Nucleic Acids

NUCLEIC ACIDS BY G . R . BARKER The University of Manchester. England . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 ..................... ..................... 286 1 . Preparation from Polynucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 a . Ribonucleosides .......................... . . . . . . . . . . . . . . . . 286 . . . . . . . . . . . . . . . . 287 b . Deoxyribonucleosides . . . . . . . . . . . . . . . . . . . . 2 . Synthesis . . . . . . . . . . . . ............................. a . Ribonucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 b . Deoxyribonucleosi ............................. 3 . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 a . Ribonucleosides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Deoxyribonucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 1. Ribonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 a . Isolation and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 h . Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 c . Cyclic Phosphates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 d . Dinucleotides . . . . . . . . . . . . . .................................... 302 .................................... 305 2 . Deoxyribonucleotides . . . . . . . . a . Isolation . . . . . . . . . . . . . . . . . . . .................................... 305 b . Pyrimidine Deoxyribonucleoside Diphosphates . . . . . . . . . . . . . . . . . . . . . 305 c . Structure and Synthesis., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 IV . Polynucleotides .................................................... 307 1. Occurrence and Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 a . Isolation of Ribonucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 b . Isolation of Deoxyrihonucleic Acids. . . . . ....................... 310 c . Separation of Riho- and Deoxyribo-nuclc cids . . . . . . . . . . . . . . . . . . . 311 d . Biologically Active Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 2 . Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 a . Methods of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b . Composition of Deoxyribonucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . 315 c . Composition of Ribonucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 3 . Structure ............................................... 317 a . Ribonucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 b . Deoxyribonucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 c . Macromolecular Structure ........ .................... 331

I. INTRODUCTION The term “nucleic acid” has been used with different meanings by different authors . In its widest sense, it comprises compounds which conform 285

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G . R. BARKER

to the “nucleotide” pattern, consisting of a phosphoric ester of a N-l-deoxyglycoside; many of these compounds possess well-defined, coenzyme functions. In a narrower sense, the term is applied to polynucleotides, characterized by either D-ribose or 2-deoxy-~-erythro-pentose(“2-deoxy-~-ribose”) as the carbohydrate component, and these are the materials with which this review will be concerned. Since the chemistry of nucleic acids was last discussed in this Series,’ publications on the subject have appeared at an unprecedented rate. Degradation products have been further investigated and their structures are more firmly established. Moreover, studies of the properties of these materials have led to a fuller understanding of the behavior of polynucleotides. Emphasis will be laid on the organic chemistry of nucleic acids, and many physicochemical investigations will not be discussed. The period under review has seen the beginning of an understanding of the biosynthesis of nucleic acids, but space does not allow of a consideration of this aspect of the subject. It is intended that this review should be read in conjunction with the previous article by Tipson,’ and for this reason, older work is, for the most part, either omitted or only briefly mentioned. I n some cases, however, recapitulation has been necessary in order to throw into relief the essential features of new concepts. 11. NUCLEOSIDES 1. Preparation from Polynucleotides a. Ribonucleosides.-For the preparation of nucleosides, hydrolysis of ribonucleic acids is normally carried out in alkaIine medium a t elevated temperatures. Dilute sodium hydroxide or ammonium hydroxide have been commonly used, but lead hydroxide has been suggested as a satisfactory reagent; zinc hydroxide causes incomplete mineralization of the phosphoric acide2Boiling aqueous pyridine, however, is probably the most convenient reagent for laboratory use.8 The method of Bredereck and coworkers is considerably improved by separation of uridine and cytidine by ion-exchange chromatography.41 If it is desired to isolate only the pyrimidine nucleosides, hydrolysis of the nucIeic acid may be carried out in acid medium.6 This process, however, entails extensive deamination of cytidine to uridine. The pyrimidine (1) R. S. Tipson, Advances i n Carbohydrate Chem., 1, 193 (1945). (2) K. Dimroth, L. Jaenicke and Doris Heinzel, Ann., 666, 206 (1950). (3) H. Bredereck, Annelise Martini and F. Richter, Ber., 74, 694 (1941). (4) R. J. C. Harris and J. F. Thomas, J . Chem. SOC.,1936 (1948). (5) D. T. Elmore, J. Chem. SOC.,2084 (1950). (6) H. S. Loring and J. M. Ploeser, J . Biol. Chem., 178, 439 (1949).

NUCLEIC ACIDS

287

nucleosides may also be obtained in good yield from the corresponding nucleotides by heating at pH 10 in the presence of lanthanum nitrate? b. Deoxyribonuc1eosides.-Deoxyribonucleic acid has also been hydrolyzed by means of lead hydroxide, to yield nucleosides which were isolated by continuous, countercurrent partition.8 However, for the most part, hydrolysis by intestinal phosphatase is used, as in the earlier w ~ r k . ~ # ~ O 2-Deoxyguanosine* may be separated from the hydrolysis products by partition between water and a chloroform-alcohol mixture. 2-Deoxyadenosine is crystallized from the mother liquor, and the pyrimidine deoxyribonucleosides are separated by adsorption chromatography on alumina.ll Thymidine may also be isolated, by making use of its solubility or as its 3,5-di-0in alcohol and acetone,12by means of the ~hromatopile,'~ benzoyl derivative.13aAll four nucleosides may be separated by a combination of ion-exchange chromatography and partition chromatography on starch.14These methods are unsuitable when used on a large scale. However, a satisfactory procedure has been devised,16in which silver ions are added to destroy deaminase activity in the intestinal phosphatase preparation used to hydrolyze the deoxyribonucleic acid, thus avoiding deamination of 2-deoxyadenosine. The products are first separated on a column of a basic anion exchanger (Dowex-2) which does not cause hydrolysis of the purine nucleosides. Only the pyrimidine nucleosides, which are more resistant to hydrolysis by acids, are separated by means of an acidic cation-exchanger (IRC-50). Using this method, some 2-deoxyuridine has been obtainedl6besides the expected 2-deoxycytidine,but its presence was shown to be due to deamination of the cytosine residue during isolation of the nucleic acid and not during the hydrolysis of the polynucleotide. (2-Deoxy-~-ribosyl)5-methylcytosine (5-methylcytosine 1,2-dideoxy-~-riboside),also, has been obtained from wheat-germ nucleic acid, using this procedure."

* In such names, numerals will refer to carbon atoms of the sugar moiety, and primed numerals to the positions on the nitrogenous base; compare Ref. 1, pp. 200 and 208. Pyrimidines and purines are numbered by the Chem. Abstracts system. (7) J. E. Bacher and F. W. Allen, Federation PTOC., 9, 148 (1950). (8) F. Weygand, A. Wacker and H. Dellweg, 2. Naturforsch., 88, 130 (1951). (9) P. A. Levene and E. S. London, J . B i d . Chem., 81, 711 (1929); 83, 793 (1929). (10) W. Klein, Hoppe-Seyler's 2. physiol. Chem., 224, 244 (1934); 266, 82 (1938). (11) 0. Schindler, Helv. Chim. Acta, 32,979 (1949). (12) T. G . Brady, Biochem. J . (London), 47, v (1950). (13) W. Drell, J. Am. Chem. SOC., 76, 2506 (1953). (13a) F. Weygand and W. Sigmund, 2.Nalurjorsch., 9b, 800 (1954). (14) P. Reichard and B. Estborn, Acla Chem. Scand., 4, 1047 (1950). (15) W. Andersen, C. A. Dekker and A. R. Todd, J. Chem. SOL,2721 (1952). (16) C. A. Dekker and A. R. Todd, Nature, 106,557 (1950). (17) C. A. Dekker and D. T. Elmore, J . Chem. Soc., 2864 (1951).

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G . R. BARKER

2. Synthesis a. Ribonuc1eosides.-The synthesis of ribonucleosideshas been reviewed,lfi and attention will here be confined to certain aspects only. Outstanding in this field has been the contribution of Todd and his coworkers. The significance of their work has been, first, that it has led to successful syntheses of adenosine, guanosine, cytidine, and uridine. Equally important, however, has been its contribution toward the final confirmation of the structures of the nucleosides. This may be illustrated in the following way. By the route I to V, which had been developed for the syiithesis of pentosylpurines (purine 1-deoxypentosides), it was possible to obtain 9-/3-D-mannopyranosyladenine (V), in which the position of the carbohydrate residue was known with certainty.lg This nucleoside, on oxi-

MeS

Nq -5 h I NH,

MeSAN

I

MeS

NHC,H,,O,

I1

111

IV

yJJ>

V

1

Np>

-

HoH2cd ‘N

HoH2iYo9 CHO

OHC

HO

OH

VI

N

vIr

(18) R . W. Jeanloz and H. G . Fletcher, Jr., Advances i n Curbohydrate Chem., 6 , 135 (1951). (19) J. Baddiley, B. Lythgoe and A. R. Todd, J. Chem. Soc., 571 (1943);B. Lythgoe, H.Smith and A. R. Todd, ibid., 355 (1947).

NUCLEIC ACIDS

289

dation with sodium metaperiodate, yielded the same dialdehyde (VII) as that obtained under similar conditions from natural adenosine (VI), Thus, it could be concluded that the D-ribose residue of adenosine is also located at position 9 of adenine. Furthermore, the dialdehyde (VII) was obtained ako from D-glucosyladenine (adenine 1-deoxy-D-glucoside)prepared via interaction of tetra-0-acetyl-cu-D-glucopyranosylbromide and the silver salt of a purine derivative.20 This synthetic nucleoside bad been known for many years,21 and although the allocation of the D-glucosyl residue to position 9 of the purine depended only on spectroscopic evidence,22it was known from the method of formation that the glucosidic linkage had the p-D configuration. Since the glycosidic linkage is unaffected during the oxidation with sodium metaperiodate, it is evident that natural adenosine had been correctly designated 9-B-D-ribofuranosyladenine. The application of this type of nucleoside synthesis t o the preparation of l-deoxy-D-ribofuranosides proved troublesome owing to the difficulty of maintaining the sugar in the five-membered ring form.23However, by blocking the C5 hydroxyl group of D-ribose with the benzyl group, isomerization was prevented and products identical with natural adenosine and guanosine were obtained.24Nevertheless, for preparative purposes, the synthetic route first developed by Fischer and HelferichZ1is to be preferred. Improvements in the method, such as the use of chloromercuri derivatives instead of silver salts, have been reviewed,’a and it has been reported further that this method is also suitable for the synthesis of pyrimidine nucleosides.26 b. Deoxyribonucleosides.-So far, no natural deoxyribonucleoside has been synthesized. Attempts have been made by the following two routes, but with little success.26*27 I n the first, silver theophylline was condensed with the “acetohalogeno” derivative of either a 2-deoxypentose or a 2chloro-2-deoxypentose. Not only were very low yields obtained, but the products consisted of mixtures of a and ,B anomers. In the second method, the epoxide rings of 2,3-anhydropentosides were opened by means of sodium thiomethoxide, followed by desulfurization with Raney nickel, or aluminum lithium hydride. 1,3-Dideoxypentosides were, however, formed almost exclusively. (20) J. Davoll, B. Lythgoe and A. R. Todd, J . Chem. Soc., 833 (1946). (21) E. Fiseher and B. Helferich, Ber., 47, 210 (1914). (22) J. M. Gulland and L. F. Story, J . Chem. Soc., 259 (1938). (23) G. W. Kenner, H. J. Rodda and A . R. Todd, J . Chem. Soc., 1613 (1949). (24)(a) G. W. Kenner, C. W. Taylor and A. R. Todd, J . Cheni. Soc., 1620 (1949). (b) J. Davoll, B. Lythgoe and A. R. Todd, ibid., 1685 (1948). (25) J. J. Fox, N. Chang and J. Davoll, Federation Proc., 13, 211 (1954). (26) J. Davoll and B. Lythgoe, J . Chem. SOC.,2526 (1949). (27) J. Davoll, B. Lythgoe and S. Trippett, J . Chem. Soc., 2230 (1951).

