Chemistry and Biology of Pteridines: 9 Zurich, Switzerland, September 3–8, 1989 9783110889000, 9783110121995

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Chemistry and Biology of Pteridines 1989 Pteridines and Folic Acid Derivatives

Chemistry and Biology of Pteridines 1989 Pteridines and Folic Acid Derivatives Proceedings of the Ninth International Symposium on Pteridines and Folic Acid Derivatives Chemical, Biological and Clinical Aspects Zurich, Switzerland, September 3-8,1989 Editors H.-Ch. Curtius • S. Ghisla • N. Blau

w G DE

Walter de Gruyter • Berlin • New York 1990

Editors

Prof. Dr. Hans-Christoph Curtius Dr. Nenad Blau Division of Clinical Chemistry Department of Pediatrics University of Zurich Steinwiesstr. 75 CH-8032 Zurich Switzerland Library of Congress Cataloging-in-Publication

Prof. Dr. Sandro Ghisla Faculty of Biology University of Konstanz D-7750 Konstanz Federal Republic of Germany

Data

International Symposium on Pteridines and Folic Acid Derivatives : Chemical, Biological, and Clinical Aspects (9th : 1989 : Zurich, Switzerland) Chemistry and biology of pteridines, 1989 : pteridines and folic acid derivatives: proceedings of the Ninth International Symposium on Pteridines and Folic Acid Derivatives, Chemical, Biological, and Clinical Aspects, Zurich, Switzerland, September 3-8,1989 / editiors, H. Ch. Curtius, S. Ghisla, N. Blau. XXXVI, 1340 p. 2 4 x 1 7 cm. Includes index. ISBN 0-89925-609-0 - ISBN 3-11-012199-9 1. Pteridines—Congresses. 2. Folic acid—Derivatives—Congresses. I. Curtius, H.Ch.(Hans-Christoph) 1923- . II. Ghisla, S. (Sandro), 1942- . III. Blau, N. (Nenad), 1946- . IV. Title. [DNLM: 1. Folic Acid-analogs & derivatives—congresses. 2. Pteridinescongresses. QU1881616c 1989] OP801.P69I585 1980 612'.0157-dc20 DLC 90-3597 for Library of Congress CIP

CIP-Titeiaufnahme der Deutschen Bibliothek Chemistry and biology of pteridines 1989 : pteridines and folic acid derivatives; proceedings of the Ninth International Symposium on Pteridines and Folic Acid Derivatives, Chemical, Biological and Clinical Aspects, Zurich, Switzerland, September 3-8,1989 / ed. H.-Ch. Curtius . . . - Berlin ; New York : de Gruyter, 1990 ISBN 3-11-012199-9 NE: Curtius, Hans-Christoph [Hrsg.); International Symposium on Pteridines and Folic Acid Derivatives, Chemical, Biological and Clinical Aspects

© Copyright 1990 by Walter de Gruyter & Co., D-1000 Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Dieter Mikolai, Berlin. - Printed in Germany.

The F. Gowland Hopkins Lectures 1989

Zurich, Switzerland

Gene M. Brown

1986

Montreal, Canada

Stephen J. Benkovic

1982

St. Andrews, Scotland

Frank M. Huennekens

Committee members and guests on the terrasse of the ETH faculty club. September 5, 1989

Preface It has been several months since the 9th International Symposium on Pteridines and Folic Acid Derivatives was held in Zurich, and looking back we are proud to have had the opportunity to organize this meeting. More than 300 participants attended the symposium, a number not expected from the preliminary registrations. Possibly many colleagues were motivated at the last minute either by the attractiveness of the scientific program ... or by that of the scenery offered by Zurich and Switzerland. There were several highlights at the conference: First we would like to mention the F. Gowland Hopkins lecture presented Tuesday morning by Gene Brown. It was not only a well-balanced overview of the biochemistry of tetrahydrobiopterin biosynthesis, with the Drosophila system as a focal point, but also a personal account full of interesting details and anecdotes documenting the history of this field. At the symposium banquet a medal was presented to the F. Gowland Hopkins lecturer, Gene Brown, as well as to the previous two lecturers, Frank Huennekens and in absentia Stephen Benkovic. We hope that this will become a tradition. For those interested in historic aspects of science and in particular of pteridines, a further highlight was the lecture by Adrien Albert, one of the founding fathers not only of "pteridinology" but of heterocyclic chemistry, which he presented Saturday morning, September 2, at the satellite meeting held in Konstanz in honor of one of us (H.Ch.C.). The symposium participants were welcomed at a reception in the modem building of the Hotel Zurich, which provided an appropriate architectural contrast to the more historical setting of the atrium at the University of Zurich, where the symposium banquet was held Wednesday evening. The superb buffet served on this occasion was a masterpiece prepared under the supervision of Mr. W. Kurth, chef de cuisine at the Children's Hospital. The evening was crowned by George Hitchings' recollections on his and G. Elion's work that led to their receiving the Nobel prize in 1988. Turning to the scientific side, we have the impression the choice of topics was reasonably well balanced, offering a good coverage of the most recent results also to those interested primarily in specific topics. Special thanks for a job well done go to those colleagues who presented state-of-the-art lectures. We feel, and several members of the scientific advisory committee concur with us, that lectures giving a critical appraisal of recent progress in a subfield become more and more important in view of the steadily increasing flood of scientific information. We were fortunate in being able to set up the poster display in the vicinity of the lecture hall - and the coffee bar - so that it was possible to view the posters during the entire congress. This arrangement facilitated personal contacts and offered a chance to the participants to have a look at this or that poster when they felt saturated by the oral presentations. We think this physical proximity of lecture hall, poster and industrial display, and coffee break areas proved very fruitful and might well be recommended to future organizers.

VIII

The following is a short historical recollection which might illustrate the reasons ultimately leading to the meeting being held in Zurich: In the early fifties E. Hadorn of the Zoological Institute at the University of Zurich spent some time as a visiting professor in the laboratory of H.K. Mitchell in Los Angeles, with whom he published in 1951 a paper on the isolation of two yellow fluorescent compounds from the Drosophila fly. Back in Zurich, Hadorn got in touch with M. Viscontini and P. Karrer and motivated the two to pursue the study of the Drosophila chromophores which later led to their identification as sepiapterin and isosepiapterin. In 1955 the same group discovered the compound HB2 (Himmelblau 2 = skyblue 2), which in 1958 was identified as biopterin. This work was carried out parallel to similar studies by E.L. Patterson and E.L.R. Stokstad on components of human urine, and by H.S. Forrest and H.K. Mitchell also with Drosophila. In 1959 F. Leuthardt, O. Brenner, and E. Hadorn did some of the first biochemical work on pterin biosynthesis using a Drosophila mutant. And one of today's most prominent members of the pteridine community, S. Kaufman, also studied the cofactor of phenylalanine hydroxylase as a visiting scientist in the laboratories of Hadorn here in Zurich. Not far from here, in Munich, F. Weygand and H. Simon carried out their biochemical studies on pterin butterfly pigments. Pteridine chemistry then became the subject of Viscontini's research, which led to a series of basic chemistry papers, and culminated in 1977 in the first practical synthesis of optically pure biopterin and tetrahydrobiopterin. This achievement opened the doors to a pharmacological application of these compounds. One of the advantages of Zurich is its central location in Europe, facilitating contacts with neighboring universities. We would like to take this opportunity to thank a number of researchers and groups for the contacts and fruitful ties that have been seminal for the work done in our laboratories: F. Weygand and H. Simon in Munich; A. Butenandt and H. Rembold in Tübingen, and later in Munich; W. Pfleiderer first in Stuttgart with H. Bredereck, and later in Konstanz; A. Bacher in Munich; the late P. Hemmerich, also in Konstanz; H. Wächter in Innsbruck. Incidentally, just to mention one of the fruits of such contacts, we recently came across a discussion remark by the late Peter Hemmerich, who, following Kaufman's lecture at the 1961 symposium in Stuttgart, came up with the idea that the product of tetrahydrobiopterin oxidation might have a quinonoid structure (see the proceedings of that congress). This hypothesis was later demonstrated by others to be correct, and turned out to be a breakthrough in the understanding of biopterin chemistry. Since the last symposium in Montreal two outstanding scientists, our dear colleagues and friends Alois Niederwieser and Tetsuo Shiota, have died. Niederwieser was closely involved in the discovery and characterization of most of the inborn errors of tetrahydrobiopterin biosynthesis. He initiated the first successful therapy of patients with atypical phenylketonuria and tetrahydrobiopterin deficiency. The discovery of genetic defects in tetrahydrobiopterin biosynthesis fostered the biochemical study of the latter and eventually led to the elucidation of this biosynthetic pathway. This was achieved in our groups and also in collaboration and/or friendly competition with several other laboratories around the world. Tetsuo Shiota was involved in some of the most important work on tetrahydrobiopterin biosynthesis and pioneered the study of dihydrofolate biosynthesis in bacteria. From his group came also the confirmation of the correct structure for the side

IX

chain of the important biosynthetical intermediate, 6-pyruvoyltetrahydropterin, as first proposed by A. Suzuki and M. Goto. The legacy of these two scientists is for us to continue on the path of pterin research. Adrien Albert, an initiator of the pteridines symposia and a pioneer in heterocyclic chemistry, attended the meeting in good health and admirable alertness. He told us that for him as a Swiss descendant it had been a particular pleasure to return once more to the country of his ancestors. In fact, this turned out to be his last participation at a pteridines symposium. After a brief illness he passed away at the end of December at age 82. We mourn not only an outstanding scientist, but also an inspirator and a dear friend. We are grateful to the president of the ETH (Federal Institute of Technology) for offering us the use of their facilities, and to the president of the University of Zurich for the hospitality at the occasion of the symposium banquet. Special thanks are due the members of the scientific committee for their invaluable help and cooperation, and in particular to Mrs. M. Killen, without whose skillful assistance and dedication the organization of the congress would have been a great deal more difficult. Finally, we wish to express our gratitude to the sponsors who are listed separately, and to Dr. R. Weber and his collaborators at Walter de Gruyter, for their efficient and professional assistance in publishing this volume. Zurich, April 1990

Hans-Christoph Curtius Sandro Ghisla Nenad Blau

Adrien Albert 1907 - 1989

On December 29, 1989 Adrien Albert passed away unexpectedly after a scientifically very successful and rewarding life which he devoted fully to his profession. His contributions to science are widespread and range from investigations of basic principles of heterocyclic chemistry to problems of medicinal chemistry concerning the molecular basis of "selective toxicity". His book on this subject can be regarded as a long-lived best-seller and the "introduction to heterocyclic chemistry" has to be considered as the first successful attempt to classify this large field in a systematic and easily understandable manner. The proposed basic principles, which are now generally accepted, have been derived from his pioneering work in pteridine chemistry as one of his favorite heterocyclic ring systems. All his friends and the scientific community are indebted to Adrien Albert as a great scientist and regret his loss deeply. Wolfgang Pfleiderer

Alois Niederwieser 1935 - 1987

Alois Niederwieser died February 5, 1987 after a long illness at the age of 52. We mourn the loss of an outstanding scientist of world renown, a dear colleague and friend. Niederwieser localized and characterized two of three inborn errors of tetrahydrobiopterin metabolism, i.e., GTP cyclohydrolase I deficiency and 6-pyruvoyltetrahydropterin synthase deficiency. He initiated the treatment of patients with tetrahydrobiopterin deficiency, using tetrahydrobiopterin synthesized by Viscontini et al. Niederwieser was also involved in the elucidation of the tetrahydrobiopterin biosynthetic pathway. He was the first to perform prenatal diagnosis of 6-pyruvoyltetrahydropterin synthase deficiency. In Montreal Alois Niederwieser had been nominated to organize together with me the 9th International Symposium Pteridines and Folic Acid Derivatives. I hope that we carried out the task in his spirit. Hans-Christoph Curtius

Tetsuo Shiota 1923 - 1988

Tetsuo Shiota passed away soon after his retirement from the University of Alabama, Birmingham. Shiota had pioneered the study of dihydrofolate biosynthesis in bacteria. Together with M. Akino and others he was also involved in the elucidation of tetrahydrobiopterin biosynthesis. They proposed the correct structure for the side chain of the biosynthetical intermediate, 6-pyruvoyltetrahydropterin. Shiota had looked forward to his retirement in California but was able to enjoy it for only a brief time. We mourn the loss of an outstanding scientist and a dear friend and colleague. Hans-Christoph Curtius

Contents I. CHEMISTRY OF PTERINS AND FOLATES Recent progress in pteridine chemistry. Pfleiderer W, Abou-Hadeed K, Wiesenfeldt M, Kazimierczuk Z, Mohr D, Kiriasis L, Bischler M

3

Synthesis of pteridines for immunoassay of biopterin and neopterin isomers. Matsuura S, Sugimoto T

17

Design of new mechanism-based substrates for dihydrofolate reductase. Gready JE

23

New methods for the synthesis of quinazoline antifolates and poly-y-glutamyl metabolites of folates and antifolates. Li SW, Abraham A, Edwards D, Nair MG

31

The decay of quinoid- and 7,8-dihydropterins. Ayling JE, Dillard SB, Turner AA, Bailey SW

43

Synthesis of 6-acetyl-2,4-diamino-7,8-dihydro-9//-pyrimido-[4,5-b][l,4]diazepine, an amino analog of 6-acetyldihydrohomopterin. Boyle PH, Hughes EM, Khattab HA

49

Synthetic approaches to methanopterin. Heizmann G, Pfleiderer W

55

Synthesis and properties of tetrahydropterins coupled to 1,4-dihydropyridines. Rehse J, Pfleiderer W

59

A new synthesis of biopterin and some analogues from epoxyaldehydes. Sugimoto T, Ogiwara S, Murata S

63

Synthetic studies on the molybdenum cofactor. Taylor EC, Ray PS, Darwish IS, DOtzer R

67

A new regiospecific synthesis of anapterin and primapterin. Viscontini M, Bosshard R

73

A novel and efficient synthesis of folylpolyglutamates. Dunlap RB, Silks VF

77

Reaction of acidic anhydride and pyridine with folic acid and classical antifolates. Nair MG, Nair IG

81

XVI Quinazoline antifolate thymidylate synthase inhibitors: Synthesis of analogues containing heterocyclic isosteres of the para-aminobenzoate unit. Marsham PR, Hayter AJ

86

Synthesis of tetrahydro-8-deazabiopterin. DeGraw JI, ColweU WT, Hayano T

90

Synthesis and enzymic properties of new mechanism based substrates for dihydrofolate reductase of the 8-substituted pterin class. Koen MJ, Haynes RK, Gready JE, Pilling PA

94

Imidazolines as carbon-transfer vehicles: models of the tetrahydrofolate (and other) coenzymes. Jones RCF, Nichols JR, Smallridge MJ

98

Asymmetric synthesis and absolute configuration of 5,10-dideaza5,6,7,8-tetrahydropteroic acid and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF). Barnett CJ, Wilson TM

102

Synthesis and biological evaluation of a fluorescent analogue of folic acid. McAlinden TP, Hynes JB, Patel SA, Ratnam M, Freisheim JH

106

Metal complexes of tetrahydroneopterin. Fischer B, Strähle J

110

Syntheses of lipophilic analogues of 2-desamino-2-CH3-NIO-propargyl5,8-dideazafolate targeted at the enzyme thymidylate synthase (TS). Bisset GMF, Jackman AL, O'Connor B, Jones TR, Calvert AH, Hughes LR . . . . 114 Studies in models of folate cofactors: oxidation of 5-methyltetrahydropterin revisited. Hilhorst E, Chen TBRA, Pandit UK

118

New strategy in the synthesis of neopterin phosphates. Zagalak B, Neuheiser F

122

First regiospecific synthesis of 7-iso-D-neopterin and 7-iso-D-neopterin3 '-monophosphate. Zagalak B, Neuheiser F, Borschberg HJ

126

H. ANALYSIS OF PTERINS AND FOLATES Detection of pterins in biological material using mass spectrometry. Küster Th, Curtius HCh, Richter WJ, Dahinden R, Raschdorf F

133

XVII Mass spectrometric methods for the structural determination of pteridines and folic acid derivatives. Przybylski M

140

Structural characterization of underivatized pteridines by laser desorption fourier transform mass spectrometry. Jacobson KB, Hettich RL

147

New immunoassays for determination of biopterin and neopterin in body fluids. Rokos H, Hey A

151

A new immunoassay for neopterin and biopterin. Sugimoto T, Ogiwara S, Matsuura S, Kiuchi K, Nagatsu T, Sakai M, Nagatsu I, Fujita K

155

Enzyme immunoassay for urinary neopterin. lino T, Sawada H, Tsusud M, Matsuura S, Sugimoto T, Mazda T

159

A competitive enzyme linked ligand sorbent assay (ELLSA) for quantitation of erythrocyte folates. Hansen SI, Holm J

163

Preparation of a bovine serum albumin based chiral matrix for resolution of (6R,S)-N5-formyltetrahydrofolate. Mullin RJ, Duch DS

167

Rp-HPLC methods for the isolation and chemical identification of pterins from monocytic blood cells. Zeitler HJ, Andondonskaja-Renz B, Ziegler-Heitbrock HWL

171

Cyclodextrin based HPLC separation of 6-S and 6-R diastereomers of 5,10-dideaza and 5-deazatetrahydrofolates. Shih C, Wilson GM, Osborne LM, Harrington PM, Gossett LS, Snoddy JD

177

Analysis of folate and antifolate metabolites by mass spectrometry. Nair MG, Abraham A, Weintraub S

181

A critical assessment of methods for measuring folate in human serum and red blood cells. Leeming RJ, Pollock A, Barley C, Vaughan G, Hamon CGB

188

HPLC analysis of intracellular folate compounds after enzymic conversion to the corresponding pteroates. Vitols KS, Montejano YD, Huennekens FM

192

XVIII HI. BIOSYNTHESIS AND BIOCHEMISTRY OF PTERINS The Gowland Hopkins Lecture: Biosynthesis of H4biopterin and related compounds. Brown GM

199

The structure of g-BH 2 -chemistry and biochemistry. Armarego WLF

213

Pteridines in riboflavin biosynthesis. Bacher A, Schott K, Volk R, Ladenstein R

219

Pteridine mimicking antibodies. Jennings IG, Cotton RGH

231

6-Pyruvoyl-tetrahydropterin reductases. Steinerstauch P, Leimbacher W, Curtius HCh, Wermuth B, Ghisla S

238

Analysis of 6-pyruvoyl tetrahydropterin synthase, a target gene product of su(s) suppressor in Drosophila. Yim JJ, Park YS, Kim JH, Jacobson KB

243

Synthesis of fluorinated 8-ribityllumazines as "F NMR probes and potential inhibitors of the light riboflavin synthase of Bacillus subtilis. Cushman M, Patel HH, Patrick DA, Bacher A, Schott K

249

Studies on the oxidation state and biosynthesis of molybdopterin. Rajagopalan KV, Johnson JL, Johnson ME, Gardlik S, Chaudhury M, Wuebbens MM, Pitterle D

255

Molybdenum cofactor genes in Drosophila. Finnerty V, Shelton M, Arcangeli LM, Olsen-Rasmussen M

262

DNA photolyase: role of pterin in DNA repair. Jörns MS

268

7-Substituted pterins, a new class of mammalian pteridines. Curtius HCh, Adler C, Matasovic A, Akino M

274

Methanopterin: A structural and functional folic acid analogon in the biochemistry of methanogenic bacteria. Keltjens JT, te Brömmelstroet BW, Kengen SWM, van der Drift C, Vogels GD

285

Biosynthesis of methanopterin. White RH

294

Tetrahydrobiopterin is required for the oxidation of arginine to nitric oxide by macrophages. Tayeh MA, Marietta MA

300

XIX

Biosynthesis of 7-hydroxybiopterin. Takikawa S, Nakagoshi M

306

GTP cyclohydrolase I from Escherichia coli. Molecular cloning and crystallization. Katzenmaier G, Schmid C, Bacher A

312

Molecular characterization of the GTP cyclohydrolase I gene of Drosophila: analysis of cDNA clones. O'Donnell JM, McLean JR

316

Inhibitors of bovine adrenal medullary sepiapterin reductase. Smith GK, Duch DS, Edelstein MP, Bigham EC

320

Catalytic implication of the reductase and isomerase activity of sepiapterin reductase in the biosynthesis of tetrahydrobiopterin. Katoh S, Sueoka T

324

Purification and characterization of sepiapterin reductase from human liver. Zagalak B, Neuheiser F, Redweik U

328

Biosynthesis of methanopterin and deazaflavin in Methanobacterium thermoautotrophicum. Reuke B, Eisenreich W, Schwarzkopf B, Bacher A

332

Studies on riboflavin synthases of Bacillus subtilis. Amino acid sequence of the a subunit Kellennann J, Schott K, Lottspeich F, Mehl E, Bacher A

336

Structure of the lumazine synthase/riboflavin synthase complex of Bacillus subtilis: crystallization of hollow reconstituted capsids. Schott K, Ladenstein R, König A, Bacher A

340

Biosynthesis of 6,7-dimethyl-8-ribityllumazine. Purification and properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase. Volk R, König A, Kohnle A, Bacher A

344

High resolution electron microscopy on crystals of the icosahedral lumazine synthase/riboflavin synthase complex. Weinkauf S, Bacher A, Schott K, Ladenstein R, Baumeister W, Bachmann L . . . . 348 Differential synthesis of LDH A-like proteins and of phosphoproteins in response to the modified deazaguanine-derivative queuine. Mahr U, Kersten H

352

Metabolic control in HeLa-cells mediated by the deazaguanine-derivative queuine. Langgut W, Kersten H

356

XX

On the biosynthesis of the deazaguanine-derivative queuine: structure of the tgt gene of E. coli. Reuter K, Frey B, Kersten H

.360

Changes in the level of biopterin and of queuine-containing tRNA during erythroid differentiation of murine erythroleukemia cells. Pamiak MA, Kleiman L, Marx S, Andrejchyshyn S

364

Brain contents of biopterins and monoamines after intraperitoneal injections of SUN0588, active 6R-tetrahydrobiopterin, during ontogeny in rats. Tani Y, Kawai M, Furuya M, Takehisa M, Ishihara T, Noguchi T, Watanabe Y

368

The effect of 2,4-diamino-6-hydroxypyrimidine on tetrahydrobiopterin and serotonin in the gastrointestinal tract of mice. Kobayashi T, Hasegawa H, Ichiyama A, Kaneko E, Honda N

372

Differential metabolism of tetrahydrobiopterin in central and peripheral monoamine neurons maintained in culture. Kapatos G, Hasegawa H, Hirayama K, Kemski V

376

IV. TETRAHYDROBIOPTERIN DEFICIENCY Inborn errors in tetrahydrobiopterin metabolism. Blau N, Curtius HCh

383

Molecular defects in dihydropteridine reductase deficiency. Cotton RGH

389

Screening and treatment of tetrahydrobiopterin deficiency. Ponzone A, Ferrerò GB, Guardamagna O, Ferraris S, Curtius HCh, Blau N

393

Tetrahydrobiopterin deficient phenylketonuria detected by neonatal screening in Taiwan. Hsiao KJ, Chiang SH, Liu TT, Chiù PC, Wuu KT

402

Prenatal diagnosis of 6-pyruvoyl tetrahydropterin synthase deficiency in East Asia. Shintaku H, Fujioka M, Sawada Y, Isshiki G, Yamaoka S, Oura T, Hsiao KJ, Liu TT, Chen RG

408

Heterogeneity of tetrahydrobiopterin deficiency: combined phenylalaninetetrahydrobiopterin loading test. Ponzone A, Guardamagna O, Ferraris S, Ferrerò GB, Blau N, Curtius HCh, Kierat L, Cotton RGH

414

Unusual case of atypical PKU: peripheral or central form of PPH4S deficiency. Blau N, Endres W, Guardamagna 0, Ferrera GB, Ferraris S, Ponzone A

418

XXI Development of vision in Bli, deficiency. Vitale Bravatone F, Porro G, Chiado Piat L, Fea A, Soldi A, Ferrerò GB, Ponzone A

422

Fetal guinea-pig model of tetrahydrobiopterin deficiency. Fujioka M, Shintaku H, Isshiki G, Sawada Y, Oura T

426

Unexpected favorable clinical results of therapy in late diagnosed DHPR deficiency. Cerone R, Caruso U, Maritano L, Lupino S, Schiaffino MC, Fantasia AR, Romano C

430

Systematic investigation for biopterin defects in hyperphenylalaninemic patients. Romano C, Caruso U, Cerone R, Maritano L, Zignego G, Blau N

434

Neuroradiological improvement after one year of therapy in a case of DHPR deficiency. Biasucci G, Valsasina R, Giovannini M, Brioschi M, Saleri L, Riva E

438

V. PTERINS VARIOUS Lumazine protein and the bioluminescence of photobacterium. Lee J

445

Binding of pteridine derivatives to lumazine apoprotein. O'Kane DJ, Lee J, Kohnle A, Bacher A

457

Hormonal imbalance by pteridine treatment in diapausing Pieridae induces mutations in the progeny of treated Drosophila melanogaster. L'Hélias C

462

N-acetyltransferase activity in Pieris L'Hélias C, Callebert J, Launay JM

466

brassicae.

