxymethyl or
carboxyl; R.sup.3 represents hydrogen or acyl; and X, W, Z, n, p, q
and r are as defined above, comprising the steps of phosphorolyzing
and isomerizing an anomer mixture of a 1-phosphorylated saccharide
derivative represented by formula (1): ##STR26## wherein R.sup.1,
R.sup.2, R.sup.3, R.sup.4, X, W, Z, m, n, p, q and r are as defined
in claim 2, to give .alpha. and .beta. isomers of the
1-phosphorylated saccharide derivative monomer; selectively
crystallizing one of these isomers to displace the equilibrium
between these anomers; and then removing the protective group
represented by R.sup.4.
4. A trimer, dimer or monomer of a 1-phosphorylated saccharide
derivative represented by formula (4): ##STR27## wherein R.sup.1
and R.sup.2 independently represents hydrogen, methyl,
hydroxymethyl protected with substituted benzoyl or protected
carboxyl; R.sup.4 represents hydrogen or a protective group for
hydroxy; and R.sup.3, X, W, Z, m, n, p, q and r are as defined in
claim 2, or salts thereof.
5. A 1-phosphorylated saccharide derivative monomer represented by
formula (5): ##STR28## wherein p and q represents an integer of 0
to 3; r represents 0 or 1; and R.sup.1, R.sup.2, R.sup.3, R.sup.4,
X, W and Z are as defined in claim 2; provided that p, q and r meet
the conditions of p+q+r.ltoreq.3 when Z is oxygen or sulfur and of
p+q+r.ltoreq.5 when Z is carbon, or salts thereof.
6. A 1-phosphorylated saccharide derivative monomer represented by
formula (6): ##STR29## wherein R.sup.1 and R.sup.2 independently
represents hydrogen, methyl, hydroxymethyl or carboxy; and R.sup.3,
X, W, Z, n, p, q and r are as defined in claim 2 other than natural
products, or salts thereof.
7. The 1-phosphorylated saccharide derivative monomer as claimed in
claim 6 wherein n=1 in formula (6), or its salt.
8. The 1-phosphorylated saccharide derivative monomer as claimed in
claim 7 wherein R.sup.1 is hydroxymethyl; R.sup.2 is hydrogen; p
and r are 0; and X is fluorine, or its salt.
9. A process for preparing a 1-phosphorylated saccharide
represented by formula (20): ##STR30## wherein R.sup.11 represents
protected hydroxymethyl and R.sup.14 represents a protective group
for hydroxy, comprising the steps of treating a compound
represented by formula (18): ##STR31## wherein R.sup.11 and
R.sup.14 are as defined above, with phosphoric acid in the presence
of a base to give an anomer mixture of a 1-phosphorylated
saccharide derivative represented by formula (19): ##STR32##
wherein R.sup.11 and R.sup.14 are as defined above and m is as
defined in claim 2; phosphorolyzing and isomerizing the mixture;
and displacing the equilibrium between the anomer isomers by
selectively crystallizing an .alpha.-isomer formed.
10. A process for preparing 2-deoxy-.alpha.-D-ribose-1-phosphate
comprising the steps of treating a compound represented by formula
(18): ##STR33## wherein R.sup.11 represents protected hydroxymethyl
and R.sup.14 represents a protective group for hydroxy, with
phosphoric acid in the presence of a base to give an anomer mixture
of a 1-phosphorylated saccharide derivative represented by formula
(19): ##STR34## wherein R.sup.11 and R.sup.14 are as defined above
and m is as defined in claim 2; phosphorolyzing and isomerizing the
mixture; displacing the equilibrium between the anomer isomers by
selectively crystallizing an .alpha.-isomer formed to give the
.alpha.-isomer; and then removing the protective group.
11-20. (canceled)
21. A synthetic nucleoside represented by formula (11): ##STR35##
wherein B, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W, Z, n, p, q and
r are as defined for formulas (8) in claim 11 or its salt,
excluding trifluorothymidine, ribavirin, orotidine, uracil
arabinoside, adenine arabinoside, 2-methyl-adenine arabinoside,
2-chloro-hypoxanthine arabinoside, thioguanine arabinoside,
2,6-diaminopurine arabinoside, cytosine arabinoside, guanine
arabinoside, thymine arabinoside, enocitabine, gemcitabine,
azidothymidine, idoxuridine, dideoxyadenosine, dideoxyinosine,
dideoxycytidine, didehydrodeoxythymidine, thiadideoxycytidine,
sorivudine, 5-methyluridine, virazole, thioinosine, tegafur,
doxifluridine, bredinin, nebularine, allopurinol uracil,
5-fluorouracil, 2'-aminouridine, 2'-aminoadenosine,
2'-aminoguanidine, 2-chloro-2'-aminoinosine, DMDC and FMDC.
22. A synthetic nucleoside represented by formula (12): ##STR36##
wherein B, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W, Z, n, p, q and
r are as defined for formulas (8) in claim 11 or its salt,
excluding trifluorothymidine, ribavirin, orotidine, uracil
arabinoside, adenine arabinoside, 2-methyl-adenine arabinoside,
2-chloro-hypoxanthine arabinoside, thioguanine arabinoside,
2,6-diaminopurine arabinoside, cytosine arabinoside, guanine
arabinoside, thymine arabinoside, enocitabine, gemcitabine,
azidothymidine, idoxuridine, dideoxyadenosine, dideoxyinosine,
dideoxycytidine, didehydrodeoxythymidine, thiadideoxycytidine,
sorivudine, 5-methyluridine, virazole, thioinosine, tegafur,
doxifluridine, bredinin, nebularine, allopurinol uracil,
5-fluorouracil, 2'-aminouridine, 2'-aminoadenosine,
2'-aminoguanidine, 2-chloro-2'-aminoinosine, DMDC and FMDC.
23. A nucleoside represented by formula (13): ##STR37## wherein B,
R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W, Z, n, p, q and r are as
defined for formulas (8) in claim 11 or its salt, excluding
trifluorothymidine, ribavirin, orotidine, uracil arabinoside,
adenine arabinoside, 2-methyl-adenine arabinoside,
2-chloro-hypoxanthine arabinoside, thioguanine arabinoside,
2,6-diaminopurine arabinoside, cytosine arabinoside, guanine
arabinoside, thymine arabinoside, enocitabine, gemcitabine,
azidothymidine, idoxuridine, dideoxyadenosine, dideoxyinosine,
dideoxycytidine, didehydrodeoxythymidine, thiadideoxycytidine,
sorivudine, 5-methyluridine, virazole, thioinosine, tegafur,
doxifluridine, bredinin, nebularine, allopurinol uracil,
5-fluorouracil, 2'-aminouridine, 2'-aminoadenosine,
2'-aminoguanidine, 2-chloro-2'-aminoinosine, DMDC and FMDC.
24. A 1-phosphorylated saccharide represented by formula (20):
##STR38## wherein R.sup.11 represents protected hydroxymethyl; and
R.sup.14 represents a protective group for hydroxy, or its salt.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a process for producing a
1-phosphorylated saccharide derivative. 1-phosphorylated
saccharides are widely distributed in the living world, are
reaction substrates for a variety of enzymes and are utilized
starting materials for preparing useful substances such as drugs
and nutritional foods. Synthetic 1-phosphorylated saccharide
derivatives have been expected to be used as starting materials for
preparing drugs such as antiviral agents and enzyme inhibitors.
[0003] This invention also relates to a process for producing a
nucleoside compound used as a starting material or drug substance
for medical drugs such as antiviral, anticancer and antisense
drugs.
[0004] 2. Description of the Prior Art
[0005] There are known processes for producing a 1-phosphorylated
saccharide such as:
[0006] 1) condensation of a 1-bromosaccharide with a silver
phosphate salt (J. Biol. Chem., Vol. 121, p. 465 (1937); J. Am.
Chem. Soc., Vol. 78, p. 811 (1956); J. Am. Chem. Soc., Vol. 79, p.
5057 (1957));
[0007] 2) condensation of a 1-halogenated saccharide with a
triethylamine salt of dibenzylphosphoric acid (J. Am. Chem. Soc.,
Vol. 77, p. 3423 (1955); J. Am. Chem. Soc., Vol. 80, p. 1994
(1958); J. Am. Chem. Soc., Vol. 106, p. 7851 (1984); J. Org. Chem.,
Vol. 59, p. 690 (1994));
[0008] 3) thermal condensation of a 1-acetylated saccharide with
orthophosphoric acid (J. Org. Chem., Vol. 27, p. 1107 (1962);
Carbohydrate Res., Vol. 3, p. 117 (1966); Carbohydrate Res., Vol.
3, p. 463 (1967); Can. J. Biochem., Vol. 50, p. 574 (-1972));
[0009] 4) condensation of dibenzylphoshoric acid with a saccharide
activated at 1-position by imidation (Carbohydrate Res., Vol. 61,
p. 181 (1978); Tetrahedron Lett., Vol. 23, p. 405 (1982));
[0010] 5) treatment of a saccharide activated at 1-position by
thallium or lithium alcolate with dibenzylphosphoric chloride
(Carbohydrate Res., Vol. 94, p. 165 (1981); Chem. Lett., Vol. 23,
p. 405 (1982));
[0011] 6) phosphorolysis of a nucleoside utilizing action of
nucleoside phosphorylase to form a 1-phoshorylated saccharide
derivative (J. Biol. Chem., Vol. 184, p. 437 (1980)).
[0012] These processes have the following drawbacks.
[0013] A common problem in the chemical processes described in the
above 1) to 5) is that it is difficult to establish a general
synthetic method for preparing a desired isomer with a good
selectivity due to variation in an anomer selectivity between
.alpha./.beta. anomers owing to influence of a functional group
adjacent to 1-position. For achieving selectivity and a good yield,
the presence of 2-acetoxy or acetamino group is essential. However,
since 2-deoxysaccharide is unstable, these synthetic processes may
be limited to a considerably narrow application range. Thus, it is
difficult to control anomer selectivity so that column
chromatography purification is required, leading to a poor yield
(Chem. Zvesti, Vol. 28(1), p. 115 (1974); Izv. Akad. Nauk SSSR,
Ser. Khim., Vol. 8, p. 1843 (1975)).
[0014] Of course, there have been no reports for chemical
preparation of a 1-phosphorylated 2-deoxyfuranose which is more
unstable than a 1-phosphorylated 2-deoxypyranose, resulting in more
difficult selectivity control.
[0015] In terms of 6), preparation of a nucleoside itself is
difficult except a quite limited type of rebonucleosides such as
inosine. A limited type of 1-phosphorylated saccharide derivatives
such as ribose-1-phosphate can be, therefore, prepared. In
addition, since a nucleoside itself as a starting material is
expensive, the process is not satisfactory in its cost.
[0016] As described above, the term "nucleoside phosphorylase" is a
generic name for enzymes capable of phosphorolysis of an
N-glycoside bond in a nucleoside in the presence of phosphoric
acid, which catalyze a reaction represented by the following
equation: Nucleoside+Phosphoric acid
(salt).fwdarw.Base+1-Phosphorylated saccharide derivative
[0017] The enzymes which may be generally categorized into two
groups of purine nucleoside phosphorylases and pyrimidine
nucleoside phosphorylases, are widely distributed in the living
world; they are present in tissues of mammals, birds and fish;
yeasts; and bacteria. The enzyme reaction is reversible and there
have been disclosed methods for synthesis of a variety of
nucleosides utilizing a reverse reaction; for syntheses of
thymidine (thymine, adenine or guanine) (JP-A 01-104190),
2'-deoxyadenosine (JP-A 11-137290) or 2'-deoxyguanosine (JP-A
11-137290) from 2'-deoxyribose 1-=phosphate and a nucleic-acid
base.
[0018] Furthermore, Agric. Biol. Chem., Vol. 50 (1), pp. 121-126
(1986) has described a process where by a reaction using a purine
nucleoside phosphorylase from Enterobacter aerogenes in the
presence of phosphoric acid, inosine is decomposed into ribose
1-phosphate and hypoxanthine and the former isolated using an
ion-exchange resin and 1,2,4-triazole-3-carboxamide are also
treated with a purine nucleoside phosphorylase from Enterobacter
aerogenes to prepare ribavirin as an antiviral agent.
[0019] However, as described above, an industrial process for
producing a 1-phosphorylated saccharide derivative has not been
established, and thus an industrial process for preparation of a
universally useful nucleoside utilizing a reverse reaction of a
nucleoside phosphorylase has been also not established.
[0020] Furthermore, since the reaction for forming a nucleoside
from 1-phosphorylated saccharide derivative and a base utilizing
the reverse reaction of the enzyme is reversible, there is a
technical drawback that an inversion rate cannot be improved.
SUMMARY OF THE INVENTION
[0021] An objective of this invention is to provide a highly
universal and anomer-selective process for preparing
1-phospholyrated saccharide derivative which is not influenced by
difference in a saccharide skeleton such as furanose and pyranose,
presence of a substituent such as a deoxysaccharide or a saccharide
type, i.e., natural or synthetic.
[0022] Another objective of this invention is to provide a highly
universal process for producing a nucleoside by treating a
1-phosphorylated saccharide derivative and a nucleic-acid base with
a nucleoside phosphorylase and a method for improving an inversion
rate for the nucleoside in the reaction.
[0023] In other words, the ultimate objective of this invention is
to provide a process for producing a highly pure nucleoside with a
lower cost by achieving the first and the second objectives
above.
[0024] We have intensely made attempts for achieving the first
objective. Finally, we have found that a 1-phosphorylated
saccharide derivative is present in an equilibrium with an anomer
and a dimer of the 1-pohsphorylated saccharide derivative under
certain conditions and that the conditions may be adjusted to allow
only a desired anomer to be precipitated as crystals so that the
equilibrium may be displaced toward the preferable direction to
provide the desired anomer with good selectivity and a high yield.
Thus, based on the findings, we have achieved this invention.
[0025] Specifically, this invention encompasses the following
embodiments.
[0026] (1) A process for selectively preparing either .alpha. or
.beta. isomer of a 1-phosphorylated saccharide derivative monomer
comprising the steps of phosphorolyzing and isomerizing an anomer
mixture of a 1-phosphorylated saccharide derivative to give .alpha.
and .beta. isomers of the 1-phosphorylated saccharide derivative
monomer and selectively crystallizing one of these isomers to
displace the equilibrium between these anomers.
[0027] (2) A process for selectively preparing either .alpha. or
.beta. isomer of a 1-phosphorylated saccharide derivative monomer
comprising the steps of phosphorolyzing and isomerizing an anomer
mixture of a 1-phosphorylated saccharide derivative represented by
formula (1): ##STR1##
[0028] where R.sup.1 and R.sup.2 independently represents hydrogen,
methyl, protected hydroxymethyl or protected carboxyl; R.sup.3
represents acyl; R.sup.4 represents a protective group for hydroxy;
X represents halogen, alkoxy or alkylthio; W represents oxygen or
sulfur; Z represents oxygen, sulfur or optionally substituted
carbon; m represents an integer of 1 to 3; n represents 0 or 1; p
and q represents an integer of 0 to 4; and r represents 0 or 1;
provided that p, q, r and n meet the conditions of p+r.ltoreq.n+1
and q.ltoreq.2x(n+1)-2x(p+r) when Z is oxygen or sulfur and of
p+r.ltoreq.n+2 and q.ltoreq.2x(n+2)-2x(p+r) when Z is carbon, to
give .alpha. and .beta. isomers of the 1-phosphorylated saccharide
derivative monomer and selectively crystallizing one of these
isomers to displace the equilibrium between these anomers:
[0029] (3) A process for preparing a 1-phosphorylated saccharide
derivative monomer represented by formula (3): ##STR2##
[0030] wherein R.sup.1 and R.sup.2 independently represents
hydrogen, methyl, hydroxymethyl or carboxyl; R.sup.3 represents
hydrogen or acyl; and X, W, Z, n, p, q and r are as defined for
formula (1), comprising the steps of phosphorolyzing and
isomerizing an anomer mixture of a 1-phosphorylated saccharide
derivative represented by formula (1) to give .alpha. and .beta.
isomers of the 1-phosphorylated saccharide derivative monomer;
selectively crystallizing one of these isomers to displace the
equilibrium between these anomers; and then removing the protective
group represented by R.sup.4.
