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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.