Title:
Process for the preparation of guanosine tetra- and penta-phosphates and products
United States Patent 3883508
Abstract:
The chemical synthesis of guanosine 3',5'-bis-diphosphate (Magic Spot I) and guanosine 3'-diphosphate, 5'-triphosphate (Magic Spot II) starting from the readily available guanosine 2',3'-cyclic phosphate is converted in a multistep procedure into the key intermediate guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-diphosphate which may then be converted into the respective tetra- or penta-phosphate in a single step procedure. The tetra- and penta-phosphate of guanosine are known enzyme activators particularly enzymes which inactivate antibiotics. Thus the tetra- and penta-phosphates of guanosine are useful as adjuncts in screens for compounds with antibiotic activity. These compounds also inhibit protein synthesis in plant materials.
US Patent References:
PROCESS FOR THE PREPARATION OF NUCLEOSIDE PHOSPHATE
Witzel et al. - September 1969 - 3467648
/3687808.html
Merigan, Jr. et al. - August 1972 - 3687808
PROCESS FOR THE PREPARATION OF NUCLEOSIDE PHOSPHATE
Witzel et al. - September 1969 - 3467648
/3687808.html
Merigan, Jr. et al. - August 1972 - 3687808
Application Number:
05/369657
Publication Date:
05/13/1975
Filing Date:
06/13/1973
View Patent Images:
Export Citation:
Assignee:
Hoffmann-La Roche Inc. (Nutley, NJ)
Primary Class:
Other Classes:
435/193, 536/26.300, 435/199, 536/26.120, 435/92, 536/26.260
International Classes:
C07H19/20; C07H19/00; C07D51/54
Field of Search:
260/211.5R
Primary Examiner:
Brown, Johnnie R.
Attorney, Agent or Firm:
Welt, Samuel Saxe Jon Gould George L. S. M.
Claims:
I claim
1. A process for the preparation of guanosine 3',5'-bis-diphosphate which process consists essentially of reacting in the dark guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-diphosphate with a C1 -C16 trialkyl ammonium phosphate in the presence of iodine and in dry pyridine at a temperature in the range of about 0° to 30°C. for a period of from about 12 to 40 hours.
2. The process of claim 1 wherein said trialkyl ammonium phosphate is tributyl ammonium phosphate and said guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-diphosphate is guanosine 5'--S--ethyl phosphorothioate-3'-diphosphate.
3. A process for the preparation of guanosine-3'-diphosphate, 5'-triphosphate which process consists essentially of reacting in the dark guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3-diphosphate with a C1 -C16 trialkyl ammonium pyrophosphate in the presence of iodine and in a mixture of a cyclic nitrogen base and a C1 -C16 trialkylamine at a temperature in the range of from about 0° to 40°C. for a period of from about 12 to 40 hours.
4. The process of claim 3 wherein said cyclic nitrogen base is α-picoline and said C1 -C16 trialkylamine is tributylamine.
5. The process of claim 3 wherein said guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3-diphosphate is 5'-S-ethyl phosphorothioate-3-diphosphate and said C1 -C16 trialkyl ammonium pyrophosphate is tributyl ammonium pyrophosphate.
6. Guanosine 5'--S--C1 -C7 alkyl phosphorothioate-2',3'-cyclic phosphate and its base addition salts.
7. The compound of claim 6 which is guanosine 5-S-ethyl phosphorothioate-2',3'-cyclic phosphate.
8. Guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-phosphate and its base addition salts.
9. The compound of claim 8 which is guanosine 5'-S-ethyl phosphorothioate-3'-phosphate.
10. Guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-diphosphate and its base addition salts.
11. The compound of claim 10 which is guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate.
1. A process for the preparation of guanosine 3',5'-bis-diphosphate which process consists essentially of reacting in the dark guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-diphosphate with a C1 -C16 trialkyl ammonium phosphate in the presence of iodine and in dry pyridine at a temperature in the range of about 0° to 30°C. for a period of from about 12 to 40 hours.
