This dissertation has been 65-13,266 microfilmed exactly as received

PAGNUCCO, Rinaldc Gene, 1938- SYNTHESIS OF PURINE NUCLEOSIDES OF D-GALACTOSE AND D#.GALACTURONIC ACID DERIVATIVES.

The Ohio State University, Ph.D., 1965 Chemistry, organic

University Microfilms, Inc., Ann Arbor, Michigan SYNTHESIS OF PURINE NUCLEOSIDES OF D-GALACTOSE AND

D-GALACTURONIC ACID DERIVATIVES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Rinaldo Gene Pagnucco, B.S,

The Ohio State University 1965

Approved by

Adviser Department of Chemistry ACKNOWLEDGMENT

I would like to dedicate this dissertation to my mother.

I am grateful to Professor M. L. Wolfrom for suggesting the problems and for the advice given throughout the research period.

I would like to acknowledge the helpful advice and encourage­ ment received from all of the members of Sugar Alley and in particular that from Percy McWain.

The financial assistance provided by the National Science

Foundation and the National Institutes of Health through

Professor Wolfrom is gratefully acknowledged.

ii \riTA

Il Rinaldo Gene Pagnucco» son of Valentino and Regina Pagnucco» was b o m on July 19* 1938» in Alliance» Ohio. After graduating from

Alliance High School in 1958» I entered Mount Union College in Alli­ ance» Ohio» from which I graduated, cum laude» in June 1960» with a

Bachelor of Science Degree. From June to September I960» I worked as a junior chemist for the Pittsburgh Plate Glass Company» Chemical

Division» Barberton, Ohio. In September I96O, I entered Tjie Ohio

State University Graduate School, having received from Professor

M. L. Wolfrom a research fellowship sponsored by the National Science

Foundation. In October I963» I was granted» from Professor M. L.

Wolfrom» a research assistantship under the sponsorship of the Depart­ ment of Health» Education» and Welfare» Public Health Service»

National Institutes of Health. I have accepted a research assistant­ ship from Professor 0. Th. Schmidt at the Chemical Institute of the

University of Heidelberg, Heidelberg, Germany.

iii CONTENTS Page

ACKNOWLEDGMENT...... ii

VITA ...... iii ILLUSTRATIONS...... v i ü

INTRODUCTION ...... 1

STATEMENT OF OBJECTIVES ...... 7

HISTORICAL ...... 8

lo Nucleosides as Natural Products ...... 8

Nucleosides as components of nucleic acids . . . 11

Antibiotics1 nucleosides ...... 15

Nucleosides of the coenzyme nucleotides ...... 17

Other natural sources of nucleosides ...... 21

II. Synthesis of Nucleosides ...... 21

The Fischer-Helferich method ...... 22

The Hilbert-Johnson method ...... 23

The Davoll-Lowy (mercuri) m e t h o d ...... 25

Fusion method ...... 39

Ethyl polyphosphate condensation method ...... 43

Method of fusion of sugar derivatives with trimethylsilyl substituted purines or pyrimidines ...... 44

9y condensation of poly-O-acetyl-glycosyl halide with a nitrogen heterocycle ...... 46

Methods involving cyclization of the aglycon of glycosyl derivatives...... 47

Other m e t h o d s ...... 55

iv CONTENTS (Contd.) Page

DISCUSSION OF RESULTS...... 57

Synthesis of 2,6-Diacetaraido-9-tetra-0-acetyl- P-D-galactofuranosyl)purine (III) ...... 57

Synthesis of 2-acetamido-9-P-D-galacto- fnranosyladenine ( I V ) ...... 59

Synthesis of 2,6-Diamino-9-P-D-galacto« furanosylpurine (V)...... 59

Synthesis of 9-P-D-Galactofuranosyiadenine (Dimorphous) (VI) ...... 6o

Synthesis of Penta-0-aoetyl-l-^-benzyl-l- deoxy-l-ethylthio-D-galactose Aldehydrol ( X ) ...... 62 Synthesis of Penta-O-acetyl-l-O-benzyl-1- bromo-l-deoxy-D-galactose Aldehydrol (XI) ...... 64-

Synthesis of Penta-0-acetyl-l-(9-adenyl picrate)- 1-0-benzyl-l-deoxy-D-galactose Aldehydrol (XII) . . . 64-

Synthesis of Penta-0-aoetyl-l-(9-adenyl)-1- 0-benzyl-l-deoxy-D-galactose Aldehydrol (XIII) ...... 65 Synthesis of Ir^Jldenyl)-1-0-benzyl-l- deoxy-D-galactose Aldehydrol (XIV) ...... 65

Hydrogenolysis of l-(9-Adenyl)-1-0-benzyl- 1-deoxy-D-galactose Aldehydrol (XIV) ...... , 67 Reaction of l-(9-Ader\yl)-1-0-benzyl-1- deoxy-D-galactose Aldehydrol (XIV) with Sodium in Liquid Ammonia ...... 67

Synthesis of 9-(Methyl Tri-g-aoetyl-p-D- galactopyranosyluronate)adenine Picrate (XIX) , , , ...... 68

Synthesis of 9-(Methyl Tri-O-aoetyl-P-D- galactopyranosyluronate)adenine (XX) ...... 69 CONTENTS (Contd.) Page

EXPERIMENTAL...... ?1

Preparation of penta-O-acetyl-P-D- galactofuranose ( i j ...... 71 Preparation of tetra-O-acetyl-P-D- galactofuranosyl,chloride (II) ...... 72

Synthesis of 2 ,6-diacetamido-9-(tetra-0- acetyl-P-D-galactofuranosyl)- purine (III) ...... 73

Synthesis of 2-acetamido-9-P-D-galacto- furanosyladenine (IV) ...... 74

Synthesis of 2;6-diamino-9-P-D-galacto- furanosylpurine (V)...... 75

Synthesis of 9-P-D-galactofuranosyladenine (dimorphous) (VI)...... 7&

Preparation of D-galactose diethyl dithioacetal ( V I I ) ...... 78 / Preparation of penta-O-acetyl-D-galactose diethyl dithioacetal (VIII) ...... 79

Preparation of penta-O-acetyl-l-bromo-1*1- dideoxy-l-ethylthio-D-galactose aldehydrol (IX) ...... 79

Synthesis of penta-O-acetyl-l-O-benzyl-1- deoxy-1-ethyIthio-D-galactose aldehydrol ( X ) ...... 80

Synthesis of pehta-Q-acetyl-l-O-benzyl-1- bromo-l-deoxy-D-galactose aldehydrol ( X I )...... 81

Synthesis of penta-0-acetyl-l-(9-adenyl picrate)-1-0-benzyl-l-deoxy-D- galactose aldehydrol (XII) ...... 81

Synthesis of penta-0-acetyl-l-(9-adenyl)-l- 0-benzyl-l-deoxy-D-galactose aldehydrol ( X I H ) ...... 83 vi CONTENTS (Contd.) Page

Synthesis of l-(9-adenyl)-1-0-benzyl-l- deoxy-D-galactose aldehydrol (XIV) 83

Hydrogenolysis of l-(9-adenyl)-1-0-benzyl-l- deoxy-D-galactose aldehydrol (XIV) ...... 85

Reaction of 1-(9-adenyl)-1-0-benzyl-I- deoxy-D-galactose aldehydrol (XIV) with sodium in liquid ammonia...... 85

Preparation of a-D-galacturonic acid monohydrate (XV)...... 86

Preparation of methyl o-D-galacto- uronate (XVI) ...... 87

Preparation of methyl tetra-O-acetyl- a-D-galactopyranuronate (XVII)...... 88 Preparation of methyl tri-O-acetyl-cu D-galactopyranosyluronate bromide (XVIII)...... 89

Synthesis of 9-(methyl tri-O-acetyl-P-D- galactopyranosylurona te)adenine picrate (XIX)...... 89

Synthesis of 9-(methyl t ri-O-acetyl-P-D- galactopyranosyIuronate)adenine (XX) ...... 90

SUMMARY...... 92

BIBLIOGRAPHY...... 95

vii ILLUSTRATIONS

Figure Page

1 Inosinic A c i d ...... 10

2 Nucleosides in Ribonucleic Acid ...... 12

3 Nucleosides in Deoxyribonucleic Acid ...... • • • 13

4 Puroinycin...... l6

5 Adenosine 5 '-Triphosphate ...... 18

6 Uridine Diphosphate Glucose ..... 20

7 Synthesis of 2,8-Dic)t’iloro-9-P-D-glucopyranosyladenine . . 22

8 Lactam-Lactim Tautomers ...... 23

9 Formation of C-1 — C-2-trans Nucleosides...... » 28

10 Synthesis of 9-(P-D-Glucopyranuronamide)adenine ...... 30

11 Formation and Rearrangement of an 0-Glycoside to the Corresponding Nucleoside ...... 38 12 Synthesis of an Unsaturated Sugar Nucleoside 8y a Modified Fusion Technique...... 42

13 Trimethylsilyl Derivatives of some Purines and I^rimidines...... 4-3

14 Adenine Nucleoside from a D-Mannosylamine Derivative . . 4-9

15 3-Cyanouracil Nucleoside from a Glycosylamine ...... 50

16 6-Amino-l-P-D-glucopyranosyluracil from Tetra-O-acetyl- D-glucopyranosylurea...... T ...... 52

17 Synthesis of a Thymine Nucleoside from a Glycosylurea ...... 53

18 9-P-D-XylopyranosylJcanthine from 4-»5-Dimethoxycarbonyl- l-(tri-0-acetyl-D-xylopyranosyl)imidazole 54-

Vlll ILLUSTRATIONS (Contd.)

Figure Page

19 Synthesis of 2,6-Diamino-9-P-D-galactofuranosyl- purine ( V ) ...... 6o

20 Synthesis of 9-P-D-Galactofuranosyladenine (VI) ..... ^2

21 Synthesis of 1-(9-Adenyl)-1-0-benzyl-l-deoxy- D-galactose Aldehydrol (XIV) ...... 66

22 Synthesis of 9-(Methyl tri-O-acetyl-P-D- galactopyranosyluronate)adenine (XX) ...... yo

TABLE

Table

1 Hexofuranosyl Nucleosides Prepared by the Mercuri Method ...... 32

IX INTRODUCTION

The term nucleoside» as first proposed by Levene and Jacobs (1)»

(1) P. A, Levene and W. A. Jacobs» Ber.» kZt 2474 (1909)* was used to describe the carbohydrate derivatives of purines and

pyrimidines obtained on nucleic acid hydrolysis. In recent times

it has been expanded to include all glycosyl derivatives of purines

and pyrimidines.

Interest in the synthesis of nucleosides began with the discovery

of the purine nucleoside guanosine in I885 by Schultze(2). Interest

(2) E, Schultze and E, Bossard» Z, Physiol, Ghem,» 2» 420

(1885).

in nucleosides was further kindled by the realization by Levene and

Jacobs (1) that nucleosides were components of the important nucleic

acids. Phosphate esters of the nucleosides, the nucleotides* con­

stitute the polymeric units of the nucleic acids. The nucleic acids

have been associated with such important biological processes as

growth, cell replication, and the transmission of genetic information.

It became necessary to synthesize nucleosides in order that the stxuc-

ture of the nucleic acid components could be proven.

Although much of the interest in nucleosides has stemmed from 2 the importance of the nucleic acids» the discovery of non-nucleic acid nucleosides in entities such as antibiotics (3-7) and co-

(3) K. G. Cunningham* S, A. Hutchinson* W, Manson* and F. S.

Spring* J. Chem. Soc.* 2299 (1931)»

(4) C. W. Waller* P. W. Fryth* B. L. Hutchings* and J. H.

Williams, J. Am. Chem. Soc.* 21* 2023 (1933)»

(3) N. Lufgren and B. Luning* Acta Chem. Scand.* 2» 225 (1933); N. Lufgren* B. Lining* and H. Hedstrora* ibid.* 8* 670 (193^)»

(6) H. Yunsten* K. Ohkuma* and Y. Ishii* J. Antibiotics (Tokyo)*

Ser. A.* 2* 193 (1936).

(7) E. H. Flynn* J. W. Hinman* E. L. Caron* and D. 0. Woolf *

J. Am. Chem. Soc.* 21 * 3867 (1933)»

enzymes (8) has resulted in much research on nucleoside synthesis.

(8) J. Baddiley in "The Nucleic Acids*" Vol. 1* E. Chargaff

and J. N. Davidson* ed.* Academic Press* New York* N. Y.* 1933»

pp. 177-187»

Today nucleosides have taken on a new light since the discovery

that the basic component of some nucleosides * the purines * have

proved effective for the control of neoplastic disease (9) such as

(9) C. P. Rhoads* consulting ed.* Conference on 6-Mercapto-

purine* Ann. New York Acad. Soi.* ^ * Art. 2* I83 (193^)» 3 cancer. After this discovery many workers took up the task of synthesizing numerous purine derivatives to be tested as antagonists.

It would only be natural that one class of purine derivatives, the nucleosides, should also be extensively investigated. The potential anti-cancer character of nucleosides was indeed a factor in the under­ taking of the work described herein.

In addition to their preparation by enzymic or chemical hydroly­ sis of nucleotides or polynucleotides (10), nucleosides may also be

(10) A. M. Michelson, "The Chemistry of Nucleosides and Nucleo­ tides," Academic Press, Now York, N« Y., 1963» P« 4. synthesized from their component parts, the heterocyclic base and the carbohydrate. This work shall concern itself with the latter

synthetic method.

The first synthetic nucleoside, 7-p-D-glucopyranosyltheophylline,

was prepared by E. Fischer and B. Helferich (11). The method used

(11) E. Fischer and B. Helferich, Chem. Ber., 210 (1914).

by these workers is (in a modified form) still the most widely used

for nucleoside synthesis. The silver salt of theophylline was con­

densed in hot xylene with tetra-O-acetyl-o-D-gluoopyranosyl bromide

and the product was deacetylated to give the desired nucleoside. Al­

though this method was successful with purine derivatives, when attempts

were made to prepare pyrimidine nucleosides using this procedure no

nucleoside was isolated (12). Hilbert and Johnson (13) successfully 44

(12) P» A. Levene and H. Sobotka» J. Biol. Chem,; 469

(1925). (13) G. E. Hilbert and B. Johnson; J. Am. Chem. Soc.; 52;

4489 (1930). modified the procedure by alkylating the hydroxyl groups on the pyrimidine ring in one of the tautomeric forms.

Davoll and Lowy (14) introduced two important modifications

(14) J. Davoll and B. A. Lowy; J. Am. Chem. Soc.; 22» 1&30

(1951). in the Fischer-Helferich procedure. Any basic amino group on the purine or pyrimidine ring was blocked with an acyl group and the mono-

chloromercuri (or mercuri) derivative of the purine (pyrimidine)

was used in place of the silver salt. This method has become the most

widely used procedure for nucleoside synthesis and is the one suc­

cessfully used in the present work. Newer synthetic schemes for

nucleoside synthesis will be discussed in the following section.

A United States patent describes the first synthetic purine

nucleoside of a hexuronic acid derivative (15). Recently this

(15) G. H. Hitchings and I. Goodman; U. S. Pat. 3»0?4;929

(1963); C. A.; jg; 739 (1963).

laboratory (16) reported the synthesis of the only other nucleoside (16) M. L* Wolfrom and P. McWain> Abstracts Papers Am. Chem.

Soc. Meeting, 148, 24D (1964). of this type, 9-(g-D-glucouronamide)adenine. Several hexuronic acid nucleosides of pyrimidines have also been reported (17,18).

(17) I. Goodman, Federation Proc., 12, 210 (1953).

(18) T. Kishikawa and H. Yuki, Ghem. Pharm. Bull. (Tokyo),

12, 1259 (1964).

The first nucleosides of a hexose in the aldehydo configuration, the 1-epimers of l-(9-adenyl)-l-deoxy-l-0-methyl-D-galactose alde­ hydrol (19)» were prepared in this laboratory. Several nucleosides

(19) M. L, Wolfrom, A. B. Foster, P. McWain, and A, Thompson,

J. Org. Chem., 26, 3095 (1961).

of this variety have since been reported (20,21).

(20) M. L. Wolfrom, P. McWain, and A. Thompson, J. Org. Chem.,,

^ » 3549 (1962).

(21) M. L. Wolfrom, H. G. Garg, and D. Horton, ibid., 29,

3280 (1964); i ^ . , ]0, 0000 (1965).

Todd and co-workers (22) synthesized a derivative of a hexo-

(22) K. J. M. Andrews, G. W. Kenner, and A. R. Todd, J. Chem.

