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Патент USA US3082213

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United States Patent 0 rice Patented Midli?i
l
2
3,032,203
The term “nucleoside” is used herein ‘to include deoxy
nuoleosides. Thus, “nucleoside” within the meaning of
NOVEL NUCLEOTIDE; (FOENZYMES
Leon Goldman and Joseph William Marslco, Nanuet,
N.Y., and George Washington Anderson, Upper Saddle
the present invention includes:
(4) Thymidine (or thymine deOXy?bOSidC):
River, N.J., assignors to American Cyanamid Com- 5
pany, New York, N.Y., a corporation of Maine
No Drawing. Filed Feb. 10, 1960, Ser. No. 7,744
4 Claims. (Cl. 260—-211.5)
The present invention relates to a novel method of 10
preparing nucleotide coenzymes of biological and medi
cal signi?cance.
In order to clarify the exact nature of the chemical com
pounds of the present invention a few de?nitions will
?rst ‘be given before proceeding with the description of th
invention.
15
‘
A nucleoside is an N-glycoside of 'a heterocyclic base,
generally a pyrimidine or a purine. Examples of nucleo
A nucleotide is a phosphate ester of a nucleoside and
may be a nucleoside monophosphate or a nucleoside poly
sides are:
(1) Adenosine (or adenine riboside):
phosphate. Examples of nucleoside monophosphates are:
(l1) Adenosine-'5’ phosphate (or muscle adenylic acid
or adenosine monophosphate, the latter hereinafter termed
AM‘P) :
25
40
45
mm
5°
.
OH
(3) Cytidine-S’ phosphate:
‘
55
O
‘
ff
.
H2O-F-0H
_0H
60
65
3,082,203
4
3
discovered by Harden and Young in 1904. Examples of
(4) Thymidine-S’ phosphate:
nucleotide coenzymes are:
(1) Cozymase (or diphosphopyridine nucleotide):
10
15
(2) Flavin adenine dinucleotide:
Examples of nucleoside polyphosphates are:
(l) Adenosine-S' diphosphate (hereinafter termed
ADP):
20
O
H
Oil
(3) Uridine diphosphate glucose:
Olly
35
l-IOH
O
on 2
(2) Adénosine-S' triphosphate (hereinafter termed
ATP):
\
40
H
H
(4) Coenzyme A.
O
45
OH no
_
0
0
I!
ll
u ’
| J
azo- -0- wens-g- H‘-C—l|ll>l
on on
50
/l‘i
\
>
N
‘u
011301-10
,
a,
CH?‘
HS-CHgCHzTHN_E=O
H2
. Hz
Originally, the term “nucleotide” referred only to phos 55 The preparation of adenosine-S' phosphoroimidazole
from adenosine-5' phosphate, dicyclohexylcarbodiimide
phate esters of nucleosides. Today, the term “nucleotide”
and imidazole was reported by R. W. Chambers and I. G.
is applied to phosphate esters of N-glycosides of hetero
Moffatt [1. Am. Chem. Soc. 80, 3752 (1958)]. There
cyclic bases generally. The newer de?nition, thus, in
cludes not only the simple nucleotides of the original
after, the more facile preparation of nucleotide imidazoles
de?nition, but also the nucleic acids (polynucleotides) 60 by the interaction of a salt of arnucleotide and a l,1’-car
bonyldiimidazole was discovered by L. Goldman et a1.
and such substances as adenosine-S' triphosphate and
nicotinamide nucleotide. Derivatives of riboflavin phos
as is more fully set forth in U.S. Patent No. 2,951,838.
phate, although not glycosidic in nature, are commonly
The reactivity of phosphorylated imidazoles is much
included among the nucleotides because of their similarity
greater than that of the ordinary phosphoramidates which
to, and association with, true nucleotides.
65 also contain a phosphorus-nitrogen linkage. It has been
A nucleotide coenzyme is a compound including in its
postulated that this is a result of electronic displacements
structure at least one simple nucleotide moiety. The
associated in particular with the electron attraction of
term “nucleotide coenzyme” is applied to a large and
the unsubstituted nitrogen atom. Surprisingly, however,
growing group of substances which are vital components
although the phosphorus-nitrogen bond in the nucleotide
of many enzyme systems involved in metabolic processes. 70 imidazoles is thus generally activated, it is nevertheless re
Nucleotide coenzymes function in association with spe
sistant to reaction with water. Hence, it is possible to
ci?c proteins or apoenzymes, the complete enzyme system
prepare nucleotide coenzymes and related compounds such
being made up of the combination apoenzyme plus co
as the linear and cyclic oligonucleotides from the nucleo~
enzyme. Historically, the ?rst nucleotide coenzyme dis
tide imidazoles in the presence of water as a solvent with
out the starting material reacting with that solvent. This
covered was cozyrnase, or diphosphopyridine nucleotide,
3,082,203
5
6
circumstance is of critical importance because water is the
most convenient solvent for nucleotides.