[0031] (4) A trimer, dimer or monomer of a 1-phosphorylated
saccharide derivative represented by formula (4): ##STR3##
[0032] wherein R.sup.1 and R.sup.2 independently represents
hydrogen, methyl, hydroxymethyl protected with substituted benzoyl
or protected carboxyl; R.sup.4 represents hydrogen or a protective
group for hydroxy; and R.sup.3, X, W, Z, m, n, p, q and r are as
defined for formula (1), or salts thereof.
[0033] (5) A 1-phosphorylated saccharide derivative monomer
represented by formula (5): ##STR4##
[0034] wherein p and q represents an integer of 0 to 3; r
represents 0 or 1; and R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W and
Z are as defined for formula (1); provided that p, q and r meet the
conditions of p+q+r.ltoreq.3 when Z is oxygen or sulfur and of
p+q+r.ltoreq.5 when Z is carbon, or salts thereof.
[0035] (6) A 1-phosphorylated saccharide derivative monomer
represented by formula (6): ##STR5##
[0036] wherein R.sup.1 and R.sup.2 independently represents
hydrogen, methyl, hydroxymethyl or carboxy; and R.sup.3, X, W, Z,
n, p, q and r are as defined for formula (1), or salts thereof.
[0037] (7) A 1-phosphorylated saccharide derivative monomer
represented by formula (7): ##STR6##
[0038] wherein p and q represents an integer of 0 to 3; r
represents 0 or 1; and R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W and
Z are as defined for formula (1); provided that p, q and r meet the
conditions of p+r.ltoreq.1, q.ltoreq.2-2x(p+r) when Z is oxygen or
sulfur and of p+r.ltoreq.2, q.ltoreq.4-2x(p+r) when Z is carbon, or
salts thereof.
[0039] (8) A process for preparing a 1-phosphorylated saccharide
represented by formula (20): ##STR7##
[0040] wherein R.sup.11 represents protected hydroxymethyl and
R.sup.14 represents a protective group for hydroxy, comprising the
steps of treating a compound represented by formula (18):
##STR8##
[0041] wherein R.sup.11 and R.sup.14 are as defined above, with
phosphoric acid in the presence of a base to give an anomer mixture
of a 1-phosphorylated saccharide derivative represented by formula
(19): ##STR9##
[0042] wherein R.sup.11 and R.sup.14 are as defined above and m is
as defined in claim 2; phosphorolyzing and isomerizing the mixture;
and displacing the equilibrium between the anomer isomers by
selectively crystallizing an .alpha.-isomer formed.
[0043] (9) A process for preparing
2-deoxy-.alpha.-D-ribose-1-phosphate, comprising the steps of
treating a compound represented by formula (18): ##STR10##
[0044] wherein R.sup.11 represents protected hydroxymethyl and
R.sup.14 represents a protective group for hydroxy, with phosphoric
acid in the presence of a base to give an anomer mixture of a
1-phosphorylated saccharide derivative represented by formula (19):
##STR11##
[0045] wherein R.sup.11 and R.sup.14 are as defined above and m is
as defined in claim 2; phosphorolyzing and isomerizing the mixture;
displacing the equilibrium between the anomer isomers by
selectively crystallizing an .alpha.-isomer formed to give the
.alpha.-isomer; and then removing the protective group.
[0046] We have intensely attempted for achieving the second
objective and thus have established a highly universal process for
preparing a nucleoside by utilizing a reverse reaction of
nucleoside phosphorylases widely distributed in the living world in
combination with the above preparation processes for a
1-phosphorylated saccharide derivative. We have further found that
a metal cation capable of forming a water-insoluble salt with a
phosphate ion may be present to allow a phosphate ion as a
byproduct in the reaction to be precipitated as a water-insoluble
salt, resulting in displacement of the reaction equilibrium toward
the direction for nucleoside production and thus improvement in a
reaction yield. Thus, we have achieved this invention providing a
process for preparing a highly pure nucleoside with a lower
cost.
[0047] This invention based on the above findings encompasses the
following embodiments.
[0048] (10) A process for preparing a nucleoside represented by
formula (8): ##STR12##
[0049] wherein B is a base independently selected from the group
consisting of pyrimidine, purine, azapurine and deazapurine
optionally substituted by halogen, alkyl, haloalkyl, alkenyl,
haloalkenyl, alkynyl, amino, alkylamino, hydroxy, hydroxyamino,
aminoxy, alkoxy, mercapto, alkylmercapto, aryl, aryloxy or cyano;
and R.sup.1, R.sup.2, R.sup.3, X, W, Z, n, p, q and r are as
defined for formula (1), comprising
[0050] the first procedure in the above (3) for preparing a
1-phosphorylated saccharide derivative monomer comprising the steps
of phosphorolyzing and isomerizing an anomer mixture of a
1-phosphorylated saccharide derivative to give .alpha. and .beta.
isomers of the 1-=phosphorylated saccharide derivative monomer;
selectively crystallizing one of these isomers to displace the
equilibrium between these anomers; and then removing the protective
group represented by R.sup.4; and [0051] the second procedure of
conducting an exchange reaction of the phosphate group in the
1-phosphorylated saccharide derivative obtained in the first
procedure with a base by the action of a nucleoside
phosphorylase.
[0052] (11) A process for preparing a nucleoside represented by
formula (9): ##STR13##
[0053] wherein B is as defined for formula (8); and R.sup.1,
R.sup.2, R.sup.3, R.sup.4, X, W, Z, n, p, q and r are as defined
for formula (1), comprising an exchange reaction of the phosphate
group in the 1-phosphorylated saccharide derivative monomer in the
above (6) with a base by the action of a nucleoside
phosphorylase.
[0054] (12) A process for preparing a nucleoside represented by
formula (10): ##STR14##
[0055] wherein B is as defined for formula (8); and R.sup.1,
R.sup.2, R.sup.3, R.sup.4, X, W, Z, p, q and r are as defined for
formula (1), comprising an exchange reaction of the phosphate group
in the 1-phosphorylated saccharide derivative monomer in the above
(7) with a base by the action of a nucleoside phosphorylase.
[0056] (13) A process for preparing a nucleoside represented by
formula (21): ##STR15##
[0057] wherein B is as defined for formula (8) in claim 11,
comprising
[0058] the first procedure of preparing
2-deoxy-.alpha.-D-ribose-1-phosphate in the above (12) where
R.sup.1 is hydroxymethyl, R.sup.2 is hydrogen, p and r are 0, and X
is fluorine; and [0059] the second procedure of conducting an
exchange reaction of the phosphate group in the 1-phosphorylated
saccharide derivative obtained in the first procedure with a base
by the action of a nucleoside phosphorylase.
[0060] In the embodiments of the above (10) to (13), a nucleoside
phosphorylase may be at least one selected from the group
consisting of purine nucleoside phosphorylase (EC2.4.2.1),
guanosine nucleoside phosphorylase (EC2.4.2.15), pyrimidine
nucleoside phosphorylase (EC2.4.2.2), uridine nucleoside
phosphorylase (EC2.4.2.3), thymidine nucleoside phosphorylase
(EC2.4.2.4) and deoxyuridine nucleoside phosphorylase
(EC2.4.2.23).
[0061] A nucleoside phosphorylase activity may be obtained using a
microorganism expressing at least one nucleoside phosphorylase
selected from the group consisting of purine nucleoside
phosphorylase (EC2.4.2.1), guanosine nucleoside phosphorylase
(EC2.4.2.15), pyrimidine nucleoside phosphorylase (EC2.4.2.2),
uridine nucleoside phosphorylase (EC2.4.2.3), thymidine nucleoside
phosphorylase (EC2.4.2.4) and deoxyuridine nucleoside phosphorylase
(EC2.4.2.23).
[0062] In the embodiments of the above (10) to (13), a metal cation
capable of forming a water-insoluble salt with a phosphate ion may
be present in the reaction solution during the exchange reaction of
a phosphate group in the 1-phosphorylated saccharide derivative
monomer with a base by the action of a nucleoside
phosphorylase.
[0063] The metal cation capable of forming a water-insoluble salt
with the phosphate ion in the embodiments of the above (10) to (13)
may be at least one metal cation selected from the group consisting
of calcium, barium, aluminum and magnesium ions.
[0064] Furthermore, this invention encompasses a compound
represented by any of formulas (11) to (13) and (20).
[0065] That is, this invention also encompasses:
[0066] a synthetic nucleoside, which is not naturally produced
represented by formula (11): ##STR16##
[0067] wherein B, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W, Z, n,
p, q and r are as defined for formulas (1) and (8) or its salt,
excluding trifluorothymidine, ribavirin, orotidine, uracil
arabinoside, adenine arabinoside, 2-methyl-adenine arabinoside,
2-chloro-hypoxanthine arabinoside, thioguanine arabinoside,
2,6-diaminopurine arabinoside, cytosine arabinoside, guanine
arabinoside, thymine arabinoside, enocitabine, gemcitabine,
azidothymidine, idoxuridine, dideoxyadenosine, dideoxyinosine,
dideoxycytidine, didehydrodeoxythymidine, thiadideoxycytidine,
sorivudine, 5-methyluridine, virazole, thioinosine, tegafur,
doxifluridine, bredinin, nebularine, allopurinol uracil,
5-fluorouracil, 2'-aminouridine, 2'-aminoadenosine,
2'-aminoguanidine, 2-chloro-2'-aminoinosine, DMDC and FMDC;
[0068] a synthetic nucleoside, which is not naturally produced
represented by formula (12): ##STR17##
[0069] wherein B, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W, Z, n,
p, q and r are as defined for formulas (1) and (8) or its salt,
excluding trifluorothymidine, ribavirin, orotidine, uracil
arabinoside, adenine arabinoside, 2-methyl-adenine arabinoside,
2-chloro-hypoxanthine arabinoside, thioguanine arabinoside,
2,6-diaminopurine arabinoside, cytosine arabinoside, guanine
arabinoside, thymine arabinoside, enocitabine, gemcitabine,
azidothymidine, idoxuridine, dideoxyadenosine, dideoxyinosine,
dideoxycytidine, didehydrodeoxythymidine, thiadideoxycytidine,
sorivudine, 5-methyluridine, virazole, thioinosine, tegafur,
doxifluridine, bredinin, nebularine, allopurinol uracil,
5-fluorouracil, 2'-aminouridine, 2'-aminoadenosine,
2'-aminoguanidine, 2-chloro-2'-aminoinosine, DMDC and FMDC;
[0070] a nucleoside represented by formula (13): ##STR18##
[0071] wherein B, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W, Z, n,
p, q and r are as defined for formulas (1) and (8) or its salt,
excluding trifluorothymidine, ribavirin, orotidine, uracil
arabinoside, adenine arabinoside, 2-methyl-adenine arabinoside,
2-chloro-hypoxanthine arabinoside, thioguanine arabinoside,
2,6-diaminopurine arabinoside, cytosine arabinoside, guanine
arabinoside, thymine arabinoside, enocitabine, gemcitabine,
azidothymidine, idoxuridine, dideoxyadenosine, dideoxyinosine,
dideoxycytidine, didehydrodeoxythymidine, thiadideoxycytidine,
sorivudine, 5-methyluridine, virazole, thioinosine, tegafur,
doxifluridine, bredinin, nebularine, allopurinol uracil,
5-fluorouracil, 2'-aminouridine, 2'-aminoadenosine,
2'-aminoguanidine, 2-chloro-2'-aminoinosine, DMDC and FMDC; and
[0072] a 1-phosphorylated saccharide represented by formula (20):
##STR19##
[0073] wherein R.sup.11 and R.sup.14 are as defined for formula
(18), or its salt.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0074] This invention will be described in detail.
[0075] Saccharides which may used in this invention include, but
not limited to, residues derived from D- and L-type natural
monosaccharides including 6-deoxysaccharides such as fucose,
rhamnose, digitoxose, oleandrose and quinovose, hexoses such as
allose, altrose, glucose, mannose, gulose, idose, galactose and
talose, pentoses such as ribose, arabinose, xylose and lyxose,
tetroses such as erythrose and threose, aminosaccharides such as
glucosamine and daunosamine, uronic acids such as glucuronic acid
and galacturonic acid, ketoses such as psicose, fructose, sorbose,
tagatose and pentulose, and deoxysaccharides such as 2-deoxyribose;
residues derived from synthetic pyranose and furanose saccharides;
and saccharide residue derivatives in which hydroxy and/or amino
groups in any of the above residues are protected or acylated or
saccharides having a halogenated saccharide residue in which
hydroxy is replaced with halogen such as fluorine.
[0076] In this invention, a 1-phosphorylated saccharide derivative
refers to a saccharide derivative in which among residues derived
from natural or synthetic monosaccharide, 1-hydroxy is
phosphorylated. Unless otherwise indicated, it may include a
monomer, dimer or trimer or a mixture thereof, where there are no
restrictions to its mixture ratio.
[0077] A protective group in terms of "protected hydroxymethyl" and
"a protective group of hydroxy" means that which may be removed by
an appropriate chemical process such as hydrogenolysis, hydrolysis
and photolysis, including formyl, acyl, silyl, alkyl, aralkyl,
carbonyl, preferably formyl, aliphatic acyl, aromatic acyl, silyl,
alkoxyalkyl, halogenated alkyl, aralkyl, alkoxycarbonyl and
aralkyloxycarbonyl.
[0078] Aliphatic acyl may be alkylcarbonyl and halogenated lower
alkylcarbonyl.
[0079] Examples of alkylcarbonyl include acetyl, propionyl,
butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl,
octanoyl, nonylcarbonyl, decylcarbonyl, 3-methylnonylcarbonyl,
8-methylnonylcarbonyl, 3-ethyloctylcarbonyl,
3,7-dimethyloctylcarbonyl, undecylcarbonyl, dodecylcarbonyl,
tridecylcarbonyl, tetradecylcarbonyl, pentadecylcarbonyl,
hexadecylcarbonyl, 1-methylpentadecylcarbonyl,
14-methylpentadecylcarbonyl, 13,13-dimethyltetradecylcarbonyl,
heptadecylcarbonyl, 15-methylhexadecylcarbonyl and
octadecylcarbonyl.
[0080] Examples of halogenated lower alkylcarbonyl include
chloroacetyl, dichloroacetyl, trichloroacetyl and
trifluoroacetyl.