VI. BIOLOGY OF PTERINS Pteridines and the immune response. Huber CH, Tratkiewicz J

473

Neopterin elevation and interferon induction in viral diseases. Rokos K, Pauli G, Lange W, Gelderblom H, Rokos H

485

Tetrahydrobiopterin in human T cells: control of its biosynthesis by interferon-y and interleukin 2 - modulator function in interleukin 2 perception. Ziegler I, Schott K, Schwuléra U, Schmid C, Bacher A

491

XXII

Effects of BH4 on the synthesis of serotonin in brain slices and synaptosomes: implications for the use of BH4 as a therapeutic agent. Levine RA, Wolf WA, Anastasiadis PZ, Kuhn DM

497

Regulation of cellular proliferation by tetrahydrobiopterin. Milstien S, Kaufman S, Tanaka K

505

Characterisation of the mutation in dihydropteridine reductase deficiency. Howells DW, Dahl HHM, Forrest SM, Cotton RGH

511

Urinary excretion of total pteridines in cancer. Noronha JM, Trehan S

515

Alterations of interferon-gamma-induced monocyte activation by immunomodulating compounds determined by neopterin production. Rokos K, Pauli G, Gelderblom H, Rokos H

519

Levels of neopterin, biopterin, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid of sick neonates and infants. Nishimura A, Katoh T, Fujioka M, Shintaku H, Murata R, Isshiki G, Sawada Y

523

Urinary neopterin levels in healthy children. Polo LM, Canas B, Izquierdo R, Nogales A, Jiménez JM, Panero A

527

Urinary neopterin factor in viral and bacteriological children infections. Nogales A, Garcia J, Merino JL, Polo LM, Canas B, Izquierdo R

530

Urinary neopterin levels in liver transplant. Nogales A, Merino JL, Garcia J, Polo LM, Canas B, Izquierdo R

534

The effect of IL-2, neopterin and immunomodulators on neopterin release from macrophages and PBMC. Barak M, Gruener N

538

Membrane transport of unconjugated pteridines. The model of human erythrocytes. Dhondt JL, Hayte JM, Noel C, Farriaux JP

543

Encephalopathy and abnormal cerebrospinal fluid pteridine profile. Fact or artefact? Dhondt JL, Hayte JM, Forzy G, Lambert M, Melancon S

547

Informativity of pteridine measurements in the survey of organ transplantations. Dhondt JL, Hayte JM, Noel C, Walter MP

551

Effect of triamteren on urinary excretion of pterins in different rat strains. Cremer-Bartels G, Krause K, Hanneken L, Gerding H

555

XXIII Pteridines and retinal degenerations in rats and humans. Hanneken L, Cremer-Bartels G, Gerding H, Krause K

559

Pterins in bovine, rat, quail and human retina. Gerding H, Krause K, Cremer-Bartels G, Hanneken L

563

Comparison of urinary biopterin in rats with various housing and enviromental conditions. Goldberg M, Koller M, Schramm G, Mericenschlager M

567

Differences in the quantity and distribution of urinary pterins in various species of mammals. Goldberg M, Koller M, Merkenschlager M

571

Total biopterin levels in the ventricular CSF of Parkinson's disease and essential tremor. Furukawa Y, Kondo T, Nishi K, Yokochi F, Tanabe K, Mizuno Y, Narabayashi H

575

Urinary xanthopterin and derivatives as indicators of liver disease. Mazda T, Ogasawara K, Fukuda A, Gyure WL, Tsusué M

579

Genetic control of dihydropterin oxidase activity in Drosophila Ordono E, Escriche B, Silva FJ, Ferre J

583

A controlled system for supplementation experiments in melanogaster. Escriche B, Silva FJ

melanogaster.

Drosophila 587

New eye-color mutants affecting the biosynthesis of pteridines and ommochromes in Drosophila melanogaster. Calatayud MT, Jacobson DA, Ferré J

591

Search for 6- and 7-biopterins in human saliva. Katoh S, Sueoka T, Matsuura S, Sugimoto T, Shintaku H, Akino M

595

V n . TETRAHYDROBIOPTERIN-DEPENDENT MONOOXYGENASES Regulation of the aromatic amino acid hydroxylases. Kaufman S

Expression of rat liver dihydropteridine reductase in Escherichia Hoch J, Trach K, Schneider M, Whiteley JM

601

coli.

Escherichia coli dihydropteridine reductase is a trifunctional enzyme with dihydrofolate reductase activity. Armarego WLF, Vasudevan SG

612

616

XXIV Rat liver preparations with glyceryl etherase activity. Armarego WLF, Kosar-Hashemi B

620

Purification and characterization of a membrane associated folate binding protein from human leukemic CCRF-CEM cells. Westerhof GR, Jansen G, Kathmann GAM, Schomagel JH, Rijksen G

624

On the effects of high protein diet and of starvation on phenylalanine hydroxylase activities in rats. Carty MP, Walsh GA, Donlon J

628

Effects of experimental diabetes on phenylalanine hydroxylase activities in rats. Howard K, Walsh GA, Donlon J

632

Molecular defects of PKU in Italy differ from those of other European countries. Dianzani I, Ferrero GB, Ponzone A, Camaschella C, Romeo G, Devoto M, Romano C, Cerone R, Giovannini M, Riva E, Trefz FK, Lichter-Konecki U, Woo SLC

636

The oxidation of tetrahydropteridines by phenylalanine hydroxylase. Davis MD, Kaufman S

640

Interaction of catecholamines and tetrahydropterins with bovine liver phenylalanine hydroxylase. Martinez A, Andersson KK, Dahle G, Flatmaric T, Haavik J

644

Studies of tetrahydrobiopterin uptake in rat brain synaptosomes. Anastasiadis PZ, Wolf WA, Levine RA, Kuhn DM

648

The influence of cofactor side-chain chirality on the activity and regulation of phenylalanine and tyrosine and hydroxylases. Bailey SW, Chandrasekaran RY, Dillard SB, Ayling JE

652

Limited denaturation stimulates tetrahydrobiopterin-dependent activity of rat liver phenylalanine hydroxylase. Pamiak MA

656

Effect of limited proteolysis on structure and activity of phenylalanine hydroxylase. Pamiak MA

660

Tetrahydropterin as a possible natural cofactor in the Drosophila phenylalanine hydroxylation system. Bel Y, Jacobson KB, Ferrt J

664

Interaction of substrate analog inhibitors with rat liver dihydropteridine reductase. Bray T, Webber S, Whiteley JM

668

XXV Simultaneous purification of phenylalanine hydroxylase, phenylalanine hydroxylase stimulator protein, and dihydropteridine reductase from rat liver. Pamiak MA

672

VIII. FOLATES A. DIHYDROFOLATE REDUCTASE NMR and mutagenesis studies of dihydrofolate reductase. Roberts GCK

681

Spectroscopic studies of the interaction of ligands with dihydrofolate reductase. Blakley RL, Appleman JR, Maharaj G, Selinsky BS, Perlman ME, London RE, Delcamp TJ, Freisheim JH, Piper JR, Montgomery JA

694

Comparison between human and bacterial dihydrofolate reductase structures. Winkler FK, D'Arcy A, Müller K, Stüber D, Oefner C

702

Mutagenesis of human dihydrofolate reductase structure as a probe of enzyme function. Freisheim JH, Huang S, Prendergast NJ, Tan X, Smith PL, Bulleijahn AME, Thompson P, Delcamp TJ, Appleman JR, Beard WA, Tsay JT, Blakley RL

708

Folate and dihydrofolate reduction by mouse dihydrofolate reductase. Thillet J, Adams J, Benkovic SJ

716

Kinetic mechanism of recombinant human dihydrofolate reductase. Appleman JR, Beard WA, Blakley RL, Prendergast NJ, Delcamp TJ, Freisheim JH

722

The kinetic mechanism of the reaction catalyzed by an R-plasmid (R-67) dihydrofolate reductase from E.coli. Morrison JF, Sneddon MK

728

Mechanism based inhibitors of dihydrofolate reductase. McGill J, Rees L, Suckling CJ, Wood HCS

734

Trimetrexate resistant L. casei and S. faecium. Tamura T, Freeberg LE

740

Second site revertants of E. coli dihydrofolate reductase. Howell E, Linn C, Reece L, Artman J

744

Structural and energetic aspects of the reaction pathway and transition state for hydride-ion transfer in dihydrofolate reductase. Cummins PL, Gready JE

748

XXVI Cloning of trimethoprim-resistant dihydrofolate reductase from multiresistant staphylococci and expression in E. coli. Burdeska A, Then RL

752

Role of TRP-24 in binding and catalysis for human dihydrofolate reductase (DHFR). Beard WA, Appleman JR, Blakley RL, Huang S, Delcamp TJ, Freisheim JH . . . . 756 Mutations at hydrophobic residues in dihydrofolate reductase. Schweitzer BI, Gritsman H, Dicker A, Volkenandt M, Bertino JR

760

Determination of the functional role of phenylalanine-31 of recombinant human dihydrofolate reductase by site-directed mutagenesis. Tsay JT, Appleman JR, Beard WA, Blakley RL, Prendergast N, Delcamp TJ, Freisheim J

765

Molecular modeling studies and crystal structure determination of pteridine antifolate-DHFR enzyme interactions. Cody V, Luft J

769

Allelic variants of Chinese hamster dihydrofolate reductase. Melera PW, Jastreboff MM

773

B: THYMIDYLATE SYNTHASE Thymidylate synthase - structural and functional considerations. Matthews DA, Villafiranca JE, Smith WW, Janson CA

781

Defining the catalytic role of specific amino acids in thymidylate synthase by site-specific mutagenesis. Maley F, LaPat-Polasko L, Frasca V, Maley GF

795

Studies of the effects of mutagenesis on a conserved active site arginine residue in thymidylate synthase. Zhang H, Johnson LF, Cisneros RJ, Deng W, Zapf JW, Dunlap RB, Villafranca JE, Matthews DL

805

Dual role of serine hydroxymethyltransferase in the synergy of fluorouracil and leucovorin: effects of L-serine and glycine on H4PteGlu/5,10-CH2-H4PteGlu ratios; and HJteGlu-catalyzed release of fluoride ion from alpha-fluoro-beta alanine. Spears CP, Ray M, Granger S, Diasio RB, Gustavsson BG

811

Interaction of N"-hydroxy-dCMP and N4-hydroxy-5-FdCMP with L1210 thymidylate synthase differing in sensitivity towards 5-FdUMP inhibition. Zielinski Z, Dzik JM, Rode W, Kulikowski T, Bretner M, Kierdaszuk B, Shugar D

817

XXVII Mechanism of thymidylate synthase inhibition by N'-hydroxy-dCMP. Dzik JM, Zielinski Z, Rode W, Kulikowski T, Bretner M, Kierdaszuk B, Shugar D

821

Thymidylate synthase activity in bronchoalveolar lavage cells as a possible marker in lung cancer diagnosis. Rode W, Zielinski Z, Ciesla J, Lasota A, Grubek-Jaworska H, Walajtys-Rode E, Droszcz W

825

Purification and properties of Hymenolepis diminuta and regenerating rat liver thymidylate synthases. Ciesla J, Rode W, Kempny M, Pawelczak K, Rzeszotarska B, Machnicka B . . . . 829 Influence of FdUMP on the binding of methotrexate to thymidylate synthase. Gilli R, Lopez C, Sari JC, Briand C

833

Efficient expression of mammalian thymidylate synthase in Escherichia coli. Zhang H, Johnson LF, Cisneros RJ, Dunlap RB

836

Catalytic role of histidine-147 in Escherichia coli thymidylate synthase. Dev IK, Yates BB, Atashi J, Dallas WS

840

Variation among human thymidylate synthase enzymes in folate affinity. Davis ST, Berger SH

844

Evaluation of in vivo inhibition of thymidylate synthase in patients with colorectal cancer treated with 5-fluorouracil. Peters GJ, van Groeningen CJ, Laurensse E, van der Wilt CL, Meijer S, Pinedo HM

848

Effect of leucovorin on 5-fluorouracil induced inhibition of thymidylate synthase in murine colon cancer. van der Wilt CL, Pinedo HM, Peters GJ

852

Feedback inhibition of thymidylate synthase by dihydrofolate polyglutamates cannot account for partial depletion of tetrahydrofolate cofactors by antifolates. Seither RL, Trent DF, Goldman ID

856

Kinetic studies on thymidylate synthase from Lactobacillus casei. Ghose C, Oleinick R, Matthews RG, Dunlap RB

860

C. METHIONINE SYNTHASE Studies on the cobalamin-dependent methionine synthase from Escherichia coli K12. Baneijee RV, Matthews RG

869

XXVIII

Foimyl phosphate as an intermediate in the N10-formyltetrahydrofolate synthetase reaction. Mejillano MR, Jahansouz H, Hirnes RH

873

Enzymes of homocysteine remethylation and transsulfuration in methionine or cholesterol induced atherosclerosis in rabbits. Toborek M, Chmurzynska W, Manteuffel-Cymborowska M, Sikora E, Grzelakowska-Sztabert B

877

Biochemical changes in porcine neural tissue after inactivation of methionine synthase by nitrous oxide. Molloy AM, Keating J, Scott JM, Weir DG, Kennedy G, Kennedy S, Rice D

881

Increased synthesis of AdoMet but not of methionine during testosterone stimulation of polyamine biosynthesis in mouse kidney. Manteuffel-Cymborowska M, Chmurzynska W, Grzelakowska-Sztabert B

885

D. ENZYMOLOGY AND METABOLISM Role of folylpolyglutamate synthetase in the regulation of folate and antifolate metabolism. Shane B, Lowe K, Osborne C, Kim J, Lin B

891

NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase present in transformed mammalian cells. MacKenzie RE, Belanger C, Peri KG, Hum DW, Sheffield WP

896

Gammaglutamyl hydrolases. Whitehead VM

903

Human methenyltetrahydrofolate synthetase. Jolivet J, Bertrand R

910

Folate catabolism in man and the rat. Scott JM, McPaitlin J, Geoghegan F, McNulty H, Courtney G, Weir DC

916

Effects of growth rate on y-glutamyl hydrolase in Ehrlich ascites carcinoma cells. Sikora E

922

Folylpolyglutamate synthesis in Neurospora crassa. Wild type FPGS activity and transformation of polyglutamate deficient mutants using cosmid vectors containing the met-6 gene. Chan PY, Atkinson U, Nargang FE, Cossins EA

926

Cloning and expression of the gene encoding Lactobacillus casei folylpolyglutamate synthetase in Escherichia coli. Toy J, Bognar AL

930

XXIX Effect of treatments with different anticonvulsant drugs on folate pattern in rat liver. Bovina C, Formiggini G, Sassi S, Marchetti M

934

A sensitive new microassay useful for measurement and purification of mammalian folylpolyglutamate synthetase. Antonsson B, Moran RG

938

Tissue differences in N^formyltetrahydrofolate metabolism. Mullin RJ, Keith BR, Duch DS

944

E. FOLATES IN ENDOGENOUS PURINE METABOLISM Glycinamide ribonucleotide transformylase from E. coli: characterization and inhibition. Inglese J, Johnson DL, Benkovic SJ

951

Estimation of the flux control coefficient of glycineamide ribonucleotide transformylase for purine biosynthesis de novo. Smith GK, Knowles R, Pogson CI, Salter M, Hanlon MH, Mullin RJ

957

Monocyclic 5-deazatetrahydrofolate analogues as inhibitors of de novo purine biosynthesis. Bigham E, Duch D, Ferone R, Kelley J, Smith G

961

Inhibition of glycinamide ribonucleotide transformylase, methotrexate transport, and the growth of MCF-7 cells by 5,8-dideazafolates. Hynes JB, Benkovic SJ, Banks SD, Duch DS, Edelstein MP, Ferone R, Smith GK

965

F. FOLATES, VARIOUS Formation of methylcobamide from methyltetrahydrofolate by acetogenic bacteria: enzymology and redox chemistry. Ragsdale SW, Harder SR, Lu WP, Roberts DL

971

Incorporation of the ring carbon of histidine into coenzymes of folylpolyglutamates. Stokstad ELR, Brody T

977

In vivo effect of folic acid on fragile-site expression. Tilkian S, Lefevre G, DeCilla P

981

XXX IX. ANTIFOLATES A. DEAZADERIVATIVES New synthetic studies on deazafolates. Taylor EC, Chang Z, Harrington PM, Hamby JM, Papadopoulou M, Warner JC, Wong GSK, Yoon C, Shih C

987

Molecular modeling studies on deazatetrahydrofolates. Shih C, Grindey GB, Moran RG, Taylor EC, Harrington PM

995

Biochemical pharmacology of deazatetrahydrofolates. Beardsley GP, Pizzomo G, Russello O, Cashmore AR, Moroson BA, Cross AD, Wildman D, Grindey GB

1001

Synthesis and evaluation of l-deaza-7,8-dihydropteridines and ring analogues. Temple CG

1009

In vitro and in vivo activity of the GAR transformylase inhibitor 5-deazacyclotetrahydrofolate. Smith GK, Banks SD, Bigham EC, Cohn NK, Duch DS, Edelstein MP, Ferone R, Hanlon MH, Heath LS, Humphreys J, Kelley JL, Knick V, McLean EW, Mullin RJ, Singer S, Wilson HR, Houghton J

1015

In vitro and in vivo studies with 2-desamino-2-CH3-N10-propargyl-5,8dideazafolate (ICI 198583), an inhibitor of thymidylate synthase. Jackman AL, Newell DR, Jodrell DI, Taylor GA, Bishop JAM, Hughes LR, Calvert AH

1023

Comparative studies on the activity of methotrexate and the analog 10-ethyl-10-deaza-aminopterin (10-EdAM) against head and neck carcinoma cell lines and xenografts. Braakhuis BJM, van Dongen GAMS, Brown DH, Snow GB, Peters GJ

1027

Transport of 5,10-dideazatetrahydrofolic acid (DDATHF) in CCRF-CEM sensitive and methotrexate resistant cell lines. Pizzomo G, Moroson BA, Cashmore AR, Cross AD, Beardsley GP

1031

Synthesis and structure-activity relationship studies of 5,10dideazatetrahydrofolic acid (DDATHF). Shih C, Grindey GB, Gossett LS, Moran RG, Taylor EC, Harrington PM

1035

Synthesis and antifolate properties of 5-alkyl-5-deazaaminopterin analogues modified in the 10-position. Piper JR, Montgomery JA, Sirotnak FM

1039

Synthesis and biological evaluation of 2-desamino-2-methyl-5,10dideazatetrahydrofolate. Patil SD, Kisliuk RL, Gaumont Y, Nair MG

1043

XXXI Quinazoline antifolate thymidylate synthase inhibitors: potent cytotoxic agents containing heterocyclic isosteres of the para-amino benzoate unit. Marsham PR, Hughes LR, Hayter AJ, Oldfield J, Jackman AL, Bishop JAM, O'Connor BM, Calvert AH

1048

Antifungal activity of some 2,4-diamino-5-methyl-6-alkyl quinazolines. Colwell WT, Degraw JI, Ryan KJ, Lawson JA, Cheng A

1052

Membrane transport characteristics of the thymidylate synthase inhibitor 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid. Jansen G, Schomagel JH, Westertiof GR, Rijksen G, Newell DR, Jackman AL

1056

Synthesis and potential antitumor activity of 5-deaza analogs of 5,10methylenetetrahydrofolic acid. Gangjee A, Patel J, Kisliuk RL, Gaumont Y

1060

Theoretical studies of the structures and conformations of triazine antifolates. Welsh WJ, McMillan LR

1064

In vitro and in vivo metabolism of 5-DACTHF, an acyclic tetrahydrofolate analog. Hanlon MH, Ferone R, Mullin RJ, Keith BR

1068

Measurement of retained thymidylate synthase (TS) inhibition by antifolates in whole cells in vitro. Taylor GA, Jackman AL, Balmanno K, Calvert AH, Bishop JAM, Marsham PR, Hughes LR

1072

Substitution on the benzene ring of quinazoline antifolates that inhibit thymidylate synthase. Jackman AL, Thornton T, O'Connor BM, Bishop JAM, Bisset G, Calvert AH, Hughes LR, Oldfield J, Wardleworth JM, Barker AJ, Marsham P

1076

Inhibition of de novo purine synthesis by deazatetrahydrofolates. Moran RG, Baldwin SW, Shih C, Taylor EC

1080

B. METHOTREXATES Resistance to methotrexate in experimental models and in patients. Bertino JR. Romanini A, Dicker A, Volkenandt M, Lin JT, Schweitzer B

1089

Methotrexate peptides as cancer chemotherapeutic agents. Huennekens FM, Kuefner U, Esswein A, Fan J, Montejano Y, Vitols KS

1100

Biochemical determinants of methotrexate selectivity. Femandes DJ, Sur P, Kute TE, Capizzi RL

1110

XXXII Mechanisms underlying methotrexate inactivity in human head and neck cancer xenografts. Braakhuis BJM, van Dongen GAMS, Snow GB, Peters GJ, Leyva A, JansenG

1119

Design and synthesis of novel methotrexate derivatives for antibody directed drug targeting. Khan TH, Gamett MC, Baldwin RW

1123

Methotrexate (MTX) transport and cytotoxity of MTX and trimetrexate (TMQ) for human squamous cell carcinoma (SCC) of the head and neck grown at low folate concentrations, van der Laan BFAM, Hordijk GJ, Jansen G, van Gestel JA, Kathmann GAM, Westerhof GR, Schomagel JH, Rijksen G

1127

Folate enhancement of antifolate synergism in lymphoma cells. Gaumont Y, Kisliuk RL, Emkey R, Piper JR. Nair MG

1131

Amino acid oxidation and dihydrofolate reductase inactivation photosensitized by a diaminopteridine. Aubailly M, Santus R, Valla A

1137

Synchronisation of U937 and SW626 cancer cell lines of human origin using methotrexate. Sen S, Erba E, D'lncalci M

1141

New inhibitors of plasmodial folate synthesis - comparison of cell-free, whole cell and in vivo activities for single drugs and drug combinations. Wiese M, Kansy M, Kunz B, Schaper KJ, Seydel JK, Walter R, Chandra S

1145

Effect of vincristine on methotrexate cytotoxicity and accumulation in MCF-7 cells, a human breast cancer cell line. Mandelbaum-Shavit F

1150

The y-tetrazole analog of methotrexate: design, synthesis and biological activity. Kalman TI, Jones CS, Yawman AM, Russel CA, McGuire JJ

1154

Effect of intravenous 24 hr infusions of methotrexate on unconjugated pteridine metabolism. Dhondt JL, Hayte JM, Millot F, Mazingue F, Dupriez B, Farriaux JP

1158

Covalent labeling of dihydrofolate reductase and folate transport proteins by fluorescein methotrexate. Fan J, Pope LE, Vitols KS, Huennekens FM

1162

Study of methotrexate polyglutamate formation in cultured leukemic cells. Whitehead VM, Vuchich MJ, Payment C, Hucal C, Kalman TI

1166

XXXIII C. VARIOUS ANTIFOLATES Folyl polyglutamates in metabolism and chemotherapy. Kisliuk RL

1173

Biological determination of selective antitumor action of 4-amino-folate analogs. Sirotnak FM, Barrueco JR, Rumberger B

1185

Inhibition of the thymidylate synthase cycle of leukemia cells by antifolates: The usefulness of the cellular tritium release assay. Kaim an TI

1192

Fluoroglutamate-containing folates and antifols: probing the role of polyglutamylation in vitro. Coward JK, McGuirc JJ, Galivan J

1198

Therapeutic strategies for 5-fluorouracil-leucovorin based upon cellular metabolic characteristics in human colon adenocarcinoma xenografts. Houghton JA, Williams LG, Cheshire PJ, Houghton PJ, de Graaf SSN

1203

The lack of response of human colonic tumor cell lines to biochemical modulation by folinic acid. Hughey CT, Berger SH

1209

Folinic acid as a source of intracellular 5,10-methylenetetrahydrofolate in human carcinoma cells. Eagerton DH, Berger SH

1213

Structure activity relationships of multidrug resistance. Selassie CD, Hansch C, Khwaja TA

1217

Enhancement of the cytotoxicity of fluoropyrimidines to human carcinoma cell lines by leucovorin. Moran RG, Scanlon KL

1221

D. TRANSPORT AND ABSORPTION OF FOLATES AND ANTIFOLATES Methotrexate transport via the high-affinity folate binding protein of L1210 cells. Henderson GB, Strauss BP

1227

Combined affinity and ion pair liquid chromatographies for direct analysis of tissue folate composition. SelhubJ

1238

Tumor cell sensitivity and resistance to folate analogues: the role of carrierand receptor-mediated transport systems. Jansen G, Schomagel JH

1247

XXXIV Lysosomal permeability to methotrexate polyglutamates. Barrueco JR, Sirotnak FM

1253

Folic acid metabolism in placental cells associated with neural tube defects. Habibzadeh N, Smithells RW, Schorah CJ

1257

Homologous membrane folate binding proteins in human placenta. Ratnam M, McAlinden TP, Bulleijahn AME, Smith PL, Marquardt H, Duhring JL, Freisheim JH

1262

A high-affinity folate binding protein in human amniotic fluid. Holm J, Hansen SI, Hoier-Madsen M

1268

Interaction of N-hydroxy(sulfo)succinimide active esters with the reduced folate/methotrexate transport system from human CCRF-CEM leukemia cells. Jansen G, Westerhof GR, Kathmann GAM, Schomagel JH, Rijksen G

1272

Renal uptake and metabolism of 5-methyltetrahydrofolate in the rat. McMartin KE, Eisenga BH, Collins TD

1276

Methotrexate- and folate-lipid conjugates of defined chemical structures as new antifolate analogs and transport probes. Knepper T, Przybylski M, Ahlers M, Ringsdorf H

1280

Characterization of receptor-mediated (anti)folate uptake in human CCRF-CEM cells. Westerhof GR, Jansen G, Kathmann GAM, Schomagel JH, Rijksen G

1284

AUTHOR INDEX

1289 - 1296

SUBJECT INDEX

1297 - 1318

LIST OF PARTICIPANTS

1319 - 1340

Sponsors We gratefully acknowledge financial and material support by the following companies and organizations:

Major sponsors: Milupa AG, Friedrichsdorf, FRG F. Hoffmann-La Roche & Co., Basel, CH

Others: Abbott AG, Cham, CH Bank Hofmann AG, Zurich, CH Burroughs Wellcome, Research Triangle Park, USA Ciba Geigy AG, Basel, CH Cilag AG, Schaffhausen, CH Gewerbebank Zürich, Zurich, CH Glaxo Research Laboratories, Research Triangle Park, USA Lilly Research Laboratories, Indianapolis, USA Lonza Ltd., Basel, CH Parke-Davis, Ann Arbor, USA Pharmacia-pdf-(Schweiz) AG, Dübendorf, CH Rentenanstalt, Swiss Life Insurance and Pension Co., Zurich, CH Sandoz AG, Basel, CH Schweiz. Bankverein, Zurich, CH Sibra Management AG, Wädenswil, CH Suntory Ltd., Tokyo, Japan Swissair, Zurich, CH Dr. Karl Thomae GmbH, Biberach, FRG Zürcher Kantonalbank, Zurich, CH

Exhibitors Fluka Chemie AG, Buchs, CH Henning Berlin GmbH, Berlin, FRG Milupa SA, Domdidier, CH Sapec SA, Lugano, CH Dr. B. Schircks Laboratories, Jona, CH Serva Feinbiochemica GmbH & Co., Heidelberg, FRG Walter de Gruyter & Co., Berlin, FRG

I C H E M I S T R Y OF PTERINS AND FOLATES

RECENT

W.

PROGRESS

Pfleiderer,

IN

PTERIDINE

CHEMISTRY

K. A b o u - H a d e e d ,

Kiriasis,

and

M.

M.

Wiesenfeldt,

D. M o h r ,

L.

Fakultät Postfach

für Chemie der Universität Konstanz 5560, D-7750 Konstanz / West Germany

Z.

Kazimierczuk,

Bischler

I n t r o d u c t i on

Studies

on

revealed halo

the

that

atom

reactivity

proceed

t i o n of t h e

very

caused

-

by

V

a

due

adduct,

afford

less

7-halolumazines

CH.NH,

to

more

adduct

> 0

C

vinylogous the

the

7-

stabiliza-

isomeric

drastic

have

6-halo-

reaction

condi-

formation.

