United States Patent (19) 11) 4,169,011 Horwitz et al. 45) Sep. 25, 1979 (54 FACILE SYNTHESIS OF Simonscits, et al., Biochim. Biophys. Acta 395:74-79 3'-PHOSPHOADENOSNE (1975). 5'-PHOSPHOSULFATE (PAPS) Rozhin et al., J. Biol. Chem. 249:2079–2087 (1974). 75 Inventors: Jerome P. Horwitz; John P. Neenan; Horwitz et al., Biochim. Biophys. Acta, 480:376-381 Radhey S. Misra; Jurij Rozhin; Anne (1977). L. Huo, all of Detroit, Mich.; Kirsten Primary Examiner-Alvin E. Tanenholtz D. Philips, deceased, late of Detroit, Attorney, Agent, or Firm-John S. Roberts, Jr. Mich., by Judson C. Philips, administrator (57) ABSTRACT 3'-Phosphoadenosine 5'-phosphosulfate, also known as 73 Assignee: The United States of America as PAPS, is useful in establishing sulfate transfer mecha represented by the Department of nisms in animals and may be produced by a chemical Health, Education and Welfare, process yielding 68-72% from a pure adenosine Washington, D.C. 2',3'-cyclic phosphate 5'-phosphate, which compound is initially prepared from the reaction of adenosine and 21 Appl. No.: 840,783 pyrophosphoryl chloride. In the present procedure the pure cyclic phosphate is reacted with triethylamine-N- (22 Filed: Oct. 11, 1977 sulfonic acid to produce 2',3'-cyclic phosphate 5'-phos phosulfate. Subsequently, by hydrolysis with the en (51) Int. Cli. C12D 13/06 zyme ribonuclease-T2, the desired compound is pro (52) U.S. C...... 435/72 duced. Alternatively, the 2'-phosphoadenosine 5'-phos 58) Field of Search ...... 195/28 N phosulfate, known as iso-PAPS, may be produced from (56) References Cited 2',3'-cyclic phosphate 5'-phosphosulfate by treatment with a different , PDase or spleen phosphodies U.S. PATENT DOCUMENTS terase. This latter compound, iso-PAPS, biologically 3,268,416 8/1966 Igarasi et al...... 195/28 N has only one-third the activity of PAPS, the natural OTHER PUBLICATIONS isomer. Cherniak, et al., J. Biol. Chem. 239:2986–2990 (1964). 4 Claims, No Drawings

4,169,011 1. 2

FACLE SYNTHESIS OF O O (V) 3'-PHOSPHOADENOSINE 5'-PHOSPHOSULFATE li ll Ad o-i-o--o O (PAPS) O O

The present invention relates to a practical chemical HO synthesis of 3'-phosphoadenosine 5'-phosphosulfate o--o 10 O (IV) O-S-O-P-O O Ad The biological activity of IV (PAPS) may be mea sured by sulfate transfer to 6,7-3H2estrone as mediated O O by bovine adrenal (3'-phos 15 phoadenylylsulfate: estrone 3-sulfotransferase, EC OH 2.8.2.4) and is identical with that obtained with a sample o--o of IV prepared by an established biochemical proce O dure. By the same contrast, V exhibits approximately one-third the activity of the natural isomer. 20 in yields of 68–72% from adenosine 2',3'-cyclic phos The Roman numerals designated above correspond phate 5'-phosphate with those of the schematic below.