Soc., 2302 (1949). 6 furanosyl purine nucleoside but did not give details on the nucleoside itself. The first 9-hexofuranosylpurine nucleoside was described by Baker and co-workers (23). Other hexofuranosyl nucleosides have since

(23) B. R, Baker, Kathleen Hewson, H. Jeanette Thomas, and

J. A. Johnson» Jr., J. Org. Chem.» 95^ (1937). been reported. STATEMENT OF OBJECTIVES

The work described in this dissertation is concerned with the synthesis of purine nucleosides of D-galactose and D-galacturonic acid derivatives. These compounds are of interest not only because

of the chemistry involved but also because of their potential value

as cancer chemotheraputic agents. The following problems were undertaken;

1) The synthesis of the 6-aminopurine (adenine) and 2,6-

diaminopurine nucleosides of D-galactofuranose.

2) The synthesis of the adenine nucleoside of 1-^-benzyl-

l-deoxy-D-galaotose aldehydrol.

3) The conversion of the carbohydrate moiety of the

nucleoside 1-(9-adenyl)-1-0-benzyl-l-deoxy-D-galactose aldehydrol from the aldehydo form to the cyclic furanose form.

4) The synthesis of the adenine nucleosides of D-galact­

uronic acid derivatives. HISTORICAL

I. Nucleosides as Natural Products

Nucleoside history may be said to begin with the isolation of the nucleotide (a phosphate ester of a nucleoside) inosinic acid from beef extract by Liebig (24) in 1847, However, it was more than fifty

(24) J. von Liebig, Ann., 25? (1847).

years later before the complete structure of this nucleotide was determined.

The following description of the elucidation of the structure

of inosinic acid will serve to illustrate the general method of

initial proof of structure of a number of the nucleotides found in

in the nucleic acids.

Inosinic acid on acid hydrolysis (25) yielded a carbohydrate

(25) F. Haiser, Monatsh., l6, 190 (1895).

derivative and a heterocyclic nitrogen base. Since the structure

of many of the nitrogenous heterocyclic compounds were known at this

time, it was relatively simple for Haiser (25) to show that the nitro­

gen base of inosinic acid was hypoxanthine by analysis and comparison

with the known compound. He also showed by analysis that inosinic

acid contained a phosphoric acid residue. Later Levene and Jacobs (26)

8 (26) P, A. Levene and W. A. Jacobs* Ber., 41, 2703 (1908); lbld.% 42, 1198 (1909); Ibld., 44, 746 (1911). isolated the carbohydrate component and showed it to be D-ribose ty comparison with an authentic sample. At the same time they showed that the phosphoid-c acid residue was attached to the C-5 of D-ribose by oxidation of the D-ribose phosphate obtained from hydrolysis of inosinic acid. The oxidation produced D-ribonic acid phosphate and not a phosphate of trihydroxyglutaric acid which would be expected if the phosphate were on a hydroxyl group other than the C-5 hydroxyl group. That the ring structure of D-ribose was furanose was shown ly méthylation and oxidation studies carried out on adenosine (27)

(27) P. A. Levene and R. S. Tipson, J. Biol. Ghem., 809

(1932). which can readily be converted to inosine without affecting the

carbohydrate moiety. Méthylation of adenosine followed by acid

hydrolysis yielded 2,3,5-tri-O-methyl-D-ribofuranose and not the

2,3,4-tri-O-methyl derivative. Nitric acid oxidation of this tri-

0-methyl derivative gave dimethoxysuccinic acid which would be ex­

pected if the derivative contained the furanose ring* Since ino­

sinic acid did not reduce Fehling*s solution (25), the hypoxanthine

must be attached to the glycosidic carbon atom of the sugar. The

problem of which atom of the nitrogen base was linked to the glyco­

sidic carbon was not solved correctly (28) until 1936. 3y comparison 10

(28) In 1923 Levene [P* A. Levene* J. Biol. Chem.» 437

(1923)] reported that the 7-position of hypoxanthine was linked to the glycosidic carbon atom. This was later shown to be incorrect. of the ultraviolet spectra of various substituted hypoxanthines with that of inosine (inosinic acid with the phosphoric acid moiety removed), Gulland and Holiday (29) showed that the 9-position of

(29 ) J. M. Gulland and E. R. Holiday, J. Chem. Soc., 765

(1936). hypoxanthine was linked to the glycosidic carbon atom. The glyco­ sidic linkage was shown to be P-D- ly x-ray crystallographic evidence

(30). The structure of inosinic acid is shown in Figure 1.

NH

O (HO),POCH z ^

OH OH

Fig. 1— Inosinic Acid 11

(30) s, Furberg» Acta Chem. Scand,, 4, 751 (1950)* In this paper adenosine was shown to be of the P-D configuration. Since adenosine and inosine are interconvertible without affecting the glycosidic site» the proof is valid for inosinic acid as well.

Nucleosides as components of nucleic acids. After the studies on the compound, guanosine, from nucleic acid, the term nucleoside was used by Levene and Jacobs (1) to define the glycosyl purine and pyrimidine derivatives obtained on hydrolysis of nucleic acids.

Since the beginning of this century numerous nucleosides (in the form of their polymeric phosphate esters) have been found to be present in the nucleic acids. The vast majority of the nucleic acids are composed of eight major nucleosides with minor amounts of other nucleosides being present in some cases.

Ribonucleic acid (RNA) present in the cytoplasm of the cell, has been found to contain the following nucleosides: 1-P-D-ribofurano- syluracil (uridine), 1-P-D-ribofuranosylcytosine (cytidine), 9-P-D- ribofuranosyladenine (adenosine) and 9-P-D-ribofuranosylguanine

(guanosine). Figure 2 depicts the structures of the nucleosides of

ribonucleic acid. 12

NH, ïji

CH^OH

OH OH OH OH Uridine Cytidine

NH, NH

NH

CHgOH

OH OH OH OH Adenosine Guanosine

Fig, 2.--Nucleosides in Ribonucleic Acid

The second large class of nucleic acids, deoxyribonucleic acid

(DNA), is found in the nuoleous of the cell and is composed of the nucleosides 1-(2-deoxy-B-D-erythro-pentofuranosyl)thymine, (thymidine),

1-(2-deoxy-P-D-erythro-pentofuranosyl)cytosine (2'-deoxycytidine),

9-(2-deoxy-P-D-erythro-pentofuranosyl)adenine, (2'-deoxyadenosine), 13 and 9-(2-deoxy-P-D-erythro-pentofuranosyl)guanlnei (2’-deoxyguanosine). Figure 3 shows the nucleosides of deoxyribonucleic acid. NH; O _ A HN o L o l v N - J N C H ^ O H

HO Thymidine 2 *-Deoxycytidine

NH NH

2

HO HO

2'-Deoxyadenosine 2'-Deoxyguanos ine

Fig. 3•“-Nucleosides in Deoxyribonucleic Acid

The structure of these nucleosides has been elucidated since

1900 in a manner similar to that used in the case of inosinic acid

described above. The structure of all eight has been further proven

chemical synthesis (31-39)*

(31) 0. A. Howard, B. Lythgoe» and A* R. Todd, J. Chem. Soc.,

1052 (1947). 14

(32) J. Davoll, B. Lythgoe, and A. R, Todd, ibid., I685 (1948).

(33) J' Davoll, B. lythgoe, and A. R. Todd, ibid., 96? (1948).

(34) J. J. Fox, N. Yung, I. Wempen, and I. L. Doerr, J. Am.

Chem. Soc., 22» 506o (1957). (35) D. M. Brown, D. B. Parihar, C. B. Reese, and A. R. Todd,

J. Chem. Soc., 3035 (1958).

(36) C. D. Anderson, L. Goodman, and B. R. Baker, J. Am. Chem.

Soc., 80, 6453 (1958).

(37) G. Shaw and R. N. Warrener, Proc. Chem. Soc., 8l (1958).

(38) M. Hoffer, R. Duschinsky, J. J. Fox, and N. C. Yung, J. Am.

Chem. Soc., 81, 4112 (1959).

(39) R. K. Ness and H. G. Fletcher, Jr., ibid., 81, 4?52 (1959).

Until now no sugars other than D-ribose and 2-deoxy-D-erythro- pentose have been found in these nucleic acids. However, several pyrimidine and purine derivatives other than those mentioned above have been found in small amounts in various nucleic acids. Among

these are 5-methylcytosine (40), N^-methyladenine (41), 2-methyl-

(40) R. D. Hotchkiss, J. Biol. Chem., 1%^, 315 (1948).

(41) D. B. Dunn and J. D. Smith, Nature, 125» 336 (1955);

Biochem. J., 6 0, xvii (1955); ibid., 62? (1958).

adenine (42), N,^,N^-dimethyladenine (42), 1-methylguanine (43*44)

(42) J. W. Littlefield and D. B. Dunn, Nature, 181, 254 (1958);

Biochem, J., 6Q» vii (1958); ibid., 20, 642 (1958). 15 (43) M. Adler, B. Weissman» and A. B. Gutman, J. Biol. Chem.,

230, 717 (1958). (44) D. B. Dunn and J. D. Smith, Proc. Intern. Congr, Biochem.,

4th Congr. Vienna, 1958, preprint No. 10 (1959)♦ and N^-methylguanine (43,44).

Antibiotical nucleosides. Another important group of naturally occurring nucleosides has been shown to possess antibiotic properties.

Cordycepin (3), active against avian tubercle bacillus, has been shown to be adenine (9-position) linked to the carbohydrate cordyce- pose [3-deoxy-3,3-C-bis(hydroxymethyl)-D-glyceraldehyde] (43),

(45) H. R. Bentley, K. G. Cunningham, and F. S. Spring, J. Chem.

Soc., 2301 (1951),

Nebularin, isolated from a mushroom (5), has shown activity against mycobacteria and mouse Sarcoma I80. Preliminary investi­ gations (5) suggested that nebularin was 9-3-D-ribofuranosylpurine.

Later, this assumption was proven to be correct by chemical

synthesis (46).

(46) G. B. Brown and V, S. Weliky, J. Biol. Chem., 204, 1019

(1953),

Nucleocidin, isolated from a Streptomyces, shows a broad spectrum

of antibacterial properties. A partial structure has been reported

(47)0 This structure includes, besides adenine and a reducing sugar, a sulfamic acid ester group. 16

(4?) G. W. Waller, J. B, Patrick, W. Fulmor, and W. E. Meyer,

J. Am. Chem. Soc., 22» 1011 (1957).

Puromycinthe first amino sugar containing nucleoside to be found in nature, is another broad-spectrum antibiotic nucleoside.

It has been isolated from Streptomyces alboniger (4). The structure was shown to be 9-[3-dsoxy-3-CC£“L-phenylalanyl)amino]-P-D-ribo- furanosyl]-^>N^-dimethylaminopurine (see Figure 4) by Baker and co-workers (48)•

(48) B. R. Baker, R. E. Schaub, and J. H. Williams* J. Am. Chem.

Soc., 22* 7 (1955)} B. R. Baker, R. E. Schaub* J. P. Joseph, and

J. H. Williams, ibid.. 12 (1955).

NMe-

CH OH

NHz O H.CO CHgCH-CHN OH

Fig. 4--Puromycin 17 Other antibiotic nucleosides such as Amicetin (?)» Angustmycin

A (49) and Psicofuranine (50»51) have been isolated and their structures

(49) H. J. Yflngsten, J. Antibiotics (Tokyo)» U A, 77, 79, 233

(1958).

(50) H. J. Yungsten, ibid., IIA, 224 (1958).

(51) W. Schroeder and H, Hoeksema, J. Am. Chem. Soc., 17&7

(1959). investigated•

Nucleosides of the coenzyme nucleotides. The coenzyme nucleo­ tides have been defined (52) as the "anhydrides of a nucleoside

(52) A. M. Michelson, "The Chemistry of Nucleosides and Nucleo­ tides," Academic Press, New York, N. Y., I963, p. 10.

5'-phosphate and another acid which may be pyrophosphoric acid, phos­ phoric acid, and mono-esters thereof, carboxylic acids, amino acids

and peptides» or sulfuric acid."

An extensive number of coenzymes have been isolated and investi­

gated but the aims of this work permit only a brief description and

listing of a small number of the common ones.

One group of coenzymes possess as a part of their structure the

nucleoside 9-P-D-ribofuranosyladenine (adenosine). Probably the

most important of these is adenosine 5'-triphosphate (see Figure 5)»

which is a key intermediate in the transfer of energy in living 18

N H g

o o o (HO),POP- OP -OCH, ^ 1 1 ^ O H O H

O H O H

Fig» 5•-“Adenosine ^'-Triphosphate organisms. It was first isolated from muscle extracts (53»5^)« 9y

(53) K. Lohmann» Naturwiss., 1^» 624 (1929)»

(54) C. H. Fiske and Y, Subbarow, Science» 20, 381 (1929)*

1932 its structure had been elucidated (55)•

(55) K, Lohmann, Biochem. Z., 254, 381 (1932)*

Nicotinamide adenine dinucleotide (56,57); flavine adenine

(56) M. R. Atkinson and R. K. Morton, Nature, 188, 58 (I960).

(57) H« von Euler, H. Albers, and F. Schlenk, Z. Physiol.

Chem., 128, 236 (1931): ibid., 2]], 95 (1935). 19 dinucleotide (58)» and the ^'-phosphate adenine nucleotide, coenzyme

(58) 0. Warburg and W. Christian, Biochem. Z., 298, 150 (1938).

A (59) constitute three additional important adenosine containing

(59) F. Lipman and N. 0. Kaplan, J. Biol. Chem., 162,

(1946). coenzymes.

Coenzymes containing the nucleoside uridine have been shown to be important in such processes as sugar metabolism, the biosynthesis of polysaccharides and cell wall synthesis. Three of these are uridine diphosphate glucose (60) (see Fig. 6) uridine diphosphate

(60) R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Paladini,

J. Biol. Chem., 184, 333 (1950).

glucuronic acid (6I) and uridine diphosphate N-acetylglucosamine

(61) Q. J. Dutton, Biochem. J., 693 (195&).

(2-acetamido-2-deoxy-D-glucopyranose) (62)•

(62) E. Cabib, L. F. Leloir, and C. E. Cardini, J. Biol. Chem.»

203, 1055 (1953). 20

o HN

C H ^ O H oLN O O OP-OP-OCH2 . H O H O H O OH

7 O H O H

Fig. 6.— Uridine Diphosphate Glucose

Other coenzymes containing the nucleosides cytidine (63)»

(63) D« Q. Comb, F, Shimizu, and S. Roseman, J. Am. Chem. Soc.»

81, 5513 (1959). guanosine (64), 2 '-deoxycytidine (65) and thymidine (66) have been

(64) E. Cabib and L. F. Leloir, J. Biol. Chem., 206, 779

(1954).

(65) Y. Sugino, J. Am. Chem. Soc., 22» 50?4 (1957).

(66) J. L. Strominger and S. S. Scott, Biochim. et Biophys.

Acta, 552 (1959). isolated. 21

Other natural sources of nucleosides» Other nucleosides have been isolated from yeasts (6?), sponges (68), liver (69), beef

(67) P. A. Levene and H. J. Sobotka, J, Biol. Chem., 551 ’

C1925).

(68) W. Bergmann and R. J. Feeney, J. Am. Chem. Soc., ^2, 2809

(1950); J. Org. Chem., ]^, 981 (1951)» W. Bergmann and D. C. Burke,

ibid., 1501 (1955).

(69) W. K. Joklik, Biochim., Biophys. Acta, 211 (1956).

blood (70), pseudovitamin B 12 (71), and the croton bean (72).

(70) A. R. Davis, E» B. Newton, and S. R. Benedict, J. Biol.

Chem., 5 4, 595 (1922).

(71) W. Friedrich and K. Bemhaur, Angew Chem., 68» 58O (1956); Chem. Ber., 8g, 2507 (1956).

(72) E. Cherbuliez and K. Bernhard, Helv. Chim. Acta, 15,

464 (1932).

II. Synthesis of Nucleosides

Although much information can be obtained concerning the structure

of nucleosides by dégradative, absorption spectral. X-ray crystallo-

graphic, analytical, and similar methods, final proof of structure

rests upon chemical synthesis. Therefore it became necessary to de­

velop reaction sequences for nucleoside synthesis. 22;

The Fischer-Helferich method. This method (11) for the synthe­ sis of nucleosides was used for the preparation of the first nucleo­ sides and is still (in modified form) the most widely used method,

By condensing the silver salt of an appropriate purine (for example theophylline) with a poly-O-acylglycosyl halide such as tetra-0- aoetyl-a-D-glucopyranosyl bromide, Fischer and Helferich were able to obtain, after déacylation,, the nucleoside 7-p-D-glucopyranosyltheo- phylline. In a similar manner these workers were able to prepare the

9-purinyl nucleoside (the 9-purinyl derived nucleosides being by far the most prevalent type of the naturally occurring purine nucleosides)

2,8-dichloro-9-P-D-glucopyranosyladenine (see Fig. ?)•

C H g O A c Cl

Deacetyl- *Ag ation Cl- O A c OH O A c OH

Fig. 7.--Synthesis of 2,8-Dichloro-9-P-D-glucopyranosyladenine

As recently as 1955 this method was used to prepare the first

purine nucleosid^ of a hexuronic acid derivative (15).