290

G . R. BARKER

3. StTWfUTe a. Ribonuc1eosides.-The structures of the ribonucleosides derived from polynucleotides are now fully established. Confirmation for the structures previously assigned has been obtained by various methods. X-ray examination of crystalline cytidine28confirms that the compound had been correctly described as 1-0-D-ribofuranosylcytosine (see footnote on p. 287). Uridine is found to have the same general shape, and since this compound is formed by the action of nitrous acid on cytidine, its structure is also completely reconfirmed. I n these nucleosides, the pyrimidine ring is planar, but the D-ribofuranosyl ring is not, Furthermore, the plane of the pyrimidine ring is considered to be almost perpendicular to that of the sugar, whereas from a study of ribopolynucleotides,29 it had been claimed that the two rings were parallel. The results of x-ray examination of adenosine and guanosineZ8 are consistent with formulation of the compounds as 9-B-D-ribofuranosyladenine and g-p-D-ribofuranosylguanine, but this evidence does not completely exclude other possibilities. Complete confirmation of the structures assigned to the ribonucleosides has been provided by a study of their 5-0- p -tolylsulfonyl derivatives. Whereas 2,3-O-isopropylideneuridine and 2,3-O-isopropylideneinosine give 5-p-tolylsulfonyl derivatives which, with sodium iodide, are converted to 5-deoxy-5-iodo the corresponding adenosine and cytidine

VIII

IX

derivatives behave differently.a2 Besides giving the normal, covalent, 5-p-toluenesulfonate, 2,3-O-isopropylideneadenosine yields a compound containing the p-toluenesulfonate ion. The covalent compound, on treatment, with sodium iodide, yields a product containing the iodide ion instead (28) S. Furberg, Acta Chem. Scand., 4. 751 (1950);Acta Cryst., 3, 325 (1950). (29) W.T. Astbury, Sllmposia SOC.Ezptl. Biol., 1, 66 (1947). (30) P.A. Levene and R. S. Tipson, J . B i d . Chem., 106, 113 (1934). (31) P.A. Levene and R. S. Tipson, J . B i d . Chem., 111,313 (1935). (32) V. M.Clark, A. R. Todd and J. Zussman, J. Chem. Soc., 2952 (1951).

NUCLEIC ACIDS

29 1

of the normal covalent iodide. Moreover, the covalent p-toluenesulfonate is converted slowly, on standing in acetone solution, into an ionic p-toluenesulfonate. The structure of the cation formed in these reactions has been shown by x-ray analysis to be VIII. Under similar conditions, cytidine yields a cyclic cation (IX). It can be seen that these “cyclonucleoside” salts can be formed only if the parent nucleosides are 0-n-ribofuranosyl derivatives. This provides confirmation of the conclusion drawn from structural’ and synthetic experiments. Furthermore, proof of the structure of cytidine also determines that of uridine. The ultraviolet absorption spectra of the purine nucleosides have been fully discussed by Tipson.’ Spectroscopic studies of pyrimidine nucleosides have now been made. It has been showna3that the absorptions of uridine and uridylic acid vary with pH owing to tautomeric shifts in the pyrimidine ring. At acid or neutral values, the spectra resemble that of uracil, but a t alkaline pH values there is a wide divergence between the spectrum of uracil and those of uridine and uridylic acid. This difference is ascribed to the fact that the presence of the carbohydrate residue at N1 (see footnote on p. 287) prevents the attainment of fully aromatic character by the pyrimidine ring. The differences between the spectrum of cytosine and those of cytidine and cytidylic acid were not as great, but the last two compounds did exhibit, at alkaline pH values, a small maximum around 230 mp which was absent from the spectrum of cytosine. Fox and S h ~ g a have r ~ ~ extended the study of the absorption spectra of pyrimidine nucleosides as a function of pH. They have observed, at high pH values, isosbestic points which are absent from the spectra of the parent pyrimidines, and which they ascribe to dissociations of the carbohydrate residue. They also find slight differences, at high pH, between the spectra of ribo- and deoxyribo-nucleosides occasioned by the different numbers of hydroxyl groups undergoing dissociation. They suggest this as a basis for distinguishing spectroscopically between the two classes of nucleoside. b. Deoxyribonuc1eoside.s.-Evidence for the structures of the deoxyribonucleosides is not as complete as for the ribonucleosides, but there is little doubt that the structures previously discussed by Tipson’ are substantially correct. As previously mentioned, the sugar moiety of the purine deoxyribonucleosides, which can now be readily obtained by hydrolysis with an acidic resin,gb has been fully characterized, and specs strongly suggests that it is attached at position troscopic (33) (34) (35) (36)

J. M. Ploeser and H. S. Loring, J. Biol. Chem., 178,431 (1949). J. J. Fox and D. Shugar, Biochim. et Biophys. A d a , 9, 369 (1952). S. G. Laland and W . G. Overend, Acta Chem. Scand., 8, 192 (1954). J. M. Gulland and L. F. Story, J. Chem. SOC.,692 (1938).

292

G . R. BARKER

9 of the purine ring. The fact that the 1,2-dideoxy-~-ribosidesof guanine and hypoxanthine do not affect the conductivity of boric acid solution@ indicates the absence of a cis-l,2-glycol system, which is in agreement with their formulation as furanosyl derivatives. That neither of these nucleosides is oxidized by sodium metaperiodate within 20 hours confirms this c0nclusion.3~However, Brown and L y t h g ~ eobserved ~~ that these nucleosides show a very small uptake of oxidant during the first 20 hours, and that a further very slow oxidation continues after this time. This amounted to the consumption of only 0.5 mole of reagent per mole after 400 hours, and there seems little doubt that it was due to fission of the glycosidic linkage, which is extremely labile. It can thus be said that the deoxyribonucleosides themselves are not oxidized by periodate, and therefore this technique cannot be used t o determine the configuration a t the glycosidic center as was done in the case of the ribosylpurines. However, by interaction of p - toluenesulfonyl chloride with 3-O-acetyl2 - deoxyadenosine, an ionic p - toluenesulfonate has been obtained which is formulated as 3-0-acetyl-2-deoxy-3', 5-cycloadenosine p-toluenesulfonate, analogous to the cyclonucleoside salts derived from adenosine.40 The formation of such a compound is consistent only with a 0-D-furanosyl structure for the parent nucleoside, and it also affords confirmation of the location of the carbohydrate residue at position 9 of the purine. Until recently, the sugar moiety of the deoxypentosylpyrimidines was assumed to be that of 2-deoxy-~-ribose,by analogy with that of the deoxyribosylpurines. However, these pyrimidine nucleosides have now been reduced, and the reduction products hydrolyzed, to yield 2-deoxy-~-ribose.~~~ Furthermore, an enzymic synthesis of thymidine has been carried out, using 2-deoxy-~-ribosylphosphate as a starting material.@"'The ring of the sugar is known to be furanose, since thymidine and 2-deoxycytidine neither react with sodium metaperiodate nor increase the conductivity of boric acid solutions.38. 4 o c They also react to give trityl ethers, which strongly 42 suggests the presence of a free, primary hydroxyl Until recently, the position of attachment of the carbohydrate residue (37) K. Makino, Biochem. Z . , 282, 263 (1935). (38) L. A. Manson and J. 0. Lampen, J. Biol. Chem., 191,87 (1951). (39) D. M. Brown and B. Lythgoe, J . Chem. SOC., 1990 (1950). (40) W. Andersen, D. H . Hayes, A . M. Michelson and A. R . Todd, J. Chem. SOC., 1882 (1954).

(40a) D. C . Burke, Chentistry & I n d u s t r y , 1393 (1954); J . Org. Chenz.,20, 643 (1955). (40b) M. Friedkin and D . W. Roberts, J. Biol. Chem., 207, 257 (1954). (40c) P. A . Levene and R . S. Tipson, Hoppe-Seyler's 2. physiol. Chem., 234, v (1935). . (41) P. A. Levene and R . S. Tipson, J. Biol. Chem., 109.623 (1935) (42) A. M. Michelson and A. R. Todd, J. Chem. SOC.,34 (1954).

NUCLEIC ACIDS

293

to the pyrimidine ring has rested on spectroscopic evidence only. Since the ultraviolet absorption spectra of 2-deoxycytidine and cytidine on the one hand, and of thymidine and uridine on the other, show close resemblances, it was tentatively concludedl that the glycosidic linkage is at N1 (see footnote on p. 287). These conclusions are open to objections and it was desirable to obtain confirmation by some independent means. This has been provided in the case of 2-deoxycytidine by the fact that N-acetyl3-0-acetyl-2-deoxycytidine gives, on tosylation, a cyclonucleoside salt analogous to that formed from cytidine.4O In this case, the cyclic salt was found to be rather unstable and was not obtained in crystalline form, but from its chromatographic behavior, it is almost certainly correctly formulated as in IXa. Final confirmation of the position of the NHAc

OAc

IXa

carbohydrate residue must await the development of practicable routes to the synthesis of these nucleosides. 111. NUCLEOTIDES 1. Ribonucleotides

a. Isolation and Structure.Since this subject was last reviewed in this Series,’ new conceptions have been introduced which have materially altered views concerning the structures of ribonucleotides. It was previously held that polynucleotides of the type of yeast ribonucleic acid are split by chemical or enzymic means to four nucleotides, formulated as 3-phosphate* esters of adenosine, guanosine, cytidine, and uridine. It is now known that, under various conditions, 2-, 3-, and 5-phosphate esters of these nucleosides are formed from ribonucleic acids, and that the nucleotides formerly regarded as 3-phosphate esters are in all probability mixtures of isomers. It is desirable, therefore, t o summarize briefly the arguments on which the previously accepted structures were based. The first nucleotides to be investigated were inosinic and adenylic acids * I n this Chapter, the terms “phospho” and “phosphate” are used as contractions for “dihydrogen phosphate.”