O (II) 25 Ad n-is- Ad I Ad HO O 2) EleHydrolysis o--oO O 0--0 O (pH 7.5) HO OH O O k V / O O. V / 30 P P N IIM I O O. II O. O. -- - (C2H5)3N:SO3 which is designated the starting material for this pro 35 cess. Compound II may be conveniently prepared by reacting adenosine Ö-S-O-P-O o " I O O HO Ad O O V / P IIM HO OH O. O. 45 with pyrophosphoryl chloride under hydrolysis condi III tions at pH 7.5 to provide a pure starting material. Reac T2-RNase / \De II tion of II with triethylamine-N-sulfonic acid affords O O O O || || Ada Ad adenosine 2',3'-cyclic phosphate 5'-phosphosulfate 50 O-S-O-P-O O O-S-O-P-O O I I O O O O (III) o-i-o--o O Ad p OH HO p 55 o--o o--o O O T. O O V. A IV V P NH2 IIM O. O. N N which, on treatment with ribonuclease T2-RNase, pro Ad = N N ! vides the desired compound, 3'-phosphoadenosine 5'- o phosphosulfate (PAPS) (IV). 65 The iso or II isomer may be prepared by treating III The biological test data is in keeping with the fact with spleen phosphodiesterase (PDase) which converts that it is well established that PAPS is the sulfate donor III to the 2'-phosphoadenosine 5'-phosphosulfate in the formation of sulfate esters of a wide range of 4,169,011 3 4. biological compounds found in nature for which a cor transfer to 6,7-3H2]estrone as mediated by bovine adre responding spectrum of of differing nal estrone sulfotransferase is identical with that ob specificities are needed for of such diversified tained with IV prepared by an established biochemical types of substrates as phenols, steroids, N-arylhydrox procedure. In contrast, iso-PAPS (V) exhibits approxi ylamines and glycosides. The source of active surface in mately one-third the activity of the natural isomer IV. these transfer reactions is in all cases 3'-phosphoadeno Additionally, the isolation of intermediate III allows sine 5'-phosphosulfate (IV). flexibility in this process to produce PAPS (IV) and Until now the pursuit of specificity studies of the analogs as well as the positional isomer V, depending on sulfotransferases has been handicapped by the lack of a the choice of enzyme treatment; i.e., T2 RNase of suitable synthesis to provide an adequate supply of pure 10 PDase. IV. It is noted that enzymic preparations of active sul fate from adenosine triphosphate (ATP) are tedious, GENERALIZED PROCEDURE time-consuming and convenient for only very small The phosphorylation of adenosine (I) with pyrophos quantities. The present process describes an expeditious phoryl chloride followed by neutral buffered hydrolysis approach to pure IV (PAPS) and additionally may be 15 (cf. Simonscits and Tomasz, supra.) provides a conve utilized for the synthesis of analogs of IV, such as those nient route to adenosine 2',3'-cyclic phosphate 5'-phos proceeding from guanosine or cytidine. Some addi phate (II). Purification of II was achieved in the present tional analogs of PAPS in which the present process invention by DEAE-Sephadex A-25 column chroma may be applied are 8-bromo-3'-phosphoadenosine 5'- tography using a linear gradient of triethylammonium phosphosulfate, 3'-phosphoinosine 5'-phosphosulfate, 20 bicarbonate (TEAB), pH 7.5 which afforded II in 62% 3'-phosphonebularine 5'-phosphosulfate, 3'-phos yield. photubercidin 5'-phosphosulfate, 2'-phosphotubercidin The formation of the 5'-sulfatophosphate anhydride 5'-phosphosulfate and 3'-phosphoformycin 5'-phospho moiety (III) was effected with triethylamine-N-sulfonic sulfate. acid. The requisite separation of III from the sulfating 25 PRIOR ART agent and trioctylamine was readily accomplished on a column of Sephadex G-10 and elution with TEAB. J. P. Horwitz, et al, Biochim. Biophys, Acta, Subsequent treatment of III with ribonuclease T2 gave 480:376-381 (1977)-a journal review illustrating the IV in yields of 68–72% (based on II) following column procedure of the present application. chromatography on DEAE-Sephadex A-25. The detec R. Cherniak and E. A. Davidson, J. Biol. Chem., 30 tion of 22% of adenosine 2'(3),5'-diphosphate indicates 239:2986-2990 (1964)--a production of PAPS by a that the conversion of II to III was in the order of 90%. chemical process which at the key II position produces A different enzyme PDase II hydrolyzed adenosine either 2", 5'-diphosphate or its positional isomer adeno 2',3'-cyclic phosphate 5'-sulfatophosphate (III) to V. sine 3',5'-diphosphate or a mixture of both; i.e., adeno In agreement with the assigned structures it was sine2(3), 5'-diphosphate, and there is produced a prod 35 uct which yields PAPS, as stated, as 43% overall. Addi