However, in attempting to prepare pyrimidine nucleosides using

this procedure, Fischer and later Levene and Sobotka (12) isolated

only substances which were readily hydrolyzed to the pyrimidine and 23 sugar with dilute acid. Since this is a property not common to pyrimidine nucleosides it was suggested that these were 0-glycosides rather than nucleosides. It was considered that these glycosides were the result of the lactam-lactim tautomérisation (see Fig. 8) of the pyrimidine silver salt (12,13).

OH

HN

O' N X Ag

Fig. 8.— Lactam-Lacttm Tautomers

The Hilbert-Johnson method. Later Hilbert and Johnson (13) overcame the above mentioned difficulty in preparing pyrimidine nucleosides by using pyrimidines not capable of this tautomérisation.

They condensed 2,4-dimethoxypyrimidine with tetra-O-acetyl-a-D-gluco- pyranosyl bromide and obtained the blocked nucleoside which on proper

treatment yielded 1-p-D-glucopyranosyluracil and 1-p-D-glucopyranosyl-

cytosine.

Todd and co-workers (31), using the Hilbert-Johnson method, were

the first to synthesise a naturally occurring nucleoside, cytidine.

Using a modified Fischer-Helferich procedure, the same workers were

able to synthesise two other naturally occurring nucleosides, adenosine

(33) and guanosine (32). 24

The Hilbert-Johnson procedure has been applied to the synthesis of 1-glycosyl pyrimidines with such aglycons as uracil (73-76)» thy-

(73) G. E. Hilbert, J. Am. Chem. Soc., jg, 330 (1937).

(74) T. H. Mills, i W . , 22» 2565 (1957). (75) M. Prystas, J. Farkas, and F. Sorm, Collection Czech.

Chem. Commun., 28, 3140 (19^3).

(76) M. Prystas and F. Sorm, ibid., 29, 121 (1964).

mine (74,75*77-79)» cytosine (80»8l), 5-methyluracil (77)» and ^ » f ^ _

(77) D. W. Visser, I. Goodman, and K. Dittmer, J. Am. Chem.

Soc., 20» 1926 (1948).

(78) W. W. Zorbach and S. Saeki» J. Org. Chem.» ^ » 2018 (1964).

(79) J. Farkas, L. Kaplan, and J. J. Fox, ibid., 29, 1469 (1964).

(80) Q. £. Hilbert and F. J. Jansen, J. Am. Chem. Soc.,

60 (1936).

(81) J. J. Fox and I. Goodman, ibid., 79» 3256 (1951).

dimethylcytosine (74).

The most commonly used sugars have been D-ribose (77»79)» 2-deoxy-

D-erythro-pentose (75»76), D-glucose (74,77*80,81), D-galactose (77*81),

D^xylose (.73*81)» and D- and L-arabinose (73*77*81)•

This procedure was reportedly used to prepare the first hexuronic

acid nucleosides but no details are given (17). Recently this method

was used for the preparation of the uracil and cytosine nucleosides

of D-glucuronic acid and its derivatives (18). 25 Pyrimidine nucleosides of the disaccharides lactose and cello- biose (82) and of the amino sugar 2-amino-2-deoxy-D-glucose (83,84)

(82) P. Blumbergs and C. L, Stevens, Abstracts Papers Am. Chem.

Soc. Meeting, 122» 30N (I96I).

(83) C. L. Stevens and K. Nagarajan, ibid., 135, 33-0 (1959)»

(84) C. L. Stevens and K. Nagarajan, J. Med. Pharm. Chem.,

1124 (1962). have been prepared utilizing this method. The Davoll-Lowy (Mercuri) method. Davoll and Lowy (14) greatly increased the utility of the Fischer-Helferich procedure by intro­ ducing two modifications. First, any basic amino groups on the purine or pyrimidine ring were blocked with acyl groups and second, they utilized the monochloromercuri or mercuri derivatives of the hetero­ cyclic base in place of the silver salt. The mercuri procedure has become the most widely used method for nucleoside synthesis and a vast number have been prepared using this method.

The mercuri procedure gives, in the case of pyrimidine nucleo­

sides, the 1-glycosylpyrimidines. In the synthesis of purine nucleo­

sides, the nucleoside isolated is nearly always the 9-glycosylpurine.

However, theophylline gives 7-glycosyltheophyllines and in a few

other cases (85-88) mixtures of the 7- and 9-glycosylpurines were

(85) B. R. Baker, J. P. Joseph, R. E. Schaub, and J. H. Williams,

J. Org. Chem., 12, 178O (1954). 26

(86) B. R. Baker and R. E. Schaub» J. Am. Chem. Soc.» 77» 5900

(1955). (87) B. R. Baker» J. P. Joseph» and R. E. Schaub» jbjd.» 77»

5905 (1955).

(88) H. M. Kissman» C. Pidacks» and B. R. Baker» ibid.» 77»

18 (1955). reported. Recently Robins (89) has restudied the reactions which were

(89) L. B. Townsend» R. K. Robins» R. N. Loeppky» and N. J.

Leonard» ibid.» 86» 5320 (1964).

said to give the 7-purine derivatives (85-88) and has shown these to

be the 3-purine derivatives (by spectral analysis and synthesis)

rather than the 7-derivatives,

By blocking the 3-position of the purine with a benzyl group

it was possible to prepare» using the mercuri method» 7-hypoxanthine-

(90) and 7-adenyl-nucleosides (91)»

(90) J. A. Montgomery and H. J. Thomas » J. Org. Chem.» 28»

2305 (1963). (91) J. A. Montgomery and H. J. Thomas » J. Am. Chem. Soc.»

85K 2672 (1963)»

The assignment of the glycosidic configuration to the nucleo­

sides prepared by the mercuri or the earlier Fischer-Helferich and

Hilbert-Johnson methods is based on the trans-rule of Tipson (92) 2 7

(92) R. S. Tipson, J. Biol. Chem., 1^0, 55 (1939). as extended by Baker (93)« This rule states that "condensation of

(93) B. R. Baker, Ciba Foundation Symposium, Chem. and Biol, of Purines, 120 (1957). a heavy-raetal salt of a purine or pyrimidine with an aoylated glycosyl halide will form a nucleoside with a C-1 — C-2-trans-confiKuration in the sugar moisty regardless of the original configuration at C-1 -

C-2."

This stereospecificity might be accounted for in at least two ways. In the case of C-1 — C-2-cis sugar halides attack by the purine (pyrimidine) from the side opposite the halide could occur to give the C-1 — C-2-trans..eonfiguration. In the case of the C-1 —

C-2-trans sugar halide either the halide might be anomerized to the cis-configuration by attack of halide ion or double Walden inversion

could occur by neighboring participation by the C-2 group through

the ortho ester ion followed by attack of the purine (pyrimidine) to

again give the C-1 — C-2-trans-configurâtion (93*94) (see Fig. 9).

(94) B. R, Baker, J. P. Joseph, R. E. Schaub, and J. H. Williams,

J. Org. Chem., !£, 1786 (1954). 28

— - — O - ■ o -X

-CrO I R R

Purine ^ (pyrimidine)

O Purine (pyrimidine) Purine____ (Pyrimidine y

C =o S R Fig» 9*— Formation of C-1 — C-2-trans Nucleosides

Thus sugars such as D-galactose» D-xylose» and D-ribose give

P-D-nucleosides» whereas D- and L-arabinose yield the o-D(and L)- nucleosides when used in the mercuri procedure. At the same time one would expect mixtures of a- and P- anomers from a 2-deoxysugar or from a sugar with a non-participating group at 0-2. Fox and co­ workers (38) first showed this to be true. In the synthesis of thymidine [1-(2-deoxy-P-D-erythro-pentofuranosylttwnine1 and 2’-deoxy-

cytidine by means of the mercuri procedure a mixture of a-D- and

P-D-anomers resulted in each case.

Although Baker and co-workers (95»96) have found exceptions to

(95) B. R, Baker, R. E. Schaub, and H. M. Kissman, J. Am. Chem.

Soc., 22» 5911 (1955) 2 % (96) H. M. Kissman and B. R. Baker, ibid., 22» 553*^ (1957). the trans rule, the C-1 — C-2-cis anomers, if formed, are found only in minor proportions.

Because of the large number and diversity of the nucleosides prepared by this method and because of the nature of this work, only a limited listing of nucleosides prepared ty the mercuri procedure will be given. Fox and Werapen (97)» Montgomery and Thomas (98), and

(97) J. J. Fox and I. Wempen, Advan. Carbohydrate Chem., 14,

283 (1959). (98) J. A. Montgomery and H. J. Thomas, ibid., 17, 301 (1962).

Michelson (99) have reviewed this aspect of nucleoside synthesis and

(99) A. M. Michelson, "The Chemistry of Nucleosides and Nucleo­

tides," Academic Press, New York, N. Y., I963» Chap. 2,

two of these (97»98) have tabulated large numbers of nucleosides pre­

pared by this as well as other methods.

The only reported nucleoside of a hexuronic acid derivative

prepared using the mercuri procedure is 9-(P~D-glucopyranuronamide)-

adenine which was prepared in these laboratories (16). One hexuronic

acid-containing nucleoside was prepared using the early Fischer-

Helferich procedure (15). The only other nucleosides of uronic acids

reported were prepared either by oxidation of the preformed nucleoside

(100-105) or by the Hilbert-Johnson method (17*18). 30 N H A c N N O C O M e N H A c ÔOM 1) picrate formation 2 ) picric acid + removal O Ac HgCl O A c O H O A c O A c

ÔOM, CNH

Ad OH O A c

OH OH O A c

Fig. 10.--Synthesis of 9-(P-D-Glucopyranuronamide)adenine

(100) P. A. Levene and F. B. La Forge» Ber.» ^ » 6o8 (1912).

(101) A. S. Jones and A. R. Williamson» Chem. Ind. (London)»

1624 (i960).

(102) I. Wempen» I. L. Itoerr» L. Kaplan» and J. J. Fox» J. Am. Chem. Soc.» 82, 1624 (l96o).

(103) C. B. Reese» K. Schofield» R. Shapiro» and Lord Todd»

Froc. Chem. Soc., 290 (I960).

(104) J. P. Vizsolyi and G. M. Tener» Chem. Ind. (London)»

263 (1962). 31 (105) G* P» Moss» C. B. Reese» K. Schofield» R. Shapiro» and

Lord Todd» J. Chem. Soc.» 1Ï49 (19&3)*

Nucleosides containing the sugar moiety in the acylic configura­ tion have been synthesized ty the mercuri procedure. All such nucleo­ sides to date have been reported from these laboratories. The first of these reported were a pair of anomers of l-(9-adenyl)-l-deoxy-l-0- methyl-D-galactose aldehydrol (19). The others of this type which have been reported are l-(9-adenyl)-l»l-dideoxy-l-ethylthio-D-galactose

(and D-glucose) aldehydrol (20) and four adenine nucleosides of 3»4»5*6- tetra-0-acetyl-2-deoxy-2-(2»4-dinitroanilino)-D-glucose aldehydrol (21).

A number of hexofuranosyl nucleosides have been synthesized by the mercuri procedure. The first of these reported was 6-chloro-9-

(o-L-rhamnofuranosyl)purine (23). Table I lists this and other hexo­ furanosyl nucleosides prepared utilizing this procedure* TABLE I HEXOFURANOSYL NUCLEOSIDES PREPARED BY THE MERCURI METHOD

Substituents on purine ring M.p., °C. [ojD, (ç» solvent) Refer

6-chloro-9-tt-L-rhamnofuranosyl (glass) -42°» (0.21, water) 23

6-amino-9-

2 ,6-diainino-9-a-L-rbamnofuranosyl 270-275 -80°, (0.03* O . M hydrochloric acid) 106

6-amino-9- (6-deoxy-P-D-glucofuranosyl ) 118-118.5 -59.9°* (2, water) 107

2 ,6-diamino-9-C6-deoxy-H-D-glucofuranosyl) 172-175 -27.7°, (.37, water) 107

6-amino-9-(6-deo3^-o-L-iodofuranosyl) 205-210 -36.9° (0.4, water) 108 2 ,6-diamino-9-(6-deoxy-a-L-iodofuranosyl) 210-211 -50.8° (1, water) 108

6-amino-9-P-D-glucofuranosyl 268-270 -58° (1, N hydrochloric acid) 109

6-amino-9-(6-deoxy-P-D-allofuranosyl ) 174-175 -72.2° (1.86, water) 110 2 ,6-diamino-9-(6-deoxy-P-D-allo- furanosyl) (hydrochloride) 210-212 -76.1° (1.71, water) n o

6-amino-9-(6-deoxy-o-L-talofuranosyl) (glass) -35° (0.84, water) 111

2 ,6-diamiho-9-(6-deoxy-

N TABLE I (Contd.) Substituents on purine ring M.p.»°C. [a]D, (c» solvent) Reference

6-amino-9-P-D-galactofuranosyl 224-225 -52° (0.3, water) 112

2-acetamido-9-P-D-galactofurano syl 240-241 -53° (0.18, water) 112

2,6-diamino-9-P-D-galactofuranosyl 224-225 -69° (0.23, water) 112

2-acetamido-6-amino-9-P-D-glucofuranosyl 241-242 d. -77° (0.13, water) 112

6-amino-9-P-D-gulofuranosyl 229-230 -56.2 ° (2 .03* 1 N hydrochloric acid) 113

6-amino-(5-deoxy-S-D-allofuranosyl) 231.5-232.5 -l6.4° (0.4, methanol) 114 6-mercapto-9-P-D-galactofuranosyl 16

6-mercapto-9-3-D-glucofuranosyl 16

(106) B. R. Baker and K. Hewson, J. Org. Chem., 22, 966 (1957). (107) E. J. Reist, R. R. Spencer, and B. R. Baker, ibid., 23, 1753 (1958). (108) E. J. Reist, R. R. Spencer, and B. R. Baker, ibid., 23, 1757 (1958). (109) E. J. Reist, R. R. Spencer, and B. R. Baker, ibid., 23, 1958 (1958). (110) E. J. Reist, L. Goodman, R. R. Spencer, and B. R. Baker, J. Am. Chem. Soc., 3962 (1958). (111) E. J. Reist, L. Goodman, and B. R. Baker, ibid., 80, 5775 (1958). (112) M. L. Wolfrom, P. McWain, R. Pagnucco, and A. Thongs on, J. Org. Chem., 454 (1964). (113) P. Kohn, R. H. Samaritano, and L. M. Lemer, J. Am. Chem, Soc., 86, 1457 (1964). (114) K. J. Ryan, H. Arzoumanian, E. M. Acton, and L. Goodman, ibid., 2503 (1964). 34

The adenine nucleosides of the ketoses D-psicofuranose (51»115)

(115) J, Farkas and F. Sorm» Collection Czech. Chem. Commun.»

^ » 882 (1963).

and D-fructofuranose (and pyranose) (II6) have been prepared in this

(116) E. J. Reist, P. A. Hart» and B. R. Baker» J. Org. Chem.»

24» 1640 (1959).

manner.

Wolfrom and oo-workers have reported the synthesis of nucleosides

of the disacobarides lactose (117)» cellobiose (118)» and maltose (118)

Cll?) M. L. Wolfrom, P. McWain» F. Shafizadeh» and A. Thompson,

J. Am# Chem. Soc.» ^ » 608O (1959)»

(118) M. L. Wolfrom» P. McWain » and A. Thompson» ibid.» 82» 4353

(i960).

Recently the mercuri method has been used to prepare the adenine

nucleoside of 4-thio-D-ribofuranose (119,120) and the adenine and

(119) E. J. Reist» D. E. Gueffroy» and L. Goodman » Chem. Ind.

(London) », I364 (1964).

(120) E. J. Reist», D. E. Gueffroy» and L. Goodman, J. Am. Chem.

Soc., 86» 5658 (1964)0

uracil nucleosides of 3-thio-D-xylopyranose (121). 35

(121) R. L. Whistler and Branko Urbas, Abstracts Papers Am.

Chem. Soc. Meeting» 149» l6c (1965)*

Nucleosides of amino sugars have also been prepared by this pro­ cedure. In the case of the amino pentoses, numerous nucleosides have been reported in which the amino group of the sugar moiety has been freed of blocking groups after the condensation reaction. The most commonly used amino pentoses are 3-amino-3-deoxy-D-ribofuranose (8?,

95*122-129) and 5-amino-5-deoxy-D-ribofuranose (123-125)o

(122) L. Goldman, J. W. Marslco, and R. B. Angier, J. Am. Chem.

Soc., 28, 4173 (1956).

(123) American Cyanamid Co., Brit. Pat. 782,440 (1957)î C.A.,

5 2, 2936 (1958). (124) B. R. Baker, J. P. Joseph, and R. E. Schaub, U. S. Pat.

2 ,852,505 (1958); C.A., j], 8174 (1959).