294

G . R. BARKER

from rn~scle.4~ It was early shown that muscle adenylic acid could be deaminated to inosinic acid, so that arguments concerning the location of the phosphoric acid residue of the former could also be applied to the latter. The recognition of these nucleosides as l-deoxy-5-0-phospho-~-ribosides depended on their degradation to D-ribose 5-phosphate. This conclusion was further justified by the fact that muscle adenylic acid forms a complex with boric acid, and it has been finally confirmed by oxidation No doubt now remains with sodium rnetaperi~date~~ and by synthe~is.4~ concerning the constitution of these nucleotides. Similar considerations apply to guanosine &phosphate, cytidine &phosphate, and uridine 5-phosphate, which have been recognized as products of the enzymic fission of calf-liver ribonucleic and have been separated by ion-exchange chromatography in the presence of borate.46a After hydrolysis of yeast ribonucleic acid by alkali, Levene and coworkers obtained four nucleotides, yeast adenylic acid (isomeric with muscle adenylic acid), guanylic acid, cytidylic acid, and uridylic acid. From both yeast adenylic acid and guanylic acid, a D-ribose phosphate was obtained which differed from that derived from muscle adenylic acid. Since the parent nucleosides were already known to be furanosyl derivatives,' and since the new D-ribose phosphate yielded both furanose and pyranose glycosides, the phosphate residue was known to be located a t either C2 or C3. The choice between these two positions was made by catalytic reduction of the D-ribose phosphate to a ribitol phosphate which was found to be optically inactive. The only ribitol phosphate which is internally compensated, and therefore necessarily devoid of activity, is the ribitol 3-phosphate. Quite possibly, the yeast adenylic and guanylic acids of Levene and coworkers were in fact 3-O-phosphates, but it is necessary here to note a possible weakness in the argument which led to this conclusion. It is now known (for details, see later) that phosphoric esters which possess a hydroxyl group adjacent to the ester residue isomerize readily, with migration of the phosphoryl grouping. Thus, had Levene and Harris initially obtained an optically active ribitolphosphoric acid, it could have isomerized to give a racemic mixture of all the possible isomers. In view of this, the inactivity of a ribitolphosphoric acid does not necessarily characterize it as a 3-phosphate. The same approach to the determination of the structures of the pyrimidine nucleotides cannot be made, in view of their resistance to hy(43) For references to the earlier literature, see Reference 1 . (44) B. Lythgoe and A. R. Todd, J . Chem. Soc., 592 (1944). (45) J. Baddiley and A. R. Todd, J. Chem. SOC.,648 (1947). (46) W. E. Cohn and E. Volkin, Nature, 167, 483 (1951). (46a) J. X . Khym and W. E. Cohn, Biochim. et Biophys. Acta, 16, 139 (1954).

NUCLEIC ACIDS

295

drolysis by acids. Because of this, it is possible to prepare them by acid hydrolysis of the polyn~cleotide,4~ during which process the purine nucleotides are destroyed; and it has been shown that the materials prepared in this way are identical with the nucleotides produced by alkaline hydrolyS ~ S . *However, ~ their designation as 3-O-phosphonucleosides was based entirely on a supposed analogy with the purine nucleotides. All that was definitely established was that they differed from synthetic uridine 5- and cytidine 5-phosphates. It is clear, therefore, that the structure of none of the nucleotides derived from the ribonucleic acids could be regarded as certain. This situation remained until it was demonstrated by three independent methods that the nucleotides derived f r o m yeast ribonucleic acid are not homogeneous and consist of pairs of isomers, provisionally designated “a” and “b.” Yeast adenylic acid has been resolved into two components by paper chr~matography~~; adenylic and cytidylic acids have each been separated into two fractions by recrystallizationKO. 6 2 ; and all four of the yeast ribonucleotides have been resolved into pairs of isomers by ionexchange chromatographyK0, ~ 4 66 , and by zone The bearing which these discoveries have had on the elucidation of the structure of iibopolynucleotides will be discussed later, It is important to stress here, however, that, for most purposes, the older methods of preparing nucleotides have been superseded by procedures which yield separate isomers of each. Of the techniques mentioned above, paper chromatography is mainly of analytical value, and is the most convenient method for the qualitative detection of isomeric adenylic acids. The only disadvantage of this method is that the isomers are not completely separable from muscle adenylic acid. The presence of the latter, however, can be readily detected by hydrolyzing it to adenosine by means of the specific 5-nucleotidase present in snake venoms,K6or by deamination by a specific enzyme K3s

(47) H. Bredereck and G. Richter, Ber., 71, 718 (1938). (48) G. R. Barker, J. M. Gulland, H. Smith and J. F. Thomas, J. Chem. SOC., 904 (1949). (49) C. E. Carter, J. Am. Chem. SOC.,72, 1466 (1950). (50) H. S. Loring, Nydia G. Luthy, H. W. Bortner and L. W. Levy, J . Am. Chem. Soc., 72, 2811 (1950). (51) H. S. Loring and Nydia G. Luthy, J . Am. Chem. SOC.,73, 4215 (1951). (52) P. Reiehard, Y. Takenaka and H. S. Loring, J. Bid. Chem., 198, 599 (1952). (53) C. E. Carter and W . E. Cohn, Federation Proc., 8, 190 (1949). (54) W. E. Cohn, J . Am. Chem. Soc., 71, 2275 (1949). (55) H. S. Loring, H. W. Bortner, L. W. Levy and M. L. Hammell, J. B i d . Chem., 196, 807 (1952). (55a) A. M. Crestfield and F.W . Allen, Anal. Chem., 27,424 (1955). (56) J. M. Gulland and Elisabeth M. Jackson, Biochem. J. (London), 32, 597 (1938).

296

G . R. BARKER

of skeletal Although for the preparation of a single nucleotide only, fractional recrystallization may prove more advantageous, ionexchange methods are usually to be preferred for the preparation of nucleotides, and full details of the techniques have been described.68 Cytidylic acid has been resolved into two isomers by making use of the sparing solubility of the dibrucine salt of one, and both isomers have been obtained in polymorphic forms which are distinguishable via their infrared absorption spectra.Kg The isomerism existing between the pairs of nucleotides was attributed to the different locations of the phosphoryl residues in the carbohydrate part of the parent nucle0side,4~~ since, for instance, the isomeric adenylic acids are both hydrolyzed by acids to adenine, and by alkalis or kidney phosphatase to adenosine. Neither is identical with adenosine 5-phosphate since they are not deaminated by adenylic-acid deaminase,68v6o and are both more labile to acids than is muscle adenylic acid. An alternative explanation of the isomerism was put forward by Doherty.61He was able, by a process of transglycosidation, to convert adenylic acids “a” and “6” to benzyl D-riboside phosphates which were then hydrogenated to optically inactive ribitol phosphates. He concluded from this that both isomers are 3-phosphates and that the isomerism is due to different configurations at the anomeric position. This evidence is, however, open to the same criticism detailed above in connection with the work of Levene and coworkers. Further work has amply justified the original conclusion regarding the nature of the isomerism, since it has been found that, in all four cases, “a” and “b” isomers give rise to the same nucleoside on enzymic 62. 133 It was therefore evident that the isomeric nucleotides hydrolysis.52* are 2- and 3-phosphates1 since they are demonstrably different from the known 5-phosphates. The decision as t o which of the pair is the 2- and which the 3-phosphate proved to be a difficult one. The problem is complicated by 64 the fact that the “(i” and “6” nucleotides are readily intercon~ertible.~~, Indirect evidence has been obtained by physical methods. It has been shown that the density of cytidylic acid “b” is eighteen parts per million (57) H. M. Kalckar, J. Biol. Chem., 167, 461 (1947). (58) W. E. Cohn, J. Cellular Camp. Physiol., 38, Supplement 1,21 (1951). (59) R. J. C. Harris, S. F. D. Orr, E. M. F. Roe and J. F. Thomas, J. Chem. Sac., 489 (1953). (60) N. 0.Kaplan, S. P. Colowick and Margaret M. Ciotti, J . Biol. Chem., 194, 579 (1952). (61) D. G.Doherty, Abstracts Papers A m . Chem. SOC.,118, 56C (1950). (62) H. S. Loring, M. L. Hammell, L. W. Levy and H. W. Bortner, J. Biol.Chem., 196, 821 (1952). (63) H. 2. Sable, Biachim. et Biaphys. A d a , 12, 522 (1953). (64) D. M. Brown and A. R. Todd, J . Chem. SOC.,44 (1952).

NUCLEIC ACIDS

297

greater than that of cytidylic acid ‘(a,” and this is taken to indicate that the basic and acidic groups in the “b” isomer are farther apart than in the (‘a’’ isomer. Furthermore, the same deduction is made from the fact that the pK’, value of the iiul’ isomer is 4.36, as against 4.28 for the “b” isomer.66Since it has been shown t,hat the 2-hydroxyl group of cytidine is nearer to the amino group than the 3-hydroxyl group,28the conclusion is reached that cytidylic acid “b” is cytidine 3-phosphate. Similar considerations lead to the conclusion that yeast adenylic acids “a” and “b” are the 2- and 3-phosphates respectively, and this is in agreement with x-ray data.66aSpectroscopic evidence also points to the above conclusion for cytidylic acids, since at high pH the ultraviolet absorption spectrum suggesta that in cytidylic acid “b” a free hydroxyl group is present at the 2 - p o ~ i t i o n Cytidylic .~~ acids a and b have recently been converted,88aby treatment with hydrazine, to D-ribose 2-phosphate and D-ribose 3-phosphate, respectively. In agreement with this, it is found that cytidylic acid “b” resembles synthetic 2-deoxycytidine 3-phosphate in its infrared absorption spectrum, ultraviolet absorption spectrum, and optical proper tie^.^^ The ease with which the isomeric nucleotides are interconvertible makes direct determination of structure by degradative methods very difficult. For instance, it has been shown6’0 68 that migration of phosphoryl residues takes place during methylation of nucleotides by methyl iodide and silver oxide. An attempt has been made to overcome the difficulty caused by possible migration in the following way.6g Yeast adenylic acids “a” and “b” and guanylic acids “a” and “b” were hydrolyzed to the respective base plus n-ribose phosphate by means of a sulfonated-polystyrene resin. As soon as the hydrolysis of the glycosidic linkage had been effected, the D-ribose phosphate was desorbed from the acidic resin and isomerization was thus reduced to a minimum. It was found that hydrolysis and isomerization take place at about the same speeds, and separation of the D-ribose phosphates by means of a basic resin showed that more D-ribose 2-phosphate is produced in each case from the “u” isomer and more D-ribose 3-phosphate from the “b” isomer. Thus, the same conclusion is reached for the purine nucleotides as for the pyrimidine nucleotides. This has been confirmed by synthesis, so far as the adenylic acids are concerned. (65) L. F. Cavalieri, J . A m . Chem. Soc., 74, 5804 (1952); 76, 5268 (1953). (65%)D. M. Brown, G. D. Fasman, D. I. Magrath, A. R. Todd, W. Cochran and M. M. Wolfson, Nature, 173, 1184 (1953). (66) J. J. Fox, L. F. Cavalieri and N. Chang, J . A m . Chem. Soc., 7 6 , 4315 (1953). (66a) Francoise Baron and D. M. Brown, J . Chem. Soc., 2855 (1955). (67) D. M. Brown, D. I. Magrath and A. R. Todd, J . Chem. Soc., 1442 (1954). (68) G . R . Barker, T. M. Noone, Margaret A. Parsons, Lorna Pickstock and D. C . C. Smith, J . Chem. Soc., 2005 (1955). (69) J. X. Khym and W. E. Cohn, J . Am. Chem. Soc., 76, 1818,5523 (1954).