2. The process of claim 1 wherein said trialkyl ammonium phosphate is tributyl ammonium phosphate and said guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-diphosphate is guanosine 5'--S--ethyl phosphorothioate-3'-diphosphate.
3. A process for the preparation of guanosine-3'-diphosphate, 5'-triphosphate which process consists essentially of reacting in the dark guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3-diphosphate with a C1 -C16 trialkyl ammonium pyrophosphate in the presence of iodine and in a mixture of a cyclic nitrogen base and a C1 -C16 trialkylamine at a temperature in the range of from about 0° to 40°C. for a period of from about 12 to 40 hours.
4. The process of claim 3 wherein said cyclic nitrogen base is α-picoline and said C1 -C16 trialkylamine is tributylamine.
5. The process of claim 3 wherein said guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3-diphosphate is 5'-S-ethyl phosphorothioate-3-diphosphate and said C1 -C16 trialkyl ammonium pyrophosphate is tributyl ammonium pyrophosphate.
6. Guanosine 5'--S--C1 -C7 alkyl phosphorothioate-2',3'-cyclic phosphate and its base addition salts.
7. The compound of claim 6 which is guanosine 5-S-ethyl phosphorothioate-2',3'-cyclic phosphate.
8. Guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-phosphate and its base addition salts.
9. The compound of claim 8 which is guanosine 5'-S-ethyl phosphorothioate-3'-phosphate.
10. Guanosine 5'--S--C1 -C7 alkyl phosphorothioate-3'-diphosphate and its base addition salts.
11. The compound of claim 10 which is guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate.
Description:
DESCRIPTION OF THE INVENTION
The present invention relates to a multistep process for the preparation of guanosine 3',5'-bis-diphosphate (also known as Magic Spot I) and guanosine 3'-diphosphate, 5'-triphosphate (also known as Magic Spot II) starting from guanosine 2',3' cyclic phosphate.
In a first step of the instant process guanosine 2',3' cyclic phosphate, which may be in the form of a pyridinium salt, is treated with a C 1 -C 7 S-alkylphosphorothioate such as S-ethylphosphorothioate, preferably in the pyridinium form in the presence of a chemical condensing agent such as a carbodiimide derivative. Suitable carbodiimides for this purpose include diethyl carbodiimide, di-para toluyl carbodiimide and dicyclohexyl carbodiimide. Dicyclohexyl carbodiimide is preferred. This reaction may be conducted at a temperature in the range of from about 0° to 50°C., most preferably in the range of from about 20° to 30°C. An organic nitrogen base is preferably utilized as solvent in the reaction medium. Suitable organic nitrogen bases for the reaction solvent include pyridine, dimethylformamide, picoline and the like. Pyridine is a preferred solvent. The product obtained from this reaction is guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2',3'-cyclic phosphate which may be isolated in the form of its base addition salt such as for example its ammonium, alkali metal or trialkylammonium, i.e., triethyl ammonium salt.
In the following step the aforesaid guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2,3'-cyclic phosphate is treated in an aqueous reaction medium containing tris hydrochloride buffer at a pH in the range of 7 to 8.5, preferably at about 7.6; with the enzyme ribonuclease T 1 . The total of ribonuclease T 1 utilized is in the range of about 1 × 10 4 to 1 × 10 7 units per millimol of guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2',3'-cyclic phosphate employed. This enzyme treatment is conducted at a temperature in the range of from about 25° to 45°C., most preferably in the range of from about 35° to 40°C. The length of treatment is not broadly critical but generally will be in the range of from about 12 to 60 hours. The resulting product from the enzyme treatment is guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-phosphate.
In the next reaction step of the instant invention the aforesaid guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-phosphate in admixture with a C 1 -C 16 tetraalkyl ammonium acetate such as tetraethyl ammonium acetate is treated with acetic anhydride. After dilution with water and passage through a Dowex 50 column in the pyridinium form the reaction residue, after removal of the solvents, is treated with a solution of carbonyl diimidazole in DMF. After quenching with methanol, a solution of C 1 -C 16 trialkyl ammonium phosphate such as tributyl ammonium phosphate in DMF is added. The reaction is conducted for a period of 12 to 100 hours at a temperature in the range of from 0° to 40°. After workup in a conventional manner there is obtained the desired product, guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate. This key intermediate may then be converted into either the tetra- or penta-phosphate according to the alternative procedures described below.