[0081] Aromatic acyl may be arylcarbonyl, halogenated arylcarbonyl,
lower-alkylated arylcarbonyl, lower-alkoxylated arylcarbonyl,
nitrated arylcarbonyl, lower-alkoxycarbonylated arylcarbonyl or
arylated arylcarbonyl.
[0082] Examples of arylcarbonyl include benzoyl, .alpha.-naphthoyl
and .beta.-naphthoyl.
[0083] Examples of halogenated arylcarbonyl include
2-fluorobenzoyl, 3-fluorobenzoyl, 4-fluorobenzoyl, 2-chlorobenzoyl,
3-chlorobenzoyl, 4-chlorobenzoyl, 2-bromobenzoyl, 3-bromobenzoyl,
4-bromobenzoyl, 2,4-dichlorobenzoyl, 2,6-dichlorobenzoyl,
3,4-dichlorobenzoyl and 3,5-dichlorobenzoyl.
[0084] Examples of lower-alkylated arylcarbonyl include 2-toluoyl,
3-toluoyl, 4-toluoyl and 2,4,6-trimethylbenzoyl.
[0085] Examples of lower-alkoxy arylcarbonyl include 2-anisoyl,
3-anisoyl and 4-anisoyl.
[0086] Examples of nitrated arylcarbonyl include 2-nitrobenzoyl,
3-nitrobenzoyl, 4-nitrobenzoyl and 3,5-dinitrobenzoyl.
[0087] Examples of lower-alkoxycarbonylated arylcarbonyl include
2-(methoxycarbonyl)benzoyl. Examples of arylated arylcarbonyl
include 4-phenylbenzoyl.
[0088] Silyl may be lower-alkylsilyl and aryl-substituted
lower-alkylsilyl.
[0089] Examples of lower-alkyl silyl include trimethylsilyl,
triethylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl,
methyldiisopropylsilyl and triisopropylsilyl.
[0090] Examples of aryl-substituted lower-alkylsilyl include
diphenylmethylsilyl, diphenylisopropylsilyl and
phenyldiisopropylsilyl.
[0091] Aralkyl may be benzyl, aralkyl substituted with lower alkyl,
aralkyl substituted with lower alkoxy, aralkyl substituted with
nitro, aralkyl substituted with halogen or aralkyl substituted with
cyano.
[0092] Examples of these include 2-methylbenzyl, 3-methylbenzyl,
4-methylbenzyl, 2,4,6-trimethylbenzyl, 2-methoxybenzyl,
3-methoxybenzyl, 4-methoxybenzyl, 2-nitrobenzyl, 3-nitrobenzyl,
4-nitrobenzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl,
2-bromobenzyl, 3-bromobenzyl, 4-bromobenzyl, 2-cyanobenzyl,
3-cyanobenzyl and 4-cyanobenzyl.
[0093] Aralkyloxycarbonyl may be aralkyloxycarbonyl substituted
with lower alkyl, aralkyloxycarbonyl substituted with lower alkoxy,
aralkyloxycarbonyl substituted with nitro, aralkyloxycarbonyl
substituted with halogen or aralkyloxycarbonyl substituted with
cyano.
[0094] Examples of these include 2-methylbenzyloxycarbonyl,
3-methylbenzyloxycarbonyl, 4-methylbenzyloxycarbonyl,
2,4,6-trimethylbenzyloxycarbonyl, 2-methoxybenzyloxycarbonyl,
3-methoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, 3-nitrobenzyloxycarbonyl,
4-nitrobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,
3-chlorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl,
2-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl,
4-bromobenzyloxycarbonyl, 2-cyanobenzyloxycarbonyl,
3-cyanobenzyloxycarbonyl and 4-cyanobenzyloxycarbonyl.
[0095] Alkoxycarbonyl may be lower-alkoxycarbonyl, alkoxycarbonyl
substituted with halogen or alkoxycarbonyl substituted with
alkylsilyl.
[0096] Examples of lower-alkoxycarbonyl include methoxycarbonyl,
ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, sec-butoxycarbonyl
and tert-butoxycarbonyl.
[0097] Examples of alkoxycarbonyl substituted with halogen include
2,2,2-trichloroethoxycarbonyl. Examples of alkoxycarbonyl
substituted with alkylsilyl include
2-trimethylsilylethoxycarbonyl.
[0098] Alkyl may be alkoxyalkyl such as methoxyethyl, ethoxymethyl,
2-methoxyethyl and 2-methoxyethoxymethyl; halogenated alkyl such as
2,2,2-trichloroethyl; or lower alkyl substituted with aryl such as
benzyl, .alpha.-naphthylmethyl, .beta.-naphthylmethyl,
diphenylmethyl and triphenylmethyl.
[0099] Among these, aliphatic acyl, aromatic acyl and aralkyl are
preferable; 4-toluoyl, 4-chlorobenzoyl and benzyl are more
preferable. A protective group in terms of "protected carboxyl" in
R.sup.1 and R.sup.2 refers to that which may be removed by an
appropriate chemical process such as hydrogenolysis, hydrolysis and
photolysis, including preferably lower alkyl such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl;
silylated lower alkyl such as 2-(trimethylsilyl)ethyl and
2-(triethylsilyl)ethyl; or the above aralkyl or alkoxyalkyl, more
preferably methyl, tert-butyl or benzyl.
[0100] Halogen in terms of X refers to fluorine, chlorine, =bromine
or iodine.
[0101] Alkoxy and alkylthio in terms of X may be alkoxy and
alkylthio having the above lower alkyl, aralkyl or alkoxyalkyl,
preferably methoxy, methoxyethoxy or methylthio.
[0102] Optionally substituted carbon in terms of Z refers to carbon
having one or two of the substituent represented by the formula (Xq
and NHR.sup.3) or when having no substituents, carbon having
hydrogen atoms.
[0103] Acyl in terms of R.sup.3 may be the above aliphatic acyl,
aromatic acyl, alkoxycarbonyl or aralkyloxycarbonyl, or
lower-alkanesulfonyl such as methanesulfonyl and
trifluoromethanesulfonyl or arylsulfonyl such as benzenesulfonyl
and p-toluenesulfonyl; preferably, aliphatic acyl, aromatic acyl or
lower-alkanesulfonyl; specifically, acetyl, trifluoroacetyl,
benzoyl and methanesulfonyl. When more than one of NHR.sup.3 are
used as a substituent, R.sup.3s in individual NHR.sup.3
independently represent any of the above radicals.
[0104] A protective group in "protected hydroxymethyl" and "a
protective group for hydroxy" in terms of R.sup.4, R.sup.11 and
R.sup.14 may be selected from those described for R.sup.1 and
R.sup.2.
[0105] Saccharide residues having a structure represented by any of
formulas (1) to (17) may be preferably, but not limited to, those
derived from a natural monosaccharide described above, those
derived from a synthetic saccharide, derivatives from the
saccharide residues or halogenated saccharide residues, as
described above.
[0106] Salts of a compound represented by any of formulas (4) to
(7) may be those formed by a phosphate radical in the compound.
Examples of such a salt include alkali metal salt such as sodium,
potassium and lithium salts; alkaline earth metal salt such as
magnesium, calcium and barium salts; metal salt such as aluminum
and iron salts; ammonium salt; or alkylamine salt such as primary,
secondary and tertiary alkyl amine salts.
[0107] Primary amine herein may be alkylamine such as methylamine,
ethylamine, propylamine, isopropylamine, butylamine, hexylamine and
octylamine; cycloalkylamine such as cyclohexylamine; or
benzylamine.
[0108] Secondary amine may be dialkylamine such as diethylamine,
diisopropylamine, dibutylamine, dihexylamine and dioctylamine;
dicycloalkylamine such as dicyclohexylamine; or cyclic amine such
as piperidine, morpholine and N-methylpiperadine.
[0109] Tertiary amine may be tertiary-alkylamine such as
trimethylamine, triethylamine, tripropylamine,
N-ethyldiisopropylamine, tributylamine, trihexylamine,
trioctylamine, N-ethyldicyclohexylamine, N-methylpiperidine,
N-methylmorpholine and N,N,N',N'-tetramethylethylenediamine;
aniline compound such as aniline, N,N-dimethylaniline,
N,N-diethylaniline, N,N-dibutylaniline and N,N-dioctylaniline;
pyridine compound such as pyridine, 2,6-dimethylpyridine,
2,4,6-lutidine and nicotinamide; amino acid such as glycine,
alanine, proline, lysine, arginine and glutamine; or optically
active amine such as cinchonidine, 1-(1-naphthyl)ethylamine and
1-phenylethylamine, all of which include monovalent and bivalent
salts.
[0110] A compound represented by any of formula (4) to (7) of this
invention may absorb moisture to have adsorbed water or become a
hydrate, all of which may be encompassed by this invention.
[0111] An anomer mixture of a 1-phosphorylated saccharide
derivative according to this invention may be prepared by, but not
limited to, a reaction represented by reaction formula (I):
##STR20##
[0112] In this formula, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W,
Z, m, n, p, q and r are as defined for formula (1), and Y
represents fluorine, chlorine, bromine or iodine. When m is 1, 2 or
3, phosphoric acid tri-, di- or mono-ester is provided,
respectively. These are referred to as 1-phosphorylated saccharide
derivative trimer, 1-phosphorylated saccharide derivative dimer and
1-phosphorylated saccharide derivative monomer, respectively.
Furthermore, 1-phosphorylated saccharide derivative trimer,
1-phosphorylated saccharide derivative dimer and 1-phosphorylated
saccharide derivative monomer are collectively referred to as a
1-phosphorylated saccharide derivative, for which there are no
restrictions to its mixture ratio.
[0113] A preferable phosphoric acid may be, but not limited to, one
with a lower water content such as orthophosphoric acid.
[0114] There are no restrictions to a base as long as it does not
inhibit the reaction and functions as a deoxidizer. Preferable
inorganic bases include carbonates and hydroxides of alkali and
alkaline earth metals. Preferable organic bases include tertiary
alkylamines, anilines, pyridines and optically active amines.
[0115] A dehydrating agent may be used when moisture from =a
solvent or an additive adversely affects the reaction. There are no
restrictions to a dehydrating agent as long as it has adequate
adsorptivity or reactivity with water; preferably molecular sieves
and phosphorus pentoxide.
[0116] The reaction is generally conducted in the presence of a
solvent. There are no restrictions to a solvent as long as it does
not inhibit the reaction and dissolve starting materials to some
degree. Solvents which may be used include aliphatic hydrocarbons
such as hexane and heptane; aromatic hydrocarbons such as benzene,
toluene, xylene and anisole; halogenated hydrocarbons such as
methylene chloride, chloroform, carbon tetrachloride,
dichloroethane, chlorobenzene and dichlorobenzene; esters such as
ethyl formate, ethyl acetate, propyl acetate, n-butyl acetate and
diethyl carbonate; ethers such as diethyl ether, diisopropyl ether,
tetrahydrofuran, dioxane, dimethoxyethane and diglyme; nitriles
such as acetonitrile, propionitrile and isobutylnitrile; amides
such as formamide, N,N-dimethylformamide, N,N-dimethylacetamide,
N-methyl-2-pyrrolidone, N-methylpyrrolidinone and
N,N-dimethyl-2-imidazolydinone; ketones such as acetone,
2-butanone, methyl isopropyl ketone and methyl isobutyl ketone; and
a mixture of two or more selected therefrom.
[0117] There are no restrictions to a reaction temperature;
generally -80.degree. C. to 60.degree. C., preferably -10.degree.
C. to 25.degree. C.
[0118] A reaction period may vary depending on many factors such as
starting materials, reagents, the type of a solvent and a reaction
temperature; generally 1 min to 24 hours, preferably 10 min to 2
hours for completing the reaction.
[0119] There are no restrictions to a ratio of a saccharide
derivative (14) to phosphoric acid; the reaction is generally
conducted with a ratio of compound (14): phosphoric acid=1:10 to
3:1. In this case, the product (1) may be a mixture of the
compounds whose saccharide residue number (i.e., m) coupled with
phosphoric acid is 1, 2 or 3, depending on the ratio of compound
(14): phosphoric acid.
[0120] Furthermore, a 1-phosphorylated saccharide derivative (16a)
or (16b) with either .alpha.- or .beta.-form may be prepared by a
reaction represented by reaction formula (II): ##STR21##
[0121] In this formula, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W,
Z, m, n, p, q and r are as defined for formula (1).
[0122] According to this preparation process, the 1-phosphorylated
saccharide derivative represented by formula (15) may be a monomer,
dimer or trimer or a mixture thereof in any mixture ratio because
they may be converted into the 1-phosphorylated saccharide
derivative represented by formula (16) in the reaction system.
[0123] A preferable phosphoric acid may be, but not limited to, one
with a lower water content such as orthophosphoric acid.
[0124] A base is important for forming a salt with the phosphate
group in compound (16) to selectively crystallize one of .alpha.-
and .beta.-compounds, (16a) or (16b). The most suitable base may be
selected in the light of a solvent used in the reaction;
preferably, the above inorganic bases, tertiary alkylamines,
anilines, =pyridines, amino acids and optically active amines, and
salts formed include monovalent and bivalent salts.
[0125] A dehydrating agent may be used when moisture from a solvent
or an additive adversely affects the reaction. There are no
restrictions to a dehydrating agent as long as it has adequate
adsorptivity or reactivity with water; preferably molecular sieves
and phosphorus pentoxide.
[0126] The reaction is generally conducted in the presence of a
solvent. There are no restrictions to a solvent as long as it does
not inhibit the reaction, dissolve starting materials to some
degree and promotes selective crystallization of one of the
.alpha.- and .beta.-forms, (16a) or (16b), generated by salt
formation of the phosphate group in compound (16), including the
above aliphatic hydrocarbons, aromatic hydrocarbons, halogenated
hydrocarbons, esters, ethers, nitrites, amides, ketones and a
mixture of two or more selected therefrom.
[0127] There are no restrictions to a reaction temperature as long
as it accelerates the equilibrium reaction between compounds (15)
and (16) for promoting selective crystallization of one of the
.alpha.- and .beta.-forms, (16a) or (16b), generated by salt
formation of the phosphate group in compound (16); generally
-80.degree. C. to 60.degree. C., preferably -10.degree. C. to
25.degree. C.
[0128] A reaction period may vary depending on many factors such as
starting materials, reagents, the type of a solvent and a reaction
temperature; generally 3 hours to 1 week, preferably 6 hours to 24
hours for completing the reaction.
[0129] There are no restrictions to a ratio of a saccharide
derivative (1) to phosphoric acid; the reaction is generally
conducted with a ratio of compound (1): phosphoric acid=1:10 to
3:1, where the pH of the reaction system is generally 1 to 7,
suitably in an acidic range from 1 to 4.
[0130] The 1-phosphorylated saccharide derivative (16a) or (16b)
with either .alpha.- or .beta.-form may be isolated as a phosphate
with a base other than that used in the reaction system by a
salt-exchange reaction.
[0131] Bases which may be herein used include the above inorganic
bases, primary alkylamines, secondary alkylamines, tertiary
alkylamines, anilines, pyridines, amino acids and optically active
amines, and salts formed include monovalent and bivalent salts.
[0132] The protective group may be removed by reaction formula
(III) to prepare a 1-phosphorylated saccharide derivative (17a) or
(17b). ##STR22##
[0133] In this formula, R.sup.1, R.sup.2, R.sup.3, R.sup.4, X, W,
Z, n, p, q and r are as defined for formula (1); R.sup.1' and
R.sup.2' independently represent hydrogen, methyl, hydroxymethyl or
carboxyl; and R.sup.3' represents hydrogen or acyl.