(125) L. Goldman and J. W. Marsico, U. S. Pat. 2,852,506 (1958);

C.A., 21» 8174 (1959). (126) H. M. Kissman and J. W. Weiss, J. Am. Chem. Soc., 80, 2575 (1958). (127) H. M. Kissman, A. S. Hoffman, and M. J. Weiss, J. Med. Chem.,

6, 407 (1963). (128) L. Goldman, J. W. Marsico, and M. J. Weiss, ibid., 6,

410 (1963).

(129) L. Goldman and J. W. Marsico, ibid., 6, 413 (I963). 36 A nucleoside of 3-amlno-3-deoxy-D-xylose, 9-(3-aniino-3-deoxy-P-D- xylofuranosyl)-N^ -dimethyladenino (130)» has been prepared by the

(130) R, E. Schaub» M. J. Weiss» and B, R. Baker» J. Am* Chem.

Soc.». 80» 4692 (1958).

mercuri method. Nucleosides of 3-3i»tno-3-deoxy-D-arabinose have been

prepared but the 3-aniino group could not be obtained free from its

acetyl blocking group (123»124),

To date only four amino hexose nucleosides have been prepared

(through the mercuri procedure) wherein the amino group of the sugar

moiety was free of blocking groups; these are 9-(2-amino-2-deoxy-B-D—

allopyranosyl)-N^»N^-dimethyladenine (124)» l-(2-amino-2-deoxy-p-D-

glucopyranosyl)cytosine (and uracil) (83»84)» and 9-(2-amino-2-deoxy-

P-D-glucopyranosyl)adenine (131). Both anomers of the latter nucleoside

(131) M. L. Wolfrom» H. G. Garg» and D. Horton» Chem. Ind.

(London), 930 (1964).

were formed since the non-participating 2 »4-dinitrophenyl group was

used to protect the 2-amino group; however » only one of the anomers with

the free amino group had been characterized at the time of publication.

Nucleosides of 2-(blocked)amino-2-deoxy-D-glucose have been pre­

pared with such N-blocking groups as acetyl (84»94»123»124»132),

(132) M. L. Wolfrom-nnd R. Wurmb» Abstracts Papers Am. Chem.

Soc. Meeting» 148» 6d (1964). 37 benzylsulfonyl (132), benzyloxy- and methyloxy-carbonyl (84) and

phthaloyl (94), Several adenine nucleosides of the acyclic amino sugar 2-amino-

2-deoxy~aldehydo-D-glucose have been prepared with the 2,4-dinitro-

phenyl group (21) as N-blocking agent.

Ulbricht, while investigating the mechanism of the mercuri

method as applied to pyrimidinones (133), condensed the silver salt

(133) T. L. V. Ulbricht, Proc. Chem. Soc., 298 (1962),

of N-acetylcytosine with tetra-O-acetyl-ouD-glucopyranosyl bromide

and as expected obtained the 0-glycoside (shown by liberation of

the heterocyclic base by dilute acid treatment). Later a similar

report of 0-glycoside formation from the same sugar halide reacted

with the chloromercuri salt of 4-ethoxy-2-pyrimidinone (134) appeared.

(134) C. Ukita, H. Hazatsu, and Y. Tomita, Chem, Pharm. Bull.

(Tokyo), 11, 1068 (1963).

The 0-glycoside, in each case, was treated with mercuric bromide in

hot toluene. This resulted in rearrangement of the 0-glycosides to

the glycosylamines (N-nucleosides) (see Figure 11). Ulbricht (133;

133) proposed that a similar sequence was taking place in pyrimidinone

(135) T. L. V. Ulbricht, Angew. Chem., 2ft» 76? (1962). 38

C H o O A c N H A c N H A c )— O MO A c (toluene) O A c OAc

N H A c N H A c N H A c

N

R B r H g B r

R — 2;3,4,6-tetra-0-acetyl-D-glucopyrano8yl

Fig. 11.— Formation and Rearrangement of an 0-Glycoside to the Corresponding Nucleoside ~ nucleoside synthesis by the mercuri procedure; namely that initially the 0-glycoside was formed, then a rearrangement to the nucleoside took place in the presence of mercuric bromide under the reaction conditions. When he tried mercuric chloride in place of the bromide no rearrangement took place. This high selectivity of a catalyst could also account for the formation of only the 0-glycoside in the case of the Fischer-Helferich (silver salt) method when applied to pyrimidinones. In cases where there is not the possibility of 0- glycoside formation, Ulbricht states, without any further explanation, that the nucleoside is formed directly. 39

Fusion method» In 1960 T. Sato and co-workers (136|137) reported

(136) T. Sato» T. Shimadate, and Y. Ishido» Nippon Kagaku

Zasshi, 81, 1440 (i960).

(137) T. Sato, T, Shimadate, and Y. Ishido, ibid., 81, 1442

(i960). a new synthetic route for the preparation of nucleosides.

In the first paper (136) they report that D-ribofuranose melted with theophylline in the presence of a catalytic amount of g-toluene-

sulfonic acid yielded 7-P-D-ribofuranosyltheophylline, The same

sugar was also fused likewise with 2 ,8-dichloroadenine, 2 ,6,8-tri-

chloroadenine, and 4-methylthioadenine to give the 9-P-D-ribofuranosyl-

adenine derivatives in from 3-40^ yields.

In the accompanying report (137) methyl tri-O-acetyl-D-ribofurano-

side or D-ribofuranose tetraacetate was condensed with theophylline

in acetic anhydride containing £-toluenesulfonic acid to give, after

deacetylation, 7-P-D-ribofuranosyltheophylline.

Later these workers and others modified these two methods to give

what is now known as the "fusion” method. This consists of fusing a

peracylated sugar with theophylline or a substituted purine in the

presence of an acid catalyst. Yields of nucleosides have been between

3 and 82^, with most yields on the order of 20 to 53#*

Ribofuranose tetraacetate (138-146) has been the most widely used

(138) Y. Ishido and T. Sato, Bull. Chem. Soc. Japan, 1347

(1961). 40

(139) T. Shimadate» Nippon Kagaku Zasshi» 82» 938» 1268 (1961)#

(140) R. Ishido» T. Sato» and T. Shimatachi» Jap. Pat, 13*829

(1961), C.A.» ^ » 11694 (1962).

(141) T, Sato and T. Shimatachi» Jap. Pat. 8»331 (1963)» C.A.,

60» 651 (1964).

(142) K. Miyamoto» A. Kinoachi» and S. Isome» Jap, Pat, 23*184

(1963); C.A.» ^ » 4243 (1964).

(143) T. Sato and R. Ishido» Jap. Pat. 26*183 (I963); C.A,» 60»

6918 (1964),

(144) T. Sato and R. Ishido» Jap, Pat. 26,395 (1963); C.A.» 60»

6918 (1964). ^

(145) Y. Ishido» A. Hosono» S. Isome» A. Maruyaraa» and T. Sato»

Bull. Chem. Soc. Japan* ^ » I389 (1964), (146) J, A. Montgomery and K, Hewson» J, Heterocyclic Chem,»

1» 213 (1964),

sugar derivative► while theophylline (138*140-151) and purine

(147) T. Shimadate* Nippon Kagaku Zasshi* ^ » 212 (1962),

(148) T, Shimadate* ibid., 8], 214 (1962),

(149) K. Onodera and H, Fukumi* Agri, Biol, Chem, (Tokyo)»

^ » 526» 864 (1963).

(150) K, Onodera* S, Hirano» and H. Fukumi* ibid,» 28» 173

(1964),

(151) T, Sato and Y, Ishido* Jap, Pat, 6695 (1964); C.A,» 6 1»

14772 (1964), 41 derivatives (138»l4l»l45»l49»150*152-154) have been the commonly

(152) W. W. Lee* A. P. Martinez, G. L. Tong, and L. Goodman,

Chem. Ind. (London), 200? (19&3)*

(153) E. J. Reist and L. Goodman, Biochemistry, 2» 15 (1964).

(154) M. J. Robins, W. A. Bowles, and R. K. Robins, J. Am.

Chem. Soc.* 86, 1251 (1964).

used aglycons. The catalysts, Jg-toluenesulfonic acid (139*140,146-

149*152*153) and zinc chloride (141*142,14?,149) have been widely used. Other catalysts which have been used with good results are

other aryl- and alkyl-sulfonic acids (138,143*144), sulfamic acid

(145)* sulfuric acid (139*151)* and polyphosphoric acid (and esters

thereof) (149*150). The sugar acetates penta-O-acetyl-P-D-gluco-

pyranose (14?*150)* penta-O-acetyl-P-D-galactopyranose (14?), tetra-

0-acetyl-a(and p)-D-arabinofuranose (14?), and tetra-O-acetyl-D-xylo-

furanose (152,153) have been successfully utilized for this procedure.

The amino sugar nucleoside ?-(2-acetamido-2-deoxy-P-D-glucopyranosyl)-

theophylline (I5I) and the 2-deoxy sugar nucleosides 6-chloro-9-

[2-deoxy-a(and P)-D-erythro-furanosyl(and pyranosyl)]purine (154) were

also prepared in this manner. Use of the 1-trichloroacetyl-polyl-

0-acylglycoses (145) did not inçrove the yield of nucleoside.

In nearly all cases the nucleoside anomer reported was the one

with the C-1 — G-2-trans relationship (when a participating group

was present on G-2). However, Goodman and co-workers (132) reported 42 that with the fusion of 6-alkylamidopurine with tetra-O-acetyl-D- xylofuranose a 64$ yield of the two anoraeric nucleosides was ob­ tained, 24$ P-D-anomer and 10$ of the o-D-anomer.

In a modification of the fusion method Bowles and Robins (155)

(155) W. A. Bowles and R. K. Robins, J. Am. Chem. Soc., 86, 1252 (1964).

fused, in one case, di-G-acetyl-l,2-dideoxy-D-erythro-pent-l-

enopyranose ("D-arabinal”) with 6-chloropurine in the presence of

2 -toluenesulfonic acid and obtained 6-chloro-9-(di-0-acetyl-2-deoxy-

a(and P)-D-erythro pentopyranosyl)purine. When they fused theophyl­

line under similar conditions with the glycal tri-O-acetyl-1,2-

dideoxy-D-arabino-hex-l-enopyranose ("D-glucal"), the unsaturated

sugar nucleoside 7-(4,6-di-0-acetyl-2,3-dideoxy-D-erythro-hex-2-eno-

pyranosyl)theophylline (mixture of anomers presumably) was produced

(see Figure 12),

ÇH 3 C H g O A c CH NH + H © CH

O A c CH 3 O A c

Figure 12.— Synthesis of an Unsaturated Sugar Nucleoside by a Modified Fusion Technique, 43

Ethyl polyphosphate condensation method. In 196I Schramm and co-workers (I56) described a new method for nucleoside synthesis which

(156) G. Schramm» H. Grotsch» and W. Pollmann» Angew. Chem.»

2 1» 619 (1961). required only the free sugar and the heterocyclic base. When these workers condensed D-ribose (or 2-deoxy-D-erythro-pentose) with adenine in N»N-dimethylformamide containing ethyl polyphosphate» adenosine and

2* deoxyadenosine (30^ yield) respectively» were formed. Later these same workers (157) utilized this method to synthesize 9-P-D-fructo-

(157) G. Schramm» H. Grotsch» and W. Pollmann » ibid.» 74» 53 (1962).

furanosyladenine and 9-(2-acetaraido-2-deoxy-P-D-glucopyranosyl)adenine.

The synthesis of these two above-mentioned nucleosides and 9-P-D-

ribofuranosyladenine in this manner is also described in a French

patent (I58).

(158) Farbwerke-Hoechst A.-G.» French Pat. 1»324»011 (1963)»

C.A.» 11648 (1963).

J. A. Carbon (159)1 while repeating the work of Schramm and

(159) J . A. Carbon, Chem. Ind. (London)» 529 (1963)»

co-workers (156) reported results which differed from those published 44 originally. Under the same conditions» he found that the yield of

2’-deoxyadenosine (P-D-anoraer) was only 65J (compared to Schramm's

30#) and that the o-D-anomer, which Schramm had not reported» had predominated (25^ yield)* Carbon also reported four other ultraviolet- observable products from this reaction but did not identify them.

Method of fusion of sugar derivatives with trimethylsilyl sub­ stituted purines or pyrimidines. By condensing tetrakis(trimethyl­ silyl )uric acid (160) with tri-O-benzoyl-D-ribofuranosyl bromide in

(160) L. Birkofer and A. R. Ritter, Angew. Chem.» 372

(1959). the presence of silver perchlorate, followed by hydrolysis to remove the blocking groups, Birkofer and associates (I6I) prepared 3-puD-

(161) L. Birkofer, A. R. Ritter, and H. P. Killthau, ibid., 75,

209 (1963).

ribofuranosyluric acid.

T. Nishimura and co-workers (162,163) prepared the trimethylsilyl

(162) T. Nishimura, B. Shimizu, and I. Iwai, Chem. Pharm. Bull.

(Tokyo), 11, 1470 (1963).

(163) T. Nishimura and I. Iwai, ibid., 12, 352 (1964).

derivatives of several purines and pyrimidines and utilized these for

nucleoside synthesis. In the case of uracil, thymine, cytosine. 45 adenine» 6-benzamidopurine» and hypoxanthine the bis(trimethylsilyl) derivatives shown below were formed. R OSiMe NHSiMe, HNSiMe,

N

,_N' N ^ k N ^ SiMe, MejSiO Uracil (R=H) Cytosine-,---- Adenine (&=H)0 and Thymine (It=GH~) Derivative 6»Benzamidopurine (RsCC^Hc) Derivatives Derivatives OSiMe.

N i) SiMe 2 Hypoxanthine Derivative

Fig. 13.--Trimethylsilyl Derivatives of Some Purines and Pyrimidines.

These derivatives were then fused with either tri-O-benzoyl-P-

D-ribofuranosyl chloride (l62»l64) or tetra-O-acetyl-o-D-glucopyranosyl

(l64) T. Nishimura, B. Shimizu, and I. Iwai, ibid., 12, 14?1

(1964).

bromide (162,165,166) to give, after hydrolysis, the respective

(165) T. Nishimura and I. Iwai, ibid., 12, 357 (1964).

(166) T. Nishimura and B. Shimizu, Agri. Biol. Chem. (Japan),

224 (1964). 4 6 nucleosides in yields of (mostly 40-5256). In all except one case the C-1 — C-2-trans anomer was reported. Fusion of 6-benzamido- bis(trimethylsilyl)purine with tetra-O-acetyl-a-D-glucopyranosyl bromide followed by treatment with ethanol to remove the remaining trimethylsilyl group and subsequent déacylation gave a mixture of anomeric 9-C“(and p)-D-glucopyranosyl)adenine in 295^ yield (.166).

Wittenberg (l6?) used the same procedure to prepare uracil and

(16?) E. Wittenberg, Z. Chem., 4, 303 (1964). thymine nucleosides of D-galactopyranose, D-glucopyranose, L-arabino- furanose, and D-ribofuranose# In addition to the method of fusing the reactants, Wittenberg carried out the reaction in inert solvents

(benzene, toluene, or nitromethane) with heating or in a solvent in

the presence of catalytic amounts of water, mercuric acetate, or

silver perchlorate.

By condensation of poly-O-acetylglycosyl halide with a nitrogen

heterocycle. The condensation of a purine or pyrimidine directly

with a poly-O-acetylglycosyl halide in boiling nitromethane containing

a hydrogen halide acceptor (as mercuric cyanide) has been recently

reported (168) to give high yields of the blocked nucleoside. In

(168) N. Yamaoka, K. Aso, and K. Matsuda, J. Org. Chem., 30,

149 (1965).

this report D-glucopyranose nucleosides of chloropurines, 6-benzamido-

purine, theophylline and ^-benzoylcytosine and 47 7-P-D-ribofuranosyltheophylline were synthesized in yields ranging from 43 to 94^. The anomer reported in each case is the one which would be predicted by the Baker "trans rule" (the C-1 — C-2-trans anomer)•

Methods involving cyclization of the aglycon of glvcosyl deriva­ tive*. These methods in general emphasize reactions which have been used for the synthesis of purines and pyrimidines. The yields of

free nucleosides are generally low and as a result these methods have

been used only sporadically since the development of the methods

previously described in this work. Therefore, these methods will

only be described in brief as one segment of the development of syn­

thetic methods for nucleoside preparation.

Several methods have been developed which involve derivatives

of glycosylamines. One of these, developed by Todd and co-workers

(169,170), has enabled the unequivocal assignment of the purine

(169) J. Baddiley, B. Lythgoe, and A. R, Todd, J. Chem. Soc.,

571 (1943).

(170) G. A. Howard, B. Lythgoe, and A. R. Todd, ibid., 556 (1945).

nucleosides of the nucleic acids as the 9-glycosylpurines (rather

than the 7-isomers).

While studying reactions which could lead to nucleosides, Todd

and co-workers (I69) prepared 6-amino-2-methylthio-4-[D-xylo(and

D-manno)pyranosylamino]pyrimidine by condensing the aldose with 4,6-

diamino-2-methylthiopyriraidine in the presence of ammonium chloride. 4 8

In later reports these compounds (170*171) and others (172-177) were

(171) B. lythgoe, H, Smith, and A. R. Todd, i W . , 355 (1947).