The conversion of guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate into guanosine 3',5'-bis-diphosphate can be readily accomplished by treating a dried mixture of guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate and a C 1 -C 16 trialkyl ammonium phosphate such as tributyl ammonium phosphate (preferred), trioctyl ammonium phosphate, dimethylbutyl ammonium phosphate and the like with iodine in dry pyridine in the dark. The reaction is conveniently conducted at a temperature in the range of from about 0° to 40°C. After a total of 12 to 40 hours with continuous shaking the reaction mixture is worked up in the conventional manner such as for example by column chromatography over DEAE cellulose to yield the desired guanosine 3', 5' bis-diphosphate.
Alternatively, the guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate intermediate may be converted into guanosine 3'-diphosphate, 5'-triphosphate by treating the former compound preferably as its trialkylammonium salt, with iodine and a C 1 -C 16 trialkyl ammonium pyrophosphate, such as tributyl ammonium pyrophosphate, trioctyl ammonium pyrophosphate, dimethylbutyl ammonium pyrophosphate and the like in the dark for 12 to 40 hours at a temperature in the range of from about 0° to 40°C. The reaction medium will also contain a cyclic organic nitrogen base such as a α-picoline, pyridine, or other 2-methyl pyridines such as lutidine or collidine, and optionally a trialkylamine such as tributylamine.
The tetra- and penta-phosphates of guanosine produced by the process of the present invention are useful as enzyme activators, particularly enzymes which inactivate antibiotics such as chloramphenicol acetyl transferase (CAT) and streptomycin adenylyl transferase (SAT). See for example De Crombrugghe et al., Nature New Biology, 241, 237 (1973). Thus the aforesaid compounds may be utilized in antibiotic screens wherein compounds to be tested for antibiotic activity are treated with the system comprising a guanosine tetra- or penta-phosphate and either CAT or SAT and absence of inactivation is indicative of resistance to these antibiotic inactivating enzymes.
The guanosine tetra- and penta-phosphates produced herein are also inhibitors of protein synthesis in plant material. See Manzocchi et al., FEBS Letters, 29, 309 (1973).
The term "alkyl" as used herein is meant to include branched or straight chained saturated radicals containing the number of carbon atoms indicated in the prefix. Suitable C 1 -C 7 alkyl groups include methyl, ethyl n-propyl, i-propyl, hexyl, and the like. A preferred C 1 -C 7 alkyl group is ethyl. Examples of suitable C 1 -C 16 alkyl groups include methyl, ethyl, i-propyl, n-butyl, n-octyl, n-decanyl, and n-hexadecyl. Unless otherwise indicated all alkyl groups are in the "normal" configuration.
The present invention also relates to the following novel intermediates produced in accordance with the processes disclosed herein:
guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2',3'-cyclic phosphate
guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate
guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-phosphate
guanosine 5'-S-ethyl phosphorothioate-3'-phosphate
guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate
guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate and the base addition salts thereof.
Suitable base addition salts include the alkali metal salts and the C 1 -C 16 trialkylammonium salts.