298

G . R. BARKER

b. Synthesis.-The two chief problems associated with the synthesis of nucleotides are, first, the choice of phosphorylating agent, and, secondly, the directing of the phosphoryl residue into the desired position in the nucleoside molecule. The older methods, some of which were reviewed by Tipson,’ were not always satisfactory. Earlier syntheses used either phosphorus oxychloride or diphenyl phosphorochloridate, but yields were often low. This was probably not necessarily due to ineffectiveness of tfhese reagents but possibly to the inadequate methods available for isolating the products, since more recently phosphorus oxychloride has been used successfully for the phosphorylation of riboflavine70 and adenosine?l For many purposes, however, dibenzyl pho~phorochloridate7~ is to be preferred, since the formation of complex products is avoided and the protective benzyl groups are readily removed. Other methods of phosphorylation will be referred to later. The introduction of a phosphoryl residue at the C5 hydroxyl group of a nucleoside presents little difficulty. Thus, adenosine 5-phosphate has been obtained by an unambiguous route from 2,3-O-isopropylideneadenosine or 2,3-di-O-a~etyladenosine,’~~ 74, 7 b and guanosine 5-phosphate by interaction of 2,3-O-isopropylidene guanosine with tetrakis(p-nitrophenyl) pyrophosphate?6a Great difficulty has been encountered in synthesizing nucleoside 2- or 3-phosphates owing to the necessity of blocking simultaneously the 5- and 3- or 5- and 2-hydroxyl groups. In some experiments, this difficulty was circumvented by relying on the difference in relative reactivities of the hydroxyl groups. Thus, whereas phosphorylation of unprotected nucleosides with one molar proportion of phosphorus oxychloride yields 5-phosphates when the reaction is carried out in dry pyridine,’6, l6 in aqueous baryta guanosine and adenosine give guanylic and “yeast” adenylic acids, and uridine gives a mixture of uridylic acid and uridine 5-pho~phate.~6,77 This method of phosphorylation not only gave poor yields but also afforded no information regarding the location of the phosphoryl residues. These experiments did, however, achieve one of their main objects, name!y, to show that phosphorylation regenerated the same nucleotide from which the nucleoside had been obtained in the first place. Since the C-0 bond is not broken (70) H . S. Forrest, H. S. Mason and A. R . Todd, J. Chem. SOC.,2530 (1952). (71) G. R. Barker, J. Chem. SOC., 3396 (1954). (72) F. R . Atherton, H. T . Openshaw and A. R. Todd, J. Chem. SOC.,382 (1945). (73) P. A. Levene and R . S. Tipson, J . Biol. Chem., 121, 131 (1937). (74) H. Bredereck, Eva Berger and Johanna Ehrenberg, Ber., 73, 269 (1940). (75) T. Jachimowicz, Biochem. Z., 293, 356 (1937). (75a) R. W. Chambers, J. G. MofTatt and H. G . Khorana, J . Ant. Chem. Soc., 77, 3416 (1955). (76) J. M. Gulland and G. I. Hobday, J. Chem. SOC.,746 (1940). (77) J. M. Gulland and G. R. Barker, J. Chem. SOC.,231 (1942).

NUCLEIC ACIDS

299

during the phosphorylation, it follows that no inversion takes place during dephosphorylation of the nucleotides, contrary to what had previously been im~lied.7~ I n view of the fact that “yeast” adenylic acid is a mixture of isomers, it is almost certain that the material obtained, for instance, from adenosine plus phosphorus oxychloride in aqueous baryta consisted of a mixture of adenosine 2- and 3-phosphates. These compounds have been found to be formed also by means of phosphorus oxychloride in pyridine containing one molar proportion of water.79 On the other hand, under scrupulously dry conditions, adenosine 5-phosphate is the major product. This suggests that, in presence of water, phosphorylation takes place through the agency of the following molecular species.

/ \

c1

HO-P=O

Cl

This is the first product of the action of water on phosphorus oxychloride,s0and might be expected to react with a 1,2-glycol to give a cyclic phosphate which is subsequenbly hydrolyzed to a mixture of 2- and 3phosphates. No explanation can be given as yet for the behavior of uridine. Attempts have been made to effect an unambiguous synthesis of nucleoside 2-phosphates by simultaneous blocking of the 3-and 5-hydroxyl groups by means of the benzylidene group. It is well known that benzaldehyde condenses with D-glucopyranoside derivatives to give 4,6-0-benzylidene acetals and, by analogy, it was assumed that nucleosides would yield s2 Indeed, evidence was obtained which 3,5-0-benzylidene seemed to show that 0-benzylideneguanosine, which had been known previ0usly,8~is, in fact, 3,5-0-benzylideneg~anosine.~4 It was found that the compound does not yield a trityl derivative, and acetylation followed by removal of the benzylidene group gave a mono-0-acetylguanosine which is not oxidized by sodium metaperiodate. This appeared to exclude the possibility of the compound’s being 2,3-O-benzylideneguanosine, and its formulation as 3,5-O-benzylideneguanosinewas thought to be justified by the fact that methylation yielded, after hydrolysis and reduction, an (58) R. Robinson, Nature, 120, 44 (1927). (79) G. R. Barker and G. E. Foll, to be published. (80) M. Viscontini and G. Bonetti, Helu. Chim. Acta, 34, 2435 (1951). (81) J. M.Gulland and H. Smith, J . Chem. SOC., 338 (1947). (82) J. M.Gulland and H. Smith, J . Chem. SOC.,I527 (1948). (83) H.Bredereck and Eva Berger, Ber., 7 3 , 1124 (1940). (84) J. M.Gulland and W. G. Overend, J . Chem. SOC.,1380 (1948).

300

a. R.

BARKER

optically active 0-methylribitol. Therefore, 0-benzylidenenucleosides were phosphorylated and debenzylidenated and, on the basis of this work, the products were assumed to be uridine 2-, cytidine 2-, guanosine 2-, and adenosine 2-phosphate~,~] * 82. It was subsequently shown, however, that all the nucleotides obtained in this way are actually 5-phosphates, and it follows that the 0-benzylidenenucleosides are substituted at the 2- and 3-hydroxyl groups?s In confirmation, it has now been shown that methylation and hydrolysis of 0-benzylideneguanosine actually gives 5-0-methylD-ribose.87 Obviously, the supposed analogy between D-ribofuranose and D-glucopyranose derivatives was not justified. The reason for this is apparent if it is borne in mind that, whereas the C5-C6 and C4-04 bonds in D-glucopyranose can assume an equatorial distribution, the corresponding bonds in D-ribofuranose cannot. In summary, it may be said that, up to this point, no 2- or 3-phosphates had been obtained by unambiguous synthesis. The problem was made more difficult by the fact that the nucleoside 2- and 3-phosphates are readily interconvertible, and more urgent because confirmation of the tentative structures assigned to the “a” and “b” nucleotides was desirable. Quite recently, the synthesis of adenosine 2-phosphate by a method which avoids migration of the phosphoryl residue has been reported.88Acetylation of 5-0-acetyladenosine with approximately one molar proportion of acetic anhydride yielded, besides some mono- and tri-0-acetyladenosine, a crystalline di-0-acetyladenosine which was shown by countercurrent distribution to be homogeneous. This compound, after phosphorylation and deacetylation, yielded a nucleotide identical with yeast adenylic acid “a” and completely free from any other nucleotide. No phosphoryl migration could have taken place during the synthesis, for otherwise a homogeneous product would not have resulted. The di-0-acetyladenosine from which adenplic acid “a” had been obtained was treated with p-toluenesulfonyl chloride and the product was converted by methanolysis to a tosylated methyl D-riboside. This was methylated and, after removal of the tosyl group (Ts) and hydrolysis, yielded 3,5-di-O-methyl-~-ribose. The sequence of reactions described above is, therefore, correctly shown as on p. 301. It may therefore be concluded that yeast adenylic acid “a” is adenosine 2phosphate and, hence, that yeast adenylic acid “b” is adenosine 3-phosphate. e. Cyclic Phosphates.-As has been pointed out above, the nucleoside (85) A. M. Michelson and A. R. Todd, J . Chem. SOC.,2476 (1949). (86) D. M. Brown, L. J . Haynes and A . R . Todd, J. Chem. SOC.,408, 3299 (1950). (87) G. R . Barker, T. M. Noone, D. C. C. Smith and J. W. Spoors, J . Chem. SOC., 1327 (1955). (88) D. M. Brown, G. D. Fasman, D. I. Magrath and A. R. Todd, J . Chem. SOC., 1448 (1954).

301

NUCLEIC ACIDS

AcoH2cQ HO

OH

AcoH2cQ AcO

OH

AcoH2 N T N > ‘N

I

AcO

1

N

I OTs

AcO

0--P -0. CH,Ph

H’ ‘ 0

MeoH2cc H, OMe

Meo

Me0

1

OTs

OH

2- and 3-phosphates are interconvertible in acid solution. This phenomenon is paralleled by the interconversion of 1-0-phospho- and 2-0-phosphoF9* The latter isomerization is glyceritol (a-and @-glycerophosphates) (89) Marie-Cecile Bailly, Compt. rend., 206, 1902 (1938); 208, 443, 1820 (1939). (90) P. E. Verkade, J. C . Stoppelenburg and W. D. Cohen, Rec. trau. chim., 69, 886 (1940).

302

G . R. BARKER

known to take place intramolecularly, and it is believed to proceed by way of a cyclic ester,gl glyceritol l12-0-(hydrogen phosphate). By analogy, it CHiO

i

\ No

/p\ CHO I CHzOH

OH

has been suggested that isomerization of nucleotides takes place via a cyclic phosphate intermediate.92This idea is supported by the fact that synthetic 2,3-cyclic phosphates of adenosine, cytidine, and uridine are converted by dilute acids or alkalis to mixtures of 2- and 3-pho~phates.~~ These synthetic materials were obtained by interaction of trifluoroacetic anhydride with the corresponding “a” and “b” nucleotide, and have also recently been obtained by the action of carbodiimides.93a They behave on paper chromatograms as diesters of phosphoric acid, and possess no titratable secondary phosphoryl group. The possibility of their being dinucleotides was excluded by determinations of molecular weight. Almost simultaneously with the postulation of cyclic intermediates in the isomerization of nucleotides, cyclic phosphates were reported as products of the enzymic fission of ribonucleic g5 The bearing of these results on diagnosis of the structure of polynucleotides is discussed later. d. Dinucleotides.--,41though evidence has been obtained that products of complexity greater than that of simple nucleotides are formed by enzymic fission of ribonucleic acids,98no oligonucleotideof natural origin has yet been fully characterized. Several dinucleotides of known structure have, however, been synthesized. By interaction of two molar proportions of 0-benzylideneuridine with diphenyl phosphorochloridate, Gulland and Smith obtained a compound which they assumed to be bis(uridine) 2,2(hydrogen p h o ~ p h a t e ) In . ~ ~view of the fact that the starting material is now known to be 2 ,3-O-benzylideneuridine, the product must be formulated as biduridine) 5 ,5-(hydrogen phosphate). An alternative route to this type of compound, which is suitable also for the synthesis of unsym(91) E.Chargaff, J . Biol. Chem., 146, 455 (1942). (92) D. M. Brown and A. R. Todd, J. Chem. Soc., 52 (1952). (93) D. M. Brown, D. I. Magrath and A. R. Todd, J . Chem. SOC.,2708 (1952). (93a) C. A. Dekker and H. G. Khorana, J . Am. Chem. SOC.,76,3522 (1954). (94) R.Markham and J. D. Smith, Nature, 168,406 (1951). (95) R.Markham and J. D. Smith, Biochem. J. (London), 62, 552 (1952). (96) R.Markham and J. D. Smith, Biochem. J . (London), 62, 558 (1952). (97) J. M. Gulland and H. Smith, J . Chem. SOC.,1632 (1948).

