A NEW METHOD OF PHOSPHORYLATION AND THE SYNTHESIS OF SOME PHOSPHATE ESTERS OF BIOLOGICAL INTEREST by JOHN GILBERT MOFFATT A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in CHEMISTRY THE UNIVERSITY OF BRITISH COLUMBIA September, 1956. Faculty of Graduate Studies PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of JOHN GILBERT M OFF ATT B.A. (U.B.C.), 1952 M.Sc. (U.B.C.), 1953 MONDAY, OCTOBER 15th, 1956, at 10:30 a.m. IN CHEMISTRY LIBRARY COMMITTEE IN CHARGE DEAN G. M. SHRUM, Chairman DEAN F. H. SOWARD DR. S. H. ZBARSKY DR. C. A. MCDOWELL DR. G. G. S. DUTTON DR. L. D. HAYWARD DR. Ross STEWART DR. W. A. BRYCE External Examiner: DR. C. A. DEKKER University of California A NEW METHOD OF PHOSPHORYLATION AND THE SYNTHESIS OF SOME PHOSPHATE ESTERS OF BIOLOGICAL INTEREST ABSTRACT Tetra-/?-nitrophenyl pyrophosphate, generated in situ by the reaction of di-p-nitrophenyl phosphate with di-/>-tolyl carbodiimide in anhydrous dioxane, has been shown to be a new and powerful phosphorylating agent. It has been used for the phosphorylation of various aliphatic alcohols and carbohydrate de• rivatives, particularly nucleosides, as well as of aliphatic amines and mercaptans. The removal of the protecting groups from the initially formed di-/>-nitrophenyl alkyl phosphates has been accomplished by both alkaline hydrolysis and catalytic hydrogenolysis. Various other chemical reactions of the intermediate neutral esters, as typified by di-/»-nitrophenyl methyl phosphate, have been studied in considerable detail. Included in these are: mild alkaline hydrolysis, partial hydro• genolysis, the action of anhydrous amines, acidic hydrolysis, base catalysed trans- esterifkation, and reductive cleavage with sodium in liquid ammonia. The method has been specifically applied to the phosphorylation of 2',3'- O-isopropylidine guanosine and has led to the first efficient synthesis of the bio• logically interesting guanosine-5 '-phosphate. Also the enzymatically useful guanosine-5'-mono-p-nitrophenyl phosphate and uridine-5'-mono-/>-nitrophenyl phosphate have been prepared and the formation of 3,5'-cyclo-guanosine quat• ernary salts observed. Several synthetic routes have been developed for the synthesis of 1,2-O-iso- proplyidine-D-xylofuranose-3,5-cyclic phosphate and of D-xylofuranose-3,5- cyclic phosphate. Hot alkaline hydrolysis of the former followed by acidic re• moval of the isopropylidine group has led to a mixture of D-xylofuranose-5- phosphate and D-xylopyranose-3-phosphate which were separated by ion exchange chromatography. Both products have been fully characterized and the method constitutes the first successful synthesis of D-xylose-3 -phosphate. A pre• viously reported' isolation of the latter compound has been re-examined and shown to be in error. Reinterpretation of the data has shown the reported com• pound to be in fact D-xylulose-5-phosphate. PUBLICATIONS 1. "Tetra-/>-Nitrophenyl Pyrophosphate — a New Phosphorylating Agent", J. Am. Chem. Soc. In press. 2. "The Snythesis of Guanosine-5'-Phosphate", J. Am. Chem. Soc. In press. 3. "The Synthesis of Xylose-3-Phosphate via 1,2-0-Isopropylidine-D-Xylose- 3,5-Cyclic Phosphate", J. Am. Chem. Soc. In press. 