[0134] When using the above aliphatic acyl, aromatic acyl or alkoxy
carbonyl as a protective group for hydroxymethyl in R.sup.1 and
R.sup.2 or hydroxy in R.sup.4, or using the above lower alkyl as a
protective group for carboxyl in R.sup.1 and R.sup.2 in compound
(16a) or (16b), it may be removed by treating the compound with a
base in an aqueous solvent. Bases which may be used include
preferably alkali metal carbonates such as sodium carbonate and
potassium carbonate; alkali metal hydroxides such as lithium
hydroxide, sodium hydroxide and potassium hydroxide; ammonium
hydroxides such as ammonium hydroxide and tetra-n-butylammonium
hydroxide; and the above inorganic bases, primary alkylamines,
secondary alkylamines and tertiary alkylamines.
[0135] Solvents which may be used include, with no restrictions,
those used in a common hydrolysis; preferably water; alcohols such
as methanol, ethanol, n-propanol and isopropanol; and the above
ethers. A reaction temperature and a reaction period vary, with no
restrictions, depending on many factors such as starting materials
and a base used; generally the reaction may be completed at
-10.degree. C. to 100.degree. C. for 1 hour to 5 days. The
protective group R.sup.3 may be left or simultaneously removed as
appropriate by adjusting a reaction temperature, a reaction period
and the equivalent values of reagents.
[0136] When using the above aralkyl or aralkyloxycarbonyl as a
protective group for hydroxymethyl in R.sup.1 and R.sup.2 or
hydroxy in R.sup.4 or using the above aralkyl as a protective group
for carboxy in R.sup.1 and R.sup.2 in compound (16a) or (16b), they
may be removed by, for example, catalytic hydrogenation using a
metal catalyst.
[0137] The catalyst may be preferably selected from
palladium-carbon, Raney nickel, platinum oxide, platinum black,
rhodium-aluminum oxide, triphenylphosphine-rhodium chloride and
palladium-barium sulfate. There are no restrictions to a reaction
pressure. Generally, a solvent used may be any of those used in a
common hydrolysis with no restrictions. It may be preferably
selected from water; alcohols such as methanol, ethanol, n-propanol
and isopropanol; the above ethers; and the above esters. A reaction
temperature and a reaction period vary, with no restrictions,
depending on many factors such as starting materials and a base
used; generally the reaction may be completed at -10.degree. C. to
100.degree. C. for 1 hour to 5 days. The protective group R.sup.3
may be generally left.
[0138] When using the above silyl as a protective group for
hydroxymethyl in R.sup.1 and R.sup.2 or hydroxy in R.sup.4 or using
the above silylated lower alkyl as a protective group for carboxy
in R.sup.1 and R.sup.2 in compound (16a) or (16b), they may be
removed by, for example, using a compound which can generate
fluoride anion such as tetra-n-butylammonium fluoride.
[0139] There are no restrictions to a solvent as long as it does
not inhibit the reaction; for example, the above ethers may be
used. A reaction temperature and a reaction period vary, with no
restrictions, depending on many factors such as starting materials
and a base used; generally the reaction may be completed at
-10.degree. C. to 50.degree. C. for 10 min to 10 hours. The
protective group R.sup.3 may be generally left.
[0140] In removing any protective group, a phosphate group in a
product is obtained as a salt with a base present in a reaction
system. The salt may be, if necessary, converted into a salt with
another base. In such a case, a base used may be selected from the
above inorganic bases, primary alkylamines, secondary alkylamines,
tertiary alkylamines, anilines, pyridines, amino acids and
optically active amines, and salts formed include monovalent and
bivalent salts.
[0141] A 1-phosphorylated saccharide derivative as used herein is a
saccharide or its derivative in which a phosphoric acid moiety is
coupled at 1-position via an ester linkage.
[0142] It may be specifically represented by formula (6):
##STR23##
[0143] wherein R.sup.1 and R.sup.2 independently represent
hydrogen, methyl, hydroxymethyl or carboxy; R.sup.3, X, W, Z, n, p,
q and r are as defined for formula (4).
[0144] Typical examples include, but not limited to,
ribose-1-phosphate, 2-deoxyribose-1-phosphate,
2,3-dideoxyribose-1-phosphate and arabinose-1-phosphate, but any
derivative may be used without distinction as long as it can be
obtained by any of the above highly universal and anomer-selective
preparation processes.
[0145] Examples of a saccharide derived from a natural product
which constitutes a 1-phosphorylated saccharide derivative include,
but not limited to, aldopentoses such as D-arabinose, L-arabinose,
D-xylose, L-lyxose and D-ribose; ketopentoses such as D-xylose,
L-xylose and D-ribulose; aldohexoses such as D-galactose,
L-galactose, D-glucose, D-talose and D-mannose; ketohexoses such as
D-tagatose, L-sorbose, D-psicose and D-fructose; deoxysaccharides
such as D-2-deoxyribose, D-2,3,-dideoxyribose, D-fucose, L-fucose,
D-rhamnose, L-rhamnose, D-fucopyranose, L-fucopyranose,
D-rhamnofuranose, L-rhamnofuranose, D-allomethylose, D-quinovose,
D-antiallose, D-talomethylose, L-talomethylose, D-digitalose,
D-digitoxose, D-cymarose, tyvelose, abequose, paratose, colitose
and ascarilose; aminosaccharides such as glucosamine and
daunosamine; and uronic acids such as glucuronic acid and
galacturonic acid.
[0146] There will be described a process for preparing a nucleoside
according to this invention. A base used in this process is a
natural or synthetic base selected from pyrimidine, purine,
azapurine and deazapurine, which may be substituted with halogen,
alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, alkylamino,
hydroxy, hydroxyamino, aminoxy, alkoxy, mercapto, alkylmercapto,
aryl, aryloxy and/or cyano.
[0147] Examples of halogen as a substituent include chlorine,
fluorine, bromine and iodine. Examples of alkyl include lower
alkyls with 1 to 7 carbon atoms such as methyl, ethyl and propyl.
Examples of haloalkyl include those having an alkyl with 1 to 7
carbon atoms such as fluoromethyl, difluoromethyl, trifluoromethyl,
bromomethyl and bromoethyl. Examples of alkenyl include those with
2 to 7 carbon atoms such as vinyl and allyl. Examples of haloalkyl
include those having alkenyl with 2 to 7 carbon atoms such as
bromovinyl and chlorovinyl. Examples of alkynyl include those with
2 to 7 carbon atoms such as ethynyl and propynyl. Examples of
alkylamino include those having alkyl with 1 to 7 carbon atoms such
as methylamino and ethylamino. Examples of alkoxy include those
with 1 to 7 carbon atoms such as methoxy and ethoxy. Examples of
alkylmercapto include those having alkyl with 1 to 7 carbon atoms
such as methylmercapto and ethylmercapto. Examples of aryl include
phenyl; alkylphenyls having alkyl with 1 to 5 carbon atoms such as
methylphenyl and ethylphenyl; alkoxyphenyls having alkoxy with 1 to
5 carbon atoms such as methoxyphenyl and ethoxyphenyl;
alkylaminophenyls having alkylamino with 1 to 5 carbon atoms such
as dimethylaminophenyl and diethylaminophenyl; and halogenophenyls
such as chlorophenyl and bromophenyl.
[0148] Examples of a pyrimidine base include cytosine, uracil,
5-fluorocytosine, 5-fluoro uracil, 5-chlorocytosine,
5-chlorouracil, 5-bromocytosine, 5-bromouracil, 5-iodocytosine,
5-iodouracil, 5-methylcytosine, 5-methyluracil (thymine),
5-ethylcytosine, 5-ethyluracil, 5-fluoromethylcytosine,
5-fluoromethyluracil, 5-trifluorocytosine, 5-trifluorouracil,
5-vinyluracil, 5-bromovinyluracil, 5-chlorovinyluracil,
5-ethynylcytosine, 5-ethynyluracil, 5-propynyluracil,
pyrimidin-2-one, 4-hydroxyaminopyrimidin-2-one,
4-aminoxypyrimidin-2-one, 4-methoxypyrimidin-2-one,
4-acetoxypyrimidin-2-one, 4-fluoropyrimidin-2-one and
5-fluoropyrimidin-2-one.
[0149] Examples of a purine base include purine, 6-aminopurine
(adenine), 6-hydroxypurine, 6-fluoropurine, 6-chloropurine,
6-methylaminopurine, 6-dimethylaminopurine,
6-trifluoromethylaminopurine, 6-benzoylaminopurine,
6-acetylaminopurine, 6-hydroxyaminopurine, 6-aminoxypurine,
6-methoxypurine, 6-acetoxypurine, 6-benzoyloxypurine,
6-methylpurine, 6-ethylpurine, 6-trifluoromethylpurine,
6-phenylpurine, 6-mercaptopurine, 6-methylmercaptopurine,
6-aminopurine-1-oxide, 6-hydroxypurine-1-oxide,
2-amino-6-hydroxypurine (guanine), 2,6-diaminopurine,
2-amino-6-chloropurine, 2-amino-6-iodopurine, 2-aminopurine,
2-amino-6-mercaptopurine, 2-amino-6-methylmercaptopurine,
2-amino-6-hydroxyaminopurine, 2-amino-6-methoxypurine,
2-amino-6-benzoyloxypurine, 2-amino-6-acetoxypurine,
2-amino-6-methylpurine, 2-amino-6-cyclopropylaminomethylpurine,
2-amino-6-phenylpurine, 2-amino-8-bromopurine, 6-cyanopurine,
6-amino-2-chloropurine (2-chloroadenine) and 6-amino-2-fluoropurine
(2-fluoroadenine).
[0150] Examples of an azapurine and a deazapurine bases include
6-amino-3-deazapurine, 6-amino-8-azapurine,
2-amino-6-hydroxy-8-azapurine, 6-amino-7-deazapurine,
6-amino-1-deazapurine and 6-amino-2-azapurine.
[0151] A nucleoside phosphorylase is a generic name for enzymes
capable of phosphorolysis of an N-glycoside bond in a nucleoside in
the presence of phosphoric acid and this invention utilizes its
reverse reaction. An enzyme used in the reaction may be of any type
or origin as long as it has an activity of forming a desired
nucleoside from a corresponding 1-phosphorylated saccharide
derivative and a base. The enzymes may be generally categorized
into two types, purine and pyrimidine types. Examples of a purine
type enzyme include purine nucleoside phosphorylase (EC2.4.2.1) and
guanosine nucleoside phosphorylase (EC2.4.2.15). Examples of a
pyrimidine type enzyme include pyrimidine nucleoside phosphorylase
(EC2.4.2.2), uridine nucleoside phosphorylase (EC2.4.2.3),
thymidine nucleoside phosphorylase (EC2.4.2.4) and deoxyuridine
nucleoside phosphorylase (EC2.4.2.23).
[0152] A microorganism expressing a nucleoside phosphorylase in
this invention may be, with no restrictions, any microorganism
expressing at least one nucleoside phosphorylase selected from the
group consisting of purine nucleoside phosphorylase (EC2.4.2.1),
guanosine nucleoside phosphorylase (EC2.4.2.15), pyrimidine
nucleoside phosphorylase (EC2.4.2.2), uridine nucleoside
phosphorylase (EC2.4.2.3), thymidine nucleoside phosphorylase
(EC2.4.2.4) and deoxyuridine nucleoside phosphorylase
(EC2.4.2.23).
[0153] Preferable examples of such a microorganism include strains
belonging to Nocardia, Microbacterium, Corynebacterium,
Brevibacterium, Cellulomonas, Flabobacterium, Kluyvere,
Micobacterium, Haemophilus, Micoplana, Protaminobacter, Candida,
Saccharomyces, Bacillus, thermophile Bacillus, Pseudomonas,
Micrococcus, Hafnia, Proteus, Vibrio, Staphyrococcus,
Propionibacterium, Sartina, Planococcus, Escherichia, Kurthia,
Rhodococcus, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium,
Salmonella, Klebsiella, Enterobacter, Erwinia, Aeromonas,
Citrobacter, Achromobacter, Agrobacterium, Arthrobacter and
Pseudonocardia.
[0154] Recent advance in molecular biology and genetic engineering
has allowed us to analyze molecular-biological properties, an amino
acid sequence and so on of a nucleoside phosphorylase in the above
strain for obtaining the gene for the protein from the strain, to
constitute a recombinant plasmid in which a control region required
for the gene and its expression is inserted, to introduce the
plasmid into a given host and to produce a gene recombinant strain
expressing the protein, and these processes have become relatively
easier. In the light of the recent technical level, such a gene
recombinant strain in which a gene for a nucleoside phosphorylase
is introduced in a given host shall be also included in a
microorganism expressing a nucleoside phosphorylase according to
this invention.
[0155] A control region required for expression herein may be a
promoter sequence (including an operator sequence controlling
transcription), a ribosome binding sequence (SD sequence), a
transcription termination sequence, or the like. Examples of a
promoter sequence include a trp operator in a tryptophane operon
derived from E. coli; a lac promoter in a lactose operon; a PL and
a PR promoters derived from lambda phage; a gluconate synthase
promoter (gnt) derived from Bacillus subtilis; an alkali protease
promoter (apr); a neutral protease promoter (npr); and
.alpha.-amylase promoter (amy). A uniquely modified and designed
sequence such as a tac promoter may be used. A ribosome linkage
sequence may be, for example, a sequence derived from E. coli or
Bacillus subtilis, but any sequence may be used as long as it can
function in a desired host such as E. coli and Bacillus subtilis.
For example, one can use a consensus sequence formed by DNA
synthesis, that is, a sequence with more than 4 consecutive bases
complementary to 3'-terminal region in 16S ribosome RNA. A
transcription termination sequence is not always necessary, but a
.rho.-factor independent terminator such as a lipoprotein
terminator and a trp operon terminator may be used. Desirably,
these control regions on a recombinant plasmid may be sequentially
aligned as follows; from upstream of 5'-terminal, a promoter
sequence, a ribosome linkage sequence, a nucleoside phosphorylase
coding gene and a transcription termination sequence.
[0156] As examples of a plasmid herein, pBR 322, pUC18, Bluescript
II SK(+), pKK223-3 and pSC101 having an autonomously replicable
region in E. coli; pUB110, pTZ4, pC194, .rho.11, .phi.1 and
.phi.105 having an autonomously replicable region in Bacillus
subtilis may be used as a vector. As examples of a plasmid
autonomously replicable in two or more hosts, pHV14, TRp7, Yep7 and
pBS7 may be used as a vector.
[0157] A given host herein may be typically, but not limited to,
Escherichia coli as described in Examples later, but other strains
such as Bacillus sp. including Bacillus subtilis, yeasts and
actinomyces may be used.
[0158] Nucleoside phosphorylase activity in this invention may be
obtained from, besides the above strains having the enzyme
activity, a processed material of the strain exhibiting the enzyme
activity and an immobilized product thereof. A processed material
of the strain may be, for example, acetone-dried strain or a
bacterial debris prepared by an appropriate procedure such as
mechanical destruction, ultrasonic disintegration, freezing and
thawing, pressurization and depressurization, osmotic pressure
method, autolysis, cell-wall decomposition and surfactant
treatment. If necessary, the strain may be further purified by
ammonium sulfate precipitation, acetone precipitation or column
chromatography.