303

NUCLEIC ACIDS

0

HO

OH

metrical dinucleotides, has been investigated by Elmore and Todd.g82,30-Isopropylideneadenosine (X) was phosphorylated with dibenzyl phosphorochloridate and, by removal of one benzyl group, the product (XI) was converted to 2,3-O-isopropylideneadenosine5-(benzyl hydrogen phosphate) (XII). Interact,ion of the silver salt of XI1 with 5-deoxy-5-iodo-2,3-

HoHz 0, o, C ’\ CH, CH,

XI

o,c/o ’ \

CH,

XI1

CH,

XI11

0-isopropylideneuridine (XIII), followed by removal of the protecting groups, gave adenosine-5 uridined (hydrogen phosphate) (XIV). This particular route to the synthesis of dinucleotides is capable of being applied only to the synth,esis of compounds in which a t least one uridine residue is (98) D. T.Elmore and A . R. Todd, J . Chem. SOC., 3681 (1952).

304

G . R. BARKER

esterified at the 5-position. This limitation is imposed by the fact that the precise method used for the introduction of the halogen atom cannot be applied to certain other nucleosides because of the formation of cyclonucleo-

N

0

II

HO YIV

PhCH,

I

0

0

I

PhCH,

OH

OH

0

0

I

I

HO

OH

OH

NUCLEIC ACIDS

305

side salts, and is also applicable to substitution only a t the 5-position of uridine. No dinucleotide has yet been synthesized in which one or both nucleoside residues are esterified at the 2- or 3-hydroxyl group. It is to be expected that this synthesis will be achieved through the use of a novel method of phosphorylation which is best illustrated by the synthesis of uridine 5-pyrophosphate as shown on p. 304.98,loo Another possible approach to the synthesis of this type of dinucleotide utilizes transesterification by means of carbodiimide derivative^.^^^ 2. Deoxyrabonucleolides

a. Isolation.--In the preparation of nucleotides from deoxyribonucleic acids, separation of the products can be achieved by techniques similar to those used for ribonucleotides.lol*lo9 Since, however, deoxyribonucleic acids are relatively stable toward alkalis, hydrolysis is carried out by means of enzymes. The methods used follow closely the older procedures previously reviewed,' but it is now realized that hydrolysis to nucleotides is not as complete as was previously believed.'O' Furthermore, the nucleotides initially produced are dephosphorylated to some extent, and so a pre-separation, on an ion-exchange column, of mononucleotides from unchanged polynucleotide and from nucleosides is recommended. The mononucleotide mixture is then fractionated on a second ion-exchange column. In contrast to results of similar treatment of ribonucleic acids, only one nucleotide derived from each of the bases guanine, adenine, cytosine, and thymine is obtained, and no isomeric nucleotides have been detected. 2-Deoxy-5'-methylcytidylic acid has been isolated from the deoxyribonucleic acid of thymus gland.'Os B. Pyrimidine Deoxyribonucleoside Diphosphates.-As stated by Tipson,' both the monophosphates and the diphosphates of pyrimidine nucleosides have been obtained by acid hydrolysis of deoxyribonucleic acids.lo4,I05 The presence of these diphosphates in acid hydrolyzates was confirmed by other workers,l08-10' but their existence was later questioned by Bredereck and Caro.'08 However, reinvestigation of the original material by Levene justi(99) N.S. Corby, G. W. Kenner and A. R. Todd, J . Chem. Soc., 3669 (1952). (100) G.W.Kenner, A. R. Todd and F. J. Weymouth, J . Chem. Soc., 3675 (1952). (101) E.Volkin, J. X. Khym and W. E. Cohn, J . Am. Chem. SOC.,73, 1533 (1951). (102) R.L. Sinsheimer and J. F. Koerner, Science, 114, 42 (1951). (103) W.E.Cohn, J . Am. Chem. SOC.,73, 1539 (1951). (104) P.A. Levene and W. A. Jacobs, J . B i d . Chem., 12, 411 (1912). (105) P.A. Levene, J. Biol. Chem., 48, 119 (1921). (106) S.J. Thsnnhauser and Berta Ottenstein, Hoppe-Sevler's 2. physiol. Chem., 114,39 (1921). (107) S.J. Thannhauser and J. Garcia Blanco, Hoppe-Seyler's 2.physiol. Chem., 161, 116 (1926). (108) H . Bredereck and G. Caro, Hoppe-Seyler's 2.phgsiol. Chem., 263, 170 (1938).

306

G. R. BARKER

fied their designation as deoxyribonucleoside d i p h o s p h a t e ~ .This ~ ~ ~ conclusion has been amply confirmed by the isolation, by ion-exchange chromatography, of thymidine diphosphate, 2-deoxycytidine diphosphate, and a small quantity of what was probably 2-deoxy-5‘-methylcytidine diphosphate, from herring-sperm deoxyribonucleic acid.l1° Compounds identical with the natural materials were also obtained by phosphorylation of thymidine and of 2-deoxycytidine. This indicates that their formulation by Levene as 3,5-diphosphates was correct. c. Structure and Synthesis.-In the case of the deoxyribonucleosidesmentioned above, there are only two hydroxyl groups in the carbohydrate residue which can be phosphorylated, but for the monophosphates, a choice between the 3- and 5-hydroxyl group has to be made. The problem is simpler than for the ribonucleotides, since no question of isomerization arises. In the past, when it was believed that nucleotides derived from ribonucleic acids are solely phosphorylated at the 3-position, the deoxyribonucleotides were assumed by analogy to be 3-phosphates. This analogy, however, no longer holds. Since no separation of isomeric nucleotides is observed on performing ion-exchange chromatography on them, it may be assumed that the deoxyribonucleotides are homogeneous and are either 3- or 5-phosphates. Evidence from enzymic hydrolysis suggests the latter alternative, since 5-nucleotidase, which hydrolyzes adenosine 5-phosphate but not ribonucleoside 2- or 3-phosphates, dephosphorylates all the natural deoxyribonucleotides.”’ Furthermore, 2-deoxyadenylic acid was found t o be deaminated by muscle-adenylic acid deaminase, which is inactive against adenosine 3-phosphate. It may also be significant that 2-deoxyadenylic acid can act as a receptor for “high-energy phosphate’’ in wiiro.112No further evidence concerning the structures of the purine deoxyribonucleotides has yet been obtained. The structures assigned to the pyrimidine deoxyribonucleotides have, however, been confirmed by synI1a Phosphorylation of 5-0-tritylthymidine and of 2-deoxy-5-0tritylcytidine yielded, after removal of protecting groups, thymidine 3-phosphate and 2-deoxycytidine 3-phosphate, respectively. By route XV to XIX, the same starting materials were converted into the corresponding 5-phosphates. The latter compounds were identical with the corresponding nucleotides obtained from deoxyribonucleic acids as regards their physical properties, their paper-chromatographic and ion-exchange behavior, and their hydrolysis by rattlesnake venom. The 3-phosphates are (109) P.A. Levene, J. Biol. Chem., 126, 63 (1938). (110)C. A. Dekker, A. M. Michelson and A. R. Todd, J. Chem. Soc., 947 (1953). (111) C. E.Carter, J . Am. Chem. Soc., 7 3 , 1537 (1951). (112) H.Z.Sable, P. B. Wilber, A. E. Cohn andM. R. Kane, Biochim. et Biophys. A d a , 13, 156 (1954). (113) A.M.Michelson and A. R. Todd, J. Chem. SOC.,951 (1953).

-. .;cq0)iBase -

307

NUCLEIC ACIDS

Tro~zc<~~Base OH XV (Tr = trityl)

OAc XVII

OAc

aB oBasl

O.r/POH,C HO

Ho'

HoHzc<~jase

XVI

J

O=/POH,C

XIX OH

Ho' Ho

OAc XVIII

not hydrolyzed by this enzyme and could be separated from the 5-phosphates by paper chromatography and by ion-exchange chromatography. The results not only confirm the assigned structures of the natural deoxyribonucleotides, but also justify the conclusion that no isomeric pairs of nucleotides are formed from deoxyribonucleic acids either by enzymic or by acid hydrolysis. A synthetic dithymidine dinucleotide, in which a 3 -+5-internucleotide linkage is present, has been shown to behave toa-nrd enzymes in the same way as the oligonucleotides derived from deosyribopol yn~cleotides."~"

IV. POLYNUCLEOTIDES 1. Occitrrcizce and Isolation

A detailed discussion of the modes of occurrence and biological importance of the polynucleotides is outside the scope of this article. Iio\vrver, in examining the structures of polynucleotides, it is necessary to take into consideration the origins of the materials studied. The pioneer rescardies of Casperssonl14 indicated that deoxyribonucleic acids are present csclusively in the nucleus, whereas ribonucleic acids are found chiefly in the cytoplasm and only to a small extent in the nucleus. This gcneral outline of the distribution of nucleic acids within the cell has been c~onfirmcdand extended by more recent work,116and it has been possible to isolate both types of nucleic acid from different cellular fractions of the same tissue.'la (113%) A. M. Michelson and A. It. Todd, J . Ghem. Soc., 2632 (1055). (114) For a summary of this work, see T. Caspersson, Sumposia SOC.Exptl. Riol., 1, 127 (1947). (115) R. Vendrely, Bull. S O C . chim. hiol., 32, 427 (1950). (116) K. K. Tsuboi and R. E. Stowell, Biochim. et Biophys. A d a , 6 , 192 (1950).