(172) J. Baddiley, B. lythgoe, and A. R. Todd, ibid., 318 (1944).

(173) G* W. Kenner, B. lythgoe, and A. R. Todd, ibid., 652 (1944).

(174) J. Baddiley, G. W. Kenner, B. lythgoe, and A. R. Todd, ibid., 657 (1944).

(175) A. Holland, B. lythgoe, and A. R. Todd, ibid,, 965 (1948).

(176) G. W. Kenner, C. W. Taylor, and A. R. Todd, ibid., 1620

(1949).

(177) K. J. M. Andrews, N. Anand, A. R. Todd, and A. Topham,

ibid., 2490 (1949).

subsequently converted to the purine nucleosides in the following

manner. Coupling of the 6-amino-4-(glycosylamino)pyrimidine with an

aryldiazonium salt followed by reduction gave (after 0<^cetylation)

3,6-diamino-4- ( poly-O-acetylglycosylamino ) pyrimidine ). Cyclization

to form the imidazole ring was effected by thioformylation followed

by heating in pyridine (see Figure 14). In this manner adenine

(and 2-methyl or 2-methylthio-adenine) nucleosides of the following

sugars were prepared: D-jcylopyranose (170,172,173); D-mannopyranose

(171;1?2), D-ribose (174,176), and D-glucopyranose (175)• Under the conditions of the reaction the glycose tends to form the pyranose con­

figuration. In order to prepare 9-B-D-ribofuranosyladenine (adenosine)

it was necessary to block the 5-OH group of D-ribose with a benzyl

group before the condensation with the pyrimidine derivative (I76). 49 NH- N=N-Ar NH ar-yi diazonium I salt ^ N' NHR NH R= D-mannopyranosyl OH o: 1) Zn, HOAc 2 ) O-acetylation M -

NH S il 1) HC S H 2 ) C 5H 5N a NHR

R = tetra-O-acetyl- D - ma nnopy r a no s yl OAc OAC

OAc

Fig» 14,— Adenine Nucleoside from a D-Mannosylamine Derivative

Since the adenosine prepared in this manner was identical to the natural product, adenosine was proven to be a 9-adenine rather than a

7-adenine-derivative•

The only anomers reported were those containing the C-1 — C-2- trans relationship.

Ralph and Shaw (l?8) prepared several 5-cyano-l-D-glycopyranosyl-

(178) R. K. Ralph and G. Shaw, i ^ . , 1877 (1956). uracils (and the 5-cyano-3-methyl analogs) by interaction of a glyco- pyranosylamine with o-cyano-P-ethoxy-N-ethoxycarbonylaciylamide (or the N-methyl derivative) (see Figure 15). Some of the glycosylamines •50

O ô. NC-C' NH NC II 1 NH c c=o I EtO i:Et I R m z R

R = glycosyl

Fig. 15*--5-Cyanouracil Nucleoside from a Glycosylaraine used were D-ribo-» D-galacto-, D-gluco-» and D-ocylo-pyranosylamine

(178). In a similar manner 9-P-D-ribofuranosyluracil (uridine)»

5-cyanouridine» 5-thiouridine» 5-methyi-2-thiouridine and $-cyano-2- thiouridine have been prepared (179)*

(179) G. Shaw, R, N, Warrener», M. H. Maguire, and R, K. Ralph, ibid., 2294 (1958).

Also, with this method the only nucleosides reported were the

C-1 — C-2-trans anomers, Shaw (IBO) suggests that this may be due

(180) A. Albert, G. M. Badger, and 0» W. Shoppee, "Current

Trends in Heterocyclic Chemistry," Academic Press, New York, N. Y.,

1958, Chap. 16. 51 to the fact that the C-1 — C-2-cis linear compound (before cyclization) can form an oxazoline ring with the C-2-acyl group rather than cyclizing to give the pyrimidine ring.

Pyrimidine nucleosides have been prepared utilizing glycosylureas.

Based on the observation that l-(cyanoacetyl)urea could be converted» in basic media, to 6-aminouracil (181), Johnson and Bergmann (182)

(181) W. Bergmann and T. B. Johnson, J. Am. Chem. Soc., 65,

1733 (1933).

(182) T. B. Johnson and W. Bergmann, ibid., 6o, I916 (1938).

prepared l-(tetra-0-acetyl-D-glucopyranosyl)-2-cyanoacetylurea from

tetra-O-acetyl-D-glucopyranosylurea and cyanoacetic acid with the

intention of cyclization to the uracil nucleosides. However, they

reported (182) that all cyclization attempts failed. Goodman (183)

(183) I. Goodman, Federation Proc., 1_^, 264 (1956).

later succeeded in cyclizing this compound with dilute base and

obtained 6-amino-l-p-D-glucopyranosyluracil (see Figure I6). 52

o "C CH- OH

NC NH: H^N' ^ q' /c=o HN

CH^OAc C H g O H

MOAc OAc OAc

Fig. 16.— 6-Amino-l-P-D-glMcopyranosyluracil from Tetra-0- acetyl-D-glucopyranosylurea

In i960 (184) a related approach was reported. The glycosylurea

(184) M. Hoffer, U. S. Pat. 2,949»449 (I960); C.A., $86

(1961).

reacted with p-ethoxy(or methoxy)-a-methylacryloyl chloride and the

adduct cyclized in ammonium hydroxide solution to give the pyrimidine

nucleoside (see Figure 17). In this manner Hoffer (184) was able to

prepare 1-(2-deoxy-g-D-eiybhro-pentofuranosyl)urac il and l-(2-deoxy-

P-D-erythro-pentofuranosyl)thymine (thymidine). Thymine and uracil

nucleosides of D-arabinopyranose (I85 186). D-ribofuranose (185,186)»

(185) T. Naito, T. Kawakami, M. Sano, and M. Hirato,

Chem. Pharm. Bull. (Tokyo)» 2» 249 (I96I). 53 (186) T. Naito» M. Sano» T. Kawakami» and M, Hirato» Jap.

Pat. 2877 (1964); C.A.» 60t 15975 (1964).

(187) T. Naito and M. Sano» Chem. Pharm. Bull. (Tokyo)» ^»

709 (1961).

Me .C M e NH NH, I ^ c=o N I 1 NH R R R = glycosyl R’ = methyl or ethyl

Fig. 17,--Synthesis of a Thymine Nucleoside from a Glycosylurea

D-ribopyranose (185»186) and D-glucopyranose (185»188) have similarly

(188) T. Naito» M. Hirata» T. Kawakami» and M. Sano» ibid.» £»

703 (1961).

been prepared.

Glycosylpurines have been prepared by completion of the pyrimidine

ring of glycosylimidazole derivatives. Interaction of tri-O-acetyl-

D-xybpyranosyl bromide with the silver salt of 4»5-dimethoxycarbonyl-

iraidazole produced 4 »5-dimethoxycarbonyl-l-(tri-O-acetyl-D-ocylopyrano-

syl)iraidazole (I89). This was then converted to the deacetylated 54

(189) R. A. Baxter and F. Si Spring» J. Chem. Soc.» 378 (194?). diamide with ethanolic ammonia and the diamide on treatment with alkaline potassium hypobromite cyclized to give 9-P-D-xylopyranosyl- xanthine (189) (see Figure 18). In the same report (189) the

o C O M e O M e O C Ol ^ N - A C O M e -N Br OAc MeOC' -N 0 O A c O A c

OAc

NH.

O &NH, N IL N ^ C N H , ô ^ \ KOBr .OH OH OH

OH

Fig. 18.— 9-p-D-Xylopyranosylxanthine from 4 »5-Dimethoxycarbonyl- l-(tri-O-acetyl-D-ocylopyranosyl)imidazole 55 imidazole derivatives of D-glucopyranose and L-arabinopyranose were prepared but cyclization to the nucleoside was not indicated. This method has been successfully used for the preparation of the 9-xanthine nucleosides of D-ribopyranose (190)» D-mannopyranose (190)» D-ribo-

(190) R. A. Baxter, A. C. McLean » and F. S. Spring» ibid » » 523 (1948).

furanose (191)» and D-glucopyranose (192). A 7-hypoxanthine nucleoside

(191) G. A. Howard» A. C. McLean» G. T. Newbold» F. S. Spring»

and A. R. Todd, ibid., 232 (1949).

(192) T. Baddiley, J. G. Buchanan, and G. 0. Osborne, ibid.»

3606 (1958).

was prepared ty cyèlization of 4-amino-5-carboxyamide-l-D-ribofuranosyl.

imidazole with formic acid in acetic anhydride (193)*

(193) <1* Baddiley, J. G. Buchanan, F. E. Hardy, and J. Stewart,

ibid., 2893 (1959)0

Other methods. Due to the nature of this work methods for pre­ paring nucleosides which involve conversions of the glycosyl or

aglycon moiety of the preformed nucleoside and enzymic synthesis

methods will not be discussed. For reviews of these two useful

methods see Fox and Wempen (194), Montgomery and Thomas (195) and

Michelson (I96)* 56

(194) J. J. fax. and I. Wempen» Mvan. in Carbohydrate Chem.»

14» 34-360 (1959).

(195) J. A. Montgomery and H. J. Thomas» ibid., 17» 326-335»

337-340 (1962).

(196) A. M. Michelson» "The Chemistry of Nucleosides and

Nucleotides»" Academic Press» New York» N. Ï.» I963» pp. 68-82. DISCUSSION OF RESULTS

Synthesis of 2«6-Diacetamido-9-fcetra- O-acetyl-P-D-galactofuranosyl)- purine dll')

The naturally occurring nucleosides have been linked with such

important biological processes as growth, cell replication, and the

transmission of genetic information. Some of these have been shown

to possess antibiotic and carcinolytic properties (see p. 15 of this

dissertation). Since the sugar moiety of most of the naturally occur­

ring nucleosides is of the furanose configuration, it was considered

desirable to prepare several nucleosides of D-galactose in the furanose

form. Not only would the synthesis of such nucleosides be of interest

for the chemistry involved, but also for the potential value of the

products as carcinolytes. The D-galactofuranose nucleosides were also

desired as reference compounds for future research.

In the pentose series, a sugar may be forced into its furanose

form by suitable blocking of the terminal position. As a result

numerous pentofuranosyl nucleosides have been prepared. This pro­

cedure is not applicable in the hexose series and other methods are

necessary. Relatively few hexofuranosyl nucleosides have been pre­

pared (197)» Haworth and associates (198) utilized carbonate esters

(197) See Table I, pp. 32-33 of this dissertation.

(198) W. N. Haworth, "The Constitution of Sugars," Longmans Green and Co., New York, N. Y., 1929» p . 52. 57 58 in several successful syntheses of hexofuranosides. Wolfrom and co­ workers (199) have reported the synthesis of ethyl 1-thio-ouD-galacto-

(199) M. L. Wolfrom, Z. Yosizawa, and B. 0. Juliano, J. Org,

Chem., 1529 (1959). furanoside by the partial demercaptalation of D-galactose diethyl dithioacetal. In a later report Wolfrom and co-workers (112) improved upon this procedure. Penta-O-acetyl-P-D-galactofuranose may be pre­

pared by fractional crystallization of the products obtained on the

acétylation of D-galactose (200,201).

(200) C. S. Hudson and J. M. Johnson, J. Am. Chem. Soc., 38,

1223 (1916); H. H. Schlubach and V.~Prochownick, Ber., 2298 (1930).

(201) R. K. Ness, H. G. Fletcher, Jr., and C. S. Hudson, J. Am.

Chem. Soc., 21» 3742 (1951).

The latter method (201) was chosen as the starting point for this

sequence. D-Galactose was acetylated in refluxing pyridine-acetic

anhydride and the pyranose- and furanose-pentaacetates thus formed

were separated by fractional crystallization to give pure penta-0-

acetyl-P-D-galactofuranose (I). I was then converted to the known

tetra-O-acetyl-p-D-galactofuranosyl chloride (II) (201) by treatment

with a solution of hydrogen chloride in glacial acetic acid. Reac­

tion of tetra-O-acetyl-P-D-galactofuranosyl chloride (II) with 59 2,6-diacetamido-9-chloromercuripurine (14) in refluxing toluene yielded the amorphous 2,6-diacetamido-9-(tetra-0-acetyl-p-D-galacto- furanosyl)purine (III)•

Synthesis of 2-Acetamido-9-B-D- galactofuranosyladenine (IV)

2,6-Diacetamido-9-(tetra-0-acetyl-P-D-galactofuranosyl)purine

(III) was partially deacetylated by treatment with refluxing

methanolic n-butylamine (202) to yield crystalline 2-acetamido-9-P-

(202) L. Goldman» J. W. Marsico* and R. B. Angier, ibid » » 78»

4173 (1956); E. J. Reist and B. R. Baker, J. Org. Chem.» ^ » IO83

(1958).

D-galactofuranosyladenine (IV).

Synthesis of 2»6-Diamino-9-B-D- galactofuranosylpurine (V)

2-Acet*mido-9-P-D-galactofuranosyladenine (IV) on treatment with

boiling methanolic sodium methoxide gave crude 2»6-diamino-9-P-D- galactofuranosylpurine (V). The compound V was then obtained in a

pure state in the following manner. Treatment of V with hot methanolic

picric acid yielded a yellow picrate. The picric acid moiety was then

removed by stirring an aqueous acetone solution of the picrate with

an anion exchange resin. In this manner pure V was obtained as color-

' less crystals.

The free nucleoside V was also obtained, however in lower yield,

by the complete deacetylation of 2,6-diacetamido-9-(tetra-O-acetyl-P-D-

galactof uranosyl )purine (III) with boiling methanolic sodium methoxide. 6o

NHAc NHAc

A c

HÇOAc NHAc CHgOAc OAc HgCl OAc II ecoAc I CH^OAc®^'^

III

NH

III n-C^HgNHg^ (CH3OH) ^ NHAc R IV

R= (3-D-galactofuranosyl NH

III or IV NaOCH. purification (CH 3OH)' via picrate salt

OH

HCOH OH CH^OH

Fig* 19«--Synthesis of 2»6-Diamino-9-P-D-Galactofuranosylpurine (V) 61

The blocked nucleoside III (and therefore the products of its deacetylation) was assigned the P-D configuration based on its rotation and on the trans rule of Baker (93) i which suggests the formation of a transient 1,2-orthoester carbonium ion which would then form the

1,2-trans glycoside (P-D-glycoside in this case) on attack by the

nitrogen heterocycle.

Synthesis of 9-P-D-Galactofuranosyl- adenine (Dimorphous) (VÎT The condensation of 6-benzamido-9-chloromercuripurine (203) with

(203) J ' R, Parikh, M, E. Wolff, and A. Burger, J. Am. Chem.

Soc., 22» 2778 (1957).

tetra-O-acetyl-P-D-galactofuranosyl chloride (II) in refluxing toluene

gave white amorphous 6-benzamido-9-(tetra-0-acetyl-P-D-galactofurano-

syl)purine. This blocked nucleoside was deacylated by two different

routes. In one case the blocked nucleoside was completely deacylated

with refluxing methanolic n-butylamine. This déacylation yielded

9-P_D-galactofuranosyladenine (VI) as a hygroscopic white crystalline

solid, dec. 209-212°, In the second case the crude 6-benzamido-9-

(tetra-O-acetyl-P-D-galactofuranosyl)purine was first debenzoylated.

This was accomplished by treatment of the fully blocked nucleoside

with methanolic picric a cid, followed by removal of the picric

acid moiety of the picrate thus formed with anion exchange resin (203).

The gummy 9-(tetra-0-acetyl-P-D-galactofuranosyl)adenine which re­

sulted was then deacetylated with boiling methanolic n-butylamine to 9 : 62 N H C C ^ H g o NHCC/Hc

II +

HÇOAc :HgOAc

1) picric acid (CH3OH) 2 ) anion exchange resin

n - C ^ H g N H , V (CH3OH) NH-

n-C ^ H g N H ^ (CH3OH)'

J HCOH CH OH 2

VI (dimorphous)

R = 2»3»5»6-Tetra-0-acetyl-P-D-galactofuranosyl

Fig. 20.--Synthesis of 9-P-D-Galactofuranosyladenine (VI) 63 give white crystalline VI, m.p. 224-225°. The lower melting (209-212°) hygroscopic VI was converted to the higher melting (224-225°) non- hygroscopic VI by recrystallization and nucléation with the higher melting VI.

The assignment of the P-D configuration to VI was also based on its optical rotation and the trans rule of Baker (93).

Synthesis of Penta-O-acetyl-1-0- benzyl-l-deoxy-l-ethylthio- D-galactose Aldehydrol (X)

Wolfrom and co-workers (1 9>20) have described acyclic sugar nucleosides which could be considered as derived from the aldehydrol

(hydrate) form of aldehydo-D-galactose (and D-glucose) pentaacetate.