[0159] In this invention, a metal cation capable of forming a
water-insoluble salt with phosphate ion may be, without
restriction, any metal cation which can form a water-insoluble salt
with phosphate ion as a byproduct in the reaction and may be
precipitated; for example, calcium, magnesium, barium, iron,
cobalt, nickel, copper, silver, molybdenum, lead, zinc and lithium
ions. Among these, particularly preferable are industrially
universal and safe metal ions which do not adversely affect the
reaction, e.g., calcium, barium, aluminum and magnesium ions.
[0160] A metal cation capable of forming a water-insoluble salt
with phosphate ion in this invention may be obtained by adding a
salt of a metal cation capable of forming a water-insoluble salt
with phosphate ion with at least one anion selected from chloride,
nitride, carbonate, sulfate, acetate and hydroxyl ions into the
reaction solution. Examples of such a salt include calcium
chloride, calcium nitride, calcium carbonate, calcium sulfate,
calcium acetate, barium chloride, barium nitride, barium carbonate,
barium sulfate, barium acetate, aluminum chloride, aluminum
nitride, aluminum carbonate, aluminum sulfate, aluminum acetate,
calcium hydroxide, barium hydroxide, aluminum hydroxide, magnesium
hydroxide, magnesium chloride, magnesium nitride, magnesium
carbonate, magnesium sulfate and magnesium acetate.
[0161] Such a metal cation may be present as a salt with a
pentose-1-phosphate in the reaction solution; for example,
ribose-1-phosphate calcium salt, 2-deoxyribose-1-phosphate calcium
salt, 2,3-dideoxyribose-1-phosphate calcium salt,
arabinose-1-phosphate calcium salt, ribose-1-phosphate barium salt,
2-deoxyribose-1-phosphate barium salt,
2,3-dideoxyribose-1-phosphate barium salt, arabinose-1-phosphate
barium salt, ribose-1-phosphate aluminum salt,
2-deoxyribose-1-phosphate aluminum salt,
2,3-dideoxyribose-1-phosphate aluminum salt and
arabinose-1-phosphate aluminum salt.
[0162] A reaction for preparing a nucleoside compound in this
invention may be conducted under the conditions such as appropriate
pH and temperature and within the control ranges thereof, depending
on a target nucleoside, a 1-phosphorylated saccharide derivative
and a base as substrates, a nucleoside phosphorylase or a
microorganism exhibiting the activity of the enzyme as a reaction
catalyst, and the type and the properties of a metal salt added for
removing phosphoric acid from the reaction system; generally at pH
5 to 10 and a temperature of 10 to 60.degree. C. If pH is not
within the control range, a reaction inversion rate may be reduced
due to, for example, poor stability of a target product or
substrate, reduction in enzyme activity and failure to forming a
water-insoluble salt with phosphoric acid. If pH varies in the
course of the reaction, an acid such as hydrochloric acid and
sulfuric acid or an alkali such as sodium hydroxide and potassium
hydroxide may be, when necessary, added at an appropriate timing.
The concentrations of a 1-phosphorylated saccharide derivative and
a base are suitably about 0.1 to 1000 mM. In terms of a molar ratio
between them, a molar ratio of a base to a 1-phosphorylated
saccharide derivative or its salt may be 1 to 10, preferably 0.95
or less in the light of a reaction inversion rate.
[0163] A metal salt capable of forming a water-insoluble salt with
phosphoric acid added may be added in a molar ratio of 0.1 to 10,
more preferably 0.5 to 5 to a 1-phosphorylated saccharide
derivative used in the reaction. There are no restrictions to an
addition procedure of the salt, and it may be added in one portion
or portionwise during the reaction. This invention basically uses
water as a solvent, but an organic solvent such as an alcohol and
dimethylsulfoxide used in a common enzyme reaction may be, if
necessary, added in an appropriate amount. In a reaction with a
higher concentration, a base as a substrate or a nucleoside as a
product may be not be completely dissolved in the reaction
solution. This invention may be also applied to such a case.
[0164] A nucleoside compound produced as described above may be
isolated by a common procedure such as concentration,
crystallization, dissolution, electrodialysis and adsorption and
desorption using an ion-exchange resin or charcoal.
EXAMPLES
[0165] This invention will be more specifically described with
reference to, but not limited to, Examples.
Example 1
Preparation of an anomer mixture of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribose-1-phosphate (18) and
bis[3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribos-1-yl]phosphate
(19)
[0166] To a mixture of 1.18 g of orthophosphoric acid in 51 mL of
acetonitrile were added 2.3 g of tri-n-butylamine and 5.07 g of
molecular sieves 4A, and the mixture was cooled to 5.degree. C.
with stirring. After one hour, to the mixture was added 5.07 g of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-ribosyl chloride
(purity: 85%), and the mixture was stirred for one hour to give a
solution of a mixture of the title compounds (18) and (19) [(18):
(19)=3:5, .alpha.-form/.beta.-form of compound (18)=5/2] in
acetonitrile.
[0167] For preparing a sample for analysis, these compounds were
converted into cyclohexylamine salts, which were then purified by
silica gel column chromatography to provide two anomer isomers
(19a) and (19b) of the title compound (19) from a fraction eluted
with methanol-ethyl acetate (1:10).
(19a): Less polar fraction
[0168] .sup.1H NMR (CDCl.sub.3, 270 MHz) d: 8.0-7.8 (m, 8H),
7.4-7.2 (m, 8H), 6.06 (m, 1.2H), 5.98 (m, 0.8H), 5.56 (m, 1.2H),
5.41 (m, 0.8H), 4.7-4.3 (m, 6H), 2.6-2.4 (m, 1H), 2.75-2.6 (m, 2H),
2.5-2.3 (m, 2H), 2.2-1.9 (m, 2H), 1.8-1.6 (m, 2H), 1.6-0.9 (m, 8H);
MS (APCI) m/z 883 (M-H).
(19b): More polar fraction
[0169] .sup.1H NMR (CDCl.sub.3, 270 MHz) d: 8.0-7.8 (m, 8H),
7.4-7.2 (m, 8H), 6.1-5.9 (m, 2H), 5.55 (m, 0.67H), 5.39 (m, 1.33H),
4.7-4.3 (m, 6H), 3.1-2.85 (m, 1H), 2.75-2.4 (m, 2H), 2.32 (m, 2H),
2.2-1.9 (m, 2H), 1.8-1.6 (m, 2H), 1.6-0.9 (m, 8H); MS (APCI) m/z
883 (M-H).
Example 2
Preparation of an anomer mixture of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribose-1-phosphate and
bis[3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribose-1-yl]phosphate
[0170] To a mixture of 1.11 g of orthophosphoric acid in 49 mL of
2-butanone were added 2.11 g of tri-n-butylamine and 4.9 g of
molecular sieves 4A, and the mixture was cooled to 5.degree. C.
with stirring. To the mixture was added 4.9 g of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-ribosyl chloride
(purity: 85%), and the mixture was stirred for 10 min to give a
solution of a mixture of the title compounds (18) and (19) [(18)
(19)=1:4, .alpha.-form/.beta.-form of compound (18)=7/10] in
2-butanone.
Example 3
Preparation of an anomer mixture of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-b-ribose-1-phosphate and
bis[3,5-O-bis(4-chlorobenzoyl)-2-deoxy-D-ribos-1-yl]phosphate
[0171] To a mixture of 136.8 g of orthophosphoric acid in 2 L of
2-butanone were added 90.6 g of tri-n-butylamine and 200 g of
molecular sieves 4A, and the mixture was cooled to 5.degree. C.
with stirring. After stirring for one hour, to the mixture was
added 200 g of 3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-ribosyl
chloride (purity: 85%), and the mixture was stirred for 2 hours to
give a solution of a mixture of the title compounds (18) and (19)
[(18): (19)=5:4, .alpha.-form/.beta.-form of compound (18)=5/2] in
2-butanone.
Example 4
Preparation of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-ribose-1-phosphate
(18a)
[0172] The acetonitrile solution prepared in Example 1 was cooled
to 5.degree. C. with stirring, and 2.29 g of orthophosphoric acid
was added to the mixture. After stirring for 3 hours,
crystallization was initiated and then the mixture became a thick
suspension. After 5 hours, the ratio of .alpha.-form/.beta.-form of
the title compound (18) in the reaction suspension was 10/1. The
crystals were collected as a mixture with molecular sieves. The
solid was dissolved in 100 mL of methanol and the mixture was again
filtrated to remove molecular sieves. HPLC assay showed that 3.68 g
of the title compound (18a) was contained in the methanol solution
(Yield: 74.6% after reduction from the purity of the starting
material) without the .beta.-form on HPLC.
Example 5
Preparation of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-robose-1-phosphate
(18a)
[0173] The 2-butanone solution prepared in Example 2 was cooled to
5.degree. C. with stirring, and 2.2 g of orthophosphoric acid was
added to the solution. After stirring for 1 hour, precipitation of
crystals initiated and then a thick suspension was obtained. After
20 hours, the ratio of .alpha.-form/.beta.-form for compound (18a)
in the reaction suspension was 8:1. To the suspension was added
6.33 g of tri-n-butylamine to dissolve the precipitated crystals
and molecular sieves were removed by filtration. To the filtrate
was added 250 mL of toluene, and the solution was washed with 55 mL
of water. The organic layer was ice-cooled. To the mixture was
added 2.32 g of cyclohexylamine for crystallization with stirring.
After 1 hour, the precipitated crystals were collected by
filtration and dried in vacuo at room temperature to provide 3.19 g
of a dicyclohexylamine salt of compound (16a) as a colorless powder
(Yield: 64.7% after reduction from the purity of the starting
material; .alpha.-form:.beta.-form=97.5:2.5).
[0174] .sup.1H NMR (DMSO-d.sub.6, 270 MHz) d: 8.00(d, J=8.6 Hz,
2H), 7.96 (d, J=8.6 Hz, 2H), 7.58 (d, J=8.6 Hz, 2H), 7.58 (d, J=8.6
Hz, 2H), 5.82 (dd, J=5.3, 5.3 Hz, 1H), 5.36 (d, J=8.6 Hz, 1H),
4.6-4.3 (m, 3H), 4.7-3.5 (br, 6H), 2.7-2.6 (m, 2H), 2.55-2.4 (m,
1H), 2.25 (d, J=4.2 Hz, 1H), 1.85-1.75 (m, 4H), 1.7-1.6 (m, 4H),
1.55-1.45 (m, 2H), 1.25-0.9 (m, 10H); MS (APCI) m/z 590
(M+C.sub.6H.sub.14N).
Example 6
Preparation of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-robose-1-phosphate
(18a)
[0175] The 2-butanone solution prepared in Example 3 was cooled to
5.degree. C. with stirring. After stirring for 1 hour,
precipitation of crystals initiated and then a thick suspension was
obtained. After 23 hours, the ratio of .alpha.-form/.beta.-form for
compound (18a) in the reaction suspension was 7:1. To the
suspension was added 259 g of tri-n-butylamine to dissolve the
precipitated crystals and molecular sieves were removed by
filtration. The filtrate was washed with 2.2 L of water and the
aqueous layer was extracted with 1 L of toluene. The combined
organic layer was ice-cooled. To the mixture was added 87.5 g of
cyclohexylamine for crystallization with stirring. After 1 hour,
the precipitated crystals were collected by filtration and dried in
vacuo at room temperature to provide 213 g of a dicyclohexylamine
salt of compound (16a) as a colorless powder (Yield: 78.1% after
reduction from the purity of the starting material; .alpha.-form:
.beta.-form=96.9:3.1).
Example 7
Preparation of 2-deoxy-.alpha.-D-ribose-1-phosphate (20)
[0176] To the methanol solution prepared in Example 4 was added 20
mL of an aqueous solution of ammonium hydroxide, and the mixture
was stirred at room temperature. After stirring for 28 hours, the
precipitated crystals were collected by filtration and dried in
vacuo at room temperature to provide 589 mg of an ammonium salt of
compound (20) as a colorless powder (Yield: 21.1% without the
.beta.-form on HPLC).
Example 8
Preparation of 2-deoxy-.alpha.-D-ribose-1-phosphate (20)
[0177] Compound (18a) prepared in Example 6 was suspended in a
mixture of 2.3 L of methanol and 450 mL of an aqueous ammonium
hydroxide solution, and the mixture was stirred at room
temperature. After stirring for 28 hours, the precipitated crystals
were collected by filtration and dried in vacuo at room temperature
to provide 62.0 g of an ammonium salt of compound (20) as a
colorless powder (Yield: 81.0% without the .beta.-form on
HPLC).
[0178] .sup.1H NMR(D.sub.2O, 270 MHz) d: 5.56 (s, 1H), 4.03 (m,
2H), 3.52 (dd, J=3.3, 12.2 Hz, 1H), 3.41 (dd, J=5.3, 12.2 Hz, 1H),
2.17 (m, 1H), 1.87 (d, J=13.9 Hz, 1H); MS (APCI) m/z: 213
(M-H).
Example 9
Preparation of
2,3,5-O-tris(4-chlorobenzoyl)-.alpha.-D-ribose-1-phosphate (21)
[0179] To a mixture of 3.32 g of orthophosphoric acid in 67 mL of
methyl isobutyl ketone were added 2.11 g of tri-n-butylamine and
6.6 g of molecular sieves 4A, and the mixture was cooled to
5.degree. C. with stirring. To the mixture was added 6.66 g of
2,3,5-O-tris(4-chlorobenzoyl)-.alpha.-D-ribosyl chloride. After 1
hour, precipitation of crystals initiated and then a thick
suspension was provided. After 10 hours, the ratio of
.alpha.-form/.beta.-form for compound (19) in the reaction
suspension was 10:1. To the suspension was added 6.33 g of
tri-n-butylamine to dissolve the precipitated crystals and
molecular sieves were removed by filtration. The filtrate was
washed with 55 mL of water. The organic layer was ice-cooled. To
the mixture was added 2.4 g of cyclohexylamine for crystallization
with stirring. After 1 hour, the precipitated crystals were
collected by filtration and dried in vacuo at room temperature to
provide 7.02 g of a dicyclohexylamine salt of compound (21) as a
colorless powder (Yield: 73.0%; .alpha.-form:
.beta.-form=99:1).
[0180] .sup.1H NMR (DMSO-d.sub.6, 270 MHz) d: 8.2-7.8 (m, 6H),
7.6-7.4 (m, 6H), 5.9-5.7 (m, 1H), 5.6-5.4 (m, 3H), 4.6-4.3 (m, 1H),
4.7-3.5 (br, 6H), 2.7-2.6 (m, 2H), 1.9-1.7 (m, 4H), 1.7-1.6 (m,
4H), 1.55-1.4 (m, 2H), 1.3-0.9 (m, 10H); MS (APCI) m/z 745
(M+C.sub.6H.sub.14N).
Example 10
Preparation of .alpha.-D-ribose-1-phosphate (22)
[0181] Compound (21) prepared in Example 9 was suspended in a
mixture of 105 mL of methanol and 21 mL of an aqueous ammonium
hydroxide solution, and the mixture was stirred at room
temperature. After stirring for 32 hours, the precipitated crystals
were collected by filtration and dried in vacuo at room temperature
to provide 1.90 g of an ammonium salt of compound (22) as a
colorless powder (Yield: 86.0% without the .beta.-form on
HPLC).