Our invention is based upon the discovery that the
nucleotide imidazoles readily react not only with nucleo
To the bomb contents, 0.612 g. of 1,3-dicyclohexyl
guanidine was added and the ‘resultant solution evaporated
tides, but also with organic and inorganic nucleophilic
crystals of 1,3-dicyclohexylguanidinium adenosine-S’ phos
phoramidate (solvated with Water and dimethylformam
ide), melting point 211-214” C. dec. The product was
homogeneous has shown by paper chromatography in iso
propyl alcohol-ammonia-water (7-1-2) and by paper elec
under reduced pressure to a gummy residue. Addition of
acetone followed by ?ltration gave 1.55 g. of colorless
agents to form nucleotide coenzymes of biological and
medical signi?cance. The nucleotide imidazoles readily
react, for example, with ammonia and amines, carboxylic
acids, sulfuric acid, carbonic acid, N-blocked u-amino
acids, alcohols, phosphate esters, and phosphoric acid as 10 trophoresis in pH 7.5 phosphate buffer. Recrystallization
exempli?ed by the following reaction scheme:
from aqueous acetone gave l,3-dicyclohexylguanidinium
NHZ.
—————>
O
RENE
RC 02H
II
_______._>
rrlsol
ll
______,
H2003
RCHCOzH
O
Base-sugar-O——
O
O
NHZ
ROH
Base-sugar-O—
RO—P 03112
H3130;
0
H
OH
adenosine-S' phosphoramidate, melting point 236—238‘’
wherein the Base may be either a purine or a pyrimidine
moiety, the Sugar may be either a pentose or a hexose
C. dec.
moiety such as D-ribose, D-glucose, or 2-deoxy-D-rib-ose,
EXAMPLE2
and wherein imidazole is a by-product in each case.
Preparation of 1,3-Dicycl0hexylguanidinium
These nucleotide coenzymes may be prepared by the novel
method of the present invention in aqueous or non
aqueous media, at temperatures of from ~-—20° C. up to
Adenosine-S ’ Phosphoratmidate
55
100° C., and over a period of time of from a few minutes
A solution of adenosine-S’ phosphoroimidazole in 7.5
ml. of anhydrous dimethylformamide (prepared from
1.00 g. of adenosine-S’ phosphate hydrate, 0.586 g. of
imidazole, and 0.92 g. of 1,l’ecarbonyldiimidazole) was
up to 12 hours.
The nucleotide coenzymes produced by the novel
method of the present invention are useful as vital com 6 O dissolved in 20 ml. of 0.46 N ammonium hydroxide and
heated in a stainless steel bomb at 65° C. for 10 hours.
ponents of many enzyme systems. They are useful as
organic catalytic agents in that they are capable of alter
To the bomb contents was added 0.612 gram of 1,3,
dicyclohexylguanidine and the solution was evaporated to
ing the velocity of many chemical reactions.
The following examples illustrate the novel method of
dryness under reduced pressure. Recrystallization of the
preparing nucleotide coenzymes of the present invention. 65 residue from aqueous acetone gave 1.41 g. of colorless
crystals of 1,3-dicyclohexylguanidinium adenosine-S’
phosphoramidate (solvated with water and dimethylform»
amide), melting point 236~238° C. dec.
EXAMPLE 1
Preparation of l,3-Dicycl0hexylguanidinium Azlenosine-b"
Phosphoramia'ate
A solution of adenosine-S' phosphoroimidazole in an
hydrous dimethylformamide (prepared from 1.00 g. of
adenosine-S' phosphate hydrate, 0.586‘ g. of imidazole,
EXAMPLE 3
70
Preparation of Acridinium Aden0sine-5’ Pyrophosphate
To a stirred solution of adenosine-S' phosphoroimida
and 1.38 g. of 1,l'-carbonyldiimidazole) was diluted with
zole in anhydrous dimethylforrnamide (prepared from
2 N ammonium hydroxide and tert-butyl alcohol and
0.200 g. of adenosine-S' phosphate hydrate,~0.ll7 g. of
heated in a stainless steel bomb at 92° C. for 11 hours. 75 imidazole, and 0.0886 g. of 1,1'-carbonyldiimidazole) at
3,082,203
8
7
—10° C. to —20° C. was added dropwise a solution of
EXAMPLE 5
0.24 ml. of 85% phosphoric acid in dimethylformamide.