I CH,

CH,NH,Cl e

X V l 1

(1)

of

» ' ^ n - Y S

t

I CHj

H

reactions

the

whereas

much

favoured

A

and

displacement

easily

intermediary

1 ,3-dimethyl1umazines tions

of 6 -

nucleophilic

8

0

CHj

-

H

^ U \

N

Y

H C H >

20h "

-NHCHj

CHj

Results

Analogous lumazine rious

substitution as

starting

N-nucleophiles

7-subst.

reactions

material lead

with

6,7-dichloro-1

,3-dimethyl-

support

these

findings,

selectively

under

mild

amino-6-chloro-1 ,3-dimethyl 1umazines

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter& Co., Berlin • New York • Printed in Germany

since

conditions

(2).

Prolonged

vato

4 heating

at e l e v a t e d

secondary sition.

amines

A most

temperatures

effects

also

interesting

especially

with

disubstitution

reaction

was

high-boiling

in 6 - a n d

observed

with

c h l o r o - and 6-chl o r o - 7 - m o r p h o l i n o - 1 , 3 - d i m e t h y l 1umazine tively

on b o i l i n g

in m o r p h o l i n e

yielding

6,7-dimorpholino-1,3-dimethy11umazine tive"

dehalogenation

reaction

besides

also

the

7-po6,7-direspec-

expected

in a f o r m a l

"reduc-

7-morpholino-1,3-dimethylluma-

zi ne .

H^N N i , CH,

CI

2°'

C ^ H - V O i \—< un, CH

1

h

" l "

H,C

TYIL .

o ^ n ^ N ^ nX l \—' CH j Attempts under ting ful

to s y n t h e s i z e

a broad synthon

despite

variety

The

the

fact

diamine

(4)

substitution

first

successful

was

by a n a l o g o u s of c o n d i t i o n s

achieved

that

on a m m o n i a synthesis

very

low. A more

with

treatment

effective

Pd/C

ammonia

were

interesunsuccess-

and

6,7-di-

the a n t i c i p a t e d

6,7-

directly.

1,3-dimethyl-6 ,7-1umazine

methyl-5,6-uraci 1 diamine

catalyst,

approach

7-hydroxy-1,3-dimethy11umazine, (5)

with

potentially

afforded

of

0 /

from 6,7-dibenzylamino-1,3-dimethyl 1 uma-

by h y d r o g e n o l y s i s

% yield

reactions the

N \

2,4,6,7-tetrachloro-(3)

respectively

zine

81

N N l CHj

1,3-dimethy1-6,7-1umazinediamine

chloropteridine diamino

-

was

which

and ethyl

but the y i e l d

developed

resulted

from

from

6-amino-

1,3-di-

ethoxycarbimidoformate

in a r e g i o s e l e c t i ve c o n d e n s a t i o n

was

reaction.

in

5 0 H 5 C V n >L

nh

' T I I CH,

HN

+

-V0Et COOEf

EIOH^

" ' ^ n Y V N H , O N N O H i CH,

61%

POCIj | AICI] 0 H

l

C

A V

H

'

NH,

1

A

DI0X

O^N /NH, CH,

H,C

AN 70«

2

O^N-^CI 1

CH,

75% Treatment of t h i s

n

57% i n t e r m e d i a t e w i t h P0C1^ i n p r e s e n c e of

l e d to 6 - a m i n o - 7 - c h l o r o - 1 , 3 - d i m e t h y l 1umazine in which the c h l o r i n e

atom i s

still

1 ,3-dimethyl-6 ,7-1umazinediamine

primary a l i p h a t i c

pectively

as s e c o n d a r y

number of new l u m a z i n e

i n 57 % y i e l d

reactive

at

and a r o m a t i c amines

amines g i v e

rise

and

to form

w i t h ammonia i n d i o x a n

70°C. A n a l o g o u s l y as well

highly

AlCl^

res-

to a l a r g e

derivatives.

H ^ l ^ N H ,

/

j i J O^N N NHCH, I CH]

I N CH,

H , c , N J y i L nh,

^ CH, n V O/ i

OKK^-Q '

i CH,

However, the most s i m p l e s y n t h e s i s

of

z i n e d i a m i n e was f i n a l l y

i n the c o n d e n s a t i o n

discovered

1 ,3-dimethyl-6,7-1uma-

t i o n between 1 , 3 - d i m e t h y l - 5 , 6 - u r a c i 1 d i a m i n e formimidate yield.

i n DMF at

'

and methyl

150° and a c i d c a t a l y s i s

reaccyano-

to g i v e a 60 %

6

H jC.

A •L ^1

+

I CHj

Cyclization acetic tuted

reactions

anhydride,

with

benzoyl

Formamide

purine

cyclizes

theophylline

one carbon chloride

building

or u r e a

which

can

N

NHj

O^n^N^NH, i CH,

lead be

blocks to

such

2-substi-

regarded

as

1,3-dimethyl-6,7-1umazine

analogue, which

gave

a mixture

diamine of the

to

two

1,6,8- and 3,6,8-trimethy1imidazo[4.5-g]-1umazines Their

by a n a l o g o u s

cyclization

structures

have

reactions

formamide.

pyra-

been

proven

the isoon

unambiguously

of 6 - a m i n o - 7 - m e t h y 1 a m i n o -

7 - a m i n o - 6 - m e t h y 1 ami n o - 1 , 3 - d i m e t h y l 1 u m a z i n e

in b o i l i n g

as

derivatives.

methylation. and

DMF 150-

imidazo[4.5-g]-1 umazines,

zine-stretched

meric

H,C. A

HN^OCHj I CN

respectively

7

h

HCONH^

c

'

CH,

^ Y y \ CH,

CH

0

'

I

I

CH,

1

.

-

(AN^N^NH I I

CH,

compound

condensations

with

of

CH,

.t

O^N^N^nh, I

Furthermore

I

CH,

the

CH,

new

heterocyclic

C2"Synthons

gave

rise

p y r a z i n o [ 2 . 3 - g ] - p t e r i d i ne

ring

system.

O^N^N

CH,

to t h e

1 ,2-diamino

formation

of

the

NH,

CH, jBENZIL S N \ V V , H ' j 1 J J 0 N ^ ^ h r ^ N C| H s CH,

H,C O^N^N^N*

CH,

Different

types

available

from

zines.

For

example,

carboxylate chloro

was

philes.

ethyl

converted

derivative,

leophilic acted

of h i g h e r

which

displacement

Ethyl

condensed

bifunctional

H

H

o

'C-N 7 n^ i CH,

pteridine

6,7-disubstituted

nuclei

are

7-hydroxy-1,3-dimethyl 1umazine-6by

POC1^

into

was

prone

to a b r o a d

reactions

using

the

corresponding variety

0-, S- and

of

with

ethyl

thioacetate

and

7nuc-

N-nucleo-

7-chloro-1,3-dimethyl 1umazine-6-carboxylate

in h i g h y i e l d

also

1,3-dimethyl1uma-

subsequent

rebase-

8 catalysed

Dieckmann

[ 3 . 2 - g ] - p t e r i d i ne on

the

other

condensation

ring

system.

hand allowed

led

to a c o n d e n s e d

Hydrazine

easy

access

to

and

thieno-

methylhydrazine

pyrazolo[4.3-g]-pte-

ri di n e s .

H.C..,

N VCO,EI O^^-^OC.H, I CH,

H,C

^N'*Sf'N«>r-cO, El O^-N-^N^"0" CH,

0 H»C,N>V,N CO.EI

H,C. N >yN co.E. O^N^N^SCHjCOjEt I CH,

CH, N

Cl

A n ^nhch, CH.CH, I CH,

^'^N-V^CO.EI •CHj S CH,

I NH,NH, H,C,

„

2

OH

0

n

„

^rrVco,E. O^-N

^ - V V * CH,

The

isomeric

was

derived

its

intermediary

TFA.

ethyl

6 - c h l o r o - 1 , 3 - d i m e thy 1 1 u m a z i n e - 7 - c a r b o x y 1 a t e

from ethyl

Secondary

5-oxide and treatment

amines

displace

chlorine

atom, whereas

function

forming

hydrazine razole

acts

ring

1,3-dimethyl 1umazine-7-carboxylate

the

primary

under amines

corresponding

as a b i f u n c t i o n a l

in a

[3.4-g]

fashion.

with

acetyl

chloride

mild conditions attack

also

substituted

nucleophile

via

only

the

amides.

attaching

in

the

ester Methyla

py-

9

CH,

1

7-Ch1oro-6-dichloromethyl-1 ,3-dimethyl1umazine , another tive

pteridine

derivative,

was

synthesized

dimethyl 1umazine-6-carboxaldehyde hydroxy-1,3-dimethyl 1umazine treatment.

Primary

atoms

formation

with

and of

zine-6-carboxaldehydes which

hydrolyse

(6)

secondary the most

on w o r k - u p .

(6) a n d

from

7 - h y d r o x y - 1 ,3-

6-dibromomethyl-7-

respectively amines

by

displace

PCI5/P0C13 the

chlorine

7 - s u b s t . ami no-1 , 3 - d i m e t h y l 1 u m a likely

via

the

Ethylmercaptide

intermediary allows

a

0 0-kNJLNJ.NHCH,

o ^ V o

CH,

CHCI,

0 N^^N

SC H

'»

imines,

selective

0 HiC,N>lYN^rCHBr1

0

reac-

10 substitution

at C - 7 , w h e r e a s

methyl h y d r a z i n e

yielded

1,6,8-

trimethyl-pyrazolo[4.5-g]-lumazine. A similar

series

of

reactions

cyano-1,3-dimethyl 1umazine,

was

which

achieved was

1,3-dimethyl1umazine-6-carbaldoxime The c h l o r i n e

atom

interactions

b u t the a d j a c e n t

in s u b s e q u e n t

is a l w a y s

cyclization

thieno[3.2-g]-

and

with

displaced cyano

P0C13

first

group

reactions

with

obtained by

7-chloro-6from

in g o o d

yield.

nucleophilic

may also

yielding

7-hydroxy-

be

involved

condensed

3-amino-pyrazolo[4.3-g]-1umazine

3-amino-

derivatives.

u H»C>N>4s^N CHO

u '-«Yy» O^N H oc>H* CH,

CH,

'-«•"yY

Furthermore bond

our

skeleton

of

the

l


i'"' yC-CH s Y f ' "

^

1

M

H 0 CHi^^. O^^/sch.-C-O CH, 0

•

"0iJ O N N^CH 0

S H /-v '-N-JYNu N

B

O

N —\

h n V V N ^ S V o g i u

COGIuEt2

8

7

Reagents (Yields): i, N-bromosuccinimide, (PhCOO)2, CHCI3 (70%); ii, EtNH2, MeCN (78%); ¡¡i, PhCONCS, Et3N, acetone, reflux (84%); iv, c HCl, IPA, 100 "C (99%); v, HC0C(CI)HC02Et, DMF, 100 "C (22%); vi, 1 N NaOH, 48 'C (87%); vii, GluEt2.HCI, DPPA, Et3N, DMF (47%); viii, 1 N NaOH, aq EtOH, (77%). This methodology was extended to give related antifolate analogues containing other heterocyclic p-aminobenzoate isosteres, the syntheses of which are outlined in Schemes 2 - 5 . Scheme 2 N

•N x 0 2 n ' "s C02H

02N

S

H,N

COGIuEt2

S

COGIuEtp 11

10

9 O ji HN

I "

N Ai - X iT^Y^

N

s

C0GluEt

^

2

HN

1 3

JCx S

COGIuEt2 12

\

o HN

r

N

COGIu 1 4

88 Scheme 2 (continued) Reagents (Yields): i, (COCI)2, DMF, CH2CI2; ii, GluEtj.HCI, Et3N, CH2CI2 (88%); iii, Fe, FeS0 4 , aq MeOH, reflux (60%); iv, Propargyl bromide, 2,6-lutidine, DMF, 80 "C (47%); v, CaC0 3 , DMF, 90 "C (47%); vi, 1 N NaOH, aq EtOH (70%) Scheme 3

O 3_ j

H H 3 CA'

J

S

"

S N 1

V ^

N-N

T

N

^ "

^

^

1 5

a JL.

S

N-N

J*-

18

>J '

B

H

H

s

o

17

o II h n ^ V V - N ^ : Et

N N -- N N VX S

COGIu

21 Reagents (Yields): i, Thiocarbonyldiimidazole, Et3N, CH2CI2 (70%); ii, N 2 H 4 .H 2 0, reflux (66%); iii, Et0 2 C.C0CI, Et3N, DMF (100%); iv, MeS0 3 H, toluene, reflux (60%); v, 1 N NaOH, EtOH (88%); vi, GluEt2.HCI, DPPA, Et3, DMF (70%); vii, 1 N NaOH, EtOH (57%) Scheme 4

Reagents (Yields): i, PhCH2NHCH3, N-Methylpyrrolidinone, 100 'C (87%); ii, H 2 ,10% Pd-C, CF 3 C0 2 H, EtOH, 60 "C (93%); iii, CaC0 3 , DMF, 90 "C (34%); iv, 1 N NaOH, aq EtOH (31%)

Reagents (Yields): i, DPPA, Bu'OH, Et3N, reflux (56%); ii, NaH, Propargyl bromide, DMF (86%); iii, 1N NaOH, aq EtOH (84%); iv, (COCI)2, DMF, CH 2 CI 2 ; v, GluEt.-HCI, Et3N, CH 2 CI 2 ; vi, CF 3 C0 2 H, 0 ' C (90%); vii, CaC0 3 , DMF, 100 "C (18%); viii, 1 N NaOH, aq EtOH (68%)

SYNTHESIS OF TETRAHYDRO-8-DEAZABIOPTERIN

J. I. DeGraw, W. T. Colwell, T. Hayano Bio-Organio Chemistry Laboratory, SRI International Menlo Park, California 94025

Introduction

The reduced pterin compound, tetrahydrobiopterin (BHtJ has been recognized as a vital cofactor for the hydroxylation of aromatic amino acids (1).

A deficiency of this substance may be a contributing factor in

hyperphenylalaninemia and PKU-like conditions as well as other neuropathology states such as Parkinson's disease.

It has also been

observed in recent years that urinary and serum levels of BH4 and related pterins are markedly elevated in various pathological conditions (2).

This includes viral and bacterial infections, lymphadenopathy,

AIDS and other immunodeficiencies.

Other pertinent disease states are

myopathies and amylotrophic lateral sclerosis.

During immunological

crises in transplant cases there is a strong increase in pterin levels just prior to obvious signs of tissue rejection.

While the role of BHi»

in the immune response is unclear, BH 4 has been shown to dramatically increase the rate of proliferation of T-cells via enhancement of the uptake of IL-2 into the T-cells (3). BH^ would seem to offer potential for counteraction of neurological deficits and enhancement of immunotherapy.

Analogs could also be inhib-

itory to unfavorable immune events such as those leading to sclerotic or inflammatory conditions.

A barrier to practical implementation of BH,,

and related tetrahydro pterins is their oxidative instability.

We

report the preparation of tetrahydro-8-deazabiopterin (8-deaza B H ^ as a reasonably stable analog for biological investigation.

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in G e r m a n y

91 Results The synthesis of 8-deaza BH,, is outlined in Scheme 1.

Crotonic acid

Q)

was treated with warm aqueous H 2 0 2 in the presence of a tungstic acid catalyst (1) to afford erythro-2,3-dlhydroxybutyric acid in 70? yield. The crystalline acid was acetylated by treatment with flc20-HCl at 80° to yield the diacetoxy acid (2) as a cyrstalline monohydrate (90/5).

The

anhydrous acid was obtained by heating ¿n vacuo and converted to the acid chloride by reflux in thionyl chloride.

A solution of the acid

chloride in ether was added dropwise to excess diazomethane in ether at 0° followed by exposure to the resulting diazoketone to dry HC1 to afford the chloromethyl ketone (3) in 95? yield.

Prolonged treatment of

OAc OAc CH3CH = CHCOOH

1) H 2 O 2 - W O 4 2)

AC20-HCI

OAc OAc SOCI,

C H 3 C H - CH-COOH

I

I

C H j C H - CH

CH2N2-HCI

I

COCHJCI

OH

I

OAc OAc O 1) 0 3 P 2) 5 % K 2 C 0 3

I

I

II

AcNH

ÀJ

C H 3 CH - C H - C- CH = P» 3

i CHO

OH

XX

AcNH

O OAc O A c

II I

CH = C H

1

fi OH

OH 1) H 2 R h / C 2)

HCI-MeOH

jòr

I

C CH - CH C H 3

O OR j

OR j

*

1) H 2 - P t Q 2 - H C I " 2) HCI-EtOH

CH 2 C H 2 - C CH - CH CH 3

R = Ac, H (mixture of monoacetate esters)

Scheme 1

I

J

H

N.

OR

OR

I

I

^CH - CH -CH 3

HjN i

R : AC, N

£

R = H

92 3 with an equivalent of triphenylphosphine in refluxing dimethoxy ethane gave a phosphorane salt which was readily converted (4) by 5% K 2 C 0 3 in 48% yield.

to the stable ylide

Some decomposition occurred during this

reaction probably as the result of 8-elimination of the 3-acetoxy group. 2-flcetamido-4-hydroxy-6-formylpyrimidine (5) (2) was suspended in DMF and treated with bis-trimethylsilyl trifluoroacetamide to give a solution of the silyl ether of 5 (3) which was stirred with an equivalent of the ylide 4 for 24 hours to yield (20%) the pyrimidinyl enone (6); u.v. (EtOH) 260, 332 nm.

The NMR spectrum showed the presence of the olefin

and three acetyl methyl signals.

In order to activate the 5-position of

the pyrimidine for diazonium coupling it was necessary to reduce the olefin (6) and selectively remove the 2-acetamido group.

This was

readily accomplished by hydrogénation over a Rh/c catalyst followed by treatment of the dihydroketone with 0.01 N HC1 in MeOH at 65° for 3 hr.

NMR analysis showed cleavage of the N-acetyl signal with retention

of the acetate esters.

The crude pyrimidine diester was suspended in

50% DMF and coupled with benzenediazonium chloride at pH 8-9 to afford the phenylazoketone monoacetate (7); u.v. (EtOH) 380, 420 nm.

Hydro-

génation of 7 over Pt0 2 in 0.3 N HCl-EtOH caused reductive cyclization to 8-deaza BH 4 acetate which was readily deacetylated by continued exposure to HCl-EtOH to yield 8-deaza BHi* (9). Compound 8 was shown to be a mixture of mono acetate esters by mass spectral analysis, while 9 was identified by its uv and mass spectrum, m/e = 240.

The product did not show evidence of air oxidation on

prolonged storage. The 8-deaza BH,, product obtained from this synthesis is a mixture of 4 isomers because of chirality at C-6 and the side chain.

We are

presently studying the optical resolution of erythro-2,3-dihydroxybutyric acid in order to prepare the product with the L-erythro side chain configuration, but still with a chiral mixture at C-6.

Acknowledgement Mass spectral analyses were conducted by Dr. David Thomas and certain NMR analyses by Mr. George Detre.

References 1.

Benkovic, S.J.

1980.

Ann. Rev. Biochem. 49, 227.

2.

Ziegler, I., H. Rokos. 169.

3.

Ziegler, I., V. Schwulra, J. Ellwart. 531.

4.

Mugdan M., D. Young.

5.

DeGraw, J.I., V.H. Brown.

6.

DeGraw, J.I., V.H. Brown, W.T. Colwell, M. Sato, R.L. Kisliuk, Y. Gaumont, F.M. Sirotnak. 1988. J. Med. Chem. 3_L, 150.

1986 EOS Revista Immunol. Immunofarmacol.

1949.

1986.

Exp. Cell Res. 167,

J. Chem. Soc. 2988. 1976.

J. Hetero. Chem. JJ, 439.

SYNTHESIS AND ENZYMIC PROPERTIES OF NEW MECHANISM BASED SUBSTRATES FOR DIHYDROFOLATE REDUCTASE OF THE 8-SUBSTITUTED PTERIN CLASS. M.J.Koen, R.K.Haynes Department of Organic Chemistry, University of Sydney, N.S.W, 2006,Australia. J.E.Gready, P.A.Pilling Department of Biochemistry, University of Sydney, N.S.W, 2006,Australia.

Introduction The synthesis and enzymic properties of a new class of substrates for vertebrate dihydrofolate reductase (DHFRs) are reported.

These compounds have been designed (1) on the basis

of theoretical calculations and chemical argu ments as analogues of the proposed protonated form of folate, which is suggested to be the active species in the initial enzymic reduction of folate (2,3).

The synthesis and enzymic properties of some of these

compounds will be described.

Synthesis The compounds, 8-methylpterin [£] (4), 6,7,8-trimethylpterin (5), 6,8-dimethylpterin

[4.], and 7,8-dimethylpterin

been prepared, see Figure 1.

[¿]

[¿] have

Under conditions of low

temperature and long reaction time, the bisulfite addition product of pyruvic aldehyde (methyl glyoxal) with 2,5-diamino-6N-methylamino-4-oxo(3H)- pyrimidinone hydrochloride 6,8-dimethylpterin

[1], gave

(47-60%), which was contaminated with < 5%

(!h n.m.r. 400 MHz)of the 7,8-dimethyl isomer.

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in G e r m a n y

This impurity

95

was easily removed by recrystallisation.

This synthesis

represents a successful solution to the isomer problem which is encountered when working with unsymmetrical dicarbonyl compounds.

The purity has been confirmed by high field

n.m.r. spectroscopy and ion exchange chromatography ( Partisil 10-SCX, 30% CH3CN in ammonium acetate buffer, pH 3.3).

Reaction

of this diaminopyrimidine [1] with pyruvic aldehyde dimethyl acetal gives a mixture with 7,8-dimethylpterin and 6,8dimethylpterin present in a ratio of 7 to 1 (^H n.m.r. 400MHz).

:xX>-

I • / Mtfhual / rrflai N •!• M—c—C—«

1M, C

• r r (1)

II C—CM, II CM,—C nuthanol I reflu] / l hour n-'N.'NPSh, CH, IH, C CH,—C—CHOH /HtO X y " ' methanol / 0* CtoRT ovar SO houL rs Jl »Ja" 47.ii CIH, 0

o

CH,—C—CH(OCHi), nuthanol / RT / 1 hour 4 (60%), so that an alternative tactic was necessary.

C h e m i s t r y and Biology of Pteridines 1989 © 1990 Walter d e G r u y t e r & C o . , Berlin • N e w York • Printed in G e r m a n y

99 3. Sulphenylation

& C-2

Alleviation

- in 'one-pot', with phenyl disulphide followed by primary alkyl- or aryl-lithiums: CH,Ph I N

c>

CH,Ph I N

1. BuLi 2. PhSSPh

N

3. R'Li

f

»M

CH,Ph I .N

c>= o

+

[ l ^ c j - R1

R2

N R'= (CHjJaMe 72%

Si02 chromatography

^^• N kl H

CH,Ph I N \

R 1 =(CH2) 7 Me 76% R = Ph 52%

CHoPh I N

c >-sph

Me(CH 2 ) 3 Li 70%

M

(56%)

(25%)

With the cleavage methods developed earlier, this completes a Cj-transfer at two oxidation levels. Only arylthio groups are substituted (the 2-butylthio-2-imidazoline was unreactive towards organometallics). 4. Sulphenylation

& C-2

Arvlation

- occurs with secondary alkyl lithiums, via a regiospecific sulphide contraction: CH,Ph I N

CH,Ph I N

1. BuLi

c>

2 mol. equiv. sec-BuLi

c>~

2. PhSSPh

CH,Ph I N

C>~

M

M

(69%) CHjPh

CH,Ph I 1. BuLi

o - o

2. (4-MeC 6 H 4 S) 2

Me

2 mol. equiv. sec-BuLi

> 0

(55%)

(60%)

Mechanism of sulphur extrusion: suggested single electron transfer from sec-BuLi: CH,Ph I

o

CH,Ph I

CH 2 Ph I N

o

rV/^Y

— 2H2

°

-H2S

CH 2 Ph

c ^ o -

•J

Me

KJ

CH,Ph I •N S

CH 2 Ph

OK>

Me

Ue

100 5. A Thiamine

Ylide

Mimic

Metallo-imidazoline (2) is related to the ylide (4) formed from the coenzyme thiamine. It adds to CH2Ph CH,Ph I -N -N S N c > -

x

>

-

c > N+

ri

(2)

(4)

(H, Me) Me)

(5)

carbonyl electrophiles to produce 2-(hydroxyalkyl)-2-imidazolines whose chemistry mimics the corresponding adducts of thiamine through the intermediacy of the imidazolinium ylides (5) as leaving groups. Thus aldehydes can be converted into ketones; 2-acyl-2-imidazolines, when quatemised, act as acylating agents. CH,Ph CH,Ph CH2Ph I I N N OH BuLi, r )-CH-Ri c >N c > R1= (CH2)8Me 70% -w R1= Ph 71% R1=3-MeOC6H4 58% Mn02 BuLi, R1COR2 R1=4-MeOC$H4 57% R1= Ph 67% (R1,R2*Me) aV= Pr 53% r ' = 3,4-(MeO)2C6H4 60% R CH,Ph I ^.N OH

CH,Ph BuLi, R'CCfcB R ^ (CH2)4Me R'= (CH^sMe R1=Ph

O II R1—C—Nu

N

u

C -W

H

-

C M R'= Ph; R2M= PhU 92% ^ R1= Ph, R2M= MeLi 69% R'= Ph; R2M= BuLi 59% 1 R = Ph; R2M= CH^CHMgBr 89% R1= Ph; R2M= EtMgBr 34% R1= Ph; R2M= Me(CH2)7MgBr 42% R1= (CH2)4Me; R2M= MeMgBr 56% R1= (CH2)4Me; R2M= PhMgBr 46% O II R1-C—R2

R2

Method A: CHCI3, trace H+ Method B: Mel

NuH= EtOH 73% MeOH, H+ 71% PhCH2M-l2 80% BUNH2 58% BuSH 89% NaOH aq. (25'C) 75%

Method A: R'=R 2 =Ph 99% Method B: R'= R = Ph 83% R1=Fti, R2=Me 94% R1= Ph, R2= Me 91% R1=Ph,R2=B 52% R1=Ph,R2=Bu 79% R1=Ph,R2=(CH2)7Me 83%

Co-products:

CH,Ph 1 -N N>

Method A

CH2Ph N

Qr

Me Method B

an via

loss of ylides

CH,Ph 1 N M

C

/)(H,N* Me) (5)

101 6. Tetrahvdroimidazo-quinolines

and

-isoquinolines

as tricyclic tetrahydrofolate models.