4. "Some General Observations on the Preparation and Properties of Five, Six, and Seven Membered Cyclic Phosphates", J. Am. Chem. Soc. In press. GRADUATE STUDIES Field of Study: Chemistry [ Drs. J. G. Hooley Topics in Physical Chemistry \ H. M. Daggett Y ] [ B. A. Dunell . Seminar in Physical Chemistry -Dr. C. Reid Introduction to Chemical Oceanography.. Dr. M. Kirsch Topics in Inorganic Chemistry Drs. K. Starke and J. A. Ham- Chemical Kinetics ~ Dr. W. A. BrycS Molecular Rearrangements Dr. A. Rosenthal Drs. L. D. Hayward G. G. S. Dutton ! W. A. Bryce Carbohydrates , Dr. L. D. Hayward Cellulose, Lignin and Related Compounds Dr. L. D. Hayward Physical Organic Chemistry Dr. C. C. Lee Seminar in Organic Chemistry Dr. G. G. S. Dutton Molecular Rearrangement Dr. A. Rosenthal Structure of Newer Natural Products Dr. A. Rosenthal Other Studies: Molecular Structure and Biological Function Dr. W. J. Polglase Introduction to Quantum Mechanics Dr. O. Theimer Outside Interest: Photography Faculty of Graduate Studies PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of JOHN GILBERT M OFF ATT B.A. (U.B.C.), 1952 M.Sc. (U.B.C.), 1953 MONDAY, OCTOBER 15th, 1956, at ,10:30 a.m. IN CHEMISTRY LIBRARY COMMITTEE IN CHARGE DEAN G. M. SHRUM, Chairman DEAN F. H. SOWARD DR. S. H. ZBARSKY -DR. C. A. MCDOWELL DR. G. G. S. DUTTON DR. -L. D. 'HAWARD •DR. .Ross STEWART ;DR. W. A. BRYCE External Examiner: DR. C. A. DEKKER University of California A NEW, METHOD OF PHOSPHORYLATION AND THE SYNTHESIS OF SOME PHOSPHATE ESTERS OF BIOLOGICAL INTEREST ABSTRACT Tetra-/?-nitrophenyl pyrophosphate, generated in situ by the reaction of di-p-nitrophenyl phosphate with di-/>-tolyl carbodiimide in anhydrous dioxane, has been shown to be a new and powerful phosphorylating agent. It has been used for the phosphorylation of various aliphatic alcohols and carbohydrate de• rivatives, particularly nucleosides, as well as of aliphatic-amines and mercaptans. The removal of the protecting groups from the initially formed di-/>-nitrophenyl alkyl phosphates has been accomplished by both alkaline hydrolysis and catalytic hydrogenolysis. Various other chemical reactions of the intermediate neutral esters, as typified by di-/>-nitrophenyl methyl phosphate, have been studied in considerable detail. Included in these are: mild alkaline hydrolysis, partial hydro• genolysis, the action of anhydrous amines, acidic hydrolysis, base catalysed trans- esterification, and reductive cleavage with sodium in liquid ammonia. The method has been specifically applied to the phosphorylation of 2',3'- O-isopropylidine guanosine and has led to the first efficient synthesis of the bio• logically interesting guanosine-5 '-phosphate. Also the enzymatically useful guanosine-5'-mono-p-nitrophenyl phosphate and uridine-5'-mono-/>-nitrophenyl phosphate have been prepared and the formation of 3,5'-cyclo-guanosine quat• ernary salts observed. Several synthetic routes have been developed for the synthesis of 1,2-O-iso- proplyidine-D-xylofuranose-3,5-cyclic phosphate and of D-xylofuranose-3,5- cyclic phosphate. Hot alkaline hydrolysis of the former followed by acidic re• moval of the isopropylidine group has led to a mixture of D-xylofuranose-5- phosphate and D-xylopyranose-3-phosphate which were separated by ion exchange chromatography. Both products have been fully characterized and the method constitutes the first successful synthesis of D-xylose-3-phosphate. A pre• viously reported isolation of the latter compound has been re-examined and shown to be in error. Reinterpretation of the data has shown the reported com• pound to be in fact D-xylulose-5-phosphate. PUBLICATIONS 1. "Tetra-/>-Nitrophenyl Pyrophosphate — a New Phosphorylating Agent", J. Am. Chem. Soc. In press. 