EXAMPLE 1
Guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate
A mixture of dilithium S-ethyl phosphorothioate (15.5 mmol) and guanosine 2',3' cyclic phosphate (1.55 mmol, tributylammonium salt) in 10% pyridine (150 ml.) were stirred with Dowex 50 resin (50 ml., pyridinium form) for 20 minutes and the slurry was added to a Dowex 50 column (2.3 × 35 cm, pyridinium form). The column was eluted with 10% pyridine (500 ml.) and the eluant was evaporated to dryness, and dried by evaporation of portions of pyridine (4 × 50 ml.) over the residue. Pyridine (50 ml.) and dicyclohexylcarbodiimide (4.1 g) were added, and the mixture was shaken for 19 hours. The reaction was terminated by addition of water (50 ml.) and after 4 hours the solids were filtered off and washed with 10% pyridine. The filtrate and washings were evaporated to half volume and diluted to 1 l. with water. The solution was filtered through celite, diluted to 2 l with water, adjusted to pH 7 with triethylamine, and applied to a DEAE cellulose column (4.5 × 65 cm, bicarbonate form). The column was eluted with a linear gradient of 6 l. of aqueous 0.005M triethylammonium bicarbonate, pH 7.5 in the reservoir. Fractions of 20 ml. were collected, and product guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate emerged from the column in fractions 460-570. After evaporation to dryness, the triethylammonium salt of guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate was obtained as a gum (0.76 mmol, 49%, based on ε 252 of 13700 for the guanine moiety).
EXAMPLE 2
Guanosine 5'-S-ethyl phosphorothioate-3'-phosphate
A solution of guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate (0.38 mmol) in water (15 ml.) and M tris hydrochloride buffer, pH 7.6 (3 ml.) was treated with ribonuclease T 1 (500,000 units) for 3 hours at 37°. The solution was diluted to 40 ml. with water and applied to a DEAE cellulose column (2.3 × 50 cm, bicarbonate form). The column was eluted with a linear gradient of 4 l. of 0.01M triethylammonium bicarbonate buffer, pH 7.5 in the mixing vessel and 4 l. of 0.2M triethylammonium bicarbonate buffer pH 7.5 in the reservoir. Fractions of 20 ml. were collected, and the product emerged in fractions 183-250. After evaporation of the buffer, the product guanosine 5'-S-ethyl phosphorothioate-3'-phosphate was obtained as a gum (0.33 mmol, 88%).
EXAMPLE 3
Guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate
A mixture of guanosine 5'-S-ethyl phosphorothioate-3'-phosphate (73 μmol) and tetraethylammonium acetate (200 mg.) were dried by evaporation of pyridine (3 × 2 ml.) from the mixture and treated with acetic anhydride (1 ml.) for 66 hours. The solution was cooled to 0° and methanol (0.5 ml.) was added. After 15 min. water (0.5 ml.) was added and after 2 hours the solution was diluted with water, and passed through a Dowex 50 column (1.5 × 20 cm, pyridinium form). The eluant was evaporated to dryness, dissolved in DMF (0.5 ml.) and treated with a solution of carbonyl diimidazole (58 mg.) in DMF (0.5 ml.). After 18 hours methanol (25 μl) was added, and after 1 hour a solution of tributylammonium phosphate (0.365 mmol) in DMF (0.365 ml.) was added. After 3 days the precipitate was spun down and washed with dry DMF (2 ml.) The combined supernatants were treated with methanol (0.5 ml.), followed by concentrated ammonium hydroxide (25 ml.), in methanol (25 ml.). After 40 hours the solution was evaporated to dryness, dissolved in water (50 ml.) and applied to a DEAE cellulose column 2.3 × 38 gm, bicarbonate form. The column was eluted with a linear gradient of 2 l. of 0.1M triethylammonium bicarbonate pH 7.5 in the mixing vessel and 2 l. of 0.25M triethylammonium bicarbonate pH 7.5 in the reservoir. Fractions of 20 ml. were collected and product guanosine 5'-S-ethylphosphorothioate-3'-diphosphate (26.7 μmol, 37%) was obtained in fractions 116-131.