We have developed an efficient and convenient access to the new fused imidazolines (6) and (7) and demonstrated their use in Cj-transfers: 1. H2 (30 atm.), Pt02, EtOH. HBr aq. (70%)

1. 4-MeC6H4S02CI, KCNaq. N

2. H202, KOH aq. (77%)

N

CONH2 2- BjHe (83%) MeC(OEt)-NH2* CI', EtOH (52%)

H02C— CH2- - R

BuLi, RI R1 _l

1. Mel; 2. R1M; 3. H30*

RCHj

r_ Me 78% R-fCHJyMe 93% R-CHMe2 58%

1. PhS02CI, KCN aq. 2. NaBH4, EtOH (65% overall) 3. H202, KOH aq. (82%)

H02C— CH2- - R

j^" R1 -i-

0=C-CH,--R

1. Mel; 2. R1M; H,0* 3. H 30*

jT^

|

^ '— *— N N R-Me 38% R-(CH2)7Me 84% R.CHMB2 66%

BuLi, RI CH

Me

2R 1

—N

(7)

Reduction to imidazolidines related to N5,N10-methylenetetrahydrofolate was not possible in either

00, op.

series; over-reduction to diamines of the type shown below occurs instead (cf.M.W. Anderson, R.C.F. Jones, and J. Saunders, J. Chem. Soc., Perkin Trans. 1, 1986, 1995):

H

Acknowledgements:

NR'R2

NR1R2

the support of ICI Pharmaceuticals Division (to J.R.N.) and of Reckitt &

Colman Pharmaceuticals Division (to M.J.S.), and the Science and Engineering Research Council, is gratefully acknowledged.

102 ASYMMETRIC SYNTHESIS AND ABSOLUTE CONFIGURATION OF 5,10DIDEAZA-5,6,7,8-TETRAHYDROPTEROIC ACID AND 5,10-DIDEAZA5 , 6 , 7 , 8 - T E T R A H Y D R O F O L I C A C I D (DDATHF).

C h a r l e s J. B a r n e t t * a n d T h o m a s M .

Wilson

Chemical Process Research and Development Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

Introduction The d e a z a a n a l o g s o f t e t r a h y d r o f o l i c a c i d r e p r e s e n t a n i m p o r t ant c l a s s o f f o l a t e a n t i m e t a b o l i t e s of i n t e r e s t as p o t e n t i a l o n c o l y t i c a g e n t s (1). W i t h i n t h e series, 5 , 1 0 - d i d e a z a - 5 , 6 , 7 , 8 t e t r a h y d r o f o l i c a c i d (DDATHF, 1) h a s s h o w n e x c e p t i o n a l a n t i t u m o r a c t i v i t y in a v a r i e t y o f a n i m a l m o d e l s of m a l i g n a n c y (2). P u b l i s h e d s y n t h e s e s o f D D A T H F (3,4) h a v e p r o v i d e d m i x t u r e s of C - 6 e p i m e r s a r i s i n g f r o m n o n s e l e c t i v e r e d u c t i o n of 5 , 1 0 dideazafolate precursors. The e p i m e r s h a v e b e e n s e p a r a t e d b y HPLC and by fractional crystallization of certain derivatives (4), a n d t h e s e p a r a t e d d i a s t e r e o m e r s (DDATHF-A a n d B) h a v e b e e n e v a l u a t e d s e p a r a t e l y (5). D D A T H F - B (LY264618) w a s s e l e c t e d for c l i n i c a l e v a l u a t i o n a n d is c u r r e n t l y u n d e r g o i n g p h a s e I t r i a l s . W e w i s h t o r e p o r t t h e a s y m m e t r i c s y n t h e s i s of D D A T H F (1) a n d a s s i g n m e n t of a b s o l u t e c o n f i g u r a t i o n to t h e A a n d B i s o m e r s a n d t h e r e l a t e d 5 , 1 0 - d i d e a z a - 5 , 6 , 7 , 8 - t e t r a h y d r o p t e r o i c a c i d s (1A).

Results Our synthetic plan was designed to utilize enzymatic enantiod i f f e r e n t i a t i o n of p r o c h i r a l 1 , 3 - d i o l (£) as a m e a n s o f e s t a b l i s h i n g t h e c o n f i g u r a t i o n of C - 6 in t h e t a r g e t s t r u c t u r e . Thus b o r a n e r e d u c t i o n of 4 - b r o m o p h e n y l a c e t i c a c i d (2) a n d t o s y l a t i o n of the resulting alcohol provided Q ) w h i c h was converted to t h e d i o l (£) b y r e a c t i o n w i t h s o d i u m d i e t h y l m a l o n a t e a n d h y d r i d e r e d u c t i o n of t h e r e s u l t i n g d i e s t e r (4.) • R e a c t i o n o f (¿) w i t h m e t h y l a c e t a t e in t h e p r e s e n c e o f p o r c i n e p a n c r e a t i c l i p a s e (PPL, S i g m a t y p e II, no. 3126) i m m o b i l i z e d o n H y - F l o (6) a f f o r d e d a 90% y i e l d of t h e m o n o a c e t a t e (+)-(£), (85% ee, c h i r a l HPLC) a n d 10% of the c o r r e s p o n d i n g d i a c e t a t e (2) w h i c h

103

could be conveniently removed chromatographically. The configuration of ( + )-(£) was shown to be R by acylation with (S)-(-)a-methylbenzyl isocyanate and single crystal X-ray analysis (7) of the resulting carbamate derivative (&). Br

RjO 2 R=CO,H 3 R=CH2OH 4 R=CH2CH(C02Et)2

5 R,=R 2 =H (FL)-(+)-FI R , = C H 3 C O ; R 2 =H 1 R1=R2=CH3CO s R , = C H 3 C O ; R 2 =(S)-C B H 4 CH(CH 3 )NHCO 1 5 R , = C H 3 C O ; FFE=TBS I Ê RI=H; R 2 = T B S U R , = C H 3 S 0 2 ; RJ=TBS

Br

•

^

(fl)-(+)-2 R|=OH; R2=N3 (S)-(-)-2 R,=^;R 2 =OH Ifl R,=CH(C02Et)2;F^=N3 IS Ri=I%; R2=OTBS

H

11 COR

O HN

Br

iT^T

(6S)-1 R=CONH-L-Glu (DDATHF-A) 12 R=Br 13 R=CN (S)-(+)-14 R=C02H

(6fl)i R=HN-L-Glu (DDATHF-B) (Rl-(-)-lA R=OH

Sequential mesylation of (R)-(+)-(£), treatment with sodium azide, and hydrolysis provided azidoalcohol (R) - ( + ) - {2.) , 85% ee (HPLC), which was converted to (1Q.) by tosylation and subsequent reaction with sodium diethyl malonate in the presence of sodium iodide. Reduction of (1Q) (tributylphosphine) afforded (11) which gave (12) upon reaction with the Meerwein reagent and exposure of the resulting lactim ether to guanidine (8). Reaction of (12.) with cuprous cyanide (N-methylpyrrolidinone, reflux) provided the nitrile (12). Acidic hydrolysis (6M HC1, reflux, 70h) of (12.) afforded (S) - ( + ) -5, 10-dideaza-5, 6, 7, 8tetrahydropteroic acid (11) as an amorphous hydrochloride, [a]589 +40.7° (c 1.0, IN NaOH). Acidic hydrolysis of an authentic sample of DDATHF-B (4), (98% de) in refluxing 6M HC1 (9), on the other hand, provided (-)-(14) hydrochloride, [cx] 589

104

-48.3° (c 1.0, IN NaOH), thus establishing that (-)-(14) and the B isomer of DDATHF (1) possess the (R) configuration at C6. It follows that DDATHF-B is topologically analogous to natural (6S)-tetrahydrofolic acid (10). The enantiomeric azidoalcohol, (S)-(-)-Q), could be obtained from (R)-(£) by a modification of the synthesis as follows. Silylation of (R)-( + )-(£) (90% ee) provided (IS) which upon saponification with sodium hydroxide afforded (1_£). Conversion of (1£) to the corresponding mesylate (17) and treatment with sodium azide in DMF provided (HI). Removal of the TBS group under acidic conditions completed the synthesis of (5)-(-)-(9) in 52% overall yield from (i?) - {£.) without loss of enantiomeric purity as determined by HPLC analysis. We have prepared (R)(-)-(lA), required for the synthesis of (6R)-DDATHF (1), by utilizing (S)-(-)-(2.) in the pteroic acid synthesis described herein. The synthesis of the 6R and 6S diastereomers of DDATHF (1) was completed by coupling of the pteroic acids with diethyl-Lglutamate (chlorodimethoxytriazine (11) method) and alkaline hydrolysis of the resulting ester products in 78% overall yield. Samples of (6R) and (6S)-DDATHF thus obtained were found to be identical with authentic DDATHF-B (LY264618) and -A (LY243246), respectively, by NMR and by P-cyclodextrin inclusion chromatography (12). The chromatographic results also established that no racemization had taken place during the coupling step. Acknowledegments We thank Professor Paul Wender, Stanford University, and Professor Edward C. Taylor, Princeton University, for their advice, suggestions, and encouragement of this work. References and Notes 1) Fry, D.W., R.C. Jackson. 1987. Cancer and Metastasis Rev. 5, 251. 2)

Beardsley, G. P., E. C. Taylor, C. Shih, G. A. Poore, G. B. Grindey, R. G. Moran. 1986. Proc. Am. Assoc. Cancer Res. 21, Abstr. No. 1027.

105

3)

Taylor, E.C., P.J. Harrington, S.R. Fletcher, G.P. Beardsley, R.G. Moran. 1985. J. Med. Chem. 2fi, 914; E.C. Taylor, G.S.K. Wong. 1989. J. Org. Chem. M , 3618; D.H. Boschelli, S. Webber, J.M. Whiteley, A.L. Oronsky, S.S. Kerwar. 1988. Arch. Biochem. Biophys. 2£5., 43; J.R. Piper, G.S. McCaleb, J.A. Montgomery, R.L. Kisliuk, Y. Gaumont, J. Thorndike, F.M. Sirotnak. 1988. J. Med. Chem. ¿1, 2164.

4)

Taylor, E.C., G.S.K. Wong, S.R. Fletcher, P.J. Harrington, G.P. Beardsley, C.J. Shih. 1986. in: Chemistry and Biology of Pteridines: Pteridines and Folic Acid Derivatives (B.A. Cooper, V.M. Whitehead, eds.) Walter de Gruyter: Berlin, pp 61-64.

5)

Moran, R.G., E.C. Taylor, C. Shih, G.P. Beardsley. 1987. Proc. Amer. Assoc. Cancer. Res. 23., Abstr. No. 1084; C. Shih, G.B. Grindey, P.J. Houghton, J.A. Houghton. 1988. Proc. Amer. Assoc. Cancer. Res. 22., Abstr. No. 1125.

6)

Ramos Tombo, G.M., H.P. Schar, X. Fernandez I Busquets, 0. Ghisalba. 1986. Tetrahedron Letters 22, 5707; 1986. in: Biocatalysis in Organic Media (C. Laane, J. Tramper, M.D. Lilly, eds.) Studies in Organic Chemistry, Vol. 29, Elsevier: Amsterdam, p 43.

7)

The configuration of the stereogenic carbon corresponding to the 2-position of (£.) was deduced by relative comparison with the known center in the carbamate substituent of (S.) . The X-ray analysis was performed by Mr. J. Deeter and Dr. N. Jones, Lilly Research Laboratories.

8)

Pyatin, B.M., R.G. Glushkov. 1968. Khim. Farm. Zh. 2, Chem. Abstr. 1969, 7J1, 28887p.

9)

Shih, C., Lilly Research Laboratories, Eli Lilly and Company, unpublished procedure.

17;

10) The absolute configuration of tetrahydrofolic acid has been determined. J.C. Fontecilla-Camps, C.E. Bugg, C. Temple, J.D. Rose, J.A. Montgomery, R.L. Kisliuk. 1979. J. Am. Chem. Soc. 1 M , 6114. 11) Kaminski, Z.J. 1985. Tetrahedron Letters 2SL, 2901. 12) Shih, C., G.M. Wilson, L.M. Osborne, P.M. Harrington, L.S. Gossett, J.D Snoddy. 1989. in these proceedings. We thank Ms L.M. Osborne and Dr. G.M. Wilson, Lilly Research Laboratories, for performing these analyses.

SYNTHESIS AND BIOLOGICAL EVALUATION OF A FLUORESCENT ANALOGUE OF FOLIC ACID

T.P. McAl inden", J.B. Hynes + , S.A. Patii*, M. Ratnam" and J.H. Freisheim' "Medical College of Ohio, Toledo, OH 43699, USA and 'Medicai South Carolina, Charleston, SC 29425, USA

University

of

Introduction

Fluorescent

analogues

of

methotrexate

(MTX)

have

been

synthesized

and

evaluated as probes of dihydrofolate reductase (DHFR) structure and function (reviewed in 1,2). acid

(FA) was

A fluorescein derivative of the lysine

synthesized

protein (FBP) from human placenta. can

be

reduced

to

both

analogue of folic

as probe for DHFR and a membrane folate

the

binding

This compound offers the advantage that it

corresponding

dihydro-

and

tetrahydrofolate

derivatives and, thus, potentially could serve as a sensitive probe for DHFR, other folate-dependent enzymes and membrane binding proteins.

Results and Discussion

Pteroic

acid

Subsequent

was

prepared

treatment

acetylpteroic

acid,

with which

by

the

carboxypeptidase

trifluoroacetic was

coupled

to

anhydride

G,

cleavage

yielded

of

FA.

10

N -trifluoro-

f

N -tert-butvloxvcarbonvl-L-lvsine

tert butyl ester using diethyl phosphorocyanidate.

The resulting compound was

treated

the

key

intermediate

compound

was

then

with

trifluoroacetic

acid

trifluoroacetylpteroyl)-L-lysine.

to

give

This

N a -(N 10 -

condensed

with

fluorescein isothiocyanate (isomer I) and the resultant material treated with

OH

Fig. 1 Chemistry and Biology of Pteridines 1989 © 1990 Walter d e G r u y t e r A Co., Berlin -New York -Printed in Germany

107

dilute NH40H to yield the desired compound, Na-(pteroyl)-N£(4'-fluoresceinthiocarbamyl)-L-lysine (PLF).

The FAB/MS of this compound was in complete

agreement with the assigned structure (Fig. 1).

The UV absorbance maxima and

millimolar extinction coefficients, in 0.1 M KP0„, pH 7.0, are as follows: A™, 237 (e = 59.6); 277 (e - 44.4); 497 (f - 54.5).

Excitation of PLF at 497

nm gave a fluorescence emission maximum at 517 nm.

Recombinant human (h)DHFR

purified as described by Prendergast et al. (3) was incubated with PLF.

When

the PLF concentration was held constant at 50 nM with incremental additions of hDHFR, the maximum enhancement of the fluorescence emission of PLF (517 nM) was approximately 20-fold, suggesting binding in a hydrophobic environment (Fig. 2).

Incubation of PLF with denatured DHFR showed no

fluorescence

Fig. 2 enhancement.

When a purified membrane-associated FBP from human placenta (4)

was incubated with PLF a similar enhancement of PLF fluorescence occurred. Titration of recombinant hDHFR with PLF by fluorescence emission enhancement (Fig. 3), followed by Scatchard analysis (Fig. 4) indicated a K D = 115 nM

Fig. 3

108

Fig. 4 which agrees quite well with the K D value of 111 nM for FA (5).

These data

suggest that the presence of the fluorescein moiety of PLF vs. FA does not affect binding to DHFR. /¿M.

The K ^

Competition experiments

for PLF vs. hDHFR was determined to be 4.5

with

MTX indicated that the chemotherapeutic

inhibitor could displace PLF from binding to hDHFR in virtually a 1:1 stoichiometry (data not shown). Reduction of PLF by dithionite (6) suggested that complete reduction occurred as measured by the increase in absorbance at 280 nm. PLF) appears to be a good substrate for DHFR.

The reduced compound (H2

Thus, since PLF can be reduced

to its dihydro form, it is likely to be reduced to the H4 form, thus serving as a potential fluorescent probe for a number of one-carbon tetrahydrofolatedependent enzymes and binding proteins.

Acknowledgements This research was supported, in part, by NIH grant number CA41461 (to J.H.F.) and grant number CA25014 (to J.B.H.).

During the course of this research use

was made of the Harold and Helen McMaster Recombinant DNA Laboratory.

References 1.

Freisheim, J.H. and D.A. Matthews. 1984. In: Folate Antagonists as Chemotherapeutic Agents, Vol. 1 (F.M. Sirotnak, J.J. Burchall, W.D. Ensminger and J.A. Montgomery, eds.). Academic Press, Orlando, FL, pp. 69-131.

109 2.

Freisheim, J.H., E.M. Price, A.A. Kumar, S.S. Susten, P.L. Smith and T.J. Delcamp. 1986. Biochem. Soc. Trans. 14, 371-373.

3.

Prendergast, N.J., T.J. Delcamp, P.L. Smith, and J.H. Freisheim. Biochemistry 27, 3664-3671.

1988.

4.

Ratnam, M., H. Marquardt, Biochemistry, (in press).

1989.

5.

Huang, S., T.J. Delcamp, X. Tan, P.L. Smith, N.J. Prendergast and J.H. Freisheim. 1989. Biochemistry 28, 471-478.

6.

Blakley, R.L.

1960.

J.L.

Duhring

and

J.H.

Nature (London) I M , 231-232.

Freisheim.

METAL COMPLEXES OF TETRAHYDRONEOPTERIN

B . Fischer, J . Strähle Institut für Anorganische Chemie der Universität Tübingen, D-7400 Tübingen

Introduction Pterin derivatives have been found in an increasing number of enzymes. Many of them contain a metalpterin complex. For example, Mo-co, the molybdenum cofactor found in at least 10 distinct enzymes, is proposed to consist of a tetrahydropterin, substituted in position 6 by a sulfur containing sidechain, coordinated to the molybdenum atom.(1,2) 0

H N / ^ V

H % - C = C - C - C H

H

N

^

> H

I

X

I

S

S S

/

Mo

l i .

2

O P O ;

OH

\

Figure 1. Proposed structure of the pterin component of a common molybdenum cofactor This cofactor mediates oxygen atom transfer of reaction type 1. 1

Mo(v/'02L„ + X ^

M o t ^ O L , , + XO

(L=Ligand, X=Substrate)

Oxo transfer is accompanied by the formation of a y.-oxo dimer as shown in reaction 2 (5). According to recent results the dimer is always formed, unless it is sterically prevented.

2

O

L„M0VI02

+

L„MoIV0

II L„Mo

O

v

II — O — Mo L„ v

Up to now there are two reports on well characterized metal complexes with oxidized pterins.(3,4) We have found a moIybdenum(V)-complex with tetrahydroneopterin containing the [Mo203] 4 + core.

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & C o . , Berlin • New York • Printed in G e r m a n y

111 Synthesis

To the solution of (6RS)-5,6,7,8-Tetrahydro-D-neopterin- dihydrochlorid (THN) (0.16 g, 0.5 mmol) (6) in dry and deaerated methanol a colorless solution of M0O2CI2 (0.11 g, 0.5 mmol) in methanol was added under nitrogen. The color of the solution changed rapidly to a deep purple. Methanol was removed and the purple, quite stable solid was dried in vacuum.

Results and Discussion

Absorption Spectra

The typical absorption spectrum of THN in methanol shows bands with \ m a x = 220 and 268 nm and a shoulder at about 300 nm. The molybdenum complex in methanol has absorptions at X

m a x

= 221 and

267 nm, therefore the conclusion can be made that tetrahydropterin is still present. Additionally there is an intense visible band at \ m a x = 487 nm, which seems to be typical for the [Mo203] 4+ core (5). We suggest a charge-transfer character for that transition caused by a donor atom. The transmission spectrum of the solid complex, with a very strong and broad absorption at 500 nm, supports this assumption.

Infrared Spectra

The absorptions in the spectrum of THN are broadened, due to the hydrochloride-form. The carbonyl stretch and the modes from C = C and C=N vibrations result in a strong absorption at 1677 c m - 1 and weak ones at 1579 c m - 1 and 1548 c m - 1 respectively. The molybdenum complex shows absorptions in the region of 1550-1700 c m - 1 and additionally a strong new absorption at 1502 c m - 1 due to mixed modes from C = C and C = N vibrations (4). The M02O3 core exhibits another strong band at 980 c m - 1 for the v M o = 0 vibration and a sharp band at 775 c m - 1 assigned to the vas M0OM0 vibration.(6) There is only one absorption found in the FIR region at 330 c m - 1 for the v Mo-Cl vibration, indicating that there is one chlorine atom coordinated to the molybdenum atom.

13

C-NMR Spectra

The

13

C - N M R spectrum of THN in DMSO shows only small shifts with respects to those reported for

THN in NaOD (6).

112 Therefore the shifts in the spectrum of the molybdenum complex are quite significant. The largest shifts occur in the signals of C(4a) from 85.5 ppm to 120.7 ppm and of C(6) from 53.8 ppm to 75.2 ppm. This indicates that molybdenum is coordinated by the oxygen atom attached to the 4-position and the nitrogen atom in the 5-position. The additional signal at 48.5 ppm supports the assumption, that a methanol molecule coordinates to the molybdenum atom.

EPR Spectra The EPR-spectra for the complex in liquid and frozen methanol solutions indicate, that there may be two different EPR-active molybdenum(V) species present. Perhaps this is due to the different conformation of the ligand and therefore different coordination possibilities, or to the different syn or anti conformation of the terminal oxo ligands of the M02O3 core. In good correlation with the proposed structure are the g-values of 1.943 and 1.932 in the spectrum of the molybdenum complex.(8)

Proposed Structures Dimeric molybdenum(V) complexes with the [Mo 2 03] 4+ core are generally characterized by a linear Mo-O-Mo bridge and coplanar, terminal oxoligands (5,7). These complexes are known to occur in the syn- or anti-conformation with a distorted octahedral coordination of the molybdenum atom. Figure 2. and 3. show the proposed structures. HO-CHj HO-C1H HO-CH H «S ..H-

Figure 2. Proposed structure of Mo 2 03Cl2(THN) 2 (CH 3 0H) 2 with syn-conformation of the M02O3 core.

a

T

>

a / I I0 V-^'H H'^ch, H HC-CH HC-CH CHO ,H Figure 3. Proposed structure of Mo 2 0 3 Cl2(THN) 2 (CH30H)2 with anti-conformation of the Mo2C>3 core.

The methanol molecule is in trans position to the terminal oxoligand. Because of the strong trans effect the coordinated oxygen atom of the solvent molecule has only a weak bond to the molybdenum atom. To distinguish between the syn or anti conformation a X-ray structure determination should be per-

113 formed. Using other solvents than methanol there is an additional possibility for the structure as shown in Figure 4.

CH,OH

Figure 4. Proposed structure of Mo203Cl2(THN)2 with syn-conformation of the M02O3 core. The hydroxo group at C(2') of the THN-sidechain could be coordinated to the molybdenum atom, instead of the weak bonded methanol molecule, forming a five-membered ring. This structure can be related to the proposed structure of the molybdenum cofactor.

Acknowledgements

We would like to thank Prof. M. Viscontini for his help in this, for us as inorganic chemists, quite unusual field of the pteridines and his interest in our work.

REFERENCES (1)

Johnson, J.L., Hainlaine, B.E., and Rajagopalan, K.V., J. Biol. Chem., 255, 1783, (1980)

(2)

Johnson, J.L., Hainlaine, B.E., Rajagopalan, K.V., and Arison, B.H., J. Biol. Chem., 259, 5414,(1984)

(3)

Burgmayer, S.J.N., Stiefel, E., J. Am. Chem. Soc., 108, 8310, (1986)

(4)

Burgmayer, S.J.N., Stiefel, E., Inorg. Chem., 27, 4059, (1988)

(5)

Craig, J.A., Harlan, E.W., Snyder, B.S.,Whitener, M.A. and Holm, R.H., Inorg. Chem., 28, 2082,(1989)

(6)

Schircks, B., Bieri, J.H., Viscontini, M., Helv. Chim. Acta, 59, 248, (1976)

(7)

El-Essawi, M.M., Weller, F., Stahl, K., Kersting, M. und Dehnicke, K., Z. anorg. allg. Chem., 542, 175, (1986)

(8)

Cleland, W.E., Barnhart, K.M., Yamanouchi, K., Collison, D., Mabbs, F.E., Ortega, R.B. and Enemark, J.H., Inorg. Chem., 26, 1017, (1987)

SYNTHESES O F LIPOPHILIC ANALOGUES OF

2-DESAMINO-2-CH3-NIO-

PROPARGYL-5,8-DIDEAZAFOLATE TARGETED AT THE ENZYME SYNTHASE

THYMIDYLATE

(TS).

G . M . F . B i s s e t , A . L . J a c k m a n , B. O ' C o n n o r , T . R . J o n e s , A . H . Calvert D r u g D e v e l o p m e n t S e c t i o n , I n s t i t u t e of C a n c e r R e s e a r c h , C o t s w o l d Rd, S u t t o n , S u r r e y SM2 5NG, E n g l a n d .

L.R.

15

Hughes

I.C.I. P h a r m a c e u t i c a l s , M e r e s i d e , A l d e r l e y Park, C h e s h i r e SK10 4TG, E n g l a n d .

Macclesfield,

Introduction

(DHFR)

have

(1) b e c a u s e

they

L i p o p h i l i c i n h i b i t o r s of d i h y d r o f o l a t e r e d u c t a s e b e e n t h e s u b j e c t of e x t e n s i v e i n v e s t i g a t i o n

m a y a f f o r d a s i g n i f i c a n t l y d i f f e r e n t s p e c t r u m of

anti-tumour

a c t i v i t y t o t h a t of m e t h o t r e x a t e . W e r e p o r t h e r e t h e

syntheses

of s o m e l i p o p h i l i c a n a l o g u e s a s p u t a t i v e i n h i b i t o r s o f a n o t h e r important folate target, thymidylate synthase. Previous work in o u r l a b o r a t o r y d e m o n s t r a t e d t h a t t h e l o s s of t h e r e s i d u e from CB 3717

(2-amino-N

10

glutamate

-propargyl-5,8-dideazafolic

acid, 1) , T a b l e 1, to g i v e t h e b e n z o i c a c i d a n a l o g u e (4.) r e s u l t e d in a n 8 5 - f o l d loss of b i n d i n g t o t h e i s o l a t e d TS enzyme

(2). T h e p o o r s o l u b i l i t y of

(4), a n d i n d e e d of CB 3717,

p r o m p t e d u s to w o r k w i t h t h e m o r e s o l u b l e

2-methyl-3,4-dihydro

- 4 - o x o - q u i n a z o l i n e s a f t e r the d i s c o v e r y t h a t the 2 - a m i n o g r o u p w a s n o t a b s o l u t e l y e s s e n t i a l for b i n d i n g t o TS

(3).

Fourteen

new C2-methyl analogues have been synthesised, and the

effects

of c h a n g i n g s u b s t i t u e n t s on the p h e n y l r i n g h a v e b e e n i n v e s t i g a t e d w i t h r e s p e c t to TS a n d cell g r o w t h

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in G e r m a n y

inhibition.