[0182] .sup.1H NMR(D.sub.2O, 270 MHz) d: 5.6 (m, 1H), 4.2 (m, 1H),
4.1-4.0 (m, 2H), 3.75 (m, 1H), 3.7 (m, 1H); MS (APCI) m/z: 229
(M-H).
Example 11
Preparation of
5-O-(4-chlorobenzoyl)-2,3-dideoxy-.alpha.-D-ribose-1-phosphate
(23)
[0183] To a mixture of 3.5 g of orthophosphoric acid in 33 mL of
acetonitrile were added 2.2 g of tri-n-butylamine and 3.3 g of
molecular sieves 4A, and the mixture was cooled to 5.degree. C.
with stirring. To the mixture was added 3.28 g of
5-O-(4-chlorobenzoyl)-2,3-dideoxy-.alpha.-D-ribosyl chloride. After
1 hour, precipitation of crystals initiated and then a thick
suspension was provided. After 20 hours, the ratio of
.alpha.-form/.beta.-form for compound (23) in the reaction
suspension was 10:1. To the suspension was added 6.5 g of
tri-n-butylamine to dissolve the precipitated crystals and
molecular sieves were removed by filtration. The filtrate was
diluted with 70 mL of toluene and then washed with 55 mL of water.
The organic layer was ice-cooled. To the mixture was added 2.5 g of
cyclohexylamine for crystallization with stirring. After 1 hour,
the precipitated crystals were collected by filtration and dried in
vacuo at room temperature to provide 4.56 g of a dicyclohexylamine
salt of compound (23) as a colorless powder (Yield: 71.5%;
.alpha.-form: .beta.-form=97:3).
[0184] .sup.1H NMR (DMSO-d.sub.6, 270 MHz) d: 8.2-7.8 (m, 2H),
7.6-7.4 (m, 2H), 5.9-5.7 (m, 1H), 5.6-5.4 (m, 1H), 4.6-4.3 (m, 1H),
4.7-3.5 (br, 6H), 2.7-2.6 (m, 2H), 1.9-1.7 (m, 8H), 1.7-1.6 (m,
4H), 1.55-1.4 (m, 2H), 1.3-0.9 (m, 10H); MS (APCI) m/z 374
(M+C.sub.6H.sub.14N).
Example 12
Preparation of 2,3-dideoxy-.alpha.-D-ribose-1-phosphate (24)
[0185] Compound (23) prepared in Example 11 was suspended in a
mixture of 46 mL of methanol and 10 mL of an aqueous ammonium
hydroxide solution, and the mixture was stirred at room
temperature. After stirring for 30 hours, the precipitated crystals
were collected by filtration and dried in vacuo at room temperature
to provide 1.68 g of an ammonium salt of compound (24) as a
colorless powder (Yield: 85.0% without the .beta.-form on
HPLC).
[0186] .sup.1H NMR(D.sub.2O, 270 MHz) d: 5.2 (m, 1H), 4.1-3.9 (m,
1H), 3.6-3.3 (m, 2H), 2.1-2.3 (m, 2H), 1.9-1.7 (m, 2H); MS (APCI)
m/z: 197 (M-H).
Example 13
Preparation of
2,3,5-O-tris(4-chlorobenzoyl)-.alpha.-D-arabinofuranosyl-1-phosphate
(25)
[0187] To a mixture of 3.3 g of orthophosphoric acid in 67 mL of
methyl isobutyl ketone were added 2.1 g of tri-n-butylamine and 6.6
g of molecular sieves 4A, and the mixture was cooled to 5.degree.
C. with stirring. To the mixture was added 6.6 g of
2,3,5-O-tris(4-chlorobenzoyl)-.alpha.-D-arabinofuranosyl chloride.
After 1 hour, precipitation of crystals initiated and then a thick
suspension was provided. After 8 hours, the ratio of
.alpha.-form/.beta.-form for compound (25) in the reaction
suspension was 10:1. To the suspension was added 6.3 g of
tri-n-butylamine to dissolve the precipitated crystals and
molecular sieves were removed by filtration. The filtrate was
washed with 55 mL of water. The organic layer was ice-cooled. To
the mixture was added 2.4 g of cyclohexylamine for crystallization
with stirring. After 1 hour, the precipitated crystals were
collected by filtration and dried in vacuo at room temperature to
provide 6.72 g of a dicyclohexylamine salt of compound (25) as a
colorless powder (Yield: 70.5%; .alpha.-form:.beta.-form=99:1).
[0188] MS (APCI) m/z 745 (M+C.sub.6H.sub.14N).
Example 14
Preparation of .alpha.-D-arabinofuranosyl-1-phosphate (26)
[0189] Compound (25) prepared in Example 13 was suspended in a
mixture of 94 mL of methanol and 18 mL of an aqueous ammonium
hydroxide solution, and the mixture was stirred at room
temperature. After stirring for 48 hours, the precipitated crystals
were collected by filtration and dried in vacuo at room temperature
to provide 1.72 g of an ammonium salt of compound (26) as a
colorless powder (Yield: 82.0% without the .beta.-form on
HPLC).
[0190] .sup.1H NMR(D.sub.2O, 270 MHz) d: 5.3 (m, 1H), 3.95-3.3 (m,
5H); MS (APCI) m/z: 229 (M-H).
Example 15
Preparation of (2R)-2-benzyloxymethyl-1,3-dioxorane-4-phosphate
(27)
[0191] To a solution of 1.06 g of
(2R)-2-benzyloxymethyl-4-(R,S)-acetoxy-1,3-dioxorane in 12 mL of
ether under ice-cooling was added 4 mL of a 4N solution of
hydrochloric acid in dioxane. After stirring 3.5 hours, the mixture
was warmed to room temperature. After removing the solvent by
concentration, the residue was further subject to azeotropy with
toluene to give 500 mg of (2R)-2-benzyloxymethyl-1,3-dioxoranyl
chloride as a colorless and transparent oil. To 1.1 mL of
acetonitrile were sequentially added 0.27 g of orthophosphoric
acid, 0.66 mL of tri-n-butylamine and 0.23 g of molecular sieves
4A, and the mixture was stirred for 1.5 hours. To the suspension
under ice-cooling was added 0.27 g of the previous oil, and the
mixture was stirred under ice-cooling for 5.5 hours. To the mixture
was added 0.6 mL of tri-n-butylamine. After stirring for 30 min,
the mixture was diluted with toluene and extracted with water. The
aqueous layer was extracted with n-butanol and then concentrated.
The concentrate was dissolved in toluene, and to the solution was
added cyclohexylamine to give a cyclohexylamine salt of compound
(27) as a white solid.
[0192] .sup.1H-NMR (D.sub.2O) .delta.: 0.98-1.10 (2H, m), 1.14-1.23
(6H, m), 1.47-1.51 (2H, m), 1.61-1.64 (4H, m), 1.78-1.83 (4H, m),
2.94-3.00 (2H, m), 3.46-3.60 (2H, m), 3.72-3.79 (1H, m), 3.92-4.00
(1H, m), 4.41-4.51 (2H, m), 5.01-5.03 and 5.22-5.24 (total 1H, m),
5.64-5.72 (total 1H, m), 7.24-7.30 (5H, m); MS (APCI) m/z: 390
(M+C.sub.6H.sub.14N).sup.+.
Example 16
Preparation of (2R)-2-hydroxymethyl-1,3-dioxorane-4-phosphate
(28)
[0193] In 10 mL of methanol was dissolved 0.2 g of compound (27)
prepared in Example 15. The solution was subject to hydrogenation
under an ambient pressure using 0.11 g of 10% Pd/C as a catalyst.
After removing the catalyst by filtration, the filtrate was
concentrated to give a cyclohexylamine of compound (28).
[0194] .sup.1H-NMR (D.sub.2O) .delta.: 0.99-1.06 (2H, m), 1.10-1.24
(6H, m), 1.47-1.50 (2H, m), 1.62-1.66 (4H, m), 1.80-1.85 (4H, m),
1.96-3.02 (2H, m), 3.51-3.57 (2H, m), 3.72-3.79 (1H, m), 3.93-4.00
(1H, m), 4.99-5.01 and 5.13-5.15 (total 1H, m), 5.64-5.67 and
5.70-5.73 (total 1H, m); MS (APCI) m/z: 199 (M-H).sup.-.
Example 17
Preparation of
2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-.alpha.-D-erythropentofuranose-
-1-phosphate (29)
[0195] Seventy mg of molecular sieves 4A was added to a stirred
mixture of 62 mg of orthophosphoric acid, 52 .mu.L of
tri-n-butylamine and 0.7 mL of acetonitrile at room temperature,
and the mixture was stirred in an ice-bath. To the mixture was
added 70 mg of
2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-D-erythropentofuranosyl
chloride, and the mixture was reacted at the same temperature for 1
day. Then, to the mixture were added 156 .mu.L of tri-n-butylamine
and then deionized water. The mixture was extracted with toluene
three times. To the organic layer was added 48 .mu.L of
cyclohexylamine and the mixture was stirred for 30 min. The mixture
was concentrated in vacuo, and acetone was added to form a
precipitate, which was collected by filtration. The residue was
washed with chloroform and dried in vacuo at room temperature to
give a dicyclohexylamine of compound (29) as a white solid.
[0196] .sup.1H-NMR (CD.sub.3OD) .delta.: 1.1-1.4 (10H, m), 1.65
(2H, m), 1.89 (4H, m), 1.96 (4H, m), 2.3-2.5 (2H, m), 2.91 (2H, m),
4.5 (2H, m), 4.6-4.8 (1H, m), 5.1-5.3 (1H, m), 5.97 (1H, m), 7.41
(1H, m), 7.47 (2H, m), 7.68 (2H, m), 7.75 (2H, m), 8.08 (2H, m); MS
(APCI) m/z: 496 (M+C.sub.6H.sub.14N).sup.+.
Example 18
Preparation of
2,3-dideoxy-3-fluoro-.alpha.-D-erythropentofuranose-1-phophate
(30)
[0197] To a solution of 21 mg of compound (29) prepared in Example
17 in 1 mL of methanol was added 20 .mu.L of cyclohexylamine and
the mixture was reacted for 2 weeks. The mixture was concentrated
in vacuo and diethyl ether was added. The mixture was filtered, and
the solid was dried in vacuo to give 12 mg of a dicyclohexylamine
salt of the title compound as a white solid.
[0198] .sup.1H-NMR (CD.sub.3OD) .delta.: 1.1-1.4 (10H, m), 1.66
(2H, m), 1.79 (4H, m), 1.94 (4H, m), 2.3-2.4 (2H, m), 2.88 (2H, m),
3.59 (2H, m), 4.3-4.4 (1H, m), 5.11 (0.5H, m; the other 0.5H was
undistinguishable because it was behind the peak of water), 5.89
(1H, m); MS (APCI) m/z: 215 (M-H).sup.-.
Example 19
Preparation of
2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-D-erythropentofuranose-1-phosp-
hate (31)
[0199] At room temperature 0.86 g of molecular sieves 4A was added
to a stirred mixture of 759 mg of orthophosphoric acid, 646 .mu.L
of tri-n-butylamine and 8.6 mL of acetonitrile at room temperature,
and the mixture was stirred in an ice-bath. To the mixture was
added 864 mg of
2,3-dideoxy-3-fluoro-5-O-(4-phenylbenzoyl)-D-erythropentofuranosyl
chloride, and the mixture was reacted at the same temperature for 1
day. Then, to the mixture were added 1.94 mL of tri-n-butylamine
and then deionized water. The mixture was extracted with toluene
three times and washed with purified water five times. The organic
layer was separated. To the organic layer was added 590 .mu.L of
cyclohexylamine and the mixture was stirred for 30 min. The mixture
was concentrated in vacuo. After addition of acetone, the mixture
was stirred and filtrated. The residue was further washed with
isopropyl ether and dried in vacuo at room temperature to give
compound (31) as a white solid. .alpha.-form: .beta.-form
66:34.
[0200] .sup.1H-NMR (CD.sub.3OD) .delta.: 1.1-1.4 ppm (10H, m), 1.66
(2H, m), 1.78 (4H, m), 1.98 (4H, m), 2.3-2.6 (2H, m), 2.89 (2H, m),
4.44 & 4.46 (.alpha. & .beta., 2H), 4.6-4.8 (1H, m),
5.1-5.3 & 5.3-5.4 (.alpha. & .beta., 1H, m), 5.97 &
6.00 (.alpha. & .beta., 1H, m), 7.40 (1H, m), 7.47 (2H, m),
7.68 (2H, m), 7.75 (2H, m), 8.07 (1H, m), 8.13 (1H, m).
Example 20
Preparation of
2,3-dideoxy-3-fluoro-D-erythropentofuranose-1-phophate (32)
[0201] To a solution of 0.29 g of compound (31) prepared in Example
19 in 15 mL of methanol was added 279 .mu.L of cyclohexylamine and
the mixture was reacted for 1 week. The mixture was concentrated in
vacuo and diethyl ether was added. After stirring, the mixture was
filtered, and the solid was dried in vacuo to give 185 mg of a
dicyclohexylamine salt of compound (32) as a white solid.
.alpha.-form:.beta.-form=66:34.
[0202] .sup.1H-NMR (CD.sub.3OD) .delta.: 1.1-1.4 ppm (10H, m), 1.67
(2H, m), 1.79 (4H, m), 2.2-2.4 (2H, m), 2.94 (2H, m), 3.59 &
3.62 (.alpha. & .beta., 2H, m), 3.3-3.4 (2H, m), 5.10 &
5.1-5.24 (.alpha. & .beta., 0.5H & 1H, m, 0.5H of the
.alpha.-form was undistinguishable because the signal was behind
the peak of water), 5.88 & 5.93 (.alpha. & .beta., 1H,
m).
Example 21
Preparation of 3,5-O-dibenzoyl-2-O-methylribose-1-phosphate
(33)
[0203] To 2.84 g of 1,3,5-O-tribenzoyl-2-O-methyl-.alpha.-D-ribose
was added 14.5 mL of a 4N solution of hydrochloric acid in dioxane,
and the mixture was stirred under ice-cooling. After stirring 2.5
hours, 10 mL of a 4N solution of hydrochloric acid in dioxane was
further added, and the mixture stirred for 1 hour. After
evaporating the solvent, the residue was further subject to
azeotropy with 10 mL of dioxane twice to give
3,5-O-dibenzoyl-2-O-methylribosyl-1-chloride. Separately, 2.98 g of
98% phosphoric acid was dissolved in 15 mL of 4-methyl-2-pentanone
and after adding 2.8 g of molecular sieves 4A, the mixture was
stirred for 30 min. To the mixture were added 1.42 mL of
tri-n-butylamine and then a solution of the previous
3,5-O-dibenzoyl-2-O-methylribosyl-1-chloride in 10 mL of
4-methyl-2-pentanone. After reacting the mixture at room
temperature for 20 hours, it was neutralized with 7.1 mL of
tri-n-butylamine. After removing the molecular sieves by
filtration, the filtrate was washed with 20 mL of water three
times. The organic layer was evaporated and purified by silica gel
column chromatography to give 950 mg of compound (33).
[0204] MS (APCI) m/z: 451 (M-H).sup.-; IR (KBr) cm.sup.-1: 3448,
2963, 1721, 1453, 1278, 1111, 976, 711, 558.
