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% product 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 enzyme, 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 estrone sulfotransferase (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 sulfotransferases of differing nal estrone sulfotransferase is identical with that ob specificities are needed for sulfation 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 estrogen 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).