EXAMPLE 4
Guanosine 3',5' bis-diphosphate
A mixture of guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate (15.5 μmol) and tributylammonium phosphate (150 mol) was dried by evaporation of portions of pyridine (4 × 3 ml.) from the mixture. Dry pyridine (0.5 ml.) and iodine (77 mg.) were added, and the reaction was shaken for 17 hours in the dark. The bulk of the pyridine was removed by evaporation, and the residue was dissolved in 5 ml. triethylammonium bicarbonate buffer (.075 M). The solution was extracted with ether (3 × 5 ml.), briefly evaporated to remove dissolved ether, and diluted to 65 ml. with water. This solution was applied to a DEAE cellulose column (2.3 × 38 cm, bicarbonate form) which was eluted with a linear gradient of 2 l. of 0.15M triethylammonium bicarbonate buffer pH 7.5 in the mixing vessel and 2 l. of 0.35M buffer in the reservoir. Guanosine 3',5' bis-diphosphate (2.8 μmol, 18%) was eluted from the column at a buffer molarity of 0.31M. After removal of the buffer, the product was stored in frozen aqueous solution.
The synthetic guanosine 3',5' bis-diphosphate and material from natural sources (prepared by the procedure of Cashel et al., J. Biol. Chem. 245, 2309 (1970) had identical mobilities in the following systems:
1. Polyethyleneimine cellulose thin layer chromatography (ascending), solvent - 1.5M potassium phosphate, pH 3.5.
2. paper electrophoresis, Whatman No. 3 paper, sodium citrate, 0.05M, pH 5, 1500v, 1.75 hr.
The synthetic and natural materials exhibited identical activities in stimulation of in vitro synthesis of the enzyme β-galactosidase.
EXAMPLE 5
Guanosine 3'-diphosphate, 5'-triphosphate
Guanosine 5'-S-ethyl phosphorothioate 3'-diphosphate (36.5 μmol, triethylammonium salt) and tributylammonium pyrophosphate (365 μmol) were dried by evaporation of pyridine (three 3ml portions) and treated with α-picoline (6 ml), tributylamine (0.2 ml) and iodine (191 mg) for 18 hr in the dark. The product was partitioned between aqueous triethylammonium bicarbonate buffer, pH 7 (0.05 M, 50 ml) and ether (50 ml), and the aqueous layer was washed with ether (2 × 50 ml), diluted with triethylammonium bicarbonate buffer, pH 7.5 (0.1M, 100 ml) and applied to a DEAE cellulose column (36 × 1.3 cm, bicarbonate form). The column was eluted with a linear gradient of 1 l. of 0.1 M triethylammonium bicarbonate buffer (pH 7.5) in the mixing vessel and 1 l. of 0.5 M buffer (pH 7.5) in the reservoir. The product was eluted from the column at a buffer strength of 0.33 M. The buffer was removed by evaporation and the product was stored at -15°C.
Paper chromatography
Isobutyric acid/conc ammonia/water 57:4:39 (v/v/v) for 3 days
Product Guanosine 3'-diphosphate, 5'-triphosphate Rf = 0.35
Guanosine 3',5'-bis-diphosphate Rf = 0.40
The present invention relates to a multistep process for the preparation of guanosine 3',5'-bis-diphosphate (also known as Magic Spot I) and guanosine 3'-diphosphate, 5'-triphosphate (also known as Magic Spot II) starting from guanosine 2',3' cyclic phosphate.
In a first step of the instant process guanosine 2',3' cyclic phosphate, which may be in the form of a pyridinium salt, is treated with a C 1 -C 7 S-alkylphosphorothioate such as S-ethylphosphorothioate, preferably in the pyridinium form in the presence of a chemical condensing agent such as a carbodiimide derivative. Suitable carbodiimides for this purpose include diethyl carbodiimide, di-para toluyl carbodiimide and dicyclohexyl carbodiimide. Dicyclohexyl carbodiimide is preferred. This reaction may be conducted at a temperature in the range of from about 0° to 50°C., most preferably in the range of from about 20° to 30°C. An organic nitrogen base is preferably utilized as solvent in the reaction medium. Suitable organic nitrogen bases for the reaction solvent include pyridine, dimethylformamide, picoline and the like. Pyridine is a preferred solvent. The product obtained from this reaction is guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2',3'-cyclic phosphate which may be isolated in the form of its base addition salt such as for example its ammonium, alkali metal or trialkylammonium, i.e., triethyl ammonium salt.