That the cytoplasmic nucleic acid is present in the mitochondria, the microsomes, and the non-sedimentable cell-sap is also known.117The nuclear ribonucleic acid has been reported t o be associated with the nucleolus and the chromosomes.118 It is known, moreover, that the ribonucleic acids of the different parts of the cell are biochemically distinct, since they become labeled with P32 at different rates.119 I n liver cells, the nuclear ribonucleic acid is also chemically distinct from the cytoplasmic material, since the two differ in composition.120 It is clear, therefore, that ribonucleic acids prepared from whole cells are likely to be mixtures of various molecular species. The pattern of distribution of deoxyribonucleic acid in the cell is less complex, and claims have been made that its concentration per nucleus is 121a*121b However, if, as is believed, constant for a given type of deoxyribonucleic acids of the chromosomes carry genetical specificity,'"? it is also possible that isolated deoxyribopolynucleotides are chemically heterogeneous. Indeed, there is some evidence to show that deoxyribonucleic acids are in fact h e t e r o g e n e o u ~Although .~~~ certain studies have been carried out on nucleic acids from single cellular fractions, structural investigations have for the most part been concerned with materials extracted from whole organs or even whole organisms. At present, this is of no great consequeiice since, as yet, not enough is known of the fine structures of polynucleotides. It must be borne in mind, however, that as more refined techniques become available, greater emphasis mill have to be laid on methods of isolation. a. Isolation of Ribonucleic Acids.-The large number of methods which have been used for the isolation of ribonucleic acids is a reflection of the inadequacy of most of them. A few illustrative examples will be chosen. Four processes are concerned in the isolation of a nucleic acid. First is the destruction of the tissue structure (stage 1). A nucleoprotein complex is then separated from other cellular constituents (stage 2). This complex is dissociated and the protein is removed (stage 3) ;and, finally, the nucleic acid is precipitated from the resulting solution (stage 4). Disintegration of (117) E'or i t summary of the literature, see H. Chantrenne, Biochim. et Riophgs. Acta, 1, 437 (1947). (118) For :I summary of the literature, see W. M. McIndoe and J. N. D:tvidson, Brit. J. ('(mrer, 6. 200 (1952). (119) R. hl. S. Smellie, W. M. McIndoe, H . Logmi, J. N. Davidson :tnd I. M. Dnwson, Biochem. J. (London), 64, 280 (1953). (120) G. W. Crosbie, R . M. S. Smellie and J. N. Diividson, Biorhem. .I. (I,ondon), 64, 287 (1953). (121) A. Boivin, R . Vendrely and Colette Vendrely, Coinpf.rend., 236, 1061 (1948). (121a) A. E. Mirsky :tnd H. Ris, Nalure, 163, 666 (1949). (121b) .'I C. Caldivell and C. Hinshelwood, J . Chent. SOC.,1415 (1950). (122) J. 11.Watson : i r d F. H. C. Crick, Nature, 171, 964 (1953). (123) G . J,. Brown and M W:itson, Nrzhre, 172, 339 (1953)

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animal tissues is commonly effected by mincing or homogenization, but for many materials, particularly bacteria, ultrasonic waves can be I n a number of methods, isolation of the nucleoprotein complex (stage 2 ) is avoided. I n the isolation of ribonucleic acid from beef nuclear material and cell debris are removed from a normal-saline extract of the minced tissue, which is then brought to half-saturation with sodium chloride (to dissociate the protein from the nucleic acid). After removal of the protein, the nucleic acid is precipitated with alcohol. However, the suggestion has been made126that it is more satisfactory to isolate the nucleoprotein first, and this has been carried out, for instance, in the extraction of the ribonucleic acid from fowl sarcoma GRCH 15.126Nucleoprotein complexes have also been isolated from baker's yeast127and have been separated into various fractions, the nucleic acids from which differ slightly in composition. I n addition, nucleoproteins have been isolated by complex formation with cetyltrimethylammonium bromide.12* I n the past, dissociation of the nucleoprotein complex has been brought about by salt solutions or by heat denaturation,12gbut, more recently, tlccomposition has been effected by hydrolysis with trypsin,1?6or by the use of dodecyl sodium sulfate13oor strontium nitrate.l3I Some virus nuclcoproteins are decomposed by ethyl alcohol.132This effect may be similar to that of alcohol on the ribonucleoproteins of mammalian tissues. If minced liver is denatured with alcohol, and the dried tissue powder is extracted with 10 % sodium chloride, the ribonucleoproteins are decomposed to give a soluble sodium ribonucleate while the deoxyribonucleoproteins are unaffected.133 On the other hand, extraction with 10% sodium chloride is not satisfactory unless the proteins have first been denatured with alcohol. Denaturation also serves to inactivate enzymes of the tissues which might otherwise bring about degradation of the nucleic acid during extraction. (124) M. G. Sevag, D, B. Laclrm;m and ,J. Smolens, J . Biol. Chem., 124,425 (1938)' (124a) S. E. K e r r and K. Scraidarian, J. Biol. Chem., 180, 1203 (1949). (125) J. M. Gulland, G. R. B:irker and D. 0. Jordan, Ann. Rev. Biochem., 14, 175 (1945). (126) R. N. Beale, R. J. C. Harris and E. hr. F. Roe, J . Chem. SOC.,1397 (1950). (127) Yvonne Khouvine and H. de Robichoii-Szulm:~jrstcr, Bzcll. soc. chim. hiol., 33, 1508 (1951); 34, 1050, 1056 (1952). (128) A. S. Jones, Chemisti!j & Industry, IOF7 (1951); Biochim. c>t Ih'oph!/s. r i c h , 10, 607 (1953). (129) S. S. Cohen and W M. Stsnley, J . Biol. C'heitz., 144, 589 (1942). (130) E. R. M. Kay and A . L. Dounce, J . Am. Chem. SOC.,76, 4041 (1953). (131) N. W. Pirie, Biochem. J . (London), 66, 83 (1954). (132) R. Markham and J. D. Smith, Biochem. J . (London), 49, 401 (1951). (133) J. N. Davidson and Charity Waymouth, Biochem. .I. (London), 38, 375 (1944).

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It has been pointed out126 that, although mild chemical agents are necessary for the process of extraction (to avoid degradation of the product), their use allows a greater degree of enzymic breakdown. T o overcome this difficulty, guanidine hydrochloride has been substituted for sodium chloride, thus combining a mild chemical reagent with one which denatures proteins.134, 186 The method chosen for the isolation of a nucleic acid is to some extent determined by the nature of the particular tissue under investigation. Thus, the extraction of ribonucleic acids from yeast by means of sodium chloride is ineffective unless the material is subjected to prolonged heating.136For this reason, the extraction of yeast has been carried out using such alkaline reagents as sodium hydr~xide'~'or ammonia.138The use of such reagents has the obvious disadvantage that ribonucleic acids are readily degraded by alkalis. However, i t has been shown that the use of heat during the extraction of yeast causes more extensive degradation than does the use of alkaline reagents a t low temperatures, and by a brief extraction at 0°C. with 5 % sodium hydroxide, a ribonucleic acid of high molecular weight has been obtained.139On the other hand, the most effective method of extracting plant viruses is with Duponol at lOO"C., and other, more usual, condiIt is evident that no generalization can tions were found to be un~uitab1e.l~~ be made regarding methods suitable for the isolation of ribonucleic acids, and it is therefore necessary, when comparing nucleic acids from various tissues, to take into consideration the method of extraction as ell as the biological origins. The advantages and disadvantages of the various methods have been discussed by Holden and Pirie.140* b. Isolation qf Deoxyribonucleic Acids.-The isolation of deoxyribonucleic acids involves essentially the same steps as for the ribonucleic acids, but the conditions necessary for carrying out stages 2 and 3 are slightly different. Thus, whereas ribonucleoproteins are extracted with 0.14 M sodium chloride, deoxyribonucleoproteins are insoluble under these conditions and are extracted only by more concentrated salt solutions. Furthermore, the deovyribonucleic acids appear to be more strongly attached to the protein, and greater difficulty is found in dissociating the complex. Thymus deoxyribonudeio acid has been prepared by removal of cytoplasmic material (134) (135) (136) (137) (138) (1951). (139) (140)

E. Volkin and C. E. Carter, J . Am. Chem. Soc., 7 3 , 1516 (1951). E. L. Grinnan and W. A. Mosher, J. Biol. Chem., 191, 719 (1951). G. Clark and S. B. Schryver, Biochem. J . (London), 11, 319 (1917). T.B. Johnson and H. H. Harkins, J . Am. Chem. SOC.,61, 1779 (1929). W. Diemair and G. Schwindling-Manderscheid, Z . anal. Chem., 132, 104

G. Jungner and L. G. AllgBn, Acta Chem. Scand., 4 , 1300 (1950). R . W. Dorner and C. A. Knight, J . B i d . Chem., 206,959 (1953). (140a) Margaret Holden and N. W. Pirie, Biochenz. J . (London), 60, 46 (1955).

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with 0.14 M sodium chloride, followed by extraction of the deoxyribonucleoprotein with 10% sodium chloride.141The nucleoprotein mas decomposed by shaking with chloroform and amyl and the polynucleotide was precipitated with ethanol. I n another method, thymus gland was first extracted with distilled water and then with saturated sodium chloride.142~142 This treatment appears to effect both extraction of the nucleoprotein and its decomposition, since sodium nucleate was obtained directly from the extract by precipitation with alcohol. The method has also been used for the large-scale preparation of deoxyribonucleic acid from ripe salmon testes.144The processes of extraction and deproteinization may also be 146* effected simultaneously by detergents.ld6. These three general methods (employing, respectively, 10 % sodium chloride, saturated sodium chloride, and detergents) have been studied critically, and the results of their use on one and the same tissue have been compared.14' It is found that the use of detergents involves some degradation during subsequent washing and drying; and that, with sodium chloride, although degradation is avoided, removal of protein is incomplete and the product may be considerably contaminated. The use of chloroform for deproteinization gives varying results according to the precise way in which the method is applied. It was first used for removal of protein from denatured streptococcal nu~leoprotein,'~~ but in the preparation of thymus deoxyribonucleic the nucleoprotein was shaken with chloroform without prior dissociation of the two components. There seems, therefore, to have been some confusion regarding the precise action of the chloroform, and it is believed that the compound should only be used for the separation of uncombined nucleic acid and protein. In agreement with this view, it is recordedl47that much lower yields are obtained if hydrolysis of the nucleoprotein is omitted. For complete removal of protein, a combination of several methods is rec0mrnended.~~7* c. Separation of Ribo- and Deoxyribo-nucleic Acids.-Although, by starting with a single type of cellular component, relatively pure nucleic acids of the two main classes may be obtained, some degree of contamination of the one with the other is usually encountered. However, deoxyribonucleic (141) J. M. Gulland, D. 0. Jordan and C. J. Threlfall, J . Chem. Soc., 1129 (1947). (142) E. Hammarsten, Biochem. Z.,144, 383 (1924). (143) H. Schwander and R. Signer, Helv. Chim. A d a , 33, 1521 (1950). (144) C. F. Emanuel and I. L. Chaikoff, J . Bio2. Chem., 203, 167 (1953). (145) A. M. Marko and G. C. Butler, J. Biot. Chem., 190, 165 (1951). (146) E. R. M. Kay, N. S. Simmons and A. L. Dounce, J . Am. Chem. Soc., 74, 1724 (1952). (146a) V. L. Mayers and J. Spizisen, J. Biot. Chem., 210, 877 (1954). (147) G. Frick, Biochim. et Biophys. Acta, 13, 41, 374 (1954). (147a) A. S. Jones and G. E. Marsh, Biochim. et Biophys. Acta, 14, 559 (1954).