found that acidic treatment of IV gave adenosine 3',5'- tionally, at page 2988, column 2, the statement is made diphosphate with no chromatographic indication in as follows: “The presence of about 5% of the corre either S1 or S2 of the presence of the 2',5'-isomer. The sponding 2' isomer was indicated by analysis with 3'- same conditions of hydrolysis applied to V gave adeno nucleotidase and acid hydrolysis'. This indicates a dif sine 2',5'-diphosphate as the sole product. ference in the purity of the cyclic phosphate reactant as Biological activity of IV and V was determined by well as in the PAPS product which in Cherniak is con sulfate transfer to 6,73H2lestrone in the presence of taminated with the isomer iso-PAPS. The separation bovine adrenal sulfotransferase (E.C. 2.8.2.4). and elution procedures using Cherniak are involved and The activity of IV was virtually identical to that ob time consuming. 45 tained with a sample of PAPS derived via the enzy A. Simonscits and J. Tomasz, Biochim. Biophys. Acta, matic procedure. By varying the concentration of IV 395:74-79 (1975)--a teaching is made of the use of pyro between 15-100 uM and with 6,7-3H2]estrone main phosphoryl chloride in production of cyclic phosphate tained at 100 uM, saturation curves for IV from two or in the present reaction I ->II. sources were produced which were essentially superim J. Rozhin et al., J. Biol. Chenn., 249:2079–2087 50 posable. Iso-PAPS exhibited approximately one-third (1974)-testing to show that in estrogen structural per cent the activity of the natural isomer. Moreover, V changes in all four rings of the estratriene nucleus af showed fractional inhibition of estrone-sulfation (by IV) fected sulfation and testing of PAPS or 3'-phos of 0.33 (unity=100% sulfation). phoadenosine 5'-phosphosulfate. The availability of adenosine 2',3'-cyclic phosphate As contrasted with the prior art above, the present 55 5'-phosphate (II) provides ready access to IV and V via process, which for practical purposes starts from a pure III. The use of Sephadex G-10 column chromatography cyclic phosphate obtains yields in the range 68-72% provides a facile and rapid separation of III from tri-n- from adenosine 2, 3'-cyclic phosphate 5'-phosphate. octylamine and triethylamine-N-sulfonic acid. Enzy This compound is purified in the present process on a matic cleavage of the 2',3'-cyclic phosphate ester in III column of DEAE Sephadex A-25 using a linear gradi can be achieved to afford either IV or Vusing the requi ent of triethylammonium bicarbonate to elute the prod site enzyme. Final purification of the products is conve uct II. The purity and exclusivity are the keys to the niently effected on DEAE-Sephadex A-25 employing present process and fulfill an awaited chemical synthesis linear gradient elution of the column by TEAB. of PAPS over the previous small quantity biochemical EXAMPLES procedures. In this process a highly purified reactant 65 minimizes the side effects which previously made the General Procedure production less than successful. In the production of Bovine spleen phosphodiesterase II (E.C. 3.1.4.18) PAPS (IV), the biological activity measured by sulfate and ribonuclease T2 were purchased from Sigma Chem 4,169,011 5 6 ical Company. Bovine-adrenal estrogen sulfotransferase ration from dimethylformamide at the oil pump. To the (E.C. 2.8.2.4) was isolated and purified as described by residue was added 3 ml of dimethylformamide, 3 ml of Adams et al, Biochim, Biophys, Acta, 370:160-188 dioxane and 0.6 ml of pyridine and the mixture was (1974). 35SPAPS and 6,7-3H2-estrone were pur shaken for 5 min. Then, 110 mg of triethylamine-N-sul chased from New England Nuclear Corp. 5 fonic acid (high oral toxicity) was added and the reac Thin-layer chromatography was performed in Sys tion mixture was sealed and shaken overnight at room tem S1: saturated (NH4)2SO4/0.1 M ammonium aceta temperature. A thin-layer chromatogram in S2 indicated te/2-propanol (79:19;2, v/v), and in System S2:1- that the major product was Compound III. The sol propanol/conc. NH4OH/H2O (6:3:1, v/v) on pre vents were removed at the oil pump and the residue and coated cellulose sheets (Polygram Cel 300 UV254, O suspended in 6 ml of ice-cold H2O containing 0.2 ml of Machery Nagel). Paper electrophoresis was performed 1.5 M NH4OH. The mixture was adjusted to pH 7 and in a Savant high voltage electrophoresis apparatus on applied to a Sephadex G-10 column (2.640 cm), which Whatman No. 1 paper in Solvent E1:0.02 M Na2HPO4, had been pre-equilibrated at