In the following step the aforesaid guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2,3'-cyclic phosphate is treated in an aqueous reaction medium containing tris hydrochloride buffer at a pH in the range of 7 to 8.5, preferably at about 7.6; with the enzyme ribonuclease T 1 . The total of ribonuclease T 1 utilized is in the range of about 1 × 10 4 to 1 × 10 7 units per millimol of guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2',3'-cyclic phosphate employed. This enzyme treatment is conducted at a temperature in the range of from about 25° to 45°C., most preferably in the range of from about 35° to 40°C. The length of treatment is not broadly critical but generally will be in the range of from about 12 to 60 hours. The resulting product from the enzyme treatment is guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-phosphate.
In the next reaction step of the instant invention the aforesaid guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-phosphate in admixture with a C 1 -C 16 tetraalkyl ammonium acetate such as tetraethyl ammonium acetate is treated with acetic anhydride. After dilution with water and passage through a Dowex 50 column in the pyridinium form the reaction residue, after removal of the solvents, is treated with a solution of carbonyl diimidazole in DMF. After quenching with methanol, a solution of C 1 -C 16 trialkyl ammonium phosphate such as tributyl ammonium phosphate in DMF is added. The reaction is conducted for a period of 12 to 100 hours at a temperature in the range of from 0° to 40°. After workup in a conventional manner there is obtained the desired product, guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate. This key intermediate may then be converted into either the tetra- or penta-phosphate according to the alternative procedures described below.
The conversion of guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate into guanosine 3',5'-bis-diphosphate can be readily accomplished by treating a dried mixture of guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate and a C 1 -C 16 trialkyl ammonium phosphate such as tributyl ammonium phosphate (preferred), trioctyl ammonium phosphate, dimethylbutyl ammonium phosphate and the like with iodine in dry pyridine in the dark. The reaction is conveniently conducted at a temperature in the range of from about 0° to 40°C. After a total of 12 to 40 hours with continuous shaking the reaction mixture is worked up in the conventional manner such as for example by column chromatography over DEAE cellulose to yield the desired guanosine 3', 5' bis-diphosphate.
Alternatively, the guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate intermediate may be converted into guanosine 3'-diphosphate, 5'-triphosphate by treating the former compound preferably as its trialkylammonium salt, with iodine and a C 1 -C 16 trialkyl ammonium pyrophosphate, such as tributyl ammonium pyrophosphate, trioctyl ammonium pyrophosphate, dimethylbutyl ammonium pyrophosphate and the like in the dark for 12 to 40 hours at a temperature in the range of from about 0° to 40°C. The reaction medium will also contain a cyclic organic nitrogen base such as a α-picoline, pyridine, or other 2-methyl pyridines such as lutidine or collidine, and optionally a trialkylamine such as tributylamine.
The tetra- and penta-phosphates of guanosine produced by the process of the present invention are useful as enzyme activators, particularly enzymes which inactivate antibiotics such as chloramphenicol acetyl transferase (CAT) and streptomycin adenylyl transferase (SAT). See for example De Crombrugghe et al., Nature New Biology, 241, 237 (1973). Thus the aforesaid compounds may be utilized in antibiotic screens wherein compounds to be tested for antibiotic activity are treated with the system comprising a guanosine tetra- or penta-phosphate and either CAT or SAT and absence of inactivation is indicative of resistance to these antibiotic inactivating enzymes.
The guanosine tetra- and penta-phosphates produced herein are also inhibitors of protein synthesis in plant material. See Manzocchi et al., FEBS Letters, 29, 309 (1973).
The term "alkyl" as used herein is meant to include branched or straight chained saturated radicals containing the number of carbon atoms indicated in the prefix. Suitable C 1 -C 7 alkyl groups include methyl, ethyl n-propyl, i-propyl, hexyl, and the like. A preferred C 1 -C 7 alkyl group is ethyl. Examples of suitable C 1 -C 16 alkyl groups include methyl, ethyl, i-propyl, n-butyl, n-octyl, n-decanyl, and n-hexadecyl. Unless otherwise indicated all alkyl groups are in the "normal" configuration.