Example 22
Preparation of 2-O-methylribose-1-.beta.-phosphate (34)
[0205] To 850 mg of compound (33) prepared in Example 21 was 20 mL
of 14% ammonia-methanol, and the mixture was reacted at room
temperature for 20 hours. After evaporation of the solvent,
diisopropyl ether was added to form a sludge and the crystalline
powder was collected by filtration. The powder was dissolved in
methanol. To the solution was added cyclohexylamine, and the
mixture was stirred. After evaporating methanol, diisopropyl ether
was added to the residue to form a sludge. The crystalline powder
was collected by filtration and washed with diisopropyl ether. The
desired product was extracted with water and the aqueous layer was
washed with 4-methyl-2-pentanone twice. The aqueous layer was
concentrated and to the layer was added diisopropyl ether to form a
sludge. After filtration, the crystals were washed with diisopropyl
ether to give 120 mg of a dicyclohexylamine salt of compound
(34).
[0206] .sup.1H-NMR (D.sub.2O) .delta.: 3.37 (s, 3H), 3.49 (dd, 1H,
J=4.9 Hz, 12.7 Hz), 3.62 (d, 1H, J=4.9 Hz), 3.69 (dd, 1H, J=2.7 Hz,
12.7 Hz), 3.74-3.78 (m, 1H), 4.28 (dd, 1H, J=4.6 Hz, 7.8 Hz), 5.39
(d, 1H, J=5.9 Hz); MS (APCI) m/z: 243 (M-H).sup.-.
Example 23
Preparation of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-ribose-1-phosphate
(18a)
[0207] To a mixture of 6.92 g of orthophosphoric acid in 80 mL of
acetonitrile were added 5.51 mL of tri-n-butylamine and 10 g of
molecular sieves 4A. The mixture was stirred at room temperature
for 5 hours and allowed to stand overnight. After cooling to
-7.degree. C., to the mixture was added 10 g of
3,5-O-bis(4-chlorobenzoyl)-2-deoxy-.alpha.-D-ribosyl chloride
(purity: 85%). The mixture was stirred for 9 hours and allowed to
stand at -15.degree. C. overnight. After adding 16.5 mL of
tri-n-butylamine, the molecular sieves were removed by filtration.
The filtrate was concentrated and the residue was dissolved in
4-methyl-2-pentanone and washed with water. The organic layer was
ice-cooled and 5.66 mL of cyclohexylamine was added with stirring
for crystallization. After 1.5 hours, the precipitated crystals
were filtered and dried in vacuo at room temperature to give 13.5 g
of a dicyclohexylamine salt of compound (18a).
.alpha.-form:.beta.-form=98.8:1.2).
Example 24
Preparation of 2-deoxy-.alpha.-D-ribose-1-phosphate (20)
[0208] To a solution of 7.05 g of the compound obtained in Example
23 in methanol was added 2.92 mL of cyclohexylamine, and the
mixture was stirred at room temperature. After stirring 72 hours,
the mixture was concentrated and to the residue was added ethanol
to provide a suspension which was then stirred. After collecting
the precipitated crystals, they were dried I vacuo at room
temperature to give 3.87 g of a dicylcohexylamine salt of compound
(20) (without the .beta.-form on NMR).
[0209] .sup.1H NMR (D.sub.2O) d: 5.57 (dd, J=5.1, 6.1 Hz, 1H), 4.03
(m, 2H), 3.54 (ddd, J=1.2, 2.2, 12.2 Hz, 1H), 3.42 (ddd, J=1.2,
5.1, 12.2 Hz, 1H), 3.18-2.94 (m, 2H), 2.17 (m, 1H), 1.90 (d, J=1.2,
12.8 Hz, 1H), 1.8-1.45 (m, 10H), 1.25-0.9 (m, 12H).
[0210] Anal. Calcd. for
C.sub.5H.sub.9O.sub.7P.C.sub.12H.sub.28N.sub.2, C, 49.50%; H:
9.04%; N, 6.79%; P: 7.51%,
[0211] Found C, 49.26%; H, 8.81%; N, 6.64%; P: 7.29%.
Example 25
Preparation of 2'-deoxyadenosine (1)
[0212] Fifty mL of an LB medium was inoculated with Escherichia
coli K-12/XL-10 strain (Stratagene Inc.) and it was cultured at
37.degree. C. overnight. After collection, the bacteria was lysed
with a lysis solution containing 1 mg/mL of lysozyme. The lysis
solution was treated with phenol and DNA was precipitated as usual
by ethanol precipitation. The DNA precipitate was collected with a
glass rod and washed to prepare an E. coli chromosome DNA.
[0213] Oligonucletides of SEQ ID Nos. 1 and 2 designed based on the
sequence a known deoD gene in Escherichia coli (GenBank accession.
No. AE000508 with a coding region of base numbers 11531 to 12250)
were used as primers for PCR. These primers have restriction enzyme
recognition sequences for EcoRI and Hind III near 5'- and 3'-ends,
respectively.
[0214] SEQ ID No. 1: GTGAATTCAC AAAAAGGATA AAACAATGGC
[0215] SEQ ID No. 2: TCGAAGCTTG CGAAACACAA TTACTCTTT
[0216] Using 0.1 mL of a PCR reaction solution containing 6
ng/.mu.L of the above E. coli chromosome DNA completely digested by
restriction enzyme Hind III and the primers (each at 3 .mu.M), PCR
was conducted by 30 cycles under the conditions of denaturation:
96.degree. C., 1 min; annealing: 55.degree. C., 1 min; elongation:
74.degree. C., 1 min per a cycle.
[0217] The above reaction product and a plasmid pUC18 (Takara Shuzo
Co. Ltd.) were digested by EcoRI and Hind III and ligated using
Ligation-High (Toyobo Co. Ltd.). The recombinant plasmid obtained
was used to transform Escherichia coli DH5.alpha.. The transformed
strain was cultured in an LB agar medium containing 50 .mu.g/mL of
ampicillin and X-Gal
(5-bromo-4-chloro-3-indolyl-.beta.-galactoside) to provide an
Am-resistant transformant as a white colony. A plasmid was
extracted from the transformant thus obtained and the plasmid in
which a desired DNA fragment had been inserted was designated as
pUC-PNP73. The transformant thus obtained was designated as
Escherichia coli MT-10905.
[0218] Escherichia coli MT-10905 was cultured by shaking at
37.degree. C. overnight in 100 mL of an LB medium containing 50
.mu.g/mL of Am. The culture medium was centrifuged at 13,000 rpm
for 10 min to collect the bacteria. The bacteria were suspended in
10 mL of 10 mM Tris-hydrochloride buffer (pH 8.0) and
ultrasonicated to give a homogenate which was then used as an
enzyme source.
[0219] Reaction solutions were prepared by adding calcium chloride
(Waco Pure Chemicals, Extra pure grade) at different concentrations
to a mixture of 2.5 mM 2-deoxy-.alpha.-D-ribose-1-phosphate
diammonium salt prepared in Example 8, 2.5 mM adenine (Wako Pure
Chemicals, Extra pure grade), 0.1 mL of the ultrasonic enzyme
homogenate from a purinenucleoside-phosphorylase producing strain
and 10 mM Tris-hydrochloride buffer (pH 7.4). One mL of a reaction
solution was reacted at 30.degree. C. for 24 hours. At the end of
the reaction, a white precipitate had been formed.
[0220] HPLC analysis described below for a post-reaction solution
showed a peak completely identical to the peak of 2'-deoxyadenosine
(Wako Pure Chemicals, Extra pure grade) in all the post-reaction
solutions.
HPLC Analysis Conditions
[0221] Column: YMC-Pack ODS-A312, 150.times.6.0 mm I.D.
[0222] Column temperature: 40.degree. C.
[0223] Pump flow rate: 0.75 mL/min
[0224] Detection: UV 260 nm
[0225] Eluent: 10 mM phosphoric acid:acetonitrile=95:5 (V/V)
[0226] Table 1 shows the calculation results of a reaction
inversion rate after determining a concentration of
2'-deoxyadenosine in a post-reaction solution. TABLE-US-00001 TABLE
1 Amount of calcium Reaction chloride inversion rate (mM) (%) 0.0
80.4 2.5 90.8 10.0 96.0
Example 26
Preparation of 2'-deoxyadenosine (2)
[0227] A reaction was conducted as described in Example 25 except
that aluminum chloride was added in place of calcium chloride. At
the end of the reaction, a white precipitate had been formed. HPLC
analysis for the post-reaction solutions as described in Example 25
showed a peak completely identical to the peak of 2'-deoxyadenosine
(Wako Pure Chemicals, Extra pure grade) in all the post-reaction
solutions. Table 2 shows the calculation results of a reaction
inversion rate after determining a concentration of
2'-deoxyadenosine in a post-reaction solution. TABLE-US-00002 TABLE
2 Amount of calcium chloride Reaction inversion rate (mM) (%) 0.0
80.4 (same as in Example 15) 2.5 90.2 10.0 93.3
Example 27
Preparation of 2'-deoxyadenosine (3)
[0228] A reaction was conducted as described in Example 25 except
that 10 mM of barium chloride was added in place of calcium
chloride. At the end of the reaction, a white precipitate had been
formed. HPLC analysis for the post-reaction solutions as described
in Example 25 showed a peak completely identical to the peak of
2'-deoxyadenosine (Wako Pure Chemicals, Extra pure grade) in the
post-reaction solutions. A reaction inversion rate after
determining a concentration of 2'-deoxyadenosine in a post-reaction
solution was calculated to be 92.4%.
Example 28
Preparation of thymidine
[0229] One mL of a reaction solution consisting of 2.5 mM
2-deoxy-.alpha.-D-ribose-1-phosphate diammonium salt prepared in
Example 8, 2.5 mM thymine (Wako Pure Chemicals, Extra pure grade),
12 units/mL thymidine phosphorylase (SIGMA), 0 mM or 10 mM calcium
nitrate (Wako Pure Chemicals, Extra pure grade) and 10 mM
Tris-hydrochloride buffer (pH 7.4) was reacted at 30.degree. C. for
24 hours. At the end of the reaction, a white precipitate had been
formed. HPLC analysis for the post-reaction solutions as described
in Example 25 showed a peak completely identical to the peak of
thymidine (Wako Pure Chemicals, Extra pure grade) in the
post-reaction solution. Table 3 shows the calculation results of a
reaction inversion rate after determining a concentration of
thymidine in the post-reaction solution. TABLE-US-00003 TABLE 3
Amount of calcium Amount of thymidine chloride formed (mM) (mM) 0
75.2 10.0 91.2
Example 29
Preparation of 2'-deoxyadenosine (4)
[0230] One mL of a reaction solution consisting of 100 mM
2-deoxy-.alpha.-D-ribose-1-phosphate diammonium salt prepared in
Example 8, 100 mM adenine (Wako Pure Chemicals, Extra pure grade),
0.1 mL of the ultrasonic enzyme homogenate from a
purinenucleoside-phosphorylase producing strain prepared in Example
25, 0 to 150 mM calcium chloride (Waco Pure Chemicals, Extra pure
grade) and 100 mM Tris-hydrochloride buffer (pH 8.0) was reacted at
50.degree. C. for 24 hours. At the end of the reaction, a white
precipitate had been formed. HPLC analysis for the post-reaction
solutions as described in Example 25 showed a peak completely
identical to the peak of 2-deoxyadenosine (Wako Pure Chemicals,
Extra pure grade) in the post-reaction solutions. Table 4 shows the
calculation results of determining a concentration of
2'-deoxyadenosine in a post-reaction solution. TABLE-US-00004 TABLE
4 Amount of calcium Amount of 2'- chloride deoxyadenosine formed
(mM) (mM) 0 85.0 20 90.0 60 96.5 100 97.8 150 97.5
Example 30
Preparation of 2'-deoxyguanosine
[0231] One mL of a reaction solution consisting of 100 mM
2-deoxy-.alpha.-D-ribose-1-phosphate diammonium salt prepared in
Example 8, 100 mM guanine (Wako Pure Chemicals, Extra pure grade),
0.1 mL of the ultrasonic enzyme homogenate from a
purinenucleoside-phosphorylase producing strain prepared in Example
25, 0 mM or 150 mM calcium chloride (Waco Pure Chemicals, Extra
pure grade) and 100 mM Tris-hydrochloride buffer (pH 8.0) was
reacted at 50.degree. C. for 24 hours. At the end of the reaction,
a white precipitate had been formed. HPLC analysis for the
post-reaction solution as described in Example 25 showed a peak
completely identical to the peak of 2-deoxyguanosine (Wako Pure
Chemicals, Extra pure grade) in the post-reaction solution. Table 5
shows the calculation results of determining a concentration of
2'-deoxyguanosine in the post-reaction solution. TABLE-US-00005
TABLE 5 Amount of calcium Amount of 2'- chloride deoxyguanosine
formed (mM) (mM) 0 50.0 150 97.5
Example 31
Preparation of adenosine
[0232] One mL of a reaction solution consisting of 100 mM
.alpha.-D-ribose-1-phosphate diammonium salt prepared in Example
10, 100 mM adenine (Wako Pure Chemicals, Extra pure grade), 0.1 mL
of the ultrasonic enzyme homogenate from a
purinenucleoside-phosphorylase producing strain prepared in Example
25, 0 mM or 150 mM calcium chloride (Waco Pure Chemicals, Extra
pure grade) and 100 mM Tris-hydrochloride buffer (pH 8.0) was
reacted at 50.degree. C. for 24 hours. At the end of the reaction,
a white precipitate had been formed. HPLC analysis for the
post-reaction solution as described in Example 25 showed a peak
completely identical to the peak of adenosine (Wako Pure Chemicals,
Extra pure grade) in the post-reaction solution. Table 6 shows the
calculation results of determining a concentration of adenosine in
the post-reaction solution. TABLE-US-00006 TABLE 6 Amount of
calcium Amount of adenosine chloride formed (mM) (mM) 0 86.1 150
98.4
Example 32
Preparation of 2',3'-dideoxyadenosine
[0233] One mL of a reaction solution consisting of 100 mM
2,3-dideoxy-.alpha.-D-ribose-1-phosphate diammonium salt prepared
in Example 12, 100 mM adenine (Wako Pure Chemicals, Extra pure
grade), 0.1 mL of the ultrasonic enzyme homogenate from a
purinenucleoside-phosphorylase producing strain prepared in Example
25, 0 mM or 150 mM calcium chloride (Waco Pure Chemicals, Extra
pure grade) and 100 mM Tris-hydrochloride buffer (pH 8.0) was
reacted at 50.degree. C. for 24 hours. At the end of the reaction,
a white precipitate had been formed. HPLC analysis for the
post-reaction solution as described in Example 25 showed a peak
completely identical to the peak of 2',3'-dideoxyadenosine (Sigma,
Extra pure grade) in the post-reaction solution. Table 7 shows the
calculation results of determining a concentration of
2',3'-dideoxyadenosine in the post-reaction solution.