Similar derivatives of 2-amino-2-deoxy-D-glucose have been reported

(21). It was considered desirable to synthesize an acyclic nucleoside

of D-galactose containing a benzyloxy group attached to the aldehydrol

carbon. Such an acyclic nucleoside was synthesized starting from

penta-O-acetyl-1-0-benzyl-1-deoxy-l-ethylthio-D-galactose aldehydrol

(X).

Compound X was synthesized from D-galactose in the following

manner. D-Galactose on reaction with ethanethiol in concentrated

hydrochloric acid, as described by Wolfrom (204), yielded D-galactose

(204) M. L. Wolfrom, ibid., 2464 (1930).

diethyl dithioacetal (VII). Substance VII was acetylated with acetic

anhydride in pyridine to give penta-0-acétyl-D-galactose diethyl

dithioacetal (VIII) (204). Following the method of Weygand (205)i 6 4

(205) F. Weygand, H. Ziemann» and H. J. Bestmann» Ber.,

2534 (1958). penta-O-acetyl-D-galactose diethyl dithioacetal (VIII) was converted to penta-O-acetyl-l-bromo-1,1-dideoxy-l-ethylthio-D-galactose alde­

hydrol (IX) by reaction with bromine in ether. Compound IX was then

reacted with benzyl alcohol containing silver carbonate to give the

desired crystalline penta-O-acetyl-l-O-benzyl-l-deoxy-l-ethylthio-D-

galactose aldehydrol (X).

Synthesis of Penta-O-acetyl-1-0- benzyl-l-bromo-l-deoxy-D- galactose Aldehydrol (XI) Penta-O-acetyl-1-0-benzyl-1-deoxy-l-ethylthio-D-galactose

aldehydrol (X) in ether was treated with bromine to give crystalline

penta-O-acetyl-l-O-benzyl-l-bromo-l-deoxy-D-galactose aldehydrol (XI)• This compound, as with most sugar halides, decomposes on exposure to

moisture over a short period of time. Substance XI was therefore

prepared immediately before it was used.

Synthesis of Penta-0-acetyl-l-(9-adeavl picrate)-l-O-benzyl-l-deoxv-D- galactose Aldehydrol (XII)

6-Benzamido-9-chloromercuripurine (203) was condensed with

penta-O-acetyl-1-0-benzyl-1-bromo-l-deoxy-D-galactose aldehydrol (XI)

in refluxing toluene to give the blocked nucleoside, penta-O-acetyl-

l-[9-(6-benzamidopurinyl)]-l-0-benzyl-l-deoxy-D-galactose aldehydrol.

The fully blocked nucleoside was not isolated but rather was converted 6 5 to the yellow crystalline penta-O-acetyl-1-(9-adenyl picrate)-l-0- benzyl-l-deoxy-D-galactose aldehydrol (XII) by treatment with hot methanolic picric acid.

Thin layer chromatography of a methanolic solution of the fully blocked (not isolated) nucleoside showed, under ultraviolet light, one large spot and one minor spot. However, the material from the minor spot could not be obtained in pure form.

Synthesis of Penta-0-acetyl-l-(9-adenyl)- 1-0-ben zyl-l-deoxy-D-galac tos e Aldehydrol (XIII)

The picric acid moiety was removed from penta-0-acetyl-l-(9- adenyl picrate)-l-0-benzyl-I-deoxy-D-gaIactose aldehydrol (XII) by treatment with anion exchange resin in aqueous acetone. This treat­ ment yielded white crystalline penta-O-acetyl-1-(9-adenyl)-1-0- benzyl-l-deoxy-D-galactose aldehydrol (XIII),

Synthesis of I-(9-Adeavl)-1-0-benzyl- l^eoxy-D-galactose Aldehydrol (XIV)

The complete deacetylation of penta-0-acetyl-l-(9-adenyl)-l-

0-benzyl-l-deoxy-D-galactose aldehydrol (XIII) was effected with

n-butylamine in a solution of equal parts of methanol and tetra-

hydrofuran. It was found that the addition of tetrahydrofuran greatly

increased the solubility of XIII. Solvent removal and recrystalliza­

tion of the residual solid gave crystalline l-(9-adenyl)-l-0-benzyl-

1-deoxy-D-galactose aldehydrol (XIV),

The reaction sequence leading to XIV from XI might be expected

to produce two anomeric nucleosides since reaction occurred at the

optically active C-1 site but only one form was isolated. 6 6

Br O C H ^ C ^ H s OCH-C.H.I Li O D CHSC,H. CHSC 2 H 5 CHBr I ' ' C ^ H ^ C H z O H I Br. 1 (CHOAc). A g 2»C03 (CHOAc)^ (CHOAc) I C H g O A c CHgOAc CHgOAc

IX X XI

O

HN XI + picric acid. (CH^OH) '

HgBr CHOCHgC^Hg

( CHOAc

CH.,OAc NH XII N ^ XII- NH

CHOCH,C,H

(CHOAc) CHOCH.C/H

(CH0H)4 XIII CH^OH

XIV

Figure 21,— Synthesis of l-(9-Aderiyl)-l-0-benzyl-l-deoxy- D-galactose Aldehydrol (XIV) ” 67 Hydrogenolvsis of 1-(9-Menyl)-1-0-benzyl-l- deoxy-D-galactose Aldehydrol llVl')

It was considered that cleavage of the 1-0-benzyl group of the nucleoside XIV by hydrogenolysis with a palladium catalyst might yield the nucleoside with the sugar moiety in the cyclic (furanosyl or

I^anosyl) configuration. However» when the hydrogenolysis was carried out under mild conditions only starting material could be recovered.

Under more stringent conditions, paper chromatography of the reaction products showed the presence of D-galactose, adenine, and a spot cor­

responding to galactitol. Conditions were not found under which

cleavage of the benzyl group took place without cleavage of the adenine moiety.

Reaction of 1-(9-Adenvl)-1-0-benzvl- l-deoxy-D-galactose Aldehydrol (XIV) with Sodium in Liquid Ammonia

Goodman and co-workers (206) debenzylated 9-(tri-0-benzyl-P-D-

(206) E. J. Reist, V. T. Bartuska, and L. Goodman, J. Org.

Ghem., 3725 (1964).

arabinofuranosyl)adenine successfully with sodium in liquid ammonia

without affecting the adenine ring. When XIV was treated with sodium

in liquid ammonia and the products chromatographed on thin layer plates

of raicrociystalline cellulose, two zones were found. One zone cor­

responded to adenine while the other unidentified zone (not visible

under ultraviolet light) traveled slower than D-galactose and 68 galactitol# No zone corresponding to the desired nucleoside with a cyclic sugar moiety was found.

Synthesis of 9-(Methyl Tri-O-acetyl-B-D- galactopyranosyluronate)adenine Picrate (XIX)

Although numerous varied types of nucleosides have been synthe­ sized» especially for the testing of their biological and chemotheraputic activity, only a very small number of hexuronic acid nucleosides have been reported (15-18).

Recently, a cytosine nucleoside which was isolated from a Streptococcus and which has shown broad anti-bacterial activity, has been postulated to contain a uronic acid as the sugar component

(207).

(207) H. Iwasaki, Yakugaku Zasshi, % , I358 (I962).

It was therefore considered desirable to prepare nucleosides of

D-galacturonic acid derivatives.

Substance XIX was synthesized in the following manner. o-D-

Galacturonic acid monohydrate (XV) % prepared by the enzymic hydrolysis

of citrus pectic acid as described by Pigman (208), was converted to

(208) W. W. Pigman, J. Res. Natl. Bur. Std., A, 301 (1940).

methyl o-D-galacturonate (XVI) by reaction with diazomethane in

methanol as described by Morell and Link (209) and in better yield

(209) S. Morell and K. P. Link, J. Biol. Ghem., IO8 , 763 (1935). 69 by treatment of XV with methanol containing a catalytic amount of hydrogen chloride (210). The methyl ester (XVI) was acetylated with

(210) H. B. Wood; Jr.; in "Methods of Carbohydrate Chemistry;"

Vol. II; R. L. Whistler and M. L. Wolfrom; eds.; Academic Press;

New York; N. Y.; 1963» p. 57.

acetic anhydride in the presence of zinc chloride (209) and the

acetylated methyl ester (XVII) was treated with a solution of acetic

anhydride saturated with hydrogen bromide to give the known methyl

tri-O-acetyl-a-D-galactopyranosyluronate bromide (XVIII) (211).

(211) S. Morell; L. Baur; and K. P. Link; J. Biol. Chem.; 110;

719 (1935).

Compound XVIII was condensed with 6-benzamido-9-chloromercuripurine

(203) in refluxing toluene to produce 6-benzamido-9-(methyl tri-0-

acetyl-P-D-galactopyranosyluronate)purine (not isolated). The com­

pletely blocked nucleoside was debenzoylated (203) by treatment with hot methanolic picric acid. This treatment produced yellow crystal­

line 9-(methyl tri-O-acetyl-B-D-galactopyranosyluronate)adenine picrate

(XIX).

Synthesis of 9-(Methyl Tri-O-acetyl-B-D- galadtopvranosyluronate)adenine (XX)

9-(Methyl tri-0-acetyl-P-D-galactopyranosyluronate)adenine pic­

rate (XIX) in aqueous acetone was treated with anion exchange resin

in order to remove the picric acid moiety. This treatment produced 7 0 white crystalline 9-(methyl tri-O-acetyl-P-D-galactopyranosyluronate)- adenine (XX).

NHCC£,H5

NHCC/Hc

picric acid. (CH3OH)

OAc

XVIII OAc

NH. -T- ■ N

NHg (C^HzOyNs)'

HN COCH, N'

\OAc XIX NL_ OAc

XX

R = methyl tri-0-acetyl p-D-galactopyranosyluronate

Fig. 22.— Synthesis of 9-(Methyl tri-0-acetyl-(3-D- galactopyranosyluronate)adenine (XX) EXPERIMENTAL

Preparation of penta-0-acetyl-P-D-galactofur5no3e (I). The method used for the preparation of the title compound is that described Ness, Fletcher, and Hudson (201).

Powdered D-galactose (40 g.) was slowly added to a boiling mixture of I85 ml. of acetic anhydride and 600 ml. of pyridine.

The mixture was stirred until solution was complete (3-5 min.) and

was then refluxed for a further 5-10 rain. The solution was concen­

trated under diminished pressure to a brown sirup. The sirup was

taken up in 125 ml. of chloroform and combined with a similar solu­

tion prepared in the same manner from 40 g. of D-galactose. The

combined solutions were washed with cold water, 3Ü sulfuric acid,

aqueous sodium bicarbonate? solution,, and finally dried (magnesium

sulfate). The dried solution was concentrated under reduced pressure

to a brown sirup. Traces of chloroform were removed by repeated addi­

tion of ethanol with subsequent concentration to dryness under reduced

pressure. The sirupy residue was dissolved in 300 ml. of 95^ ethanol,

seeded with penta-O-acetyl-p-D-galactopyranose and placed at 5° for

18 hr. The crystalline P-D-galactopyranose pentaacetate which formed

was removed ty filtration; yield 28.5 g* (I6.556); m.p. 135-140°, after

one recrystallization from ethanol; m.p. 143-144 ; [a] D +25.6

(0 4.3; chloroform). Reported (201) for p-D-galactopyranose

71 72 pentaacetate; m.p. 143-144°, [a]^^D +25.2° (chloroform).

The above filtrate was seeded with p-D-galactofuranose penta­ acetate and placed at 5° for 5 to 7 days. Filtration then gave

crystalline I; yield 22 g» (12.756); m.p. 98-100°» after one re­

crystallization from 95)6 ethanol; m.p. 99-100®; [a]^^D -42.0° (c

3.3» chloroform). The constants being in good agreement with the 25 reported values (201); m.p. 99-100®; [a] D -42.2® (c 0.89, chloro­ form) .

Preparation of tetra-O-acetyl-B-D-galactofuranosyl chloride (11).

The procedure described by Ness, Fletcher, and Hudson (201) was

utilized for this preparation.

An amount of 5 g* of (I) (201) was dissolved in 12 ml. of glacial

acetic acid. To this solution was added 15 ml. of a solution of hydro­

gen chloride in glacial acetic acid (856 hydrogen chloride w.w.).

After 100 min. at room temperature the reaction mixture was diluted

with 1,2-dichloroethane, washed with cold water, then with cold 3)6

aqueous sodium bicarbonate solution, and dried (magnesium sulfate).

The dried solution was concentrated under diminished pressura to a

sirup which was taken up in anhydrous ether. The ether solution was

repeatedly cooled in a solid carbon dioxide acetone bath and warmed

to room temperature until crystallization was initiated. Crystalliza­

tion was then completed at 5° overnight. Filtration gave crystalline

II; yield 2.8 g. (59.6#); m.p. 69-71°; [a]^^D -75.2° (c 2, chloroform).

The reported (201) constants for recrystallized II are: m.p. 71-73°

and [a]D -77.8° (c 3*9» chloroform). 73 Synthesis of 2>6-dlacetamldo-9-(tetra-0-acetyl-P-D-galacto- furanosyppurlne (III). An amount of 2 g. of II (201) was added to an azeotropioally dried mixture of 2»6-diacetamido-9-chloromercuri- purine (14) (2.8 g.), Cellte (212) (1 g.), cadmium carbonate (1.8 g.),

(212) A siliceous filter aid, Johns-Manville Co., New York,

N. Y. and toluene (100 ml.). The suspension was stirred 4 hr. under reflux,

the hot mixture was filtered, the filter cake was attracted with warm

chloroform, and the combined filtrate and chloroform solution was

evaporated under reduced pressure to a sirup. The sirup was extracted

with chloroform. The chloroform solution was washed with 30?^ aqueous

potassium iodide, then with water, and dried (magnesium sulfate).

Evaporation of the chloroform solution under reduced pressure yielded 20 a white amorphous solid; yield 1.24 g. (40.45&)| m.p. 110-112°; [a] D

-28.5^° (o 0.3» chloroform); absorption spectra data (213):

(213) The ultraviolet absorption analyses were made on a Cary

recording spectrophotometer. Model 15» Applied Physics Corp., Pasadena,

Calif. The infrared spectral data were obtained on a Perkin-Elmer

Model 137B Infracord Spectrophotometer, Perkin-Elmer Corp., Norwalk,

Conn. Structural assignments were based on analogous assignments in

"Infrared Absorption Spectroscopy," K. Nakanishi, Holden-Day, Inc.,

San Francisco, 1963» 74

236.2, 273.5» 279.5 mu; ^ 3.15, 3.25 (NH), 5.75 (ester carbonyl),

5.95 (amide carbonyl), 6,15, 6.25, 6.75 (NH and purine ring), 7.30

(methyl hydrogen), 9.04, 9.25, 9.6o, 9.82 p, (C-O-C).

Anal. Calcd. for C, 48.92; H, 5.06; N, 14.89.

Found; C, 49.12; 5.01; N, 15.8O.

Attempts to crystallize III were unsuccessful.

Synthesiseof 2-acetamido-9-P-D-galactofuranosyladenine (IV).

The partial deacetylation of III was effected with boiling methanolic n-butylamine (202). Amorphous III (70O mg.) was dissolved in methanol

(25 ml.) containing n-butylamine (l.O ml.) and heated 6 hr. under reflux, during which time precipitation of a colorless crystalline solid resulted. After partial evaporation and cooling, the ciystalline k material was removed by filtration; yield 340 mg. (885È); m.p. 212-213°.

The crude crystalline material was decolorized (activated carbon) in water and recrystallized from aqueous ethanol to give analytically pure material; m.p. 240-241°; -53^5° (£ 0.18, water); absorption

spectra data (213): 226.0 , 268.0 mp; 2.95, 3.15 (OH, NH),

5.88 (amide carbonyl), 6.10, 6.24, 6.44, 6.80 (purine), 8.90, 9.08,

9 .30, 9.55 P (C-O-C, C-O-H); X-ray powder diffraction data (214)

(214) Interplanar spacing, CuK^ radiation. Relative intensity estimated visually: s, strong; m, medium; w, weak; v, vary; three

strongest lines numbered (1, strongest).

10.40 s, 8.27 vs (1), 5.95 vw, 5.34 m, 4.58 m, 4.24 m, 3.97 vs (2),

3.65 vs (3), 3.43 vw, 3.19 vw, 2.96 w. 75 Anal. Calcd. for C, 44.05; H, 5.12; N, 23.72.

Found: C, 43.85; H, 5.17; N, 23.90.

Compound IV moved on paper chromatography (215) as a single

(215) Chromatographic data refer to descending chromatograms on Whatman No. 1 paper with 1-butanol-ethanol-water (40:11:19 v./v.) with indication ultraviolet light and by sodium metaperiodate and ammoniacal silver nitrate sprays according to L. Hough and J. K. N.

Jones ("Methods in Carbohydrate Chemistry," Vol. I, R. L. Whistler and M. L. Wolfrom, eds.. Academic Press, New York, N. Y., I963* p 28 ). zone, 0.49.