The present invention also relates to the following novel intermediates produced in accordance with the processes disclosed herein:
guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-2',3'-cyclic phosphate
guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate
guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-phosphate
guanosine 5'-S-ethyl phosphorothioate-3'-phosphate
guanosine 5'--S--C 1 -C 7 alkyl phosphorothioate-3'-diphosphate
guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate and the base addition salts thereof.
Suitable base addition salts include the alkali metal salts and the C 1 -C 16 trialkylammonium salts.
EXAMPLE 1
Guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate
A mixture of dilithium S-ethyl phosphorothioate (15.5 mmol) and guanosine 2',3' cyclic phosphate (1.55 mmol, tributylammonium salt) in 10% pyridine (150 ml.) were stirred with Dowex 50 resin (50 ml., pyridinium form) for 20 minutes and the slurry was added to a Dowex 50 column (2.3 × 35 cm, pyridinium form). The column was eluted with 10% pyridine (500 ml.) and the eluant was evaporated to dryness, and dried by evaporation of portions of pyridine (4 × 50 ml.) over the residue. Pyridine (50 ml.) and dicyclohexylcarbodiimide (4.1 g) were added, and the mixture was shaken for 19 hours. The reaction was terminated by addition of water (50 ml.) and after 4 hours the solids were filtered off and washed with 10% pyridine. The filtrate and washings were evaporated to half volume and diluted to 1 l. with water. The solution was filtered through celite, diluted to 2 l with water, adjusted to pH 7 with triethylamine, and applied to a DEAE cellulose column (4.5 × 65 cm, bicarbonate form). The column was eluted with a linear gradient of 6 l. of aqueous 0.005M triethylammonium bicarbonate, pH 7.5 in the reservoir. Fractions of 20 ml. were collected, and product guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate emerged from the column in fractions 460-570. After evaporation to dryness, the triethylammonium salt of guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate was obtained as a gum (0.76 mmol, 49%, based on ε 252 of 13700 for the guanine moiety).
EXAMPLE 2
Guanosine 5'-S-ethyl phosphorothioate-3'-phosphate
A solution of guanosine 5'-S-ethyl phosphorothioate-2',3'-cyclic phosphate (0.38 mmol) in water (15 ml.) and M tris hydrochloride buffer, pH 7.6 (3 ml.) was treated with ribonuclease T 1 (500,000 units) for 3 hours at 37°. The solution was diluted to 40 ml. with water and applied to a DEAE cellulose column (2.3 × 50 cm, bicarbonate form). The column was eluted with a linear gradient of 4 l. of 0.01M triethylammonium bicarbonate buffer, pH 7.5 in the mixing vessel and 4 l. of 0.2M triethylammonium bicarbonate buffer pH 7.5 in the reservoir. Fractions of 20 ml. were collected, and the product emerged in fractions 183-250. After evaporation of the buffer, the product guanosine 5'-S-ethyl phosphorothioate-3'-phosphate was obtained as a gum (0.33 mmol, 88%).
EXAMPLE 3
Guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate
A mixture of guanosine 5'-S-ethyl phosphorothioate-3'-phosphate (73 μmol) and tetraethylammonium acetate (200 mg.) were dried by evaporation of pyridine (3 × 2 ml.) from the mixture and treated with acetic anhydride (1 ml.) for 66 hours. The solution was cooled to 0° and methanol (0.5 ml.) was added. After 15 min. water (0.5 ml.) was added and after 2 hours the solution was diluted with water, and passed through a Dowex 50 column (1.5 × 20 cm, pyridinium form). The eluant was evaporated to dryness, dissolved in DMF (0.5 ml.) and treated with a solution of carbonyl diimidazole (58 mg.) in DMF (0.5 ml.). After 18 hours methanol (25 μl) was added, and after 1 hour a solution of tributylammonium phosphate (0.365 mmol) in DMF (0.365 ml.) was added. After 3 days the precipitate was spun down and washed with dry DMF (2 ml.) The combined supernatants were treated with methanol (0.5 ml.), followed by concentrated ammonium hydroxide (25 ml.), in methanol (25 ml.). After 40 hours the solution was evaporated to dryness, dissolved in water (50 ml.) and applied to a DEAE cellulose column 2.3 × 38 gm, bicarbonate form. The column was eluted with a linear gradient of 2 l. of 0.1M triethylammonium bicarbonate pH 7.5 in the mixing vessel and 2 l. of 0.25M triethylammonium bicarbonate pH 7.5 in the reservoir. Fractions of 20 ml. were collected and product guanosine 5'-S-ethylphosphorothioate-3'-diphosphate (26.7 μmol, 37%) was obtained in fractions 116-131.