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G . R. BARKER

acid containing less than 1% of ribonucleic acid can be isolated by adsorption of the latter on activated charcoal,lB8and it has been shown that the An ribonucleic acid can be eluted from the charcoal with 15% phen01.l~~ alternative method utilizes fractional precipitation of the calcium or cetyll~~ complete removal of traces of one trimethylammonium ~ a 1 t s . Virtually nucleic acid from a mixture of the two can be achieved by use of the appropriate specific nuclease.160 The two types of nucleic acid have also recently been separated by paper electrophoresis.'6' d. Biologically Active Nuckic Acids.-The first nucleic acid to have demonstrable biological activity was a pneumococcal deoxyribonucleic acid which was shown to have transforming activity toward certain bacteria.162 It is now known that deoxyribonucleic acid preparations from Bacterium c02i'~ and Hemophilus inJEuen~ae'~~ also have transforming activity, but since the materials were contaminated with ribonucleic acids, protein, and polysaccharide, the conclusion could not be drawn that the biological activity was necessarily associated with the deoxyribonucleic acid. A method of purifying the transforming factor from Hemophilus influenzae has now been developed, the effectiveness of which can be assessed by measurements of biological activity.ls6 The electrophoretic mobility of the deoxyribonucleic acid was found to be greater than that of the ribonucleic acid and polysaccharide contaminants, and, after purification by this technique, transforming activity was not impaired and indeed in some cases was very considerably increased. Unfortunately, electrophoresis does not remove protein, but since preliminary treatment with chloroform124did not diminish the activity, the results strongly suggest that the transforming activity resides in the deoxyribonucleic acid present in the extracts. It is interesting to note that a preliminary report ascribes a polynucleotide structure to the enzyme cysteinylglycinase.166 (148) S. Zamenhof and E. Chargaff, Nature, 168, 604 (1951). (149) S. K . Dutta, A . S. Jones and M. Stacey, Biochim. e t . Biophys. Acta, 10, 613 (1953). (150) See, for example, S . Lsland, W. G. Overend and M. Webb, Acta Chent. Scand., 4, 885 (1950). (151) M. Deimel, Biochem. Z.,326, 358 (1954). (152) 0.T. Avery, C. M. MacLeod and M. McCsrty, J. Exptt. Med., 79, 137 (1944). (153) A. Boivin, A. Delaunay, R. Vendrely and Y. Lehoult, Experientia, 1, 334 (1945); 2, 139 (1946). (154) Hattie E. Alexander and Grace Leidy, Proc. Soc. Exptt. Biol. Med., 73, 485 (1950); J . Exptl. Med., 93, 345 (1951). (155) S. Zamenhof, Grace Leidy, Hattie E. Alexander, Patricia L. Fitagerald and E. Chargaff, Arch. Biochem. and Biophys., 40, 50 (1952). (156) F. Binkley, Exptl. Cell Research, Suppl. 2. 145 (1952); Chem. Abstracts, 47, 11276 (1953).

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2. Composition

It was previously believed that a molecule of the nucleic acids contained one molecular proportion of each of the constituent mononucleotide residues. Furthermore, early measurements had seemed to indicate that, although deoxyribonucleic acids consist of larger molecules, yeast ribonucleic acid had a molecular weight corresponding approximately to that of a tetranucleotide. It was subsequently shown that the molecular weight is far higher,’ but the idea of a tetranucleotide had become so entrenched that the large molecules were regarded as a repeating series of tetranucleotides.’67 It was pointed out,lZShowever, that there was no real evidence for such a formulation and that the nucleotides could very well be arranged in a random fashion. The term “statistical tetranucleotide” was coined to emphasize the difference between this concept and the erroneously postulated [‘structural tetranucleotide.” However, more refined analytical techniques have since disclosed that the mononucleotide residues are possibly not present in equimolecular proportions, and so these terms lose their significance. Unfortunately, the term tetranucleotide still continues to appear in the literature, but its use for this purpose should be avoided. a. Methods of Analysis.-Various methods have been developed for determining the composition of polynucleotides. These involve fission of the material to mixtures of purine and pyrimidine bases, purine bases and pyrimidine nucleotides, purine and pyrimidine nucleosides, or purine and pyrimidine nucleotides. Resolution of these mixtures may be carried out by partition or ion-exchange chromatography or by electrophoresis, and the technique used is to some extent a matter of choice. Several important factors, however, determine the conditions used for the hydrolysis. Vischer and Chargaff first described the determination of purine and pyrimidine baseslb8which were liberated in two separate hydrolyses, purines being released by dilute sulfuric acid, and pyrimidines by formic acid (after removal of purines as their hydrochlorides). This two-stage procedure was adopted because it was found that conditions which are stringent enough to split off the pyrimidine bases cause destruction of purines. Liberation of both purines and pyrimidines in one stage from ribonucleic acids, using hydrochloric acid,169 is found not to be s a t i ~ f a c t o r y . It ’ ~ has ~ ~ been possible, however, to release both the purine and the pyrimidine bases from deoxy(157) H . Bredereck and I. Jochmann, Ber., 76, 395 (1942); H. Bredereck and Eva Hoepfner, ibid., 76, 1086 (1942). (158) E. Vischer and E. Chargaff, J . B i d . Chem., 168, 781 (1947) ;176, 703 (1948). (159) R. D. Hotchkiss, J . Bid. Chem., 176, 315 (1948). (159a) M. M. Daly, V. G . Allfrey and A. E. Mirsky, J . Gen. Physiol., 33,497 (1950).

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ribonucleic acids by means of formic acid.lso This method is more convenient and gives essentially the same results as the two-stage process.lB1 Perchloric acid has been suggested as suitable for hydrolyzing both riboand deoxyribo-nucleic acids to purine and pyrimidine bases.162 In view of the difficulty of hydrolyzing the pyrimidine nucleosidic linkages, ribonucleic acids have been hydrolyzed to a mixture of purine bases and pyrimidine nucleotides which is then separated by paper chromatogr a p h ~ . ~164~This ~ ~ method has been employed extensively for the analysis of ribonucleic acids, and gives reproducible results,165but it has not been used to any great extent for deoxyribonucleic acids, probably because, under these conditions of hydrolysis, they yield some pyrimidine deoxyribonucleoside diphosphates.166 For the analysis of ribonucleic acids, hydrolysis to a mixture of nucleotides is probably more reliable, since, in this case, fission takes place smoothly and unif~rrnly.'~'For analytical purposes, separation of isomeric nucleotides is unnecessary, and so the nucleotides have been converted enaymically to nucleosides which are then separated by partition chromatographyl681 IZs; or, the nucleotides may be separated by paper electrophoresis whereby the pairs of isomers are not reso1ved.l69 This latter procedure is probably the most promising for the analysis of ribonucleic acids. It is not directly applicable t o deoxyribonucleic acids since they are not degraded t o nucleotides under the same conditions. Deoxyribonucleic acids have, however, been hydrolyzed by deoxyribonuclease and snake-venom diesterase to a mixture of nucleotides which was analyzed by ion-exchange chromat~graphy.l'~ The method might well be made more convenient by the use of paper electrophoresis instead of ion-exchange chromatography, but it is not likely to be used extensively until the necessary enzymes become more readily available. Some samples of deoxyribonucleic acid have been found to contain appreciable quantities of phosphomonoesterase, and this might vitiate the results of the method unless suitable precautions are taken. (160) G. R. Wyatt, Biochem. J. (London), 48, 584 (1951). (161) E. Chargaff, Rakoma Lipshitz, Charlotte Green and M. E. Hodes, J. B i d . Chem., 192,223 (1951). (162) A. Marshak and H. J. Vogel, J. Biol. Chem., 189,597 (1951). (163) J. D. Smith and R. Markham, Biochem. J. (London), 46,509 (1950). (164) H. S. Loring, J. L. Fairley, H. W. Bortner and H. L. Seagran, J . Bid. Chem., 197, 809 (1952). (165) C. A. Knight, J . Biol. Chem., 197,241 (1952). (166) L. L. Weed and T. A. Courtenay, J. Biol. Chem., 206, 735 (1954). (167) J. Montreuil and P. Boulanger, Compt. rend., 231, 247 (1950). (168) P. Reichard, J . B i d . Chem., 179, 763 (1949). (169) J. N. Davidson and R. M. S. Smellie, Biochem. J . (London), 62, 594 (1952). (170) R. 0. Hurst, A. M. Marko and G. C. Butler, J . Biol. Chem., 204, 847 (1953.)

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b. Composition of Deoxyribonucleic Acids.-It is clear from the above that, since systematic errors may arise in some methods of analysis, comparison of one nucleic acid with another is allowable only if the same techniques are used on each. For this reason, it is proposed t o consider certain, only, of the published results and some tentative generalizations which have been made. A summary of the earlier work"' revealed that deoxyribonucleic acids of different species differ in composition, whereas those from different organs of the same species show no significant difference. It has further been sugg e ~ t e d lthat ? ~ the molecular ratios of adenine to thymine and of guanine t o cytosine are approximately unity, and that differences in composition as between one species and another are restricted to variations in the ratio (adenine thymine) :(guanine cytosine). The ratio of purines to pyrimidines is usually approximately unity. Accordingly, deoxyribonucleic acids are classified into two main groups, the one containing a preponderance of adenine and thymine (AT type) and the other, which comprises mostly those of microbial origin, containing a greater proportion of guanine and cytosine (GC type). Differences in composition between large numbers of deoxyribonucleic acids of human ,1728 mammalian,173 ~ l a n t , 1 7and ~ ~ microbia1174origins are found to conform to the above generalizations. It is also of interest to note that, whereas the deoxyribonucleic acids of three strains of Escherichia coli showed no significant differences in composition,17bthose derived from the sperm of four species of the same genus of sea-urchin had ratios of adenine :guanine varying from 1.64 to 1.93, and of thymine: cytosine from 1.58 to 1.85. In each case, the ratios adenine:thymine and guanine :cytosine were approximately unity.'7B Similar variations in composition have also been observed between deoxyribonucleic acids of different viruses,177 which apparently differ in composition from that of the deoxyribonucleic acids of the host.'78, '79 On the other hand, it is doubtful whether

+

+

(171) E. Chargaff, Ezperientia, 6, 201 (1950). (172) E. Chargaff, Federation Proc., 10, 654 (1951). (172a) L. L. Uaman and C. Desoer, Arch. Biochem. and Biophys., 48, 63 (1954). (173) E. Chargaff and Rakoma Lipshitz, J. Am. Chem. Soc., 76,3658 (1953). (173a) A. J. Thomas and H. S. A. Sherratt, Biochem. J. (London), 62, 1 (1956). (174) S. Zamenhof, G . Brawerman and E. Chargaff, Biochim. et Biophys. Acta, 9, 402 (1952). (175) B. Gandelman, S. Zamenhof and E. Chargaff, Biochim. et. Biophys. Acta, 9, 399 (1952). (176) E. Chargaff, Rakoma Lipshitz and Charlotte Green, J. Biol. Chem., 196, 155 (1952). (177) G. R. Wyatt, J. Gen. Physiot., 36, 201 (1952-3). (178) J. D. Smithand M. G. P. Stoker, Brit. J. Exptl. Puthol., 32, 433 (1951). (179) G.R. Wyatt and S. S. Cohen, Nature, 170, 846 (1962).