Preparation of P1-Aden0sine-5' PZ-UridiMe-S’
Pyrophosphate
The mixture was allowed to warm to room temperature
during one-half hour. The gummy solid, which separated
on chilling, was dissolved in dilute sulfuric acid and 5
treated with ethanolic acridine to yield 0.222 gram of
acridinium adenosine-S' pyrophosphate as yellow crystals,
melting point 2l2°-2l5° C. dec. Recrystallization from
water gave yellow needles, melting point 216°-217° C.
dec. Paper chromatography in 5% disodium phosphate
isoamyl alcohol and in isopropyl alcohol-|1% ammonium
A solution of adenosine-S' phosphoroimidazole in an
hydrous dimethylformamide (prepared from 0.0903 g.
of adenosine-5 phosphate hydrate, 0.0528 g. of imidazole,
and 0.132 g. of 1,l’dcarbonyldiimidazole) was added to
0.1045 g. of imidazolium uridine-St' phosphate. The re
10 action was diluted with 1.0 ml. of anhydrous pyridine
sulfate (3-2), and paper electrophoresis in 0.02 M potas
sium dihydrogen phosphate, showed as the only impurity
a trace of adenosine-S’ phosphate.
EXAMPLE 4
Preparation of P1, PZ-DiQdBIZOSiIIE-S' Pyrophosphate
To a stirred solution of 1.00 gram of adenosine-5’
and stirred for 74 hours at room temperature. When an
aliquot was removed and subjected to paper electro
phoresis ‘in an acetate buffer of pH 4.8, the major com
ponent, P1-adenosine-5’ PZ-uridine-S’ pyrophosphate,
traveled 15.7 cm. towards the anode, whereas the minor
components, adenosine-5' phosphate, uridine-S' phos
phate, and P1, PZ-(diuridine-S') pyrophosphate traveled
9.3, 13.0 and 18.7 cm., respectively.
We claim:
1. The method of preparing nucleotide coenzymes
phosphate monohydrate and 0.586 gram of imidazole in 20
which comprises reacting a nucleotide imidazole with a
7.5 ml. of anhydrous dimethylformamide at 0° to —5° C.
nucleophilic agent selected from the group consisting of
was added 0.922 gram of 1,1’-carbonyldiimidazole. After
five minutes the temperature of the reaction mixture was
lowered»o —10" to -20° C., and after 10 minutes 1.00
ammonia, primary amines, secondary amines, carboxylic
was added.
imidazole with aqueous ammonia at a temperature be
tween 65° C. and 95° C.
acids, sulfuric acid, carbonic acid, N-blocked a-amino
gram of adenosine-S' phosphate was added, with stirring. 25 acids, alcohols, phosphate esters and phosphoric acid.
2. The method of preparing adenosine-S’ phosphorami~
After 15 minutes the reaction mixture was warmed to
date which comprises reacting adenosine-S’ phosphoro
room temperature, and after 26 hours 3 ml. of pyridine
After 47 hoursvthe resulting solution was
worked up by chromatography on Dowex-l (formate)
[S. M. H. Christie, D. T. Elmore, G. W. Kenner, A. R. 30
Rodd, and F. J. Weymouth, 1. Chem. Soc., 2947 (1953)]
to yield, by evaporation of the 0.5 N formic acid eluate,
1.06 gram of P1, PZ-diadenosine-S' pyrophosphate ses
quihydrate as colorless crystals, which were homogeneous
by paper chromatography in 5% disodium phosphate
isoamyl alcohol and in n-butyl alcohol-acetic acid-water
(5-2-3), and by paper electrophoresis in 0.02 M potas
sium dihydrogen phosphate.
3. The method of preparing adenosine-S’ pyrophosphate
which comprises reacting adenosine-?' phosphoroimida
zole with phosphoric acid at a temperature between -20°
C. and 30° C.
4. The method of preparing P1, W-diadenosine-S' pyro
phosphate which comprises reacting adenosine-S’ phos
phoroimidazole with adenosine-S' phosphate at :1 mm
perature between —20° C. and 30° C.
No references cited.
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