EXAMPLE 4
Guanosine 3',5' bis-diphosphate
A mixture of guanosine 5'-S-ethyl phosphorothioate-3'-diphosphate (15.5 μmol) and tributylammonium phosphate (150 mol) was dried by evaporation of portions of pyridine (4 × 3 ml.) from the mixture. Dry pyridine (0.5 ml.) and iodine (77 mg.) were added, and the reaction was shaken for 17 hours in the dark. The bulk of the pyridine was removed by evaporation, and the residue was dissolved in 5 ml. triethylammonium bicarbonate buffer (.075 M). The solution was extracted with ether (3 × 5 ml.), briefly evaporated to remove dissolved ether, and diluted to 65 ml. with water. This solution was applied to a DEAE cellulose column (2.3 × 38 cm, bicarbonate form) which was eluted with a linear gradient of 2 l. of 0.15M triethylammonium bicarbonate buffer pH 7.5 in the mixing vessel and 2 l. of 0.35M buffer in the reservoir. Guanosine 3',5' bis-diphosphate (2.8 μmol, 18%) was eluted from the column at a buffer molarity of 0.31M. After removal of the buffer, the product was stored in frozen aqueous solution.
The synthetic guanosine 3',5' bis-diphosphate and material from natural sources (prepared by the procedure of Cashel et al., J. Biol. Chem. 245, 2309 (1970) had identical mobilities in the following systems:
1. Polyethyleneimine cellulose thin layer chromatography (ascending), solvent - 1.5M potassium phosphate, pH 3.5.
2. paper electrophoresis, Whatman No. 3 paper, sodium citrate, 0.05M, pH 5, 1500v, 1.75 hr.
The synthetic and natural materials exhibited identical activities in stimulation of in vitro synthesis of the enzyme β-galactosidase.
EXAMPLE 5
Guanosine 3'-diphosphate, 5'-triphosphate
Guanosine 5'-S-ethyl phosphorothioate 3'-diphosphate (36.5 μmol, triethylammonium salt) and tributylammonium pyrophosphate (365 μmol) were dried by evaporation of pyridine (three 3ml portions) and treated with α-picoline (6 ml), tributylamine (0.2 ml) and iodine (191 mg) for 18 hr in the dark. The product was partitioned between aqueous triethylammonium bicarbonate buffer, pH 7 (0.05 M, 50 ml) and ether (50 ml), and the aqueous layer was washed with ether (2 × 50 ml), diluted with triethylammonium bicarbonate buffer, pH 7.5 (0.1M, 100 ml) and applied to a DEAE cellulose column (36 × 1.3 cm, bicarbonate form). The column was eluted with a linear gradient of 1 l. of 0.1 M triethylammonium bicarbonate buffer (pH 7.5) in the mixing vessel and 1 l. of 0.5 M buffer (pH 7.5) in the reservoir. The product was eluted from the column at a buffer strength of 0.33 M. The buffer was removed by evaporation and the product was stored at -15°C.
Paper chromatography
Isobutyric acid/conc ammonia/water 57:4:39 (v/v/v) for 3 days
Product Guanosine 3'-diphosphate, 5'-triphosphate Rf = 0.35
Guanosine 3',5'-bis-diphosphate Rf = 0.40