2. "The Snythesis of Guanosine-5'-Phosphate", J. Am. Chem. Soc. In press. 3. "The Synthesis of Xylose-3-Phosphate via 1,2-0-Isopropylidine-D-Xylose- 3,5-Cyclic Phosphate", J. Am. Chem. Soc. In press. 4. "Some General Observations on the Preparation and Properties of Five, Six, and Seven Membered Cyclic Phosphates", J. Am. Chem. Soc. In press. GRADUATE STUDIES Field of Study: Chemistry [ Drs. J. G. Hooley Topics in Physical Chemistry - \ H. M. Daggett V 1 [ B. A. Dunell Seminar in Physical Chemistry Dr. C. Reid Introduction to Chemical Oceanography '. Dr. M. Kirsch Topics in Inorganic Chemistry Drs. K. Starke and J. A. Harris Chemical Kinetics Dr. W. A. Bryce Molecular Rearrangements Dr. A. Rosenthal ( Drs. L. D. Hayward Topics in Organic Chemistry..... 1 G. G. S. Dutton [ W. A. Bryce Carbohydrates. Dr. L. D. Hayward Cellulose, Lignin and Related Compounds Dr. L. D. Hayward Physical Organic Chemistry Dr. C. C. Lee Semihar in Organic Chemistry Dr. G. G. S. Dutton Molecular Rearrangement ...Dr. A. Rosenthal Structure of Newer Natural Products Dr. A. Rosenthal Other Studies: Molecular Structure and Biological Function Dr. W. J. Polglase Introduction to Quantum Mechanics Dr. O. Theimer Outside Interest: Photography %ht Jfetircrsttg at ^rtttslt (EalumMa Faculty of Graduate Studies PROGRAMME OF THE. FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Of: JOHN GILBERT M OFF ATT B.A. (U.B.C.), 1952 M.Sc (U.B.C.), 1953 MONDAY, OCTOBER 15th, 1956, at 10:30 a.m., IN CHEMISTRY LIBRARY COMMITTEE IN CHARGE. DEAN G. M. SHRUM, Chairman DEAN F. H. SOWARD DR. S. H-. ZBARSKY DR. C. A. MCDOWELL DR. G. G. S. DUTTON, DR. L, D. HAYWARD, DR. ROSS. STEWART DR. W. A. BRYCE. External Examiner: DR. G. A. DEKKER University of California A NEW METHOD OF PHOSPHORYLATION AND THE SYNTHESIS OF SOME PHOSPHATE ESTERS OF BIOLOGICAL INTEREST ABSTRACT Tetra-/7-nitrophenyl pyrophosphate, generated in situ by the reaction of di-p-nitrophenyl phosphate with di-/>-tolyl carbodiimide in anhydrous dioxane, has been shown to be a new and powerful phosphorylating agent. It has been used for the phosphorylation of various aliphatic alcohols and carbohydrate de• rivatives, particularly nucleosides, as well as of aliphatic amines and mercaptans. The removal of the protecting groups from the initially formed di-/?-nitrophenyl alkyl phosphates has been accomplished by both alkaline hydrolysis and catalytic hydrogenolysis. Various other chemical reactions of the intermediate neutral esters, as typified by di-/>-nitrophenyl methyl phosphate, have been studied in considerable detail. Included in these are: mild alkaline hydrolysis, partial hydro• genolysis, the action of anhydrous amines, acidic hydrolysis, base catalysed trans- esterifiration, and reductive cleavage with sodium in liquid ammonia. The method has been specifically applied to the phosphorylation of 2',3'- O-isopropylidine guanosine and has led to the first efficient synthesis of the bio• logically interesting guanosine-5'-phosphate. Also the enzymatically useful guanosine-5'-mono-p-nitrophenyl phosphate and uridine-5'-mono-/>-nitrophenyl phosphate have been prepared and the formation of 3,5'-cyclo-guanosine quat• ernary salts observed.