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any significant difference exists between the compositions of deoxyribonucleic acids of tumor cells and corresponding normal cells.180 For various reasons, the generalizations mentioned above must be regarded as strictly provisional. Analyses utilizing formic acid indicate the presence of more than one phosphorus atom per purine or pyrimidine residue. This discrepancy, it is pointed out, could equally well result from an apparent “deficiency” of bases, due to error in the analytical technique.lsO It is also necessary to consider that some nucleic acids are now known to contain more bases than was previously realized. Thus, 5-(hydroxymethy1)la2 and 5-methylcytosine occurs in cytosine is present in various virusesl1s1# various animal and plant deoxyribonucleicacids but is absent from those of microbial origin.”. l6o. 184.186 Certain microbial deoxyribonucleic acids also contain 6-rnethylaminop~rine.~a~~ Various bacteriophage deoxyribonucleic acids have been found to contain a component which is believed to consist of a D-ghcoside186bof 5’-(hydroxymethy1)cytidylic acid. In this connection, it must also be borne in mind that the deoxyribonucleic acida subjected to analysis have probably not been homogeneous. Deoxyribonucleic acids have been fractionated by making use of their different solubilities in normal saline,186 by extracting thymus nucleo-histone with sodium chloride solutions of increasing concentration,’m by ion-exchange,’ms and also by adsorption of the polynucleotide onto histone immobilized on a kieselguhr support.lZ3It is possible, however, that these are artefacts, since it has been shown that deoxyribonucleic acid fractions extracted from calf-thymus nucleohistone may or may not vary in composition according to the previous treatment of the material.’% The present position regarding the composition of deoxyribonucleic acids may thus be summarized by stating that although there are strong sugges(180) D.L. Woodhouse, Biochem. J . (London), 68, 349 (1954). (181) G.R.Wyatt and S. S. Cohen, Nature, 170, 1072 (1952). (182) G.R. Wyatt and S. S. Cohen, Biochem. J . (London), 66, 774 (1953). (183) G.R. Wyatt, Nature, 188, 237 (1950). (184) G.R.Wyatt, Biochem. J . (London), 48, 581 (1951). (185) G.Brawerman and E. Chargaff, J . Am. Chem. SOC.,73, 4052 (1951). (185a) D.B.Dunn and J. D. Smith, Biochem. J . (London), 80, xvii (1955);Nature, 176, 336 (1955). (185b) E. Volkin, J . Am. Chem. Soc., 78, 5892 (1954); R. L. Sinsheimer, Science, 120, 551 (1954). (186) A. Bendich and P. J. Russell, Federation Proc., 12, 176 (1953);A. Bendich, P.J. Russell and G. B. Brown, J . Biol. Chem., 203,305 (1953). (187) E.Chargaff, C.F. Crampton and Rakoma Lipshitz, Nature, 172, 289 (1953); C.F. Crampton, Rakoma Lipshitz and E. Chargaff, J . B i d . Chem., 211, 125 (1954). (187a) A. Bendich, J. R. Fresco, H . S. Rosenkranz and S. M.Beiser, J . Am. Chem. Soc., 77, 3671 (1955). (188) J. A. Lucy and J. A . V. Butler, Nature, 174, 32 (1954).

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tions that regularities exist among the nucleic acids of different origins, it is too early to express them in too general a way. Although evidence points to the constancy of the ratios adenine: thymine and guanine:cytosine, it is possible that these correlations are over-simplificationsof a situation which is far more complex. c. Composition of Ribonucleic Acids.-Comparisons between ribonucleic acids of different origins cannot be made with any degree of certainty on the basis of analytical values taken from the literature. This is partly due to the fact that a variety of analytical procedures has been used and partly because, ribonucleic acids being readily degraded, pronounced differences in composition of the isolate may arise owing to the use of different methods of extraction. Thus, the ribonucleic acid of beef pancreas was believed to contain a much higher proportion of purines than is present in that of yeast. It has been shown, however, that these supposed differences are actually due to degradation (by the enzyme ribonuclease) during extractjon.'*QCompared with the deoxyribonucleic acids, few analyses of ribonucleic acids have been published, but it has been recently claimed that certain regularities in composition can be discerned, provided that the material analyzed is ~ n d e g r a d e d . 'It ~ ~appears that uracil takes the place held by thymine in deoxyribonucleic acids, and the ratios adenine :uracil and guanine: cytosine are each found to be approximately unity. However, since there is as yet no satisfactory way of assessing the degree to which any specimen of ribonucleic acid is degraded, and since ribonucleic acids from different cellular fractions of the same tissue may vary in compositi~n,'~Oa any generalization must be accepted with caution.

3. Structure a. Ribonucleic Acids.-In the previous review of this subject,' the older ideas regarding the structures of ribonucleic acids were discussed. For a number of years, it had been believed that the polynucleotide chains consist of nucleosides joined, through phosphoric acid, by ester linkages. That ester linages are concerned is evident from the fact that some survive hydrolysis of the polynucleotide to mononucleotides. However, positive evidence for the existence of a second ester group on each D-ribose residue was lacking. An alternative structure has been proposed in which the core of the molecule consists of a phosphoric anhydride polymer, singly esterified with (189) J. E. Racher and F. W. Allen, J. Biol. Chem., 183, 641 (190) D. Elson and E. Chargaff, Nature, 173, 1037 (1954); A d a , 17, 367 (1955). (190n) P. S . Olmsted and C. A . Villee, J . Biol. Chem., 212, I,. W. Trent and E. Chargaff, Biochim. et B i o p h y s . A d a , 17, 362

(1950).

Biochim. el R i o p h y s . 179 (1955); D. Elson, (1955).

318

G . R. BARKER

nucleosides a t appropriate positions.191 Some ~omments,’9~ irrelevant t o the the present discussion, have been made on this suggestion, but since, as will be shown later, it has now been demonstrated that diester linkages do in fact exist, the postulation of such a structure becomes superfluous. [The original suggestion has been modified to allow of diester linkage^,'^^ and the molecule is regarded as having “ a sprinkling of all types of linkages” based on the phosphoric anhvdride structure. However, in view of recent studies on ribopolynucleotides, this type of formulation will not be discussed further.] Before considering newer conceptions concerning the structures and properties of ribonucleic acids, it will be necessary to summarize the previous position. Electrometric titration indicated the presence of one acidic dissociating group per phosphorus atom, and after hydrolysis with cold, dilute alkali, one further group was measured. This was consistent with the existence of a diester residue, but hydrolysis to a monoester took place far more readily than is the case with known diesters of phosphoric acid, and no explanation could be given for the lability of the internucleotide linkages. At this period, it was thought that the sole products of alkaline hydrolysis were nucleoside 3-phosphates1 and it therefore appeared that the lability was inherent in the linkage joining these nucleotides together. I n fact, it was claimed that enzymic degradation produced fragments which contained only the alkalilabile phosphate linkage, since they were dephosphorylated by cold, dilute alkali.l9* (It should be mentioned that this observation is not satisfactorily explained on the basis of present-day views.) Evidence for and against linkages other than to the carbohydrate residue of an adjacent nucleotide have already been reviewed’ and will not be discussed here. The possibility of a second ester linkage, located a t the 5-position, has been investigated. Russell’s viper venom, which contains a phosphodiesterase and 5-nucleotidase but no non-specific phosphomonoesterase,660lg6 effected partial dephosphorylation of yeast ribonucleic acid.lg6This is consistent with the presence of 5-ester linkages, but on the other hand, it is known that gucleoside 5-phosphates are resistant to hydrolysis by acids, and no 5-phosphates are formed by acid hydrolysis of ribonucleic acids. Evidence for the 2-hydroxyl group as the location of the unstable linkage was equally unsatisfactory. It was argued (by analogy with, for instance, ether linkages) that a phosphoryl residue a t the 2-hydroxyl group would be inherently (191) E.Ronwin, J. Am. Chem. Soc., 73, 5141 (1951). (192) L.Pauling and V. Schomaker, J . Am. Chem. Soc., 74, 1111,3712 (1952). (193) E. Ronwin, Science, 118, 560 (1953). (194) J.M.Gulland and E. 0. Walsh, J . Chem. Soc., 172 (1945). (195) J. M. Gulland and Elisabeth M. Jackson, Biochem. J. (London), 32, 590 (1938). (196) J. M.Gulland and Elisabeth M. Jackson, J . Chem. Soc., 1492 (1938).

NUCLEIC ACIDS

319

but this is not borne out by the facts. Furthermore a 2,3-diester linkage is unlikely for stereochemical reasons.29 Thus, no satisfactory decision could be reached. The position was somewhat clarified by the isolation of 2- and 3-O-phosphonucleosides from ribonucleic acid hydrolyzates in 92 to 100% yields,’34 and also by the demonstration that 5-O-phosphonucleosides are also present in enzymic digests.46,lS7 Yet this information gave no indication of the nature of the alkali-labile linkages. Thus, while the majority of the experimental evidence pointed to the phosphoryl residues as being doubly esterified with adjacent nucleosides, two facts remained apparently inexplicable on the basis of this type of structure. First, ready fission by alkalis, and secondly, the absence of 5-phosphates from alkaline hydrolyzates and their presence in enzymic digests. Both these facts have been explained by Brown and Todd in the following ~ a y . 9 ~ It has been mentioned earlier (p. 301) that ribonucleoside 2- and 3-phosphates are readily interconvertible in acid solution with the intermediate formation of a cyclic phosphate. Ribonucleoside 2- and 3-(benzyl hydrogen phosphates) are readily hydrolyzed both in acid or alkaline medium, with loss of benzyl alcohol and the formation of a mixture of isomeric nucleotides.64This hydrolysis also is believed to take place via a cyclic intermediate, since ribonucleoside 5-(benzyl hydrogen phosphates) do not behave in it is knownlg8that triesters of phosphoric acid are the same ~ a y . ~866 Now, * readily hydrolyzed to diesters by acids or alkalis. It is to be expected, therefore, that the cyclic. intermediate (XX) will readily undergo fission a t one of the three ester linkages. If either of the bonds marked a and b is broken, one of the original diesters (XXI or XXII) would be formed. The only fission which will produce any observable effect is, therefore, the removal of benzyl alcohol as shown. This affords the same cyclic phosphate (XXIII) as is formed during the isomerization of nucleoside 2- and 3-phosphates, and this is readily opened to give a mixture of isomeric nucleotides (XXIV and XXV). This sequence of reactions is similar to that taking place when glyceritol l-(hydrogen methyl phosphate) is hydrolyzed by alkalis t o a mixture of glyceritol 1- and 2-phosphates plus methanol.199It is clear that hydrolysis (by acids or alkalis) of a dinucleotide containing 5- and 3(or 2 ) ester linkages would, by analogy, be expected to give a mixture of nucleoside 2- and 3-phosphates but no nucleoside 5-phosphate. Furthermore, if, in a polynucleotide chain, phosphoric acid is esterified with adjacent nucleosides in this way, hydrolysis of the polynucleotide would also yield 2- and (197) W.E.Cohn and E. Volkin, J . Biol. Chem., 203, 319 (1953). (198) G.M.Kosolapoff, “Organophosphorus Compounds,” John Wiley and Sons, Inc., New York, N. Y.,1950,p. 232. (199) 0. Bailly and J. GaumB, Bull. S O C . chim. (France), 151 2,354 (1935).

320

110H2cc G . R. BARKER

Base

HO

H

0

HO-P

/

XXI

Base

XX

/

HoH2cY3 0,

Base

OH

/ XXIII

‘OCH2Ph XXII

~

Base