Synthesis of 2,6-diamino-9-P-D-galactofuranosylpurine (V).

Crystalline 2-acetamido-9-P-D-galactofuranosyladenine (IV), 200 mg., was dissolved in hot absolute methanol, treated with 20 mg. of sodium raethoxide and the solution was refluxed for 6 hr. The solution was

cooled, neutralized with glacial acetic acid and treated with 5 ml.

of I05È methanolic picric acid. The mixture was refluxed for 0 .5 hr., then cooled to room temperature at which time a yellow picrate de­

posited. The picrate was cooled, separated by filtration and washed o with absolute methanol; yield 157 mg., dec. 220-225 . The picrate

was dissolved in hot aqueous acetone and treated with enough Dowex-1 ••2 (CO^** ) (216) anion exchange to result in a colorless solution. The

(216) A product of the Dow Chemical Co., Midland, Mich. 76 solution was concentrated under reduced pressure to a sirup and the sirup taken up in hot methanol. On cooling and concentrating white 20 crystalline V deposited; yield 73 mg. (42)^); m.p. 224-225°; [a] D

-64° (c 0.23; water); absorption spectra data (213): 258; 281 mp;

3.04 (OH; NH); 6.04; 6.3O; 6.88 (NH and purine ring); 9.14» 9.70 p,

(C-OH); X-ray powder diffraction data (214); 7.53 m; 6.07 W; 5.74 m»

5.22 m; 4.82 m; 4.46 m; 4.26 s (3); 3.8I s (1), 3.48 s (2), 3.25 w;

3.07 VW; 2.95 VW; 2.?6 VW.

The material moved as a single spot; O.3I» on paper

chromatography (215).

Anal. Calcd. for C11H16N6O5: C; 42.30; H; 5.17; N; 26.91.

Found: 0; 42.13; H; 5.74; N; 26.22.

The compound V was also prepared by the complete deacetylation

of 2 ;6-diacetamido-9-(tetra-0-acetyl-P-D-galactofuranosyl)purine (III) with boiling methanolic sodium methoxide in 34?^ yield.

Synthesis of 9-B-D-galactofuranosyladenine (dimorphous) (VI).

(A) Crystalline tetra-O-acetyl-p-D-galactofuranosyl chloride (II»

1.6 g*) (201) was added to an azeotropioally dried mixture of 6-benzamido-

9-chloromercuripurine (2.07 g.) (203)» Celite (l g.) (212)» cadmium

carbonate (1.8 g.)» and toluene (I50 ml.). The suspension was stirred

for 4.25 hr. under reflux» the hot mixture was filtered» the filtrate

extracted with warm chloroform» and the combined extracts were evapo­

rated under reduced pressure to a sirup. The sirup was extracted with

chloroform» washed with 30$ aqueous potassium iodide » then with water»

and dried (magneisum sulfate). Evaporation of the chloroform under

reduced pressure gave a white amorphous solid; yield I.7I g. (69.0$); 77 m.p. (range) 67-87°; [oJ^^D -15° (c 1.25» chloroform); absorption spectra data (213); 2,8-3,0 (broad* NH), 3*33 (C-H), 5'&5

(ester carbonyl)» 5*82 (amide carbonyl), 6.05, 6.18, 6.28, 6.6, 6.7»

6.86 (NH» purine ring, benzene ring), 7*29 (methyl hydrogen), 9.4-

9.7 M* (broad, C-O-C).

Déacylation of 6-benzaraido-9-(tetra-0-acetyl-P-D-galactofurano-

syl)purine was effected with boiling methanolic n-butylamine (202). The acylated nucleoside (0.26 g.) was dissolved in methanol (20 ml.)

containing n-butylamine (0.5 ml.) and the solution was refluxed for

6 hr. The sirup obtained on solvent removal under reduced pressure

was dissolved in methanol and ether added to incipient turbidity.

Crystals formed on standing in the refrigerator. Filtration of the

fine white crystals was effected in a drybox because of their extremely

hygroscopic nature; yield 50 mg. (37^); dec. 209-212°; [a]^^D -52t3°

(0 0.3, water); absorption spectra data (213): xJJgO 262.5 ^max

3.05, 3.15 (OH, NH), 6.15, 6.25, 6.40, 6.85 (NH, purine ring), 9.15»

9 .4 5, 9 .70, 9.95 (C-O-C» C-OH); X-ray powder diffraction data (214):

9.35 m, 6.63 m, 5.77 m, 5*47 s (3)» 5.13 s (2 ), 4.6o m, 4.20 w,

3.88 m, 3.63 s (1), 3.39 s, 3.27 m, 3.12 w, 2.98 vw, 2.81 w, 2.74 w,

2.41 m, 2.29 w, 2.19 w.

Anal. Calcd. for : C, 44.43; H, 5.09; N, 23.54.

Found: C, 44.13; H, 5*42; N, 22.50.

(B) Crude 6-benzamido-9-(tetra-0-acetyl-P-D-galactofuranosyl)-

purine (0 .5 B») was dissolved in 10 ml. of absolute ethanol, and 2 ml. of a 10% ethanolic picric acid solution added (203). The mixture was refluxed for 4 min. during which time yellow crystals separated from 78 the solution and were removed by filtration of the hot solution; yield 0.33 g. This product was recrystallized three times from ethanol; m.p. 208-209°; X-ray powder diffraction data (214): 8.89 vw, 8.01 w,

7.41 m, 6.44 vw, 6.09 m, 5»70 vw, 5*28 w, 5.01 w, 4 .60 vw, 4.44 m (3)»

4.05 w, 3*87 w, 3*34 m (1), 3*21 m (2). Removal of the picrate anion

(203) iv stirring in an aqueous acetone solutionthe 0-acetylated

picrate (134 mg.) with Dowex-1 (C0^“^) (2l6) anion exchange resin gave

a white gummy material which failed to crystallize from common sol­

vents. The gummy 9-(tetra-0-acetyl-P-D-galactofuranosyl)adenine was

deacetylated with boiling methanolic n-butylamine as described in A.

A white crystalline solid precipitated from the hot solution. Upon

cooling and filtering, pure 9-P-D-galactofuranosyladenine (VI) was

obtained; yield 43 mg. (755^); m.p. 224-225°; [a]^^D -52^2° (c 0.3, water); spectral and X-ray powder diffra ction data identical with

those cited in A.

A recrystallization of the hygroscopic VI prepared in A (dec.

209-212°) utilizing the higher melting VI (m.p. 224-225°) as nucleating agent, yielded white crystals, m.p. 224-225°.

Preparation of D-galactose diethyl dithioacetal (VII). D-Galac-

tose was converted to VII following the method described by Wolfrom

(204).

D-Galactose (280 g.) was dissolved in 235 ml. of concentrated

hydrochloric acid. The solution was then cooled in an ice-water

bath and 240 ml. of ethanethiol was added in portions with vigorous

shaking. After shaking for 5-10 min. a solid crystalline mass re­

sulted. Cold water was added and the solution was filtered and washed 7 9 with cold water. The crystalline VII was recrystallized from hot

absolute ethanol; yield 101 g. (44^); m.p. 14-1-142°; [a]^^D -4-.l° 20 (c, 0.90» water). Reported (217) values for VII: m.p. 142-14-3°; [a] D

-4.8° (c 1.09» water).

(217) R* Harm» W. D. Maclay, and C. S. Hudson, J. Am. Ghem.

Soc.» 61, 1270 (1939).

Preparation of penta-O-acetyl-D-galactose diethyl dithioacetal

(VIII). The acétylation of VII was carried out following the procedure

described by Wolfrom (204).

An amount of 100 g. of D-galactose diethyl dithioacetal (VII)

(204) was dissolved in pyridine (280 ml.) and the solution was cooled

in an ice-water bath. Acetic anhydride (250 ml.) was added in por­

tions to the cold solution with shaking after each addition. The

mixture was placed at room temperature overnight, then poured into

2 1. of a mixture of crushed ice and water. The crystalline mass which resulted was removed by filtration,, washed with water and re-

crystallized from hot absolute ethanol; yield 158 g. (92/6); m.p. 77-

78°; +9.4° (c 2.5, chloroform). Reported (204) for VIII:

m.p. 77.5-78.5°; +9 .7O (chloroform).

Preparation of penta-0-acetyl-l-bromo-l,l-dideoxy-l-ethyl- thio-D-galactose aldehydrol (IX). The compound IX was prepared from

penta-O-acetyl-D-galactose diethyl dithioacetal (VIII) by the method

of Weygand and co-workers (205).

Penta-O-acetyl-D-galactose diethyl dithioacetal (VIII, 27.0 g.) 80

(204) was dissolved with stirring in 250 ml. of dry ether. To the stirred solution was added dropwise a solution of 8.6 g, of bromine in 90 ml. of dry ether. A white solid separated after 15-20 min. of addition. The mixture was stirred a further 30 min. after addition was completed then cyclohexene was added to remove the excess bromine.

Petroleum ether (b.p. 65-110°) was added to aid precipitation and the solution was kept at 5° overnight. The crystalline IX was removed by filtration; yield 23.5 g. (84$); m.p. 102-104°; [a]^^D -16.3° (c 1.2, chloroform). The chloroform solution of IX tended to become cloudy after standing for a short period of time, thus making an accurate reading of the specific rotation difficult. Weygand and co-workers

(205) have reported for IX; m.p. 104-105°; [oJ^^D -17.5 "* +28.0°

after 3 hr. (c 3» chloroform).

Synthesis of penta-O-acetyl-l-O-benzyl-l-deoxy-l-ethylthio-

D-galactose aldehydrol (X). An amount of 23.5 g. of penta-O-acetyl

l-bromo-l,l-dideoxy-l-ethylthio-D-galactose aldehydrol (IX) (205)

was dissolved in 200 ml. of dry benzyl alcohol. Silver carbonate

(31*0 g.) was added and the mixture was stirred for 24 hr. in the

absence of light. The salts were then removed by filtration, the

filter cake was washed with hot ethanol, the washings and filtrate were combined and taken to dryness under diminished pressure. The

residue was extracted with excess hot ethanol, filtered and placed

in the refrigerator. The long needles which deposited were filtered

and recrystallized from ethanol; yield I6 .8 g. (68$); m.p. 117-118°;

[a]^^D -8.0° (c 6, chloroform); absorption spectra data (213):

3.42-3.30 (CH, aromatic), 5*72 (acetate carbonyl), 6.23, 6.64 81

(phenyl)» 9*17-9*77 (C-O-C)» 12.93» 13*20» 14.19 H (substituted phenyl);

X-ray powder diffraction data (214); II.63 m» 9*94 s (1)» 7.50 vw»

7*08 VW» 6.15 s (2)» 5.90 m» 5.34 s (3)» 4.90 m» 4.35 m (broad)» 3*97 m»

3.71 m» 3*58 w» 3*36 w.

Anal. Calcd. for Cg^H^Og^^S: C» 55*34; H» 6.23; 3» 5.92 .

Found: C» 55*20; H» 6.50; S» 5*95*

Synthesis of penta-O-acetyl-l-O-benayl-l-bromo-l-deoxy-D—

galactose aldehydrol (XI). Five grams of penta-O-acetyl-l-O-benzyl-

1-deoxy-l-ethylthio-D-galactose aldehydrol (X) was dissolved with

stirring in 50 ml. of dry ether. To this stirred solution was added

dropwise a solution of I.5 g* of bromine in 20 ml. of dry ether. After

the addition had proceeded for 15-20 min.» a white precipitate appeared

in the mixture. After completion of the bromine addition» the stir­

ring was continued for 45 min. Excess bromine was removed by the addi­

tion of cyclohexene and» after the addition of petroleum ether (b.p.

65-110°)» the solution was placed at 5° for crystallization; yield

4 .9 g* (94^); m.p. 112-113°; [af^D +73*6° (c 1*53» chloroform). This product was unstable and decomposed readily on exposure to air.

Anal. Calcd. for C» 49.20; H» 5.20; Br» 14.23.

Found; C» 49*43; H» 5*26; Br» 14.82. Synthesis of penta-0-acetyl-l-(9--adenyl picrate)-1-0-benzyl-l-

deoxy-D-galactose aldehydrol (XII). A mixture » 2.0 g. of Celite (212)»

1.7 g. of cadmium carbonate» and 5*3 g* of 6-benzamido-9-chloromercuri-

purine (203) in 400 ml. of toluene was dried ly azeotropic distillation.

The solution was cooled to 6o-70°and a solution of 4.8 g. of penta-0-

acetyl-l-O-benzyl-l-brorao-l-deoxy-D-galactose aldehydrol (XI) in 82 azeotropically-dried toluene was then added. The mixture was refluxed with stirring for 4 hr. The hot solution was filtered and the filter cake was extracted with hot chloroform (300 ml.). The filtrate and chloroform extracts were combined and concentrated to a glass under diminished pressure. The residual glass was extracted with 300 ml. of warm chloroform and the extract was filtered. The filtrate was washed with a 30^ aqueous potassium iodide solution, water, and dried

(magneisum sulfate). The dried solution was concentrated to a glass

under diminished pressure and the glass was dissolved in 75 ®1. of

methanol. Thin layer chromatography (2l8) of this solution showed two

(218) Thin layer plates (50 x 120 mm.) of silica gel 0 (0.25 mm.)

(E. Merck, Darmstadt, Germany) utilizing a solution of benzene-ethyl

acetate-methanol (90:0.5:0.5 v./v.) as developer. Indication of spots was by ultraviolet light and cone, sulfuric acid spray.

spots under ultraviolet light: a large spot at 0.24 and a minor

one at 0.55* The material in the minor spot could not be obtained

in pure form.

To the above methanol solution was added 14 ml. of a 10)^ methanolic

picric acid solution and the mixture was refluxed for 1 hr. A yellow

solid precipitated from the hot solution. The solution was kept at 5°

to complete precipitation, and the yellow picrate was removed by fil­

tration; yield 3*5 g* (52j6 based on XI); m.p. 174-175° (from methanol);

[a]^®D -40° (c 3*4, chloroform); X-ray powder diffraction data (214)

10.28 w, 8.67 m (1), 7*63 m, 6.19 m, 5.6I w, 5*16 w, 4.62 w, 4.3I m,

3.97 m, 3.69 m (2), 3.15 m. 83 Anal. Calcd. for G^H^O^gNg: C, 46.00; H, 4.18; N, 13.25»

Found; C, 46.26; H, 4.43; N, 13.11.

Synthesis of penta-0-acetyl-l-(9-adenyl)-1-0-benzyl-l-deoxy-

D-galactose aldehydrol (XIII). The above picrate (XII» 3.5 g.) was dissolved» with stirring» in aqueous acetone and sufficient Dowex-1 O (coy" ) (216) was added to attain a colorless solution. The resin was then removed by filtration and the filtrate was allowed to con­ centrate by evaporation at room temperature until crystals appeared.

The mixture was kept at 5° to complete crystallization; yield 1.4 g.

(54.9^); m.p. 225-226°; [a]^^D -7.4° (0 2 .6» chloroform); absorption spectra data (213); ^^ax ^59 mi 4 a g 3.01» 3.I6 (0H» NH)» 3.41 (CH),

5.69 (ester carbonyl)» 5.97» 6.10» 6.23 (purine and phenyl)» 9 .I6-

9 .30» 9*58 (C-O-C)» 13.18» 14.27 (substituted jphenyl); X-ray powder diffraction data (214); 14.49 m» 9.72 vw» 7.08 s (1)» 6.42 w»

5.75 w» 5.04 m (2)» 4.7 2 ra (3)» 4.48 w» 3.87 w» 3.48 w» 3.27 w» 2.98 w.

Anal. Calcd. for C28 H33O6N5: c» 5^.63; H» 5.43; N» 11.37.

Found: C» 54.46; H» 5.53; N» II.65,

Synthesis of l-(9-adenyl)-l-0-benzyl-l-deoxy-D-galactose aldehydrol (XIV). An amount of 1.4 g. of penta-0-acetyl-l-(9-adenyl)-

1-O^benzyl-l-deoxy-D-galactose aldehydrol (XIII) was dissolved in a solution of 1;1 methanol-tetrahydrofuran and 1 ml. of n-butylamine was added. The solution was refluXed for 6 hr. It was then concen­

trated under diminished pressure to near one-half volume and placed

in the refrigerator to effect crystallization; yield 0.7I g. (54.656).