115

Results a n d discussion Anilines

(5) (X = 4-H, F, O C H 3 , C 0 2 E t , C O ^ B u ; 3,4-Cl; 3,4,5-

OMe) w e r e alkylated using propargyl bromide in dry DMF w i t h K 2 C 0 3 as base, Scheme 1. Mono alkylamines

(6)

w e r e separated

away from any dialkylamines formed by column chromatography. Propargylamines

(6) w e r e condensed w i t h 2 - m e t h y l - 6 - b r o m o -

methyl quinazoline -N

10

(2)

(4) in dry DMF / C a C 0 3 to give 2-methyl

- p r o p a r g y l quinazolines

(8) to (14) in good yield. t-Butyl

ester (8) w a s cleaved to the acid (15) by treatment w i t h trifluoroacetic acid. 2 - D e s a m i n o - N 1 0 - p r o p a r g y l b e n z o i c acid (16) w a s obtained by carboxypeptidase G 2 cleavage desamino-N

10

-propargyl-5,8-dideazafolic

acid (2)

(5) of 2 (3).

Scheme 1. CHsBr

DMF / CaCOj

(8—15)

R — propargyl

(17. 18)

R = ethyl

(19-22)

R = methyl

(23. 2+)

R -

hydrogen

© 3,4-Dichloro and 3,4,5-trimethoxy anilines were alkylated u s i n g either methyl iodide or ethyl iodide in DMF / K 2 C 0 3

to

give the corresponding ethyl- (6, R = Et) or m e t h y l a m i n e s

(6,

R = Me). These in turn were condensed w i t h the 6-bromomethyl quinazoline

(7) to give N 1 0 - e t h y l or N 1 0 - m e t h y l

quinazolines

(17) to (20). Coupling of (7) w i t h 4-carboxy or 4-nitro N methyl anilines quinazolines

(6, R = Me, X = C 0 2 H , N 0 2 ) gave N 1 0 - m e t h y l

(21) and (22.) . Because of the p o o r solubility

characteristics of all these compounds, column chromatography w a s n o t possible a n d purification was eventually effected by recrystallisation from a large volume of h o t ethyl acetate

116

( a p p r o x i m a t e l y 800 m i s / g m ) . D i r e c t c o u p l i n g o f or 3,4,5-trimethoxy anilines with

3,4-dichloro

(7) g a v e p r o d u c t s

w h i c h w e r e t o o i n s o l u b l e t o p u r i f y o r t e s t for

(23) f (24)

inhibition.

T a b l e I. I n h i b i t i o n o f T h y m i d y l a t e S y n t h a s e a n d o f L 1 2 1 0

Cell

Growth

TS I

Growth

Inhibition

Inhibition

IC50

IC50

(HM)

(KM)

1

NH 2

2

H

CH2C•CH

4-COGlu

0.16

0.4

3

CH3

CH2C.CH

4-COGlu

0.04

0.085

4

NH 2

CH2C•CH

4"C02H t

CH2C.CH

4-COGlu

8

CH3

CH2C.CH

4-C02 Bu

9

CH3

CH2C•CH

4-C02Et

10

CH3

CH2C.CH

11

CH3

12

CH3

0.02

1.7

3.4

>200

24

5

1.6

'100

4-H

>3.8

>100

CH2 C•CH

4-F

>3.8

>100

CH2C•CH

4-OMe

>3.8

>100 >

13

CH3

CH2 C•CH

3,4-Cl

>3.8

14

CH3

CH2 C•CH

3,4,5-OMe

17.3

25

15

CH3

CH2C.CH

4-C02H

2.7

>100

16

H

CH2C.CH

4-C02H

7.9

56

17

CH3

CH2CH3

3,4-Cl

11.8

" 10

" 4.3

19

CH3

CH3

3,4-Cl

18

CH3

CH2CH2

3,4,5-OMe

20

CH3

CH3

3,4,5-OMe

21

CH3

CH3

4-C02H

22

CH3

CH3

4-N02

25

NH 2

CH2C.CH

4-N02

15

50

7.5 28

"66.7

30

8.9

>100

20.7 0.62

8.8 2.4

117

Modifications on the phenyl ring failed to produce any significantly active TS inhibitors, although the C2-methyl ethyl ester (9) was equipotent to our original C2-amino lipophilic lead (4). 3,4-Dichloro and 3,4,5-trimethoxy substitutions were clearly disfavoured. 4-Nitro N 1 0 - m e t h y l quinazoline

(22.) was a surprisingly worse TS inhibitor by

comparison w i t h the 4 - N 0 2 compound (25) from Warner Lambert (6). TS activity dropped off considerably with the introduction of the sterically bulky t-butyl ester group in (8). Compounds possessing the more lipophilic substituents, e.g. 3,4-dichloro, 3,4,5-trimethoxy and 4 - C 0 2 t B u , were generally more active against growth of L1210 cells in tissue culture although none were significantly active. Removal of the glutamate residue from either (3.) or (2) resulted in a 68 fold or 49 fold decrease in TS activity respectively,

confirming

glutamate is important for good enzyme binding. Replacing the 2-amino group in (4) with methyl did not give the substantial increase in cytotoxicity observed for the parent folates. Overall, the N 1 0 - p r o p a r g y l group remained the b e s t for retention of TS activity. References 1. Werbel, L.M. 1984. In: Folate Antagonists as Therapeutic Agents, (F.M. Sirotnak, J.J. Burchall, W.B.Ensminger, J.A. Montgomery, eds.). Academic Press, Inc., Volume 1, p. 261. 2. Jones, T.R., M.J. Smithers, R.F. Betteridge, M.A. Taylor, A.L. Jackman, A.H. Calvert, L.C. Davies, K.R. Harrap. 1986. J. Med. Chem. 29, 1114-1118. 3. Hughes, L.R., P.R. Marsham, J. Oldfield, T.R. Jones, B.M. O'Connor, J.A.M. Bishop, A.H. Calvert, A.L. Jackman. 1988. Proc. Amer. Assoc. Cancer Res. 29, 286. 4. Hughes, L.R. 1986.

U.K. Patent 8607683.

5. Sherwood, R.F., R.G. Melton, S.M. Alwan, P. Hughes. Eur. J. Biochem. 148. 447-453.

1985.

6. Fry, D.W., D.J. McNamara, L.M. Werbel, E.M. Berman. 1989. Proc. Amer. Assoc. Cancer Res. .30, 479.

STUDIES IN MODELS OF FOLATE COFACTORS: OXIDATION OF 5-METHYLTETRAHYDROPTERIN REVISITED.

E. Hilhorst, T.B.R.A. Chen and U.K.Pandit Organic Chemistry Laboratory, University of Amsterdam, Nieuwe Achtergracht 129,1018 WS Amsterdam, The Netherlands

Introduction

The biosynthesis of methionine involves the overall transfer of the methyl group of 5-CH3-H4 folate to homocysteine, resulting in the formation of methionine and H 4 folate (Scheme I).

Scheme I The reaction is catalyzed by two different classes of methionine synthases, viz. those which are cobalamine-dependent and those which do not require cobalamine for the operation of their function (1). The mechanistic details of the methionine synthase reaction are not yet fully elucidated. In the case of the cobalamine-dependent enzyme, an oxidative activation of the 5-CH 3 -H 4 folate appears to be necessary for the formation of a methylcobalamine ( B 1 2-CH3) intermediate. In this context it has been recently suggested that copper, found in the homogeneous enzyme from E.coli B, may play the role of a redox system (2). As a part of our current interest in mechanistic investigations with models of tetrahydrofolate cofactors (3), we have undertaken a systematic study of the oxidation of the model system 5,6,7-trimethyl5,6,7,8-tetrahydropterin (la), in order to develop a closer insight into the molecular events during the methionine synthase reaction.

Results Oxidation of 1a by oxygen Initial reports (4) on the oxidation of l b by oxygen , claimed that the main oxidation product was the corresponding 4a-hydroxy derivative 2. (Scheme II). However, it was later shown by Jongejan et al. (5)

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in G e r m a n y

119 that 1 b underwent a complex course of reactions under oxidative conditions and led to products in which the pyrazlno-triazine derivative a , resulting from a skeletal rearrangement, was predominant. A second type of rearrangement products corresponded to the spiro systems 4a.b.

lb H

H

1a. R = C H a 1b. R = Ph.

Scheme II

4a RI = H, R 2 = CH 3 4b RI = CH3, R 2 = H

W e have studied the oxidation of l a . 2 H C I . H 2 0 in buffered solutions at room temperature (~293 K), in the range pH 2 - 1 3 . The reactions were monitored by H P L C till the substrate had been consumed (3 days). At low p H ' s (2.9 and 4.5) no reaction was observed. At pH's above 7 the main oxidation product ( ~ 8 0 % )

w a s pyrazino-triazine 5 ( Scheme III). Detectible amounts of the demethylated

pterins fiand Z were also formed. The fully aromatic s y s t e m ^ is obviously a further oxidation product of

The latter compound is presumably formed by oxidation of the methyl group, followed

by the loss of formaldehyde. The formation of 5 . from l a , for which a mechanism has been proposed (5), Is In accordance with the results reported by Jongejan and coworkers (6). While no demethylation products have been reported during the oxidation of 1a. Mager (7) has shown that 1,3,5-trimethyl-tetrahydrolumazines do undergo oxidative demethylation, involving loss of formaldehyde. Oxidation of 1a by hydrogen peroxide The results of oxidation of l a - 2 H C I . H 2 0 b y H 2 0 2 a t pH's above 7, were similar to the ones observed for the aerobic reaction. Once again, compound 5. was the predominant product, while the formation of small amounts of £ and Z could be demonstrated.

120 O

CM,

o

o +

+

o ot

i

1

i

1

i

1 Scheme III

AtlowpH's (~2) oxidation of la.2HCI.H 2 0 by H 2 O 2 ledt0 mixtures of 5 and Z . containing substantial amounts of the demethylated product Z ; the ratio of 5 to Z attaining a value of 1.5:1. That the methyl group was lost as formaldehyde, following an oxidation step, was attested by trapping HCHO as its dimedone derivative. Oxidation of 1a bv Cuf2+Vions It was demonstrated in orientation experiments that la.2HCI.H 2 0 underwent oxidative demethylation in heterogeneous aqueous systems under Influence of cupric salte. For a systematic study, the Cu(2+) complex £ ( Scheme IV) was synthesized and employed for oxidation of la-2HCI.H 2 0 in acetonitrile solutions. When two equivalents of the complex were employed , the tetrahydropterin la.2HCI.H 2 0 underwent a rapid reaction during which the reduction of Cu(2+) could be observed by the change of colour of the reaction mixture. Analysis of the mixture showed it to consist mainly of £ , accompanied by small amounts of Z • It is noteworthy that no triazinefi was observed in the oxidation products. When three equivalents of & were used in an analogous experiment, the reaction mixture was found, as anticipated, to contain a significantly higher proportion of compound ZThe oxidative demethylation of l a can, in principle, follow two pathways : (a) a direct transfer of the methyl group , from a species such as_2or_LQ, to an appropriate nucleophile, or (b) loss of formaldehyde from an intermediate of type H (Scheme IV). The operation of pathway (b) was demonstrated by the qualitative identification of formaldehyde (as its dimedone derivative ), during the demethylation reaction. In view of the lack of quantitative data it is not clear at this stage whether pathway (a) contributes to any extent to the demethylation reaction . The search for "methyl transfer" from 2. or 1Q. or yet another intermediate, in the Cu(2+) mediated oxidation of la, is currently in progress. The difference between the oxygen and the hydrogen peroxide oxidations on one hand and that mediated by Cu(2+) -complex on the other, is highly significant in that no pyrazino-triazine 5. is formed during the oxidation with the Cu(2+)-complex &. Coupled with the fact that the pyrazino-triazine derivative of 5-CH3-H4 folate is biologically inactive, it follows that studies of oxidation of 5-methyltetrahydropterins by Cu(2+)-complexes may be of relevance to the understanding of the biological oxidative demethylation of 5-CH3-H4 folate.

121 [Cu (2,9 - dimethyl - phenanthrollne)2 H 2 0 ] 2 * ( CI0 4 ' ) 2 fi

Scheme IV References 1. Matthews, R.G. .1984. In: Folates and Pterins, vol I ( R.L. Blakley and S.J. Benkovic, eds.). Wiley New York, p. 497. 2. Matthews, R.G., D.A. Jencks , V. Frasca, K.D. Matthews. 1986. In: Chemistry and Biology of Pterldines (R.L. Blakley and S.J. Benkovic, eds. ). Walter de Gruyter en co. Berlin , p.697. 3. Pandit, U.K. .1988. Recl.Trav. Pays-Bas 107 ,111. 4. Viscontini, M., T. Okada. 1967 . Helv. Chim. Acta 5Q., 1492. 5. Jongejan , J.A., H.I.X. Mager, W. Berends. 1975. Tetrahedron 2 1 , 534. 6. Jongejan, J.A., H.I.X. Mager, W. Berends . 1979 . In: Chemistry and Biology of Pteridlnes ( R.L. Kisliuk and G.M. Brown, eds.). Elsevier North Holland, p.241. 7. Mager, H.I.X., W.Berends. 1976 . Tetrahedron 3 2 , 2303.

NEW

STRATEGY

B. Z a g a l a k ,

IN T H E

F.

SYNTHESIS

OF NEOPTERIN

PHOSPHATES

Neuheiser

D i v i s i o n of C l i n i c a l C h e m i s t r y , D e p a r t m e n t of P e d i a t r i c s , U n i v e r s i t y of Z u r i c h , S t e i n w i e s s t r a s s e 7 5 , C H - 8 0 3 2 - Z u r i c h , Swi t z e r l a n d

Introduction

D-7,8-Dihydroneopterin-3'-triphosphate cial

substrate

therefore

biochemical from

not

This

biosynthesis

chemical

interest.

guanosine-5

recently are

in the

a simple

1

suitable

is d u e

to

(epimerase)

7,8-H,,-NTP

the

for

the

can

presence

of C - 3 ' - p h o s p h a t e s be

prepared

preparations

kinetic of GTP

or

and

of

invariably

ficult

to

led

separate

to m i x t u r e s

of

we

7,8-H,,-NTP,

enzymatically

of p h o s p h a t e s

is

I. A s

spectroscopic

L-7,8-H2-monapterin-3'-triphosphate.

theses

cru-

enzymatical1y

and G T P - c y c l o h y d r o l a s e

enzymatic

precise

is a

L-tetrahydrobiopterin,

synthesis

-triphosphate

observed,

of

(7,8-H2-NTP)

study. formed

Previous which

are

syndif-

(1,2).

Results

We

reinvestigated

using

modern

observed,

and

that

results

phates.

The

classical

spectroscopic

of u n p r o t e c t e d phosphoryl

the

the

in the

chromatographic

phosphorylation

D-neopterin

chloride

and

phosphorylation

with

in t r i e t h y l formation

phosphorylation

of with

of

primary

primary

(3)

and

P0C1, gave:

D-neopterin

techniques hydroxyl

polyphosphoric phosphate

of

acid

secondary 74.6%

Chemistry and Biology o f Pteridines 1989 © 1990 Walter de G r u y t e r & C o . , Berlin • N e w York • Printed in G e r m a n y

group

(1,2)

is n o t

of

and

or

specific monophosC-3',

123

M-H-ft H C^ I H-C-OH I H-C-OH I H-C-OH B I M CHjO-P-OH H\

OH

OH

OH

0

C — C —CH»0-P-0H I I I H OH 1. HCOOH 2. Et 3 N 3. 1,1' -Carbontjldlimldazole in DMF

C — CH20-P-N

N

Ov

H,N

Et.N® 1. [ H 3 P 2 0 7 • B U 3 N H ]

in

DMF

2. pH 8 3. DEAE-Sephadex A - 2 5 , LiCl

OH

H,N

OH

O

O

O

C — C —CH»0-P-0-P-0-P-0LI I I ' i l l H H OLI OLI OLI 1. PdO : Hg Cor sodium h y p o s u l f i t e ) 2. DEAE-Sephacel, LiCl

OH OH 0 0 0 ll^ i i ii ii n "^-c—c—CH2O-P-O-P-O-P-OLI I I ' i l l H H OLI OLI OLI H

Fig.

1 Chemical s y n t h e s i s of D - 7 , 8 - d i h y d r o n e o p t e r i n - 3 ' - t r i phosphate from p h e n y l h y d r a s o n e of D - r i b o s e - 5 - m o n o p h o s p h a t e and 2 , 5 , 6 - t r i a m i n o - 4 - o x o - d i h y d r o p y r i m i d i n e .

124 23.6%

C-2 1 and

of

We a l s o is n o t

observed

a) A T P

1

that

b) A T P

+ GMP the

-triphosphate

kinase

(EC

two (EC

are

notoriously

difficult

we

primary

and and

investigated

4-oxo-dihydropyrimidine 5-monophosphate

sults

(Ba

under

(after

SP-1080

H

+

acidic

of

(Ba

1

3 - NT P w a s

form).

salt)

phosphorylation

Similar

low y i e l d

date

tective

ceeds

the

groups

with

found

the

that

chain the

a good yield

equivalents

of

methoxypropane.

ion

of

D-ribose-

before

the

of

result

of

re-

3'-NMP Lewa-

2,5,6-triaminoof

and

D-ri bose-5-trino t r a c e

Unfortunately

of the

1

1 ,1 -carbonyl-

n-tributylammonium Probably

con-

and

exchanger

this

salt

is d u e

(4) to

end

the f a s t

1

D-neopterin-3 -monophosphoimihydroxyl

cyclization have of

tried the

group(s) of

neopterin

samples acid

that another

the

to i n s e r t s e v e r a l

1

the

of

3'-NMP-imidazoli-

1'-2 -ketalization

when

prepara-

2,5,6-triamino-

in the f o r m a t i o n on

which

phenylhydrazone

purified

3 -NMP using

p-toluenesulfonic We f o u n d

of

1

of f o r m e d

we

in

derivatives

condensation

neighbouring the

of

phenylhydrazone

3-TNP.

groups

into

separate

is C - 6 - r e g i o s p e c i f i c

a negative

to a v o i d

hydroxyl

a n d we h a v e

of

reaction

In o r d e r

with

with

pyrophosphate

with

salt,

D-neopterin

monophosphates,

to

purified

of u n p r o t e c t e d

up

chain.

kinase

kinase.

unprotected

chromatographica11y.

and

dazolidate

of

condensation

NaNC^)

(twice

gave

diimidazol

cyclization

or

systems:

+ PEP + PEP

+ PEP

hydrazine

The

with

18%

observed

in v e r y

enzymatic

condensation

condition

4-oxo-dihydropyrimidine phosphate

+ PEP

tedious

the

with

salt).

oxidation

in overall y i e l d tit

2.7.4.4)

secodary

D-ribose-5-monophosphate.(Ba

densation)

different

phosphorylation

of

3

by

2.7.4.8)

to m i x t u r e s

of

to D - n e o p t e r i n - 3 - d i p h o s p h a t e

or,

classical

scale,

(3-NMP)

1

further

led

tive

(NMP).

D-neopterin-3 -monophosphate

+ nucleoside-P1-kinase

2.7.1 .40)

Since

of C - T - m o n o p h o s p h a t e 1

phosphorylated

D-neopterin-3

(EC

11.8%

were

monophosphate of 3 ' - N M P refluxed

in e x c e s s

suitable

0-pro-

of

prowith

2,2-di-

protective group

125 like

the

0°-10°c

1',2'

ethoxyethylether

using v i n y l e t h y l e t h e r

p-toluenesulfonic can

easily

gave

(5).

mild

and

the

chance

at

triphosphate

avoiding

is p r e s e n t e d

the

in F i g u r e

1. T h e

neopterin-3 -triphosphate active and

in the

in

triphosphate

the

and

was

enzymatic

found

formic

diesters

RT,

2 h

yield

10-18%.

Our

synthesis

epimerization

to

can

be

of

3-NMP room

cleaved

observation

The

to

synthesis

D-7,8-dihydro-

preparations

of

at of

at

of 3 - N M P

reaction.

overall

that

acid

. This

phosphorylation

cyclization

1

pterin

mono-

be p r e p a r e d

3 equivalents

have

anhydrous

pH 8 . 5 - 9 . 0 ,

of f u r t h e r

can

and

F i n a l l y , we

produced

condition

us the

fully

(solvent)

be 0 - f o r m y 1 a ted w i t h

temperature under

acid

of 3 ' - N M P

were

L-tetrahydrobio-

7,8-dihydromonapterin-3'-

(6,7).

Acknowledgment This work was supported by the Swiss National Science Foundation, project no. 3.159-0.88

References

1„

Sugiura, Jap. 45,

K . , H. Y a m a s h i t a , 3564.

2. V i s c o n t i n i ,

M.,

Y. F u r u t a .

3. Y o s h i k a w a , M . , T. Letters, 5065.

Kato,

4. H o a r d ,

Ott.

D.E.,

5. K o z a r i c h , 6o

D.G.

J.W.

et a l .

Z a g a l a k , B. et a l . 1 5 2 , 1 1 93 .

7. Z a g a l a k ,

B.

1988.

M. G o t o .

1972.

1973. Helv.

T. T a k e n i s h i .

1 965.

J. A m e r .

Bull.

Chim.

Acta

Biol.

Biochem.

Chem.

56,

Soc.

1891.

1967.

Tetrahedron

Chem.

S o c . 87^, 1 7 8 5 0

1 9 7 5 . B i o c h e m i s t r y J_4,

1988.

Chem.

981.

Biophys.

Res.

Commun.

Hoppe-Seyler

369,

535.

F I R S T R E G I O S P E C I F I C S Y N T H E S I S O F 7-I S O - D - N E O P T E R I N 7-IS0-D-NE0PTERIN-3'-M0N0PH0SPHATE

B. Z a g a l a k ,

F.

Neuheiser

D i v i s i o n of C l i n i c a l C h e m i s t r y , D e p a r t m e n t U n i v e r s i t y of Z u r i c h , S t e i n w i e s s t r a s s e 7 5 ,

H.-J.

AND

of P e d i a t r i c s , CH-8032-Zurich

Borschberg

O r g a n i c C h e m i s t r y L a b o r a t o r y , F e d e r a l I n s t i t u t e of U n i v e r s i t a t s t r a s s e 16, C H - 8 0 9 2 - Z u r i c h , S w i t z e r l a n d

Technology,

Introduction

Very

recently,

small

amounts

cantly

higher

In t h i s

which

are

two

to

?. F o r an

reference

formed known

via

investigation such

of

syntheses

substituted tedious sation

to of

D-ribose

invariably

pterins

(2,3)

separate.

led

which

to

Therefore,

we

in

signifi-

patients a) w h a t

(1).

are

the ?,

pathways

L-tetrahydrobiopterin

controlled

C-6/C-7-isomer-

problems

7-substituted are

to m i x t u r e s

are

in

these metabolites

7-iso-D-dihydroneopterin-31-triphosphate, previous

and

arise:

of t h e s e as

detected

biosynthetic

route

enzymatica11y

compounds,

been

subjects

questions

configurations

the

have

in p h e n y l k e t o n u r i c

important

7-iso-pterins parallel

pterins

of h e a l t h y

concentrations

a subsequent,

ization pure

urine

and absolute

the

or via

in the

context

relative b) a r e

C-7-substituted

pterins

needed. of

notoriously

2,5,6-triamino-4-oxo-dihydropyrimidine

Chemistry and Biology o f Pteridines 1989 © 1990 Walter de G r u y t e r & C o . , Berlin • N e w York • Printed in G e r m a n y

and

However,

C-6- and difficult

reinvestigated

derivatives.

isomerically

C-7and

the

conden-

with

various

127 Results

In the c o u r s e synthesis

of

of

our

study

7-iso-pterins

"¿tnhi

directed

towards

we

that

^

found

0 © r V " '

^

HN'JSjj--ii">

0 Tvc

the

a

regiospecific undesired

forma-

rix"'

NLCi.aa^Hn.ic titc

« EX (J

• IV

- NH j C - OM

?

I© M'C -Oi

0 I 1

n

H'^M

" lQl-H-/

"i"

HH'

°

I

' ]

C-OM

M

C«0 VJ (•'•HI

III

C-7 SUBSTITUTED PTERINS

C r 4 SUBSTITUTED PTERINS - C — C - CHjO -

OH OH

Figure.

,

P r o p o s e d m e c h a n i s m of the f o r m a t i o n C-7-substituted pterins.

of

C-6-

and

128 tion

of 6 - s u b s t i t u t e d

employed

by e a r l i e r

isomers

workers

of D - r i b o s e - 5 - p h o s p h a t e densed (see The

with

less

structure by

(4-7).

(_I_V) w a s

to an e x c e s s

Indeed,

purified

of

if the

of

(VJ

than

the

0.2%

of

isolated

(H_I)

was

formed

product

was

shown

hydrazine,

hydrazone

before

it w a s

2,5,6-triamino-4-oxo-dihydropyrimidine

Figure),

sented

is d u e

con-

a t pH

2.3

(8). to be

7-iso-D-neopterin-3'-monophosphate

repre-

on t h e

basis

i of

its

H-NMR

hydrolyzed T a b l e . ^"H-NMR

spectrum

(see T a b l e )

and

by a l k a l i n e

phosphatase

to

spectral

(chemical

cpd.

solvent

A

D2O

B

D2O

data

shifts

H-C(6) -

IN

DC1

D

IN

DC1

-

of

some

6- and

H- C ( 2 ' )

h

A

-C(31)

4 .20

c a . 4.1

c a . 4.1

ca . 4

ca. 4

8.79

4 . 86

3 .95

3 .66

3.55

6.6

-

4 .81

3 .90

3 .64

3 . 54

6.8

of

6-D-neopterin-3'-monophosphate

of

7-iso-D-neopterin-31-monophosphate

C:

6-D-neopterin

D:

7-iso-D-neopterin

(V_U

to

(III), when (9).

it w a s

regioselectivities

and

7.8

(III) (V)

(^presumably (H.) w h i c h

eventually

findings, only

explanation

rearrangement

takes

for

is p r e s e n t e d first

furnishes

hydrazone

the

to

the

(III);

and

thus

the

in the

rearranges the

corresponding

these

to

relevant on

the

(j_V)is a t t a c k e d

place

to

6-D-neopterin-3'-mono-

subjected

represents

corresponding

(Na10^/KMnO^)

acid.

(J) f u r n i s h e d

mechanistic

11)

degraded

the a b o v e

conditions

noketone

was

carboxylic

A possible

hydrazone

7.4

(VI)

a confirmation,

gent

J(1•/2 • )

ca . 4

salt

phosphate

3

HB-C(3-)

4 .82

salt

phenylhydrazone

pterins,

5 .01

Disodium

the c o n t r a s t

(VI).