TABLE-US-00007 TABLE 7 Amount of calcium Amount of 2',3'- chloride
dideoxyadenosine formed (mM) (mM) 0 82.4 150 96.4
Example 33
Preparation of adenine-9-.beta.-D-arabinoside
[0234] One mL of a reaction solution consisting of 100 mM
.alpha.-D-arabinofuranosyl-1-phosphate diammonium salt prepared in
Example 14, 100 mM adenine (Wako Pure Chemicals, Extra pure grade),
0.1 mL of the ultrasonic enzyme homogenate from a
purinenucleoside-phosphorylase producing strain prepared in Example
25, 0 mM or 150 mM calcium chloride (Waco Pure Chemicals, Extra
pure grade) and 100 mM Tris-hydrochloride buffer (pH 8.0) was
reacted at 50.degree. C. for 24 hours. At the end of the reaction,
a white precipitate had been formed. HPLC analysis for the
post-reaction solution as described in Example 25 showed a peak
completely identical to the peak of adenine-arabinoside (Sigma,
Extra pure grade) in the post-reaction solution. Table 8 shows the
calculation results of determining a concentration of
adenine-9-.beta.-D-arabinoside in the post-reaction solution.
TABLE-US-00008 TABLE 8 Amount of calcium Amount of
adenine-9-.beta.-D- chloride arabinoside formed (mM) (mM) 0 79.4
150 93.4
Example 34
Preparation of
2-amino-6-chloropurine-2'-deoxy-.beta.-D-riboside
[0235] One mL of a reaction solution consisting of 10 mM
2-deoxy-.alpha.-D-ribose-1-phosphate diammonium salt prepared in
Example 8, 10 mM 2-amino-6-chloropurine (Tokyo Kasei), 100 mM
Tris-hydrochloride buffer (pH 7.5) and 50 .mu.L of the ultrasonic
enzyme homogenate from a purinenucleoside-phosphorylase producing
strain prepared in Example 25 was reacted at 50.degree. C. for 4
hours. At the end of the reaction, a white precipitate had been
formed. HPLC analysis for the post-reaction solution under the
conditions below showed a peak of
2-amino-6-chloropurine-2'-deoxy-.beta.-D-riboside. A reaction
inversion rate was calculated to be 20.9% after determining the
concentration of 2-amino-6-chloropurine-2'-deoxy-.beta.-D-riboside
in the post-reaction solution.
HPLC Analysis Conditions
[0236] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0237] Column temperature: 40.degree. C.
[0238] Pump flow rate: 1.0 mL/min
[0239] Detection: UV 254 nm
[0240] Eluent: 25 mM potassium dihydrogen phosphate methanol
875:125 (V/V)
Example 35
Preparation of 2,6-diaminopurine-2'-deoxy-.beta.-D-riboside
[0241] A reaction was conducted as described in Example 34 except
that 2,6-diaminopurine (Tokyo Kasei) was added in place of
2-amino-6-chloropurine. HPLC analysis for the post-reaction
solution as described in Example 34 showed a peak of
2,6-diaminopurine-2'-deoxy-.beta.-D-riboside. A reaction inversion
rate was calculated to be 75.5% after determining the concentration
of 2,6-diaminopurine-2'-deoxy-.beta.-D-riboside in the
post-reaction solution.
Example 36
Preparation of 6-mercaptopurine-2'-deoxy-.beta.-D-riboside
[0242] A reaction was conducted as described in Example 34 except
that 6-mercaptopurine (KOUJIN) was added in place of
2-amino-6-chloropurine. HPLC analysis for the post-reaction
solution as described in Example 34 showed a peak of
6-mercaptopurine-2'-deoxy-.beta.-D-riboside. A reaction inversion
rate was calculated to be 57.2% after determining the concentration
of 6-mercaptopurine-2'-deoxy-.beta.-D-riboside in the post-reaction
solution.
Example 37
Preparation of 2-amino-6-iodopurine-2'-deoxy-.beta.-D-riboside
[0243] A reaction was conducted as described in Example 34 except
that 2-amino-6-iodopurine was added in place of
2-amino-6-chloropurine. HPLC analysis for the post-reaction
solution as described in Example 34 showed a peak of
2-amino-6-iodopurine-2'-deoxy-.beta.-D-riboside. A reaction
inversion rate was calculated to be 69.2% after determining the
concentration of 2-amino-6-iodopurine-2'-deoxy-.beta.-D-riboside in
the post-reaction solution.
Example 38
Preparation of
2-acetylamino-6-hydroxypurine-2'-deoxy-.beta.-D-riboside
[0244] A reaction was conducted as described in Example 34 except
that 2-acetylamino-6-hydroxypurine (Tokyo Kasei) was added in place
of 2-amino-6-chloropurine. HPLC analysis for the post-reaction
solution under the conditions described below showed a peak of
2-acetylamino-6-hydroxypurine-2'-deoxy-.beta.-D-riboside. A
reaction inversion rate was calculated to be 48.7% after
determining the concentration of
2-acetylamino-6-hydroxypurine-2'-deoxy-.beta.-D-riboside in the
post-reaction solution.
HPLC Analysis Conditions
[0245] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0246] Column temperature: 40.degree. C.
[0247] Pump flow rate: 1.0 mL/min
[0248] Detection: UV 254 nm
[0249] Eluent: 25 mM potassium dihydrogen phosphate methanol=75:25
(V/V)
Example 39
Preparation of
2-amino-6-cyclopropylaminopurine-2'-deoxy-.beta.-D-riboside
[0250] A reaction was conducted as described in Example 34 except
that 2-amino-6-cyclopropylaminopurine was added in place of
2-amino-6-chloropurine. HPLC analysis for the post-reaction
solution as described in Example 38 showed a peak of
2-amino-6-cyclopropylaminopurine-2'-deoxy-.beta.-D-riboside. A
reaction inversion rate was calculated to be 87.6% after
determining the concentration of
2-amino-6-cyclopropylaminopurine-2'-deoxy-.beta.-D-riboside in the
post-reaction solution.
Example 40
Preparation of 2',3'-dideoxy-3'-fluoro-D-guanosine
[0251] One mL of a reaction solution consisting of 7.0 mM
2,3-dideoxy-3-fluoro-D-erythropentofuranose-1-phosphate prepared in
Example 18, 10 mM guanine (Tokyo Kasei), 100 mM Tris-hydrochloride
buffer (pH 7.5) and 0.1 mL of the ultrasonic enzyme homogenate from
a purinenucleoside-phosphorylase producing strain prepared in
Example 25 was reacted at 50.degree. C. for 114 hours. HPLC
analysis for the post-reaction solution as described in Example 34
showed a peak of 2',3'-dideoxy-3'-fluoro-D-guanosine. A reaction
inversion rate was calculated to be 47.7% after determining the
concentration of 2',3'-dideoxy-3'-fluoro-D-guanosine in the
post-reaction solution.
Example 41
Preparation of 2',3'-dideoxy-3'-fluoro-D-guanosine
[0252] One mL of a reaction solution consisting of 7.0 mM
2,3-dideoxy-3-fluoro-D-erythropentofuranose-1-phosphate prepared in
Example 18, 10 mM guanine (Tokyo Kasei), 100 mM Tris-hydrochloride
buffer (pH 7.5) and 0.1 mL of the ultrasonic enzyme homogenate from
a purinenucleoside-phosphorylase producing strain prepared in
Example 25 was reacted at 50.degree. C. for 47 hours. To the
solution was added calcium chloride to a final concentration of 20
mM and the mixture was reacted at 50.degree. C. for additional 67
hours. HPLC analysis for the post-reaction solution as described in
Example 34 showed a peak of 2',3'-dideoxy-3'-fluoro-D-guanosine. A
reaction inversion rate was calculated to be 84.4% after
determining the concentration of
2',3'-dideoxy-3'-fluoro-D-guanosine in the post-reaction
solution.
Example 42
Preparation of 6-chloro-9-(.beta.-D-ribofuranos-1-yl)purine
[0253] One mL of a reaction solution consisting of 10 mM
6-chloropurine (Aldrich), 50 mM D-ribose-1-phosphate (22) prepared
in Example 10, 0.1 mL of the ultrasonic enzyme homogenate from a
purinenucleoside-phosphorylase producing strain prepared in Example
25 and 100 mM Tris-hydrochloride buffer (pH 7.5) was reacted at
50.degree. C. for 20 hours. After completion of the reaction, HPLC
analysis for the reaction solution under the conditions described
below showed a peak of the title compound. A reaction inversion
rate was calculated to be 62.4% after determining the concentration
of 6-chloro-9-(.beta.-D-ribofuranos-1-yl)purine in the
post-reaction solution.
HPLC Analysis Conditions
[0254] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0255] Column temperature: 40.degree. C.
[0256] Pump flow rate: 1.0 mL/min
[0257] Detection: UV 254 nm
[0258] Eluent: 25 mM potassium dihydrogen phosphate methanol=75:25
(V/V)
Example 43
Preparation of
1-(2-deoxy-.beta.-D-ribofuranos-1-yl)-1H-imidazo[4,5-b]pyridine and
3-(2-deoxy-.beta.-D-ribofuranos-1-yl)-1H-imidazo[4,5-b]pyridine
[0259] One mL of a reaction solution consisting of 10 mM
2-deoxy-.alpha.-D-ribose-1-phosphate ammonium salt prepared in
Example 8, 10 mM 4-azabenzimidazole (Aldrich), 100 mM
Tris-hydrochloride buffer (pH 7.5) and 50 .mu.L of the ultrasonic
enzyme homogenate from a purinenucleoside-phosphorylase producing
strain prepared in Example 25 was reacted at 50.degree. C. for 17
hours. HPLC analysis for the post-reaction solution under the
conditions described below showed two peaks of the title compounds.
Reaction inversion rates were calculated to be 3% and 7.2% after
determining the concentrations of the products in the post-reaction
solution.
HPLC Analysis Conditions
[0260] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0261] Column temperature: 40.degree. C.
[0262] Pump flow rate: 1.0 mL/min
[0263] Detection: UV 254 nm
[0264] Eluent: 25 mM potassium dihydrogen phosphate methanol=50:50
(V/V)
[0265] LC-MS analysis data: MS(APCI) m/z: 236 (MH).sup.+
Example 44
Preparation of 8-aza-2'-deoxyadenosine
[0266] A reaction was conducted as described in Example 43 except
that 8-azaadenine (Aldrich) was used in place of
4-azabenzimidazole. HPLC analysis for the post-reaction solution
under the conditions described below showed a peak of
8-aza-2'-deoxyadenosine. A reaction inversion rate was calculated
to be 4.8% after determining the concentration of
8-aza-2'-deoxyadenosine in the post-reaction solution.
HPLC Analysis Conditions
[0267] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0268] Column temperature: 40.degree. C.
[0269] Pump flow rate: 1.0 mL/min
[0270] Detection: UV 254 nm
[0271] Eluent: 25 mM potassium dihydrogen phosphate
methanol=875:125 (V/V)
[0272] LC-MS analysis data: MS(APCI) m/z: 253 (MH).sup.+
Example 45
Preparation of 8-aza-2'-deoxyguanosine
[0273] A reaction was conducted as described in Example 43 except
that 8-azaguanine (Tokyo Kasei) was used in place of
4-azabenzimidazole. HPLC analysis for the post-reaction solution as
described in Example 44 showed a peak of 8-aza-2'-deoxyguanosine. A
reaction inversion rate was calculated to be 36.1% after
determining the concentration of 8-aza-2'-deoxyguanosine in the
post-reaction solution.
Example 46
Preparation of 2-chloro-2'-deoxyadenosine (Cladribine)
[0274] A reaction was conducted as described in Example 43 except
that 2-chloro-4-aminopurine was used in place of
4-azabenzimidazole. HPLC analysis for the post-reaction solution
under the conditions described below showed a peak of the title
compound. A reaction inversion rate was calculated to be 96% after
determining the concentration of 2-chloro-2'-deoxyadenosine in the
post-reaction solution.
HPLC Analysis Conditions
[0275] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0276] Column temperature: 40.degree. C.
[0277] Pump flow rate: 1.0 mL/min
[0278] Detection: UV 254 nm
[0279] Eluent: 25 mM potassium dihydrogen phosphate
methanol=875:125 (V/V)
Example 47
Preparation of
1-(.beta.-D-ribofuranos-1-yl)-1,3,4-triazole-3-carboxamide
(Ribavirine)
[0280] A reaction was conducted as described in Example 43 except
that 1,2,4-tosyazole-3-carboxamide was used in place of
4-azabenzimidazole. HPLC analysis for the post-reaction solution
under the conditions described below showed a peak of the title
compound. A reaction inversion rate was calculated to be 69% after
determining the concentration of
1-(.beta.-D-ribofuranos-1-yl)-1,3,4-triazole-3-carboxamide in the
post-reaction solution.
HPLC Analysis Conditions
[0281] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0282] Column temperature: 40.degree. C.
[0283] Pump flow rate: 1.0 mL/min
[0284] Detection: UV 210 nm
[0285] Eluent: 25 mM potassium dihydrogen phosphate
Example 48
Preparation of
1-(.beta.-D-ribofuranos-1-yl)-5-aminoimidazole-4-carboxamide
(Acadesine)
[0286] A reaction was conducted as described in Example 43 except
that 5-aminoimidazole-4-carboxamide was used in place of
4-azabenzimidazole. HPLC analysis for the post-reaction solution
under the conditions described below showed a peak of the title
compound. A reaction inversion rate was calculated to be 46% after
determining the concentration of
1-(.beta.-D-ribofuranos-1-yl)-5-aminoimidazole-4-carboxamide in the
post-reaction solution.
HPLC Analysis Conditions
[0287] Column: Develosil ODS-MG-5, 250.times.4.6 mm I.D.
[0288] Column temperature: 40.degree. C.
[0289] Pump flow rate: 1.0 mL/min
[0290] Detection: UV 254 nm
[0291] Eluent: 25 mM potassium dihydrogen phosphate methanol=93:7
(V/V)
Example 49
Preparation of 2'-deoxyguanosine
[0292] To 20 g of purified water were added 3.22 g of
2-deoxyribose-1-phosphate di(monocyclohexylammonium) salt prepared
in Example 24 (7.72 mmol), 1.11 g of guanine (7.34 mmol) and 0.67 g
of magnesium hydroxide (11.48 mmol). The reaction mixture was
adjusted to pH 9 with a 20% aqueous solution of sodium hydroxide.
To the mixture was added 0.1 mL of the above enzyme solution (0.1
mL), and the mixture was reacted with stirring at 50.degree. C. for
8 hours. HPLC analysis for the reaction mixture after 8 hours
indicated that the desired 2'-deoxyguanosine was provided with a
reaction yield of 99%.
Example 50
Preparation of 2'-deoxyadenosine
[0293] To 20 g of purified water were added 3.22 g of
2-deoxyribose-1-phosphate di(monocyclohexylammonium) salt prepared
in Example 24 (7.72 mmol), 1.01 g of adenine (7.47 mmol) and 0.67 g
of magnesium hydroxide (11.48 mmol). The reaction mixture was
adjusted to pH 8.6 with a 20% aqueous solution of sodium hydroxide.
To the mixture was added 0.1 mL of the above enzyme solution (0.1
mL), and the mixture was reacted with stirring at 50.degree. C. for
3 hours. HPLC analysis for the reaction mixture after 8 hours
indicated that the desired 2'-deoxyadenosine was provided with a
reaction yield of 99%.
INDUSTRIAL APPLICABILITY
[0294] As described above, this invention is quite useful as an
anomer selective process for producing a 1-phosphorylated
saccharide derivative or a nucleoside and may be expected to used
in a variety of applications.
* * * * *