Thin layer chromatography (219) of the product before recrystallization 8 4

(219) Chromatography was on Avicel (Technical Grade) (micro- crystalline cellulose) produced by the EMC Corp.» American Viscose

Division» Marcus Hook» Pa.) plates (0.25 mm. thick» 50 x 120 mm.) following M. L. Wolfrom» D. L. Patin» and R. M. de Lederkremer»

Chem. Ind. (London)» IO65 (1964); J. Chromatog.» in press. The de­ veloper was a solution of 1-butanol-ethanol-water (40:11:19 v./v.). Location of spots was by ultraviolet light and with a spray of a solu*

tion consisting of 1 part of a solution of potassium permanganate in 25& sodium carbonate and 4 parts of a solution of 2$ sodium periodate,

revealed two components » R^ 0.57 (minor) and R^ O.63. The minor

component » which was absent after three recrystallizations from

methanol-water, could not be obtained in sufficient amount for

characterization. " After three recrystallizations from methanol-water, XIV moved

as a single spot on thin layer chromatography (219)» m.p. 212.5-

213.5°; -13° (c 2.1» dimethyl sulfoxide); absorption spectra

data (213): aJiJ 261 m;i; x g j 2.92-3.1? (OH, NH), 3.42 (CH)» 6.09,

6.23, 6.35, 6.72 (purine» phenyl)» 9*22—9*66 (C—0—C» C-O-H), 13*29™

13*40» 13*71 4 (substituted phenyl); X-ray powder diffraction data

(214): 11.19 m, 8.59 m, 6.19 w, 5.61 vs (1), 4.6? vs (3)» 4.42 m,

4.21 m, 4.06 m, 3*65 vs (2), 3.45 vw, 3*36 s, 3.21 m, 2.97 m, 2.89 vw

2.81 m.

Anal. Calcd. for 03^6^23%^^* C» 53*31; H» 5*72; N» 17*2?.

Found: G» 53*52; H» 5.92 ; N, 17*37* 85

Hydrogenolysis of l-(9-adeavl)-l-0-benzyl-l-deoxy-D-galacto3e aldehydrol (XIV)» An amount of 45 mg. of XIV was dissolved in 40 ml. of methanol and 10 mg. of 105& palladium on charcoal catalyst was added.

The suspension was shaken in a Parr hydrogenation apparatus under 42 p.s.i. of hydrogen for 24 hr. The suspension was filtered and the filtrate was chromatographed on paper (215)« 8y comparison with

authentic samples, adenine, D-galactose, and galactitol were found

in the filtrate. No spots were found corresponding to the desired

9-P_D-galactofuranosyladenine (112) or 9-P-D-galactopyranosyladenine

(19). When the same hydrogenolysis conditions were used with shorter

reaction times (2-4 hr.), only unreacted starting material could be

detected. Use of water as solvent or 5?^ palladium on charcoal

catalyst with various pressures (20-48 p.s.i.) of hydrogen produced no

detectable changes in the kinds of products obtained.

Reaction of l-(9-adenyl)-l-0-benzyl-l-deoxy-D-galactose

aldehydrol (XIV) with sodium in liquid ammonia. Liquid ammonia (25 ml.)

was added to a cooled (solid carbon dioxide-acetone) flask containing

50 mg. of XIV. The solution was stirred and sodium metal was added

(3-4 mg. pieces) until the blue color persisted (total 20-30 mg.)

(206). Ammonium chloride was then added until the blue color dis­

appeared. The solvent was allowed to evaporate under a stream of

nitrogen. The solid which remained was dissolved in water and

chromatographed (219) • A spot corresponding to adenine was found

along with a spot (R^ 0.14) which traveled slower than D-galactose

or galactitol. No spots were found corresponding to 9-P-D-galacto-

furanosyladenine (112) or 9-P-D-galactopyranosyladenine (19)* 86

Preparation of a-D-galacturonlc acid monohydrate (XV)« The compound (XV) was prepared according to the method described by

Pigman (208)*

To 1.4 1, of water at 40° was added 140 g. of citrus pectic

acid (220) followed by the addition of 20? ml. of 3 N sodium

(220) Citrus pectic acid produced by the Fruit Growers Exchange»

Ontario, Calif.

hydroxide. To the stirred solution was added 11 g. of pectinase (221)

(221) Pectinase (Fungal), C grade was purchased from Calbio-

chem., Los Angeles, Calif.

and the mixture was maintained at 38° for 14 days. An amount of

207 ml. of 3 N sulfuric acid was added, the solution was stirred

vigorously, filtered, and the filter cake was washed with water.

The filtrate and washings were combined, treated with carbon and

concentrated under reduced pressure to a brown gum. The gum was taken

up in 1 1. of boiling methanol, filtered, the filtrate was concen­

trated under diminished pressure to 300-350 ml., seeded and placed at* 5° for 2 days. The crystalline material was then removed by fil­

tration; yield 81.3 g* (585^); dec. (softens 109-110°) 125-130°

[a] D +95*3° (3 min.) +51*9 (equilibrium value) (o 3» water).

Reported (208) for o-D-galacturonic acid monohydrate; m.p. 109-112°

(unrecrystallized); [a]^^D +51*5° (Ç 4, water) (equilibrium value). 87

Preparation of methyl a-D-galacturonate (XVI)# (A) The proced­ ure of Morell and Link (208) was utilized for this preparation of XVI»

a-D-Galacturonic acid (5 g.) from a-D-galacturonic acid mono­ hydrate (208) dried at 78° under diminished pressure over phosphorus pentaoxide for 8 hr.» was dissolved in 100 ml, of dry methanol and cooled in a salt-ice-water bath. An ethereal solution of diazomethane

(222) was then added to the above cooled solution in portions until

(222) The solution of diazomethane was prepared by the method of De Boer and Backer [T. J, De Boer and H. J, Backer» Rec, Trav,

Chim.» 22.» 229 (195^)] from 21.5 g* of N-methyl-N-nitroso-£-toluene- sulfonamide; yield 2,7-2.9 g. of diazomethane. no further gas was evolved. A small amount of solid (shown to be

0 .9 g. of a-D-galacturonic acid) was removed by filtration and the

filtrate was concentrated under diminished pressure to a sirup. The

sirup was taken up in 50 ml. of hot dioxane» filtered» and the fil­

trate was allowed to stand at room temperature overnight in an open

vessel'. The crystalline XVI which appeared was removed by filtration;

yield 2.4 g. (55# including recovery of 0.9 g. of starting material);

m.p. 134-135°; +76.8° (2 min.) +38.8° (equilibrium value)

(0 1.8» methanol). Morell and Link (209) report; sinters 135°» m.p«

145-147°; [a]^^D +75.5° -* +38.0° (after 90 min.) (0 I.3» methanol). (B) Methyl a-D-galacturonate (XVI) was also prepared according

to the method described by H. B. Wood» Jr. (210).

An amount of 60 g. of a-D-galacturonic acid (208) (from the 88 monohydrate as described in A) was added to 1.8 1. of cold (0-5°) dry methanol containing 0.8 g./l. of diy hydrogen chloride and the mixture was kept at 5° for 66 hr. The cold solution was then neutralized with excess silver carbonate» filtered, and the filtrate was concentrated under reduced pressure to a sirup. The sirup was taken up in 2 volumes of hot dioxane and the solution was filtered and placed at room temperature for 24 hr. The crystalline XVI was then filtered and washed with cold ether; yield 5^.7 g. (855^)» identi­ cal with XVI prepared in A.

Preparation of methyl tetra-O-acetyl-o-D-galactopyranuronate

(XVII)> Acétylation of XVI was carried out following the method described by Morell and Link (209),

Methyl a-D-galacturonate (XVI, 10 g.) (209) was slowly added to a cooled (salt-ice-water bath) solution of 4.0 g. of freshly fused zinc chloride in $6 ml. of acetic anhydride. The mixture was allowed to warm until solution was complete, then recooled andJnaintained at o 0 for 5 hr. The mixture was then poured into 225 ml. of chloroform

containing 50 g. of chipped ice, then shaken vigorously. The chloro­

form layer was washed with water and dried (magnesium sulfate). The

solvent was then removed by distillation under reduced pressure and

the resulting sirup was dissolved in dry ether (75 ml.). After a

short time at room temperature crystallization began and the solution was placed in the refrigerator overnight. The crystalline XVII was

filtered and washed with cold dry ether; yield 14.0 g. m.p.

143°; +142.6° (c 1.6, chloroform). Literature (209) values

for methyl tetra-O-acetyl-a-D-galactopyranuronate are, m.p. 142-143°, [a]^^D +143° (£ 1, chloroform). 89 Preparation of methyl trl-0-acetyl-

Dried methyl tetra-O-acetyl-ouD-galactopyranuronate (XVII,

9*0 g.) (209) was added to a cooled (0°) solution of 42 g, of acetic anhydride containing 20 g. of dry hydrogen bromide. The cold solu­ tion was then saturated with hydrogen bromide gas, the flask stoppered tightly and placed at room temperature for 12 hr. The solution was then concentrated under diminished pressure to a light brown sirup.

The sirup was dissolved in chloroform, sodium sulfate and charcoal were added and the mixture was placed in the refrigerator. The dried solution was then concentrated to 15-20 ml., ether (20 ml.) added and the mixture was placed at 5° overnight. Crystalline XVIII was removed by filtration and washed with ether; yield 8.5 g* (89#); m.p. 129-130°, [a]^^D +248.6° (c 1.4, chloroform). Reported (211) constants for XVIII: m.p. 129.5-130.5°; +248° (c 1, chloro­ form).

Synthesis of 9-(Methyl tri-O-acetvl-B-D-galactopyranosyluronate)-

adenine picrate (XIX). A mixture of 4.9 g. of 6-benzamido-9-chloro-

mercuripurine (203), 1«5 g* of cadmium carbonate, and 2 g. of Celite

(212) in 300 ml. of toluene was dried azeotropic distillation and

cooled to 60-70°. To the cooled suspension was added a solution of

3.3 g» of methyl tri-O-acetyl-o-D-galactopyranosyluronate bromide

(XVIII) (211) in 40 ml. of dried toluene and the whole was refluxed

with stirring for 4 hr. The hot solution was filtered, the filter

cake was extracted with hot chloroform, and the combined washings 90 and extracts were concentrated to a glass under diminished pressure.

The glass was extracted with warm chloroform (300 ml.)» the chloro­ form solution was filtered, the filtrate was washed with 30^5 aqueous potassium iodide solution and water, then dried (magnesium sulfate).

The dried solution was concentrated to dryness under diminished pres­ sure and the residue was taken up in 100 ml. of hot methanol. Methan­ olic picric acid (35 ml. of a 10^^ solution) was added to the hot mixture and the whole was refluxed for 1 hr. On cooling to room temperature 0 the yellow picrate precipitated and the mixture was placed at 5 for completion of crystallization. The picrate was removed by filtration and washed with methanol; yield of XIX 3*2 g. (57^ based on XVIII); dec. 154-158°. Recrystallization from methanol with high loss gave yellow crystals; dec. I62-165®; +45.4° (c 3*4, chloroform); absorption spectra data (213); 2.99-3.26 (NH), 3.40 (CH), 5«69

(ester carbonyl), 5*90 (aromatic amine salt), 6.10, 6.20» 6.40* 6.67

(purine), 9*12-9*45 4 (C-O-C)j X-ray powder diffraction data (214):

10.45 vw, 6.80 m (1)» 4.61 vw, 3.92 w (diffuse), 3*4? w (diffuse). Anal. Calcd. for C, 42.35; H, 3*56; N, 16.46.

Found: C, 42.54; H, 3.94; N, 15,70.

Synthesis of 9-(methyl tri-O-acetyl-B-D-galactopyranosyl-

uronate)adenine (XX). An amount of 2.8 g. of the above picrate (XIX)

was dissolved with stirring in 50 ml. of aqueous acetone and suffici­

ent Dowex-1 (C0y"2) (216) was added to produce a nearly colorless

solution (203) (the solution remained slightly yellow-orange even on

repeated treatment with resin). The resin was removed by filtration,

the filtrate was concentrated at room temperature until the 91 appearance of colorless crystals» then the mixture was placed at 5° overnight. Crystalline XX was removed hy filtration; yield 0.89 g. iMr&jh); m.p. 200-262°; [a]^^D +19*4° (c 0.8 , tetrahydrofuran); absorption spectra data (213): 258 m|i; 3.00» 3.12

(NH)» 3*41 (OH), 5*70 (ester carbonyl)» 6,02» 6.23» 6,82 (purine)»

9 .16» 9 *23» 9*35» 9*80 p (C-O-C); X-ray powder diffraction data

(214): 16.07 m» 8.42 vs» (2)» 7,69 vw» 6.86 s» 5.68 vs (1)» 5.40 vs

(3)» 5*10 m» 4.70 m» 4.31 vw» 4.13 m» 3.95 m» 3*75 s» 3*62 s.

Anal. Calcd. for 0» 47*89; H» 4.7O; N» 15.50.

Found: 0» 47*90; H» 4.66; 16.28.

The substance moved as a single zone» 0.79» on thin layer

chromatography (218)» using a solution of benzene-methanol (9:1 v./v.) ascbveloper. SUMMARY

1. 2 »6-Diacetamido-9-(tetra-0-acetyl-P-D-galactofuranosÿl)- purine was synthesized by the reaction of 2,6-diacetamido-9-chloro- inorcuripurine with tetra-O-acetyl-p-D-galactofuranosyl chloride.

2. 2-Acetamido-9-P-D-galactofuranosyladenine was synthesized by partial deacetylation of 2,6-diacetamido-9-(tetra-0-acetyl-P-D- galactofuranosyl)purine with methanolic n-butylamine.

3. 2,6-Diamino-9-P-D-galactofuranosylpurine was synthesized by deacetylation of both 2-acetamido-9-P-D-galactofuranosyladenine and

2 »6-diacetamido-9-(tetra-0-acetyl-p-D-galactofuranosyl)purine with methanolic sodium methoxide followed by purification through picrate

formation and subsequent removal of the picric acid moiety.

4. 9-B-D-Galactofurano8yladenine was synthesized by reaction

of 6-benzamido-9-chlororaercuripurine with tetra-O-acetyl-P-D-

galactofuranosyl chloride followed by deacetylation of the crude

6-benzamido-9-(tetra-0-acetyl-P-D-galactofuranosy].)purine thus formed with methanolic n-butylamine.

5* 9-P-D-Galactofuranosyladenine was also synthesized by

débenzoylation of crude 6-benzamido-9-(tetra-0-acetyl-p-D-galacto- furanosyl)adenine with picrate formation and subsequent depicration

followed by deacetylation with methanolic n-butylamine.

9-p_D-Galactofuranosyladenine was dimorphous. The lower

melting (dec. 209-212°, hygroscopic) form was converted to the

92 93 higher melting (224-22$°) form by recrystallization and nucléation with the higher melting form.

6 * Penta-0-ac etyl-l-O-benzyl-l-deoxy-l-ethylthio-D-galac to se

aldehydrol was synthesized ty the reaction of penta-O-acetyl-l-bromo-,

lil-dideoxy-l-ethylthio-D-galactose aldehydrol with silver carbonate

in benzyl alcohol.

7. The synthesis of penta-O-acetyl-l-O-benzyl-l-bromo-1-

deoxy-D-galactose aldehydrol was effected by the reaction of bromine

with an ethereal solution of penta-O-acetyl-l-O-benzyl-l-deoxy-1-

ethylthio-D-galactose aldehydrol.

8» Penta-^-acetyl-l-(9-adenyl picrate)-1-0-benzyl-l-deoxy-D-

galactose aldehydrol was synthesized by interaction of 6-benzamido-9-

chloromercuripurine with penta-O-acetyl-l-O-benzyl-l-bromo-l-deoxy-D-

galactose aldehydrol followed by treatment of the resulting product

with methanolic picric acid.

9# Penta-0-acetyl-l-(9-adenyl)-l-Q-benzyl-l-deo%y-D-galactose aldehydrol was synthesized from penta-0-acetyl-l-(9-adenyl picrate)-l-

0-benzyl-l-deoxy-D-galactose aldehydrol by treatment with anion ex­ change resin.

10. Synthesis of 1-(9-adenyl)-1-0-benzyl-l-deoxy-D-galactose

aldehydrol was effected by deacetylation of penta-0-acetyl-l-(9-adenyl).

1-0-benzyl-l-deoxy-D-galactose aldehydrol with methanolic n-butylamine.

11. The catalytic hydrogenolysis of l-(9-adenyl)-1-0-benzyl-l-

deoxy-D-galactose aldehydrol produced adenine> galactitol; and 94

D-galactose rather than the desired 9-P-D-galactofuranosyladenine or

9-P-D-galactopyranosyladenine.

12. Treatment of l-(9-adenyl)-l-0-benzyl-l-deoxy-D-galactose aldehydrol with sodium in liquid ammonia yielded adenine and another unidentified product rather than the desired 9-P-D-galactofuranosyl­ adenine or 9-P-D-galactopyranosyladenine.

13» 9-(Methyl tri-O-acetyl-p-D-galactopyranosyluronate)adenine picrate was synthesized by treatment of the product of interaction of methyl tri-^-acetyl-o-D-galactopyranosyluronate bromide and 6-benzamido-

9-chloromercuripurine with methanolic picric acid.

14. 9-(Methyl tri-0-acetyl-p-D-galactopyranosyluronate)adenine

was synthesized ty treatment of 9-(methyl tri-O-acetyl-P-D-galacto-

pyranosyluronate)adenine picrate with anion exchange resin. CHRONOLOGICAL BIBLIOGRAPHY

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