-

Disodium

In

is

8.84

8.64

7-pteryl

(V)

Hz)

A:

known

that

7-subst i tuted

in

B:

As

fact

7-iso-neopterin

constants

H-•C(L • )

H-C(7)

8 . 36

C

( 300 MHz)

i n pptn; c o u p l i n g

the

leeds

same

remerkably Figure the

diver-

: phenyl -

phenylhydrazi-

electrophile other

before to

reaction

(V^)

hand,

an

(10,

the

analogous

instead.

129 Acknowledgment This work was supported by the Swiss National Science Foundation, project no. 3.159-0.88

References

1. C u r t i u s , H . - C b . , T. K ü s t e r , A . M a t a s o v i c , N. B l a u , J . - L . Dhont. 1988. B i o c h e m . Biophys. Res. Commun. 153, 715. 2. K a r r e r , P 0 , Chim. Acta

R. S c h w y z e r , 1031.

B.

Erden, A.

3 . P a t t e r s o n , E . L . , R. M i l s t r e y , Chem. Soc. 80, 2018. 4. F o r r e s t ,

H.S., J. Walkers.

5. F o r r e s t , 4865.

H.S.,

6. V i s c o n t i n i ,

M.,

H.K.

7. R e m b o l d ,

H., L. B u s c h m a n n .

8.

B.

Zagalak,

1988.

Biol.

Stokstad.

1949. J. Chem.

Mitchell.

H. R a s c h i g .

E.L.R.

Siegwart.

1955. J. Am.

1958. 1963.

Chem.

Helv. Chem.

1947.

1958.

Soc.,

Soc.

Chim. Acta

Hoppe-Seyler

369,

J.

Am.

79.

Chem.

Ber. 96,

Helv.

41^,

77,

108.

1406. 535.

9 . Z a g a l a k , B . , F. N e u h e i s e r , R. B o s s h a r d , U . R e d w e i k . 1 9 8 7 . In: U n c o n j u g a t e d P t e r i n s a n d R e l a t e d B i o g e n i c A m i n e s ( H . - C h . C u r t i u s , N. B l a u , E d s . ) . W a l t e r de G r u y t e r , B e r l i n , p. 13. 10. P f l e i d e r e r , W . 1 9 8 3 . In: B i o c h e m i c a l a n d C l i n i c a l of P t e r i d i n e s . W a l t e r de G r u y t e r ( H . - C h . C u r t i u s , d e r e r , H. W ä c h t e r , E d s . ) . B e r l i n , V o l . 2, p . 3 .

Aspects W. Pflei-

11. V i s c o n t i n i , M . 1 9 8 4 . In: B i o c h e m i c a l a n d C l i n i c a l A s p e c t s of P t e r i d i n e s . W a l t e r de G r u y t e r (H. W ä c h t e r , H . - C h . C u r t i u s W . P f l e i d e r e r , E d s . ) . B e r l i n , V o i . 3, p. 19.

II ANALYSIS OF PTERINS AND FOLATES

DETECTION OF PTERINS IN BIOLOGICAL MATERIAL USING MASS SPECTROMETRY

Th. Küster, H.-Ch. Curtius Div. of Clin. Chem., Department of Pediatrics, University of Zurich Switzerland W. J. Richter, R. Dahinden, F. Raschdorf Central Research Division, Ciba-Geigy, CH- Switzerland

Introduction Over the past years, the interest on pterins in various fields of resarch has continously increased. On the other hand however, the methods of analysis remain more or less restricted to well established

techniques such as radioimmunoassays

and, most

widely

applied reversed phase HPLC with either fluorometric or electrochemical detection. Whereas all these methods are very sensitive they have the inherent lack of selectivity which can be circumvented by mass spectrometry. The following examples may show the application of some mass spectrometric techniques in the field of pterins.

Results 1. Characterization of 7-substituted pterins 1987, Dhondt (1) reported on a patient with mild hyperphenylalaninemia whose neopterin to biopterin ratio was elevated by a factor of ten. BH^-loading resulted in a significant decrease of the blood phenylalanine level. Further, he observed an unknown peak in urine besides biopterin. After purification by SEPAK and HPLC (2) we derivatized a fraction of the patient's urine to yield

the

trimethylsilyl derivatives and analyzed the sample by mass spectrometry .

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin New York Printed in G e r m a n y

134

Figure 1 shows the total ion current chromatogram of that fraction.Besides 6-biopterin two unknown peaks appear. The mass spectrum of peak 1 is very similar to that of biopterin but

time

was

about 30 sec shorter and

the

retention

did

not match any of the hitherto investigated

references.

The massspectrometric results may be summarized as follows: Electron impact yields a tentative

molecular

weight

of

525 and mass 409 typical for rearrangement

a

process

served in all aromatic

obpte-

rins bearing a OR-function in position 2 of the side chain.

Figure 1: Total ion current chromatogram of the patient's urine fraction Chemical ionization with methane under positive and negative ionization conditions showed the MH+ -ion at m/z 526 and the M~- ion at m/z 525 respectively. The retention times in methylene units were

23.95

for

the

unknown

and

24.88

for

the

reference

6-biopterin. The available data concerning position isomers all show a shorter retention

time for

the

7-substituted

isomer

and

support the

assumption that the unknown has a 7-biopterin structure. The analysis of

the reference compound

7-biopterin

showed

an

identical behaviour to the unknown peak under all aspects of analysis. Figure 2 shows a mass spectral library search of the unknown

against

entries.

The

7-biopterin.

a best

"home—built"

data set

containing

around 120

fit with a practically identical spectrum is

135

leee simf

409

J ^ C21.H43.03.HS.S14

525 , 1'

7-BIOPTERIH and A,- on scheme 1.

138

m / z 238

Figure 4 : FAB-MSMS spectra of m/z 238 from the intermediate, sepiapterin and biopterin The

sepiapterin

tautomer

A^

can

be

ruled

out

because

its

chromophore is incompatible with the observed UV-adsorption. A^ on the other hand which is "UV-compatible" is very unlikely since its FAB-MS behaviour contrasts sharply that of the quinoid-Bh^ reference which

rapidly

takes up two hydrogen atoms prior or

during analysis, whereas in the case of the intermediate no such H-uptake was observed. Therefore,

the PPH^-structure A^ which has

NMR-evidence spectrometry.

by

Ghisla

(4)

is

fully

been

derived

supported

by

from mass

139 References 1. D h o n d t , J . L . , P. G u i b a u d , M . O . R o l l a n d , C . D o r c h e , S . A n d r e , G . F o r z y a n d J . M . H a y t e . 1 9 8 7 . E u r . J. P e d i a t r . 1 4 7 , 1 5 3 . 2. K ü s t e r , T h . a n d A . N i e d e r w l e s e r . 1 9 8 3 . J. C h r o m a t o g r . 2 7 8 ,

245.

3. R i c h t e r , W . J . , F . R a s c h d o r f , R. D a h l n d e n , A . N i e d e r w i e s e r , H . C h . C u r t i u s , W . L e l m b a c h e r a n d T h . K ü s t e r . 1 9 8 7 . In: U n c o n j u g a ted Pterins and related biogenic amines (H.-Ch. Curtius, N . B l a u a n d R . A . L e v i n e , e d s . ) . W a l t e r d e s G r u y t e r , B e r l i n , p . 39. 4. G h i s l a , S . , P. S t e i n e r s t a u c h , C u r t i u s , i b i d . p. 6 7 .

Th.

Hasler,

N.

Blau

and

H.-Ch.

MASS SPECTROMETRIC METHODS FOR THE STRUCTURAL DETERMINATION OF PTERIDINES AND FOLIC ACID DERIVATIVES

M. Przybylski Fakultät für Chemie, Universität Konstanz, D-7750 Konstanz, FRG

Introduction

The relatively mass

high thermal

spectrometry

(MS),

stability of pteridines

in

principle,

a

renders

sensitive

and

selective analytical tool both for the identification of biological metabolites and the characterization of new synthetic derivatives.

A recent literature survey shows that a variety

of MS methods have been applied unconjugated conjugates GC-MS

pteridines

(Table 1).

have

been

to

to structures

highly

polar

applied

to

from

folate-glutamyl

Gas-phase ionization MS

extensively

ranging

(EI, CI) and

the

study

of

pteridines (1, 2) but often failed in the analysis of pteroyl and folate derivatives

(3).

Studies of the latter compounds

have mainly become amenable by the development

of

condensed

phase ionization/desorption methods, such as FAB-MS and FD-MS (3,

4)

which

determination emphasis

of

provide without

this

molecular limitations

summary

is

on

weight of

and

structure

volatility.

applications

of

The direct

ionization MS methods which - especially when combined with liquid chromatographic

techniques - have much contributed

to

structure determinations of complex and high molecular weight pteridine and pteroyl derivatives.

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in Germany

141 Table 1. Ionization and chemical derivatization methods employed in the mass spectrometric analysis of pteridine and folic acid derivatives.

Pteridlne derivative

Ionization®

Derivatization

unconjugated pteridines

EI EI, CI EI FAB

+ permethylation + trimethylsilylation + ++

EI EI CI FD FAB LD

methyl ester permethylation

pteroic acid derivatives , pteroylglutamates

M

Fragments

Ref.

(+) (+)

1.5 1.6

2.

+c

(+)

8

(+) (+)

6

2, 9 3, 4, 10, 11 4, 12-15

(+) ++ + ++ tetrahydrofolate). Some c h r o m a t o g r a p h i c s y s t e m s , moreover, f a i l to e f f e c t complete separation of a l l f o l a t e s , t h e r e b y n e c e s s i t a t i n g t h e use of m u l t i p l e columns. To o b v i a t e these d i f f i c u l t i e s , we are attempting to develop an a l t e r n a t e p r o c e d u r e in which the f o l y l p o l y g l u t a m a t e s a r e converted at neutral pH ( v i a the r e a d i l y a c c e s s i b l e , highly p u r i f i e d c a r b o x y p e p t i d a s e G2 (CPG 2 )) t o the corresponding pteroates which, in turn, can be f u l l y resolved by HPLC. F i g u r e 1 i l l u s t r a t e s t h e p r o c e d u r e u s i n g a m i x t u r e of standard f o l a t e compounds as a model system. Resolution of a representative pterin (6-hydroxymethylpterin), 3 f o l a t e compounds ( f o l a t e , 5 - m e t h y l t e t r a h y d r o f o l a t e , and 5 - f o r m y l t e t r a h y d r o f o l a t e ) and a folylpolyglutamate (pteroylpenta—y-L-

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in Germany

193

Folate 24.5% 9.9%

22.4% .. 5-CHj-FH* 19% 5-CHO-FH, 23.1%

100

Retention Time (min)

F i g . 1. HPLC o f f o l a t e compounds b e f o r e and a f t e r treatment with CPG2. Panel I . The mixture contained i n 56 nL: 6-hydroxymethylpterin (6-CH2OH-Pterin), 10.9 nmoles; 5-formyltetrah y d r o f o l a t e (5-CHO-FH4), 9 . 5 n m o l e s ; f o l a t e , 11.4 nmoles; 5-methyltetrahydrofolate (5-CH3-FH4), 9 . 7 n m o l e s ; and p t e r o y l pentaglutamate ( f o l a t e + 4G), 10.8 nmoles. Panel I I . The same m i x t u r e was t r e a t e d w i t h 0 . 2 9 mg o f CPG2 i n 56 /iL o f 100 mM T r i s / 0 . 2 mM ZnS0 4 , pH 7 . 3 . A f t e r 30 m i n a t 3 7 ° , excess p r o t e i n was removed by h e a t i n g ( 1 0 0 ° , 3 min) and c e n t r i f u g a t i o n . The s o l u t i o n s ( 5 6 nL) w e r e a n a l y z e d by HPLC ( U l t r a sphere ODS C 1 8 column; UV d e t e c t i o n a t 280 nm u s i n g a Beckman M o d e l 166 d e t e c t o r and a Model 427 integrator. M o b i l e Phase: Pump A, 5 mM t e t r a b u t y l ammonium phosphate (TBAP)/10 mM NH 4 H 2 P0 4 , pH 6 . 2 ; Pump B, 5 mM TBAP/10 mM pH 7 . 2 , i n 50% N H 4 H 2 P O 4 , F1ow rate : 1 CH 3 O H . ml/min. E l u t i o n : 100% A, 4 min; l i n e a r gradient from 40% t o 100% B from 4 t o 65 m i n ) .

g l u t a m a t e ; f o l a t e +4G) i s shown i n P a n e l I . Following i n c u b a t i o n w i t h CPG2, t h e p t e r i n was unchanged, but t h e t h r e e f o l a t e s and t h e p o l y g l u t a m a t e w e r e c o n v e r t e d t o t h e corresponding p t e r o a t e s (Panel I I ) . R e p r o d u c i b i l i t y was e x c e l l e n t , and i n t e g r a t i o n o f t h e peak a r e a s i n d i c a t e d t h a t enzymic c o n v e r s i o n was q u a n t i t a t i v e . A l l manipulations were p e r f o r m e d under a r g o n , o b v i a t i n g t h e need f o r o t h e r antioxidants. Degradation products ( e . g . , p t e r i n s or p-aminobenzoylglutamate), which are r e a d i l y detected,

194 T a b l e 1. PTEROATES

RETENTION TIMES

FOR FOLATE

Compound

COMPOUNDS AND

DERIVED

Retention Times,

min

Folate

Pteroate

6-Hydroxymethylpterin 5,10-Methenyltetrahydrofolate p-Aminobenzoylglutamate Tetrahydrofolate 10-Formyltetrahydrofolate 5-Formyltetrahydrofolate Dihydrofolate 5,10-Methylenetetrahydrofolate Folate 5-Methyltetrahydrofolate Methotrexate

9.4 15.1 26.0 28.5 28.9 31.1 33.2 33.8 35.2 38.6 46.0

10.9 12.4 16.7 18.0 18.9 21.5 23.4 24.1 26.4 37.4

F o l a t e + 1G F o l a t e + 2G F o l a t e + 4G M e t h o t r e x a t e + 2G

46.6 52.0 56.9 60.5

24.1 24.2 23.3 37.4

—

H P L C a s d e s c r i b e d in t h e l e g e n d f o r F i g . 1. Treatment with C P G 2 w a s for 3 0 m i n at room t e m p e r a t u r e using excess enzyme in 400 ¿iL c o n t a i n i n g - 0 . 5 m M f o l a t e c o m p o u n d . were not present. R e t e n t i o n times for the p t e r o a t e s , compared to t h o s e of t h e i r f o l a t e p r e c u r s o r s , a r e s h o w n in T a b l e 1. The pteroates are resolved better than the folates, and retention times are 10-13 min shorter. The order of e l u t i o n for t h e f o l a t e c o m p o u n d s is r e p r o d u c e d w i t h t h e pteroates. A l t h o u g h C P G 2 is c o n s i d e r e d t o b e a n e x o p e p t i d a s e , it a c t s a s an endopeptidase toward folylpolyglutamates. No folate +3G, + 2 G , +1G, o r f o l a t e is s e e n in Fig. 1 (Panel II), e v e n t h o u g h a small a m o u n t of u n r e a c t e d f o l a t e + 4G r e m a i n s ; TLC i n d i c a t e s t h a t f r e e g l u t a m a t e is a l s o a b s e n t ( d a t a n o t shown). E n d o - and exopeptidase activities of C P G 2 , however, d i f f e r b y ca. 1 0 4 - f o l d . A t 37°, a c t i v i t i e s of 19.2, 3.5, a n d 4.7 n m o l e s p t e r o i c a c i d p e r m g p r o t e i n a r e o b t a i n e d w i t h folate +1G, folate +2G, and folate +3G, respectively; these v a l u e s m a y b e c o m p a r e d t o 130 jjmoles/min/mg for f o l a t e . The a v a i l a b i l i t y o f h i g h l y p u r i f i e d e n z y m e (3), h o w e v e r , a l l o w s t h e p o l y g l u t a m a t e s t o b e u s e d as s u b s t r a t e s .

195

In adapting the pteroate procedure for analysis of intracellular folates, we have examined each of the specific steps. The following sequence constitutes our current protocol: (A) Folate compounds are labeled by growth of L1210 cells on 1 ¿iM [3H]folate (3.66 x 108 cpm/mmole), the lowest concentration capable of supporting maximal growth (4), for two 72-h transfers. Cells are harvested at late-log phase. (B) Extracts are prepared by subjecting cells to 3 freeze/thaw cycles under argon and in the presence of 2 0 mM sodium ascorbate, pH 7.0. Recovery of labeled folates is 9599%. (C) Proteins are precipitated by addition of '3 volumes of cold acetone or heat treatment. (D) The supernatant is treated with CPG2, as in Fig. 1. The latter step, however, is hampered by the presence of an unidentified endogenous component(s) that is capable either of sequestering the "sticky" folate polyglutamates or inhibiting CPG 2 . In a representative experiment, 6.1 and 5.2 nmoles of folate were hydrolyzed by CPG 2 in the absence and presence of extract (16% inhibition). This effect appears to be time-dependent; when the extract was frozen overnight with the substrate, inhibition increased to 32%. Work is in progress to identify (and remove) the responsible component(s). This work was supported by an Outstanding Investigator grant (CA-39836) from the National Cancer Institute and a grant (CH-31) from the American Cancer Society. The authors are indebted to Dr. R. Sherwood for a generous gift of recombinant CPG2 and to Susan Burke for preparation of the manuscript. References 1.

Bunni, M., M.T. Doig, H. Donato, V. Kesavan and D.G. Priest. 1988. Cancer Res. 48, 3398-3404.

2.

Duch, D.S., S.W. Bowers and C.A. Nichol. 1983. Anal. Biochem. 130. 385-392.

3.

Sherwood, R.F., R.G. Melton, S.M. Alwan and P. Hughes. 1985. Eur. J. Biochem. 148. 447-453.

4.

Fujii, K., T. Nagasaki and F.M. Huennekens. 1981. J. Biol. Chem. 256. 10329-10334.

Ill BIOSYNTHESIS AND BIOCHEMISTRY OF PTERINS

BIOSYNTHESIS OP H BIOPTERIN AND RELATED COMPOUNDS

fi. M. Brown Massachusetts Institute of Technology Cambridge, Massachusetts 02139

Introduction

When I was invited to give the Gowland Hopkins lecture at this symposium Dr. Ghisla informed me that I was expected to give an historical account of the work that led to our present understanding of the biosynthesis of H^biopterin and related compounds.

It is a great honor to be asked to

give the Hopkins Lecture, and I am particularly pleased to have been asked because it has provided the justification for me to attend and participate in this symposium. My intention is to review this subject in approximately the order that the relevant work has been presented at past symposia in this series. Space limitation precludes an extensive review and I apologize in advance to my fellow scientists who have made important contributions to this area of research for presenting the work from my own laboratory in greater detail than the work of others.

Another limitation that I have

imposed is that I shall review material presented before and during the Symposium in Montreal in 1986.

I expect that relevant information

obtained in the past three years will be presented by other participants at this meeting.

Enzymatic Synthesis of Dihydrofolic Acid

An historical treatment of the enzymatic synthesis of naturallyoccurring pterins must begin with a discussion of how folic acid is made.

Folate was the first of the pterins whose enzymatic synthesis was

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in Germany

200 investigated.

The biosynthetic pathway for the formation of ^folate is

presented in Fig. 1. 0 A Ï

N

I

HCO2

M

>

"

I Ribose-Rj be

V--CH

0H

do2

0P

X

3

" - ¿ - ¿ - C H 20P

H2NTP AMP ATP - -

^

OH

H

GTP

I

?H ?H

•• ©

®Lp|

CH2-CH0 I OH

N

-VCI H . O*-H ^-N

OH OH ^ ¿-I— - ¿I.— - UC H H A

^J

®

H

2

0 H

H2Neopterin

P-AB

© L

• PPi Glutamate

-m^ -N^ H

ATP ATP ADP+Pi ADrT rI (!)

H 2 Pteroate

_ CH

H

I CH2 I

H2Folate

co;

Fig. 1. Biosynthetic pathway for the formation of ^folate. The investigations that led to the elucidation of this set of enzymatic reactions were carried out primarily in my own laboratory and in Shiota1s laboratory.

Since most of the results of these investigations were

presented in a previous volume of this series (1, 2), no extensive discussion of the work will be presented here. are worth noting.

However, a few aspects

Three of the seven enzymes (in E. coli) involved in

the transformation of GTP to ^folate are relatively heat stable.

GTP

cyclohydrolase (enzyme 1 in Fig. 1) has a half-life of 7 minutes when heated at 82°C.

H2neopterin aldolase (enzyme 4) is stable to heating at

100°C for 5 minutes, and CH2OH-H2pterin pyrophosphokinase (enzyme 5) can be heated at 100°C for 5 minutes with retention of 75 percent of its activity.

Of the seven enzymes involved in this pathway, GTP

cyclohydrolase is the most interesting because of its properties and

201 because we now know that it is the only one of the seven that functions in the production of other naturally-occurring pterins.

Evidence

summarized previously (1,2,4) suggests that this enzyme catalyzes four chemical reactions as shown in Fig. 2. NH2

XI>

H 2 N '' ^ M ^

M

Ribose-P3

Ribose-P3

Ribose-P3

GTP

0

OH OH ....

C-C-CH

OH OH

II 2

0P

3

^

HNX Y

I

I

C - C - C - C H •CH2 H

2

0 P

H

H2NTP

Fig. 2.

Probable chemical reactions catalyzed by GTPCH.

The first two are hydrolytic reactions with the probable production of the formylated intermediate shown in Fig. 2.

This is followed by an

Amadori rearrangement to allow the ribose triphosphate moiety to undergo concomitant reduction on carbon 1 and oxidation on carbon 2, and finally ring closure yields dihydroneopterin triphosphate (H^NTP), the final product of the action of the enzyme.

Although the compounds shown in

Fig. 2 cannot be detected as free intermediates, their existence as enzyme-bound intermediates can be inferred from accumulated experimental evidence (1,2).

GTP cyclohydrolase has been purified to homogeneity from some of its properties have been determined (3).

coli and

The molecular weiqht

of the native enzyme has been estimated at 210,000.

It consists of four

identical subunits, and each subunit contains two identical polypeptide chains.

Thus, the fully constituted and active form of the enzyme

3

202 consists of eight identical polypeptides.

The heat stability of this

enzyme is surprising because of its relatively high molecular weight. Before leaving this subject, I should mention that E. coli contains another OTP cyclohydrolase that is responsible for the first enzymatic reaction in the biosynthetic pathway for riboflavin.

To distinguish it

from the enzyme committed to folate synthesis, this enzyme has been given the name of GTP cyclohydrolase II (5).

Properties that

distinguish it from the first enzyme are: its molecular weight 2+ (52,000), its requirement for Mg , its lability to heat, and the products of its action, which are: PPi, formate and 2,5-diamino-6-oxy-4• (5 -phospho-ribosylamino)pyrimidine. Presence of GTP Cyclohydrolase in Drosophila melanogaster Consideration of the chemical structures of many of the naturally occurring pterins, especially those that occur in the fruit fly, D. melanogaster, suggested that these compounds may be produced biosynthetically from neopterin or an appropriately reduced and/or phosphorylated form of this compound.

Thus, production of these end

products could be visualized as various modifications of the 3-carbon side chain of neopterin or as complete removal of the side chain, followed in some cases by hydroxylation of the pteridine ring system. It, therefore, seemed likely that the biosynthesis of all naturallyoccurring pterins begins with the conversion of GTP to H^NTP through the action of the enzyme GTP cyclohydrolase.

We decided to test this

possibility by the study of the biosynthesis of some of these end products. We settled on the fruit fly, D. melanogaster, as a source of enzymes because this organism contains a variety of pterins, many of them as eye pigments.

Our first goal was to determine whether or not Drosophila

contains the enzyme, GTP cyclohydrolase.

Our efforts indicated that

extracts of Drosophila heads were active in the conversion of GTP to H^NTP (6), and further investigations established that an enzyme is present in Drosophila heads that can be described as GTP cyclohydrolase (7).

This enzyme resembles the bacterial GTP cyclohydrolase in that the

products (formate and H^NTP) are the same and it has a relativelv high molecular weight (ca. 345,000).

203 Enzymatic Synthesis of Sepiapterin The discovery that RTP cyclohydrolase is present in Drosophila strongly sugqested that H^NTP is a key intermediate in the biosynthesis of a variety of pterins present in flies.

In our initial efforts to understand

how these pterins are made, we chose to investigate the possible enzvmatic conversion of H 2 NTP to sepiapterin (6-lactoyl-7,8-dihydropterin) because of its likely importance as a precursor of tetrahydrobiopterin

(H^biopterin).

At the time this work was undertaken, the set of reactions thought to be responsible for the biosynthesis of H4biopterin was:

sepiapterin — 7 , 8 - H 2 b i o p t e r i n — >

H^iopterin

The reduction of sepiapterin to 7,8-H2biopterin, in the presence of NADPH, is known to be catalyzed by an enzyme that has been named sepiapterin reductase (8), and the NADPH-dependent reduction of 7,8H2biopterin to H^biopterin is catalyzed by H2folate reductase.

Our

initial investigations established that H NTP can be converted to 2+ sepiapterin in the presence of Mg and an enzyme preparation from Drosophila (9).

Either NADP + or NADPH was needed for maximal synthesis

and H2neopterin could not replace H 2 NTP as substrate.

At approximately

the same time, Shiota and coworkers (10) reported that homogenates of various organs (liver, brain, kidney, lung) of the Syrian golden hamster promoted the conversion of either GTP or H 2 NTP to biopterin (or H2biopterin). Either NADP + or NADPH was needed for optimal conversion. This work was followed by additional findings by the same group that 2+

NADPH and Mg

were essential for the formation of H2biopterin from H -

NTP in the presence of an enzyme preparation from hamster kidney (11) and, somewhat later, these workers reported that two enzymes from chicken kidney are needed for the formation of sepiapterin from H 2 NTP (12) .

In

the meantime, we had extended our investigations on the formation of sepiapterin from H 2 NTP by enzymes from Drosophila.

We were able to

separate the system into two protein fractions, both of which were required for the transformation (13, 14).

The two enzymes were named

"sepiapterin synthase A" and "sepiapterin synthase B", or "enzyme A" and "enzyme B" for short. Mg 2+ was found to be essential for the activity of

204 enzyme A which catalyzed the conversion of H^NTP to a phosphate-free product which was used as substrate by enzyme B for the NADPH-dependent formation of sepiapterin. by enzyme

Since H2neopterin can not be used as substrate

A, it was concluded that during its action the removal of the

phosphate groups was likely to be an elimination reaction.

Enzvmes A

and B were partially purified and their molecular weights were assessed at 82,000 and 36,000, respectively.

Since the substrate, H^NTP, and the

end product, sepiapterin, are at the same oxidation state, a simple set of rearrangements (after the elimination of the phosphates) can account for the chemistry involved in the overall transformation, as shown in Fig. 3.