room temperature with 0.1 pH 7 at 30 V.cm -1 for 1.5 h. Thin-layer electrophoresis M triethylammonium bicarbonate in 20% ethanol. Elu was performed in a Brinkmann Desaga apparatus on 15 tion was continued until 60 ml had been collected. Six pre-coated cellulose F plates (E. Merck) in Solvent E2: 20 ml fractions then were collected and fractions 1-4 0.025 M sodium citrate, pH 5.4 at 15 V-cm for 2 h. were pooled to give 2880 A259 units. Buffer was re RF values and electrophoretic mobilities are summa moved in the usual manner. To the residue was added 5 rized in Table I. ml of ice-cold H2O and the pH was adjusted to 5.9 with TABLE I 20 1.5 M NH4OH. Crude Compound III was used in the Thin-Layer Chromatography and Electrophoresis of Compounds enzymatic transformations without further purification. - RE - - Mobility Compound Sl S2 E1. E*** EXAMPLE 3 Adenosine 5'-phosphate - - 0.58 0.19 25 Preparation of 3'-phosphoadenosine 5'-phosphosulfate II 0.33 0.32 0.82 0.72 (Compound IV) III 0.33 0.48 0.99 - Adenosine 3',5'-diphosphate 0.50 0.15 0.85 0.59 To the solution of Compound III was added 0.2 ml IV 0.53 0.27 1.00 .00 V 0.56 0.24 1.00 1.08 (100 units) of an aqueous solution of ribonuclease T2. Adenosine 2',5'-diphosphate 0.59 0.15 0.85 - 30 After 20 hat room temperature a thin-layer chromato Relative to mobility of Compound V. gram in System S2 showed complete conversion of "Paper electrophoresis. Compound III to Compound IV (cf Table I, supra). ***Thin-layer electrophoresis. Chromatography on DEAE-Sephadex A-25 in the EXAMPLE 1. manner described for Compound II (Example 1) gave three major peaks. Peak 1 (fractions 104-122) contained Preparation of Adenosine 2',3'-cyclic phosphate 5'-3 623 A259 units (22%) of adenosine, 3',5'-diphosphate phosphate chromatographed C. on a (Nina H2O=259 nm). Peak 2 (fractions 140-159) con This compound was prepared from adenosine (Com tained 1932 A259 units (68%) of Compound IV. Peak 3 pound I, 0.67 g, 2.5 mmol) using about 2.5 mmol of (fractions 166-180) contained 196 A259 units (7%) of an pyrophosphoryl chloride in tenfold relationship to unidentified compound, which exhibited an anomalous adenosine at 0-3°C. with stirring and without moisture 40 ultraviolet spectrum (Nnaxh2O=262 nm). by a modification of the method of Simoncsits and Fractions containing Compound IV were pooled and Tomasz, supra. After hydrolysis of the reaction mixture the buffer was removed in the usual manner. The prod with 1.0 M triethylammonium bicarbonate at a pH 7.5 uct was redissolved in approx. 3 ml of H2O and the below 10° C., the buffer was removed by concentration solution was adjusted to pH 7 with dilute NH4OH. to a small volume, followed by several evaporations 45 Treatment of a small aliquot of the latter with an equal with ethanol under reduced pressure at 30° C. The volume of 0.2 MHCl at 37° C. for 1.75 h gave adenosine white residue was dissolved in 37.5 ml of 0.05 M trie 3',5'-diphosphate as the sole detectable product in Sys thylammonium bicarbonate and stored at -15 C. A 6 tems S1 and S2. Compound IV was homogeneous in S1, ml portion of the crude product (5525 A259 units, one A259 unit is that amount of material in 1 ml of solution 50 S2, E1 and E2. It exhibited a typical adenine nucleotide that has an absorbance of 1.0 when it is measured with spectrum (Nina H2O=259 nm, Amin H2O=227), and it a 1.0 cm optical path at 259 nm) was chromatographed could be frozen for storage or kept indefinitely in 50% at 4 C. on a 2.5.35 cm column of DEAE-Sephadex ethanol at -20° C. Compound IV gave yields of A-25 with a linear gradient of 21 each of 0.05-1.0 M 68-72% based on Compound II. Consonant with the triethylammonium bicarbonate, pH 7.5. Fractions 55 assigned structures, it was found that acidic treatment of (approx. 20 ml each) 78-97 contained 3800 A259 units Compound IV gave adenosine 3',5'-diphosphate with (62% yield, based on adenosine and enahz0 = 15 400 no indication of the presence of the 2',5'-isomer. for AMP) of Compound II. The buffer was removed in the usual manner and the product was frozen for stor EXAMPLE 4 age in 5 ml of H2O. 60 Preparation of 2'-phosphoadenosine 5'-phosphosulfate (Compound V) EXAMPLE 2 To a solution of 790 A259 units of crude Compound Preparation of adenosine 2',3'-cyclic phosphate III in 2 ml of H2O, adjusted to pH 6 with 1.5 M 5'-phosphosulfate (Compound III) NH4OH, was added 0.4 ml (9 units) of a solution of To a solution of 2890 A259 units of Compound II in 65 spleen phosphodiesterase II in 0.05 M potassium ace 9.1 ml of ethanol was added 0.34 ml of trioctylamine. tate, pH 6. After 18h at room temperature, chromatog The ethanol was removed under reduced pressure and raphy in S1 and S2 showed approx. 40% formation of the residue was rendered anhydrous by repeated evapo Compound V. An additional 4.5 units of enzyme were 4,169,011 7 8 added and the mixture was kept at room temperature in 10 ml of Bray's dioxane scintillation solution. Methy for 18 h to complete the reaction. Compound V was lene blue did not affect counting efficiency. The amount isolated in 89% yield following chromatography on ofsteroid sulfate ester produced was calculated utilizing DEAE-Sephadex A-25 in the usual manner. It was the specific activity of Hestrone. homogeneous in S1, S2, E1 and E2 and exhibited a typi 5 The effect of Compound V as an inhibitor of estrogen cal adenine nucleotide spectrum (Anax = 259 nm, sulfotransferase was determined by adding the inhibitor Amin = 227 nm). It moved slightly faster than Compound (0.1 mM) to an incubation mixture containing 22 M IV in S1 and E2 but had nearly the same mobility as Compound IV and 6,7-3H2estrone (1.3.106 dpm/22 Compound IV in Systems S2 and E1. After adjustment nmol). Results are expressed as fractional inhibition. of pH to 7, Compound V was frozen for storage at 10 -15'. Hydrolysis of Compound V under acid condi I claim: tions gave adenosine 2',5'-diphosphate as the sole prod 1. The preparation of 3'-phosphoadenosine 5'-phos uct and the biological activity as compared with the phosulfate (IV) by a process which comprises reacting natural isomer IV was about 33%. adenosine (I) with pyrophosphoryl chloride to produce 15 pure adenosine 2',3'-cyclic phosphate 5'-phosphate (II) EXAMPLE 5 and subsequently reacting said cyclic phosphate (II) Comparative sulfation of estrone by Compounds IV with triethylamine-N-sulfonic acid to produce adeno and V sine 2',3'-cyclic phosphate 5'-phosphosulfate (III) and Sulfation of estrone with synthetic Compound IV treating (III) with the enzyme ribonuclease-T2 to pro (PAPS) was determined using the procedure described 20 duce the desired 3'-phosphoadenosine 5'-phosphosul in the article by Roshin et al, ante. Sulfation by Com fate (IV). pound V was as follows. The ability of Compound V to 2. The preparation of 2'-phosphoadenosine 5'-phos donate its sulfate to estrone was determined with 6,7- phosulfate (V) by a process which comprises reacting 3H2)estrone (1.3.106 dpm/4 nmol) by modification of adenosine (I) with pyrophosphoryl chloride to produce the above-mentioned Rozhin et all method in which 5 pure adenosine 2',3'-cyclic phosphate 5'-phosphate (II) Compound V (0.11 mM) is now substituted for Com and subsequently reacting said cyclic phosphate (II) pound IV. with triethylamine-N-sulfonic acid to produce adeno After termination of the incubation, excess free sine 2',3'-cyclic phosphate 5'-phosphosulfate (III) and 3H]estrone was extracted three times with 0.3 ml of treating (III) with the enzyme spleen PDase to produce ethyl ether and the aqueous sample adjusted to 0.5 ml 30 2'-phosphoadenosine 5'-phosphosulfate (V). with redistilled methanol. An aliquot (25 ul) was ap 3. The process according to claim 1 wherein the plied, together with 15ug of methanolic pyrophosphoryl chloride is substantially pure pyro marker, to type SG chromatography media (Gelman phosphoryl chloride. Instrument Co., Ann Arbor, Mich.) and developed with 4. The process according to claim 2 wherein the chloroform/acetone/acetic acid (110:35:6). Estrone 35 pyrophosphoryl chloride is substantially pure pyro sulfate was visualized by spraying with methylene blue phosphoryl chloride. and the spot punched out with a cork borer and counted

45

50

55

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UNITED STATES PATENT OFFICE Page 1 of 2 CERTIFICATE OF CORRECTION Patent No. 4, 69, Oll Dated Sept. 25, 1979 Inventor (S) Jerome P. Horwitz et al. It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below: Column 2, lines 25-58, formulas I, II, III, IV, & V

Ad O HO - O - 1) PO Cl O-P-O- -- y | 2) Hydrolysis2 - /A - (pH 7.5) HO OH

I I / (C2H5). So, Ad

III

UNITED STATES PATENT OFFICE Page 2 of 2 CERTIFICATE OF CORRECTION Patent No. 4, 169, Oll Dated Sept. 25, 1979

Inventor(s) Jerome P. Horwitz et all It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below: Column 2, lines 25-58, formulas I, II, III, IV & V (cont) try Note I Z O-S-O-P-O--- Q Ad O O S/

W eigned and Sealed this Eleventh Day of November 1980 SEAL

SIDNEY A. DAMOND Attesting Officer Commissioner of Patents and Trademarks