0

A HN ^

N

OH OH I

I

W - C - C H

2

0 P

^

3

H

p

H2NTP

.XjO"

C-C-CH-. II I

0

OH

\

J

S

3

1 1

N. 55

OH OH I I

>J-C-C=CH2

H I

OH

,N S I

OH OH

I

H

II

0

H

Sepiapterin

Fig. 3. Chemistry of conversion of H^NTP to sepiapterin. However, because NADPH is required, and it has been shown that tritium from tritiated NADPH is incorporated into sepiapterin during the enzymatic reaction (14), the set of reactions shown in Fig. 3 clearly could not account for the enzymatic reactions.

A major problem in

trying to elucidate the mechanism of this overall reaction is that the product made from H NTP through the action of enzyme A is very labile.

3

205 In an extension of their work with enzymes from chicken kidney, shiota and coworkers (15) proposed a set of reactions to account for the conversion of H^NTP to sepiapterin which included 6-pyruvoyl-H2pterin as the unstable intermediate.

This proposal is attractive in that it

explains the instability of the intermediate (6-pyruvoyl-H2pterin would be expected to be susceptible to degradation by hydrolytic reactions), and the incorporation into sepiapterin of tritium from tritiated NADPH. However, it is necessary to include an oxidative reaction in the proposal and since no source of oxidant is required this explanation seemed untenable.

The critical information that led to the elucidation of the

nature of this intermediate and to an understanding of the formation of sepiapterin came from investigations on the biosynthesis of H^biopterin as described in the next section.

Enzymatic Synthesis of H^biopterin In 1983, some surprising observations by Nichol and coworkers (16, 17) strongly suggested that H4biopterin can be produced from GTP and H^NTP without 7,8-H2biopterin as an obligate intermediate.

To summarize briefly,

these workers found that f^biopterin can be made from GTP or H^NTP in the presence of enough methotrexate to inhibit completely the action of H^folate reductase, an enzyme that must function to convert 7,8-H2biopterin to H^biopterin.

The obvious conclusion is that 7,8-H2biopterin is not an

obligate intermediate in the formation of H^biopterin from H 2 NTP and that, therefore, there are two separate pathways that can operate for the conversion of H 2 NTP to H^biopterin:

one which includes sepiapterin and

7,8-H2biopterin as intermediates and requires the action of t^folate reductase, and an alternative pathway that does not include these substances. These observations stimulated proposals from several laboratories almost simultaneously (18, 19, 20, 21, 22, 23) that in the alternative pathway biosynthetic intermediates from H 2 NTP to H^biopterin are tetrahydropterins rather than dihydropterins.

The initial experimental evidence in support

of these proposals was supplied by work from our laboratory (20) and by Smith and Nichol (21) .

We provided spectrophotometry evidence to indicate

206 that under anaerobic conditions a tetrahydropterin was produced from HjNTP by incubation with the Drosophila enzyme, sepiapterin synthase A, and that this product could be converted to another tetrahydropterin by incubation (again, under anaerobic conditions) with NADPH and a second Drosophila enzyme, sepiapterin synthase B.

This second product could be readily

oxidized to sepiapterin non-enzymatically by exposure to 0,,.

These

observations suggested that enzyme A catalyzes both an internal oxidationreduction reaction and the elimination of the triphosphate group from the H 2 NTP to produce 6-pyruvoyl-H4pterin and that, in the presence of enzyme B and NADPH, this product is reduced to another tetrahydropterin, 6-lactoylH^pterin.

Finally, it was observed that incubation of either 6-pyruvoyl-

H4pterin or 6-lactoyl-H4pterin with NADPH and sepiapterin reductase resulted in the formation of H^biopterin.

Later work from our laboratory

(24, 25) provided evidence for the identities of 6-pyruvoyl-H^pterin and 6-lactoyl-H^pterin as the enzymatic products and that the three phosphate groups of H^NTP are removed as triphosphate during the action of enzyme A. Smith and Nichol (21) presented evidence that, in a mammalian system, H^NTP can be converted to H^biopterin directly without either sepiapterin or 6-lactoyl-H^pterin as intermediates.

Milstein and Kaufman (18)

observed at about the same time that 6-lactoyl-H^pterin can be converted to H^biopterin by incubation with NADPH and sepiapterin reductase.

At the immediately preceding symposium in this series held in Montreal in 1986, four presentations were made on the enzymatic events involved in the conversion of H,,NTP to H^biopterin with enzyme systems derived from the following four sources:

Drosophila melanogaster (25), human

liver (26), bovine adrenal tissue (27), and rat brain (28).

There was

remarkable agreement about the basic steps involved in these four different systems.

All agreed that the first step is the formation of

6-pyruvovl-H^pterin from H^NTP; that 6-pyruvoyl-H^pterin can be reduced directly to H^biopterin in the presence of NADPH and the enzyme, sepiapterin reductase; that a second reductase exists in all four systems for the reduction of 6-pyruvoyl-H^pterin to 6-lactoyl-H^pterin; and that the latter compound is capable of being reduced to H^biopterin in the presence of NADPH and sepiapterin reductase.

By mutual agreement,

it was decided to call the enzyme that catalyzes the conversion of

207 H^NTP to 6-pyruvoyl-H^pterin "pyruvoyl-H^pterin synthase" and the enzyme that catalyzes the reduction of 6-pyruvoyl-H^pterin to 6-lactoyl-H^pterin "pyruvoyl-H^pterin reductase".

It was clear from these presentations and

also from the work of Katoh and Sueoka (29) that sepiapterin reductase possesses dicarbonyl reductase activity.

When this enzyme functions for

the reduction of 6-pyruvoyl-H pterin to H biopterin the sequence of i ^ t ** reductions of the 1 -keto and the 2 -keto groups is not yet clear. Perhaps relevant to this point, however, is that in his presentation at the i i Montreal symposium, Smith presented evidence that the 1 -hydroxy-2 -keto compound (see Fig. 4) is produced during the conversion of 6-pyruvoylH^pterin to H^biopterin by an enzyme system in bovine adrenal tissue. Milstien (in his presentation) and Smith agreed that sepiapterin reductase i catalyzes the reduction of the 1 -oxo group at a faster rate than the i 2 -oxo group is reduced. The various reactions thought to be involved in the conversion of H^NTP to H^biopterin are summarized in Fig. 4.

In the initial chemical events,

whether the rearrangement to form the tetrahydropterin occurs before the elimination of the triphospho group (as shown in Fig. 4) or whether the reaction sequence is reversed can not be decided.

A case can be made for

either sequence of events (26).

As indicated in Fig. 4, the evidence is clear that in all systems studied an enzyme exists for the reduction of 6-pyruvoyl-H^pterin to 6lactoyl-H^pterin and it has also been firmly established that the latter compound can be reduced to H^biopterin in the presence of sepiapterin reductase and NADPH.

It is also equally clear that sepiapterin reductase

can catalyze the reduction of 6-pyruvoyl-H^pterin directly to H^biopterin. The auestion that arises is: (LPH^)?

of what importance is 6-lactoyl-H^pterin

A possibility is that the LPH^ pathway operates in certain tissues

in the synthesis of H4biopterin.

Another possibility is that if

sepiapterin is important for reasons other than in the synthesis of H^biopterin, LPH 4 would be important because it is the precursor (bv oxidation) of sepiapterin.

The question of the importance of the LPH^

pathway can only be answered after further experimentation.

208 OH HN

OH

H

^C — C —

H,N

H

•N' H

ch2 - o p 3

u

+ OH

^ ^

H

OH

C — C —CHj

OP,

H

'N

Hz NTP

- c = ch2

Tc'0 N

—

J N-^

H+

c 1 1

OH I A — C^y1c C H j — 0 P3

0

H

H /

y

C -C-CH, Il II •N ^ 0 0 H

NADPH+H+ NADP +

y

C — C -CH3 I II OH 0

H I -C —CH3 I 0 OH

or N

PPH4

r

NADP H + H +

r

NADPf

^

H I C — C - CH, II I OH 0

N A D P H + Hh -

v NADP*

NADPH H

HNg

H I C

Y Y OH ^~ î OH

LPH4

Fiq. 4.

NADPH + H +

-CH,

H4Blopterin Enzymatic conversion of H 2 NTP to H biopterin. 4

Unfortunately, space limitation does not permit a thorouqh presentation and discussion of the details of the important contributions to our present understanding of the enzymatic synthesis of H^biopterin made by a number of research qroups, especially the Wellcome Research Laboratories group, the Curtius group, and Milstien and Kaufman.

The reader is

209 referred to the articles presented in the proceedings of the Montreal symposium (25, 26, 27, 28) for much of this important information. Biosynthetic Importance of 6-Pyruvovl-H^Pterin It now seems clear that 6-pyruvoyl-H^pterin is a common precursor for all of the pterins found in Drosophila.

In addition to its role, described

above, as a precursor of H^biopterin and sepiapterin, it is known to be the substrate for an enzyme that catalyzes the formation of a pyrimidodiazepine (PDA) which, in turn, is thought to be a precursor of the drosopterins, the red eye pigments of Drosophila.

In our laboratory we

have purified this enzyme, named "PDA synthase", to near homoqeneity and have shown that it catalyzes the conversion of 6-pyruvoyl-H^pterin to PDA (30).

The only other component needed for this transformation is

reduced glutathione (GSH).

This transformation can be envisioned to

involve a number of chemical events, including the openinq of the 6-membered pyrazine rinq, some rearrangements, ring closure to form the 7-membered diazepine ring, and reduction. the reduction.

The GSH is probably needed for

The complexity of this transformation can be appreciated

by contrasting the structure of 6-pyruvoyl-H^pterin with that of PDA, shown in Fig. 5.

GSH

Fig. 5.

Enzymatic conversion of P p H^ to PDA.

Since PDA contains the same 7-membered ring structure found in the drosopterins (31, 32), it is a likely precursor of these pigments.

In

our laboratory, we have shown that at acid pH values (2.5-5.9) drosopterins can be made nonenzymatically from a mixture of ^pterin and

210 PDA.

(33).

A speculative

is shown in F i g .

set of r e a c t i o n s

to a c c o u n t for this

synthesis

6.

Drosopterin Fig.

6. p r o p o s a l f o r t h e n o n e n z y m a t i c a n d P D A to d r o s o p t e r i n .

conversion of

H^pterin

So far, no e v i d e n c e h a s b e e n o b t a i n e d for an e n z y m e - c a t a l y z e d of the drosopterins

from H^pterin and

Pyruvoyl-

Lactoyl - H4Pterin

Fig.

4

Pterin

H2Pterin

H4Biopterin

Sepiapterin Fiq.

H

Pterin

7, 8 - H 2 B i o p t e r i n

Drosopterin

7. S u m m a r y o f t h e r o l e o f P P H ^ a s a p r e c u r s o r o f number of pterins.

7 summarizes

the

likely

lactoyl-H^pterin to H ^ p t e r i n ,

Isoxanthopterin a

central role of 6 - p y r u v o y l - H ^ p t e r i n

biosynthesis of a variety of pterins found that catalyze

synthesis

PDA.

in Drosophila.

all of the reactions

to sepiapterin,

Enzymes have

in

except for the o x i d a t i o n of

the conversion of

and the p r o d u c t i o n o f d r o s o p t e r i n s

the

been 6-

6-pyruvoyl-H^pterin

from PDA and

H^pterin.

211 All three of these reactions are known to occur readily in the absence of enzymes.

Whether or not enzymes exist to speed up the rates of these

reactions must await further work.

If these enzymes do not occur, it

seems possible that these reactions may take place nonenzymaticallv to an extent that could account for the formation of the relevant products. References: 1.

Brown, G.M. 1970. In: Chemistry and Biology of Pteridines (K. Iwai, M. Goto and Y. Iwanami, eds.). International Academic Printing Co. Ltd., p. 243.

2.

Shiota, T., R. Jackson, C.M. Baugh. 1970. In: Chemistry and Biology of Pteridines (K. Iwai, M. Akino, M. Goto and Y. Iwanami, eds.). International Academic Printing Co. Ltd., p. 265.

3.

Yim, J.J. and G.M. Brown. 1976. J. Biol. Chem. 251, 5087.

4.

Brown, G.M. 1985. In: Chemistry and Biochemistry of Pterins, vol. 2 (R.L. Blakley and S.J. Benkovic, eds.). John Wilev and Sons, p. 115.

5.

Foor, F. and G.M. Brown. 1975. J. Biol. Chem. 250, 3545.

6.

Brown, G.M. and C.L. Fan. 1975. In: Chemistry and Biology of Pteridines (W. Pfleiderer, ed.). Walter de Gruyter, p. 265.

7.

Fan, C.L. and G.M. Brown. 1976.

8.

Matsubara, M., S. Katoh, M. Akino, S. Kaufman. 1966. Biochim. Biophys. Acta. 122, 202.

9.

Fan, C.L., G.G. Krivi, G.M. Brown. 1975. Commun. 67, 1047.

Biochem. Genet. 1£, 259.

Biochem. Biophys. Res.

10.

Fukushima, K., I. Eto, T. Mayumi, W. Richter, S. Goodson, T. Shiota. 1975. Chemistry and Biology of Pteridines. (W. Pfleiderer, ed.). Walter de Gruyter, p. 24.

11.

Eto, I., K. Fukushima, T. Shiota. 1976.

12.

Tanaka, K., M. Akino, Y. Hagi, T. Shiota. 1979. In: Chemistry and Biology of Pteridines (R.L. Kosliuk and G.M. Brown, eds.). Elsevier North-Holland, p. 147.

13.

Krivi, G.G. and G.M. Brown. 1979.

14.

Brown, G.M., G.G. Krivi, C.L. Fan, T.R. Unnasch. 1979. In: Chemistry and Biology of Pteridines (R.L. Kisliuk and G.M. Brown, eds.). Elsevier North-Holland, p. 81.

J. Biol. Chem. 251, 6505.

Biochem Genet.

371.

212 15.

Tanaka, K. , M. Akino, Y. Hagi, M. Doi, T. Shiota. 1981. Chem. 256, 2963.

J. Biol.

16.

Nichol, C.A., g.K. Smith, D.S. Duch. 1983. in: Chemistry and Biology of Pteridines. (J.A. Blair, ed.). Walter de Gruyter and Co., p. 759.

17.

Smith, G.A., and C.A. Nichol. 1983. Arch. Biochem. Biophvs. 227, 272.

18.

Milstien, S. and S. Kaufman. 1983. 115, 888.

19.

Culvenor, A.J., L.P. Miller, R.A. Levine, W. Lovenberg. 1984. J. Neurochem. 42, 1707.

20.

Switchenko, A.C., J.P. Primus, G.M. Brown. 1984. Biochem. Biophvs. Res. Commun. 120, 754.

21.

Smith, G.K., and C.A. Nichol. 1984. 120, 761.

22.

Heintel, D., S. Ghisla, H.-Ch. Curtius, A. Niederwieser, R.A. Levine. 1984. Neurochem. Int. 6, 141.

23.

Masada,M., M. Akino, T. Sueoka, T. Katoh. Acta. 840, 235.

24.

Switchenko, A.C. and G.M. Brown. 1985.

25.

Brown, G.M., J.P. Primus, A.C. Switchenko. 1986. In: Chemistry and Bioloqv of Pteridines (B.A. Cooper and V.M. Whitehead, eds.). Walter de Gruyter, p. 125.

26.

Curtius, H.-Ch., S. Takikawa, A. Niederwieser, S. Ghisla. 1986. In: Chemistry and Biology of Pteridines (B.A. Cooper and W.M. Whitehead, eds.). Walter de Gruyter, p. 141.

27.

Smith, G.K., D.S. Duch, C.A. Nichol. 1986. In: Chemistry and Biology of Pteridines (B.A. Cooper and V.M. Whitehead, eds.). Walter de Gruyter, p. 151.

28.

Milstien, S. and S. Kaufman. 1986. In: Chemistry and Biology of Pteridines (B.A. Cooper and V.M. Whitehead, eds.). Walter de Gruyter, p. 169.

29.

Katoh, S. and T. Sueoka. 1984. Biochem. Piophys. Res. Commun. 118, 859.

30.

Wiederrecht, G.J. and G.M. Brown. 1984. J. Biol. Chem. 259, 14121.

31.

Theobald, N. and Pfleiderer, W. 1977.

32.

Theobald, N. and Pfleiderer, W. 1978. Chem. Ber. Ill, 3385.

33.

Paton, D.R. and G.M. Brown. 1986. In: Chemistry and Biology of Pteridines (B.A. Cooper and V.M. Whitehead, eds.). Walter de Gruytej; p. 295.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

1984.

Biochim. Biophvs.

J. Biol. Chem. 260, 2945.

Tetrahedron Lett. 10, 841.

THE STRUCTURE OF «-BH 2 -CHEMISTRY AND BIOCHEMISTRY Wilfred L.F. Armarego Division of Biochemistry and Molecular Biology, JCSMR, ANU, GPO Box 334, Canberra City, ACT 2601, AUSTRALIA In his pioneering studies of the phenylalanine hydroxylating system, Dr Seymour Kaufman found that the cofactor involved was a non-conjugated pteridine (1). His studies further showed that the cofactor was a tetrahydropterin which was oxidised to a transient pterin which was at the dihydro level of reduction. This dihydropterin was the substrate for the sheep liver enzyme which is now known as dihydropteridine reductase (pHPR) (2). These same transient species could also be produced by chemical oxidation with oxidants such as bromine, ferric iron and 2,6-dichlorophenolindophenol. These species were unstable in the presence of oxygen and finally gave the fully oxidised pterin. O

H

(3) O

(j HI Hl 2

HiN^^N^^^N^ H ®0/J - j

PPH4 S

Mg a *

0 HN

PPH4

NADPH NADP

SR

0 0 I I

-c-c-

PPH4 R

CHj NADPH 4 NADP

H0 1 1 I I HN ->'' ^-C-C-CHj C-C-CHj I OH HjN'^KJ'^M'^ OH NADPH NADP BH«

SR,

H.N^,

NADPH NADP*

HH I I C-C-CH.

Figure 1 . Biosynthesis of tetrahydrobiopterin (BH4): GTPCH, GTP c y c l o hydrolase I ; NH2TP, dihydroneopterin triphosphate; PPH4, 6-pyruvoyl t e t r a hydropterin; PPH4R, 6-pyruvoyl tetrahydropterin reductase; PPH4S, 6-pyruvoyl tetrahydropterin synthase; Pyd-P3, pyrimidine nucleoside triphosphate; SR, sepiapterin reductase; and J), 2 ' - k e t o and l ' - k e t o tetrahydropterin intermediates. Table 1 i l l u s t r a t e s

our findings in patients and controls as well

1 i v e r of man and mouse.

as in

276 Table 1.

HPLC and GC-MS data of 6- and 7-substituted pteridines in the

urines of two patients, their family members, healthy and

hyperphenylala-

ninemic controls, and in human and mouse liver homogenates. Taken from (5) N*

7-N

B*

7-B

%1-W*

1041

864

2.0

%7-B**

umol/mol creatinine Patient P.P.

1278

26 13

45 7.0

Mother

P.

464

588

45

2.7

Father

P.

159

0.5

429

17

0.3

3.8

Brother P. 7y

462

7

832

41

1.5

4.6

Brother P. 9y

220

1.4

596

24

0.6

3.6

410

390

nm

49

840

nm

37

30

Patient J.S.

1674

nm

Brother T.S. 2y

1350

nm

1430

Father

S.

260

nm

550

Mother

S.

410

nm

960

nm

5

trace

nm

-

827

58

0.7

238

116

0.7

Controls Control 5y

805

6.0

6.6

PPH4S deficiency

8010

DHPR deficiency

4260

7

7622

42

0.2

0.6

PKU1 (Phe 200 umol/1) 1654

14

3378

121

0.8

3.5

1840

105

0.02

5.4

54

PKU2 (Phe 538 umol/1) 5816

1.0 N*

B*

Children 2-12m

1100--4000

470- 3000

Children 3-14y

200--1700

530- 2700

Adults

100-- 500

220- 1100

Normal range

N* Liver homogenate Human liver Mouse liver

7-N

B*

7-B

X7-N

33

%7-B

pmol/g tissue 3650 8 .3

50 0.48

4300

140

1835

3.8

1 .3

3.1

0.2

2.7

B, biopterin; 7-B, 7-biopterin; DHPR, dihydropteridine reductase; N, neopterin; 7-N, 7-neopterin; nm, not measured; uria;

PKU, classical

phenylketon-

PPH4S, 6-pyruvoyl tetrahydropterin synthase; *calculated by HPLC.

** In % of the sum of 6- and 7-substituted isomers.

277 It

is

of

special

interest

that

after

BH4 loading 2 p a t i e n t s

showed

in-

creased u r i n a r y e x c r e t i o n not only o f b i o p t e r i n but also of p r i m a p t e r i n . F i g . 2 shows n e o p t e r i n , b i o p t e r i n , and phenylalanine concentrations i n the urine of p a t i e n t J . S . before and during B H 4 therapy.

Biopterin, Primapterin

(mmol/mol creat.)

Neopterin

Age Figure 2. Excretion of b i o p t e r i n ( o ) , primapterin ( * ) , and neopterin ( • ) i n the urine of p a t i e n t J . S . a f t e r o r a l a d m i n i s t r a t i o n of 2 mg/kg/d of B H 4 . Taken from ( 4 ) .

The mother of one of the p a t i e n t s and the brother of another p a t i e n t also showed elevated e x c r e t i o n of p r i m a p t e r i n . e r r o r of metabolism.

This f i n d i n g suggests an inborn

278 Investigation of blood cells In order to check whether GTPCH or PPH4S are involved in the formation of 7-substituted pterins, G. Schoedon from our group investigated the leukocytes and erythrocytes of the patient P.P. for GTPCH and PPH4S activity as well

as

for SR and dihydropteridine

reductase

(DHPR) activity

(5).

SR

activity was 1.0 mU/g Hb (control: 0.6-2.2 mU/g Hb), the activity of GTPCH was 0.9 ull/mg protein (control: 0.3-1.2 uU/mg protein), that of PPH4S was 22.2 uU/g Hb (control: 11-29 ull/g Hb), and for DHPR we found an activity of 3.2 mU/mg Hb (control: 2.0-5.0 mU/mg Hb) in the patient's blood cells.

All enzyme activities were found to be normal.

A deficiency of one of the

known BH4 biosynthetic enzymes can therefore be excluded.

During the GTPCH

reaction we only observed about 2% anapterin formation, which is in agreement with normal controls.

No primapterin was found.

tion of about 2% found also different GTPCH reaction.

in normal

The anapterin forma-

controls might be explained by a

This hypothetical

pathway is shown in Fig. 3.

On the other hand, the relatively high concentration of primapterin in our patients can not be explained by this pathway (5).

Investigation of feces One

possibility

for

the

formation

of

7-substituted

pterins

isomerization of the side chain by a yet unknown isomerase.

would be

Such an iso-

merization reaction might also be catalyzed by a gut bacterial zyme.

flora en-

But after sterilizing a patient's intestine by a two-day treatment

with neomycin primapterin excretion was not measurably suppressed (5). measured 6- and 7-biopterin in fecal Control

an

We

specimens of patients and controls.

feces showed a biopterin:primapterin

ratio of about 1:1, whereas

the ratio in patients was 1:12. After

BH4 loading

of a control,

Niederwieser

found only

biolumazine

higher concentrations and only very small amounts of biopterin.

in

His find-

ings showed that a desaminase reaction leading to lumazines must occur in

279 0 HN^l N XJ vJ LX j J HjN

N

" INI

GTP

P30i^0. OH OH

o" HN'VNH2 • i r Ho N

N

Pyd-P3

NH

OH OH o jr H ,, OH OH OH H2N^-N^N •H' •|j

H H H

H

OH OH OH

H

»

H H H

-H*Jf "

NH

2

OHOHOH

N= C-C-C-C-CH,OP-,

i i i

h

OHOHOH

* u H H H

" H * Il Ï

1

i

J

l

rr

OHOHOH H i i i N=C-C-C-C-CH,OP, i i i

NH-

H H

H

t

J

H H H

-h2O!

•h2O| OH OH i i

-C-CH2OP3

H 6-NH2TP

N

R?"

C H ? 0 P 3

H H 7"NH2TP

Figure 3. GTP c y c l o h y d r o l a s e I r e a c t i o n : P r o p o s e d Amadori r e a r r a n g e m e n t s l e a d i n g t o 6 - and 7 - s u b s t i t u t e d n e o p t e r i n t r i p h o s p h a t e . GTP, g u a n o s i n e t r i p h o s p h a t e ; Pyd-P3, p y r i m i d i n e n u c l e o s i d e t r i p h o s p h a t e ; 6/7-NH2TP, 6 / 7 di hydroneopteri n t r i phosphate.

280 the gut bacterial

flora.

It is possible that the supposed desaminase lead-

ing to lumazines is very specific and does not catalyze the desamination of 7-biopterins.

Based on these results it is of special

interest that in the

patient's feces we traced mostly primapterin and only very small biopterin.

amounts of

Therefore it seems that primapterin accumulates in fecal

mens and that it cannot be metabolized.

speci-

The investigation of lumazines is

in preparation.

Incubation

studies

of

BH4

with

feces

under

anaerobic

conditions

are

in

preparation.

When

investigating

a patient's

feces

for parasites

we

found lamblia.

On

first sight we assumed that the lamblia might directly or indirectly be the cause

of the

fecal

primapterin

culture and of two control

formation

but

investigation

patients with lamblia infection

of lamblia

in

showed no prim-

apteri n at al 1.

We also investigated the urine of one of our patients using HPLC and electrochemical

detection

and

we

were

able

to

show

that

20% of

about

apterin and 20% of biopterin were excreted in the tetrahydro form.

prim-

This is

in definite contrast to normal controls where about 80% of biopterin is excreted in the tetrahydro

The

cause

of

form.

hyperphenylalaninemia

relatives is still unclear.

in

these

patients

the BH4 biosynthetic enzymes showed normal bition of the BH4 biosynthetic enzymes

factors:

6-biopterin excretion

and

may

be

competition

showed

no inhibition values

Km

of

by tetrahydro

for

their

primapterin.

Never-

might be explained by two

6-BH4

and 7-BH4

in the

two

phenyl al ani ne-4-hydroxyl ase

and 5 times higher, respectively.

range

enzymes

Inspite of the fact that 6-BH4 also

of the enzymes phenylalanine-4-hydroxylase

7-BH4

of

is at the lower limit of the normal

between

phenylalanine-4-hydroxyl ase and DHPR.

the

some

values and we observed no inhi-

theless, the occurrence of hyperphenylalaninemia

there

and

Following our investigations the activities of

and

and DHPR,

DHPR

were

20

281 A further possibility would be a partial

defect of phenylalanine

hydroxy-

lase caused by a K m mutant. This could explain the light hyperphenylalaninemia in the patients and the fact that phenylalanine can be normalized by BH4 loading.

As we had no liver biopsy material

at our disposal, we

were not able to directly test the above mentioned hypothesis.

As a model

system we examined uncoupled or partially uncoupled phenylalanine hydroxylation expecting formation of a 7-isomer during the reaction via 4a-carbinolamine (6,7).

In such a partially uncoupled reaction the 4a-carbinolamine

may accummulate and then split off H2O.

A double-cleavage of the pyrazine

ring to form amino-2-alloxane and a 1,2-diamino compound and a reverse ring closing might explain the formation of 7-substituted pterins. tions

have already been observed in chemical

systems under

Such reacphysiological

conditions (7) (see Fig. 4).

We used the following Viscontini's

two model

systems:

earlier non-enzymatic work

The first is an adaptation of

(8); according to this method we

incubated BH4 with phenylalanine Fe++ and EDTA in a phosphate buffer at pH 6.8 and 8.2.

For the second experiment we used an enzyme preparation of

rat liver, either as a crude extract or a Sephadex G-25 treated crude extract, and incubated with BH4, and for partial inhibition with para-chlorophenylalanine or tyrosine. the

non-enzymatic

(8) or

Using HPLC we detected no 7-isomers either in in the enzymatic

investigating liver biopsy material partial

mutation

of phenylalanine

(7) experiment.

But without

of the patients, the possibility of a hydroxylase

caused by a K m mutant can

not be ruled out. In nature some 7-substituted pterins such as erythropterin, ekapterin, and chrysopterin are known

in insects.

pterin

function

ring

derivatives

nucleus.

and have an oxygen

This

ring oxygen

function

7-position for a nucleophilic attack.

leads

They all

lepidopterin, are xantho-

in the 6-position of the to an activation

of

the

Forrest et al. (9) demonstrated the

enzymatic synthesis of erythropterin from xanthopterin and oxalacetic acid. In all

the

naturally

occurring

7-substituted

pterins

an

addition

of

an

aliphatic keto acid to the 7-position of the ring nucleus has to be postulated.

282

0 H H H N ^ y * HN'tM ^ N 1 H 1-h2O 0 h

HN

n

^

V

N

If

R

N H

0 H N - ^ N C R

h2n I 0

H N ^ ^ O HoN^N^O

H2N N»20 ( K e q = 0.44 M) d 5 , 10-methylene-Hi»MPT + F 1 ) 2 o H 2 » s 5-me thy 1-H „ MPT + F ^ o ( K e q = 2) d 5-methyl-Hi.MPT + HS-CoM -»• CH 3 S-CoM + H i, MPT

a

E q u i l i b r i u m constants are given for pH 7 and at 60°C, for reaction (3) which was determined at 37°C. ^Calculated from data presented in (9). c T a k e n from DiMarco et al (12) . d B . W . te Brommelstroet, unpublished results

except

Table 2. Properties of Hi,MPT-dependent Enzymes in Methanogene s is a Reaction k 1 2 3 4 5 a

Enzyme formylmethanofuran:Hi,MPT formyltransferase 5,10-methenyl-H^MPT cyclohydrolase 5,10-methenyl-H^MPT cyclohydrolase 5,10-methylene-Hi.MPT dehydrogenase 5, 10-methylene-Hi, MPT reductase

Mr Ref . Spec .Act c (subunit) (purif.factor) -160 000 539 (a 1, ; 4 1 . 0 0 0 )(261) 82.000 130 (128) (a 2 ;41.000) 82.000 470 ( a 2 ; 4 1 . 0 0 0 )(313) 2 16.000 736 ( a 6 ; 3 6 . 0 0 0 ) (151) 35.000 7.4 (35.000) ( 22)

9 12 d d d

T h e enzymes were purified from Methanobaotevium thermoautotvophiaum, except the cyclohydrolase catalyzing reaction (3), which was obtained from Methanosaroina barkeri . ^Reaction numbers are those as given in Table 1. c S p e c i f i c activities (umol/min.mg protein) of the x-fold purified homogeneous enzyme preparations were all determined for the reversed reactions of Table 1, except for formyltransferase . d B . W . te Brommelstroet, unpublished results.

289 comparison

to the one described

uses different substrates, viz different methanofuran

for M. thermoautotrophicum Hi,SPT and a

derivative

structurally

(15), and which will

1 O-formyl-Hi, SPT , is not further characterized Reduction

reactions of the one-carbon

Hi»folate biochemistry

as yet.

serves as the

studied

in M. thermoautotrophicum

quite specifically coenzyme F u o i

reduc-

and M.

barkeri

low-potential

electron carrier in methanogens, mediates (18). As such it can be considered

derivative

(E 0 = -350 mV)

in hydride

transfer

as a functional analogon

cofactors. Both the dehydrogenase

contrast

to the 5 , 10-methy lene-Hi» folate dependent reaction, which in the direction of methylene

(19,20). The methanogenic

reductase

is further

by the absence of a flavin prosthetic Brommelstroet, unpublished

is

reduction

characterized

(B.W. te

results) contrary

Hi^folate reductases purified sources

group

of

and the

reductase reactions are reversible. This latter is in known only to proceed

is

(4,16,17, B.W. te

results). The deazaflavin

which is a central

the nicotinamide

and

(4,16,17) . The compound

used in the reactions

Brommelstroet, unpublished

and

electron

carrier, in the 5 , 10-methy lene-Hi»MPT dehydrogenase is the co-substrate

to

to 5-me thy 1-H i,MPT

1). Whereas in eubacterial

systems NAD(P)(H) usually

coenzyme F ^ o

to

5 , 1 0-methenyl-Hi,MPT is to be reduced

(reactions 4 and 5 in Table

tase reactions

produce

unit. Analogously

5 , 1 0-methylene-Hi,MPT and subsequently eukaryotic

(9)

from eubacterial

to

5,10-methylene-

and

eukaryotic

(19,20,21) .

Methyl group transfer reactions. The next step in the CO2 reduction

to methane

involves a methyl transfer

Ht*MPT to the thiol group of coenzyme M ethanesulfonic

acid) resulting

2-methy1thioethanesulfonic

(HS-CoM,

in C H 3 S - C 0 M

acid)

(methylcoenzyme

M,

1) . From

thermoautotrophicum

the involvement of an enzyme-bound

tive, 5-hydroxybenzimidazoly1 cobamide

5-methyl-

2-mercapto-

(reaction 6, Table

experiments with crude cell extracts of M. and M. barkeri

from

(B12-HBI)

B12

deriva-

in CH 3 S-CoM

290 synthesis was suggested published

(22,23

; W. van de Wijngaard,

un-

results). This latter could be confirmed now with

highly-purified

corrinoid-containing

therrnoautotrophiaum

enzyme preparations

(S.W.M. Kengen, unpublished

of M.

results).

In

fact, the 5-methyl-HitMPT : HS-CoM me thy 11 r an s f e r a s e reaction composed of two subsequent enzymic

Hi,MPT-bound methyl group to enzyme-bound Co-methy1-B12:HS-CoM bvyantii

in 1974 from

(24). Upon purification

corrinoid

protein

Bi 2-HBI followed by a

methyl transfer. The latter

enzyme was already purified

is intimately

is

steps: the transfer of the oxygen-stable

Methanobaeterium

the extremely associated with

oxygen-labile high-molecular

weight cell fragments, presumably membranes, and it may be identical to the membrane-bound

corrinoid

function described by Fuchs et al Like in the corrinoid-containing

protein of

unknown

(25 , 26) . methionine

catalyzes an otherwise mechanistically

synthetase, which

related reaction.

the methyl transfer of 5-me thy 1-H t, folate to a thiol methanogenic

methy1transferase

the prosthetic state

is catalytically

(27) , the

active, when

group is present in the highly-reduced

(22,23). Artificially

Physiologically

this proceeds

phosphate

in an as yet little

action of ATP

(CoM-S-S-HTP) of HS-CoM and

B12S

the corrinoid protein may be

activated by the strong reducing agent Ti(III)citrate way by a combined

(22,23) and the

(23).

understood

heterodisulfide

7-mercaptoheptanoy1threonine

(HS-HTP), the electron donor of the terminal

methanogenesis

(Fig. 1)

Viz

(S.W.M. Kengen, unpublished

step of

results) .

The activation mechanism may be the same as the one in CO2 activation

and reduction

In methanogenesis recently

from acetate by M. barkeri

shown to act as a methyl carrier

activation moiety

(28,29). Hi^MPT

(30). Subsequent

to yield acetyl-CoA, the C-C bond of the

is cleaved by CO hydrogenase

are formed at the CO and methyl

a high affinity

for the pterin

= 4 uM)

units

(30, and to Hi,MPT

methyltransferase, which (apparent K m

to

acetyl

and two one-carbon

sta.te of reduction

references herein) . The methyl group is transferred by a not further characterized

(H^SPT) was

shows

(30). M.

291 barkeri

grown

to g e n e r a t e (Fig. by

reducing

1) . T h e

a methyl

HS-CoM

on m e t h a n o l

is n o t

intermediate

to o x i d i z e

equivalents

oxidation

transfer

has

pathway

for may

from m e t h a n o l

required

suggesting

(J.T. K e l t j e n s ,

part

of the

the m e t h y l proceed

group

via

to H it M P T ;

reduction

5-methyl-Hi,MPT

in t h i s

that C H 3 S - C 0 M

unpublished

substrate

transfer

is not

an

results).

Acknowledgements

The ship

research

of J . T .

Keltjens

was

of the R o y a l N e t h e r l a n d s

(KNAW).

The work

Foundation

of

S.W.M.

of F u n d a m e n t a l

supported

Academy

Kengen

of A r t s

was made

Biological

by a s e n i o r and

Sciences

possible

Research

fellow-

by

the

(BION).

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1. W o l f e ,

R.S.

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K a y , L . D . , M . J . O s b o r n , Y. J. B i o l . C h e m . 235, 195.

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15 1, 459.

BIOSYNTHESIS OF METHANOPTERIN

R. H. White Department of Biochemistry and Nutrition Virginia Polytechnic Institute and State University Blacksburg, Virginia 24061-0308

Introduction Current biosynthetic evidence indicates that methanopterin, a coenzyme involved in C^ metabolism in the methanogenic archaebacteria

(1), is the first example of a naturally occurring,

structurally modified folic acid coenzyme in cells.

(2,3) that functions as a

Despite these structural modifications,

current evidence indicates that methanopterin

functions

biochemically in these bacteria in the same way folic acid does in other cells.

In fact, it appears to have taken over

all the normal functions of folic acid in these cells, which appear to lack folic acid

(4,5) .

Thus, methanopterin, not

folic acid, serves as the Ci carrier for serine hydroxymethyltransferase, N 5 , N 10 -methylenetetrahydromethanopterin genase, and N5, Nio-methenyltetrahydromethanopterin hydrolase.

dehydro-

cyclo-

In order to understand how and why this change in

the structure of the folic acid coenzyme arose, the details of the reactions involved in the biosynthesis of methanopterin have been explored.

Results Recent work on the biosynthesis of the individual units of methanopterin has shown that GTP is a likely precursor to the pterin, as is the case in folic acid biosynthesis

Chemistry and Biology of Pteridines 1989 © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in G e r m a n y

(3,6). The

295 aniline portion of the

5-(p-aminophenyl)-1,2,3,4-tetrahydroxy-

pentane was shown to arise from p-aminobenzoic acid, which was, in turn, produced via the shikimic acid biosynthetic pathway

(2).

from a pentose

The five-carbon side chain was shown to arise (6), which was proposed to be ribose on the

basis of the stereochemistry of the polyol side chain

(7).

That both of the methyl groups of methanopterin are derived from methionine was established by growing volta

with

[methyl-2H 3 ]-methionine

Methanococcus

(0.3 mg/ml) and measuring

the extent of deuterium incorporation into the 6-ethyl-7methylpterin, which was derived from the methanopterin by reductive cleavage with zinc dust in the presence of acid. The M+-15 m/z 34 9 fragment in the mass spectrum of the ditrimethylsilyl of this methylated pterin, recorded by gas chromatography-mass spectrometry

(GC-MS), showed molecules

with 2C2H3.

Cell extracts of M. volta were found to readily convert neopterin to 7,8-H2-6-hydroxymethylpterin

7,8-H 2 -

in a reaction

presumably catalyzed by dihydroneopterin aldolase, a wellcharacterized reaction involved in folic acid biosynthesis. That the resulting 7,8-H2~6-hydroxymethylpterin

can be

converted to methanopterin was confirmed by growing M. in the presence of

2

[methylene- !!]-6-hydroxymethylpterin

mg/500 ml of medium) and measuring its incorporation

volta (3.8

into

methanopterin, to an extent of 29%, using the reductive cleavage method outlined above. demonstrated the incorporation of methylpterin into methanopterin, 2

H 3 ]-6-[(IRS)hydroxy-ethyl]pterin

In addition to having [methylene-2H]-6-hydroxyI have shown that

[methyl-

is incorporated to an extent

of 8.9% when fed to M. volta at a level of 30 mg/500 ml of

296 medium. Pterins containing a 7-methyl were not found to be incorporated into methanopterin by growing cells of M. volta. These results indicated that the C-9 methyl group of methanopterin is added to 7,8-H2-6-hydroxymethylpterin before it is coupled with methaniline, the side-chain structure of methanopterin, and that the C-7 methyl group is added at a later stage in the biosynthesis.

In order to test this idea,

extensive work was undertaken to establish the methylation of 7,8-H2-6-hydroxymethylpterin, and, since it is involved in folic acid biosynthesis, and, therefore, likely to be involved in the biosynthesis of methanopterin, its pyrophosphate ester was tested as well.

However, incubations of

3

these substrates and C H3-SAM (22 mCi/mmol) with cell-free extracts of M. volta and Methano-bacterlum formicicum produced no evidence of the formation of labeled methylated pterins or labeled methanopterin, thereby indicating that 7,8H2-6-hydroxymethylpterin must condense directly with an arylamine before any methylation takes place. The next question to be addressed concerned the chemical nature of the arylamine that is coupled to the pterin.

Since

the methanogenic archaebacteria contain no pteric acid or folic acid (4,5), and since all archaebacteria, except for the halo-bacteria, are completely resistant to sulfonamides (8), one could argue that the normal condensation of 7,8-H2-6hydroxymethylpterin-PP with p-aminobenzoic acid (pAB) is not operative in methanogenic bacteria.

This argument is further

supported by the observation that some methanogenic bacteria contain large amounts of methaniline (3).

All these observa-

tions suggested that methaniline is biosynthesized from pAB which is then coupled with H2-6-hydroxymethylpterin-PP.

That

this pathway does, in fact, occur was confirmed when cell

297 extracts of M. volta incubated with pAB, ATP, and ribose-P were found to produce, in addition to 5-(p-aminophenyl)1,2,3,4-tetrahydroxypentane, an anionic compound currently considered to be the ribose-P derivative of 5-(p-aminophenyl) -1, 2, 3, 4-tetrahydroxypentane . This work, then, strongly suggests that the pAB is converted to the complete arylamine before its condensation with the pterin. The condensation of 7,8-H2-6-hydroxymethylpterin with methaniline would generate a demethylated dihydromethanopterin which would-then be methylated at the C-7 and C-9 positions to produce dihydromethanopterin.

This condensation reaction has

now been confirmed by the isolation of demethylated methanopterin from cell extracts of M. formicicum incubated with [methylene-3^]-7,8-H2-6-hydroxymethylpterin, ATP, and methaniline.

In addition, cell extracts of the same cells were able

to methylate the isolated demethylated methanopterin to methanopterin in the presence of SAM.

(It is presumed that

the demethylated methanopterin is reduced by the cell extract to the dihydropterin before the methylations occur, and that the produce is oxidized back to methanopterin during the workup.) On the basis of these and other observations, a pathway for the biosynthesis of methanopterin is proposed (Scheme 1). The reactions marked by currently have data supporting their occurrence, in methanogenic bacteria, either in cell extracts or in whole cells. This first reported occurrence of the methylation of a coenzyme further expands our knowledge on nature's use of methylation reactions to control the biochemical reactivity and specificity of biomolecules.

298

8

H

i"'

NADP-E-BH4 > BH4, NADP-E [

isoraerization.

In this speculation, both CI'-keto PH4 and C2'-keto PH4 are successive intermediates of SPR function as follows: PPH4 [Cl'-keto PH4 C2'-keto PHJ — BH4 Nevertheless, only C2'-keto PH4 was appeared as the intermediate during the reduction of PPH4 to BH4 by SPR at pH 8.6 (7) (Fig. 3B) or at pH 7.4 as previously recognized by Smith(3). More recently, however, when we examined the effect of pH on the reduction of PPH4 by SPR (Fig. 3A), we found that pH optimum of PPH4 reduction by SPR was pH 5.5 (while pH 6.2 for BH4 formation), and at pH higher than pH 5.5 only C2'-keto PH4 was found besides the substrate and the product as PH4, while at lower pH than pH 5.5, Cl'-keto PH* besides them was detectable (9) (Fig. 3A). Then the PPH4-reduction was performed at pH 3.8 with varied amounts of SPR for short period (lOmin) to observe complete conversion without decomposition of PH4. In that case, sequential increase and decrease of Cl'-keto PH4 and C2'-keto PH4 and gradual increase of BH4 were observed (9)(Fig. 3C).

(A)

(C)

Seplopterln

Reductase

(mU)

Fig. 3. PPH4-reduction by SPR in the presence of 50 mM DTE. (A)Effect of pH, (B)in Tris-HCl buffer, pH 8.6, (C)in glycine-HCl-NaCl buffer, pH 3.8.

327 Fig. 4. Enzymatic mechanisms for the conversion of PPH4 to BH4 by SPR and lactoyl PH4 synthase.

GTP

I

NHzP3 ^ PK«- Ç - Ç - CH»

Ó 0

pyruvoyl PH«

-I 3

PH«-C Ö

- CHa C '

Itomarlzatlon |

CI'- ke

PH«-C - C - CHj ÒH 0

PH«-C ->0 995

DOM ol SUNOS6a ( mpftg I.p. )

Fig. I

E f f e c t or repeated a d u i n i s t r a l i o n s of SUN0586 on brain t o t a l

biopterin

contents in r a t s 1 p +1 Irt cm' CM

+1 to

CO CM CT) — PO CM* O o +1 +1 +1 +1 +1 •V «o o» m

o

®

CO •V*

(«•' —•* ai

-

,

t-

cm' cm' CM cm' cm' +1 +1 +1 +1 +1 m c- O CM CM OS o o» o e

o> co o e

•V ift

f-

© o* o o +1 +1 +1 +1 « « m m o ¿ ï CM

cm m

*

CM

[

0Í —« CO CM o

o +1 m o CM

?

C0

CM* — CM +1 +1 +1 +1 +1 m o * e * od eo' r-' CM «D co

1 —

O —4 mg/dL) were recalled immediately by phone calls.

A second sample was requested in borderline positive

404 (Phe 2-4 mg/dL) cases and was collected by the sample collecting system or the follow-up system, which consists of public health nurses in every county on this island.

Seventeen

positive cases and 117 borderline cases were detected in the first screening samples.

No positive cases were found in the

99 rechecked second samples for the borderline cases. 17 positive cases were recalled successfully. cases were confirmed.

All the

From them 6 PKU

Typical abnormal urinary PKU metabo-

lites were identified by gas chromatography in all the PKU cases.

Among the 6 PKU cases-detected, two cases (P014 &

P023) were found with defective synthesis of BH^ and one case was caused by DHPR deficiency (Table 1).

Reduced total biop-

terin ratio (B/(B+N)) and positive BH^ oral loading test were found in the two BH^ synthesis deficient cases.

Blood DHPR

activity was not detectable in the DHPR deficient case (P026) and he only partially responded to BH 4 oral loading tests. Up-to-date, no false negative screening result has been identified.

All of the PKU cases detected by our neonatal

screening program were differentially diagnosed and treated accordingly within 37 days after birth (Table 2).

The

classical ones were treated with restricted diet (20-50 mg/kg/day).

BH^ (1.6-2 mg/kg/day) and neurotransmitters,

including L-dopa (10-14 mg/kg/day), 5-hydroxytryptophan (5HTP; 1-2.7 mg/kg/day), and carbidopa (1-1.4 mg/kg/day), replacement therapy were used for the BH^ synthesis deficient cases.

However, the mono-therapy with BH^ was initially used

in the first BH . deficient case (P014) for 8 months and the 4 neurotransmitter replacement was not started until the baby developed involuntary movement and irritable signs at 9 month of age.

For the DHPR deficient case, in addition to BH^ plus

neurotransmitters replacement therapy, the dietary intake of Phe was also under control.

Except very mild mental

development delay (IQ 77) of the first BH^ deficient case (P014), the physical and mental developments of these classical and BH^ deficient PKU cases are apparently normal at the present time (Table 2).

The mild mental impairment of the

405 W H CO O

4H 0) Q

O

C

•

2


i

H

0

(Il

0

(NI m

«

CN 2

(1)

0

•H -P W TD G

•

(pmol/ml) 150

Fetus 1 Fetus 2 Fetus 3 Controls Homozygote

• A

2

o

¿D Neopterin Fig.

1.

Pteridine

• Biopterin

N/B

l e v e l s in a m n i o t i c f l u i d .

( 5

Ratio ; Mean ± SD)

(Fig. 2). The PTPS a c t i v i t y in the e r y t h r o c y t e s of a l l p a r e n t s of the four f e t u s e s was in or near the range f o r a d u l t heterozygotes (Table 2). The a c t i v i t y in f e t u s 2 measured in e r y t h r o c y t e s from cord blood had decreased to 6 . 8 ii U/g Hb a t b i r t h . The a c t i v i t y in i n f a n t 1 had decreased t o 4 . 2 (iU/g Hb a t 9 days of b i r t h . The PTPS a c t i v i t y in f e t u s 3 was 56% of the mean control a d u l t value. Fetus 4 had t h r e e - f o l d the PTPS a c t i v i t y of the control a d u l t s . This a c t i v i t y was on the same level a s the mean control f e t a l value. The PTPS a c t i v i t y of f e t u s e s 1 and 2 a s a percentage of age-matched c o n t r o l s was c l o s e to t h a t of a d u l t s heterozygotes (values f o r p a r e n t s and s u b j e c t s r e p o r t e d in the l i t e r a t u r e ) .

412

o Fetus 1 A Fetus 2 Fetus 3 • Fetus 4

(xU/gHb)

•

60 o

•

1

Mean± SD

50

40-

30-

20

">!•

o

•

*

A

o

Control Fetuses

Cord Blood

Hetero Homo Control Zygotes Zygotes Adults

Fig. 2. PTPS a c t i v i t y in e r y t h r o c y t e s of c o n t r o l f e t u s e s , c o n t r o l cord blood, h e t e r o z y g o t e s . homozygotes, and c o n t r o l adults.

Discussion The p r e n a t a l d i a g n o s i s of t h r e e h e t e r o z y g o t e s of PTPS d e f i ciency and one h e a l t h y i n f a n t was confirmed p o s t n a t a l l y . All

413

of the infants are developing normally. Because the N/B ratio was 2 to 3 times that of the controls, we diagnosed these fetuses as being heterozygotes for PTPS deficiency, and the pregnancies was allowed to continue. In a homozygote with PTPS deficiency described previously (2), the low biopterin level and the high neopterin level gave a very high N/B ratio. The neopterin level and N/B ratio in these fetuses were between those of the homozygote and the controls (Fig. 2). The PTPS activity for these probands, except fetus 4, were 14-22% of the mean, which was the same relationship as between heterozygotes and controls in the adults. However, the activity for these heterozygotes was 40-73% of the mean control adult value (Table 2), and higher than the mean for the heterozygotes from the literature (Fig. 3), because of the high PTPS activity in young erythrocytes (3), abundant in fetal and cord blood. As the proportion of reticulocytes decreased to within the adult range, the enzyme activity in these probands decreased. Even a heterozygote of PTPS deficiency can be diagnosed prenatally by analysis of the pterins in the amniotic fluid.

Acknowledgments This work was supported in part by a Grant-in-aid for Matherand-Child's Health Foundation 1988 and by a Grant for Maternal and Child Health Research from the Ministry of Health and Welfare, Japan, 1988.

References 1. Shintaku, H., A. Niederwieser, W. Leimbacher, H.-Ch. Curtius. 1988. Eur. J. Pediatr. 147, 15. 2. Niederwieser, A., H. Shintaku, Th. Hasler, H.-Ch. Curtius, H. Lehmann, 0. Guardamagna, H. Schmidt. 1986. Eur. J. Pediatr. 145, 176. 3. Shintaku, H., T. Ueda, K. Hatanaka, M. Suzuki, R. Murata, M. Matsumoto, Y. Sawada, G. Isshiki, T. Oura. 1989. In: Pteridines and Biogenic Amines in Neuropsychiatry, Pediatrics, and Immunology, (R.A. Levin, S. Milstien, D.M. Kuhn and H.-Ch. Curtius, eds.). Lakeshore Publishing Company, Grosse Pointe, Michigan, p. 307.

HETEROGENEITY OF TETRAHYDROBIOPTERIN DEFICIENCY: PHENYLALANINE-TETRAHYDROBIOPTERIN LOADING TEST

COMBINED

A.Ponzone, 0.Guardamagna, S.Ferraris, G.B.Ferrerò Istituto di Clinica Pediatrica, Università di Torino, Piazza Polonia 94, 1-10126 Torino, Italia N.Blau, H.-Ch. Curtius, L. Kierat Division of Clinical Chemistry, Department of Pediatrics, University of Zürich, CH-8032 Zürich, Switzerland R.G.H.Cotton Royal Children's Hospital, Parkville, Victoria, Australia

Introduction Three inborn errors of tetrahydrobiopterin (BH4) metabolism are known to cause hyperphenylalaninemia (HPA) with dopamine and serotonin deficiency (1). Recently a new form of atypical phenylketonuria (PKU), primapterinuria, with excretion of 7-sub— stituted pterins in two patients with HPA has been recognized (2). All newborns with HPA should be rescreened for BH4 deficiency, by using different methods of investigation, alternatively based on analysis of pterins in urine, measurement of specific enzyme activities, analysis of phenylalanine (PHE) and tyrosine (TYR) in serum before and after oral BH4 loading (3). The latter test is always reliable in patients with GTPCH and PPH4S deficiency, but it misses some cases of "non-responding" DHPR deficiency, namely those with CRM+ mutation (4). This limitation can be overcome in most cases by increasing the dose of the administered cofactor (5), or even better by performing a combined PHE+BH4 loading test as described below.

Patients andi Methods Two patients with PPH4S deficiency and 3 patients with DHPR deficiency (two of whom ; with the CRM-i- mutation, had previously not responded to the oral loading test with 7.5 mg/Kg BH4) were loaded orally with PHE (100 mg/Kg b.w.) and after 1 hour with BH4 (20 mg/Kg b.w.), while on PHE-restricted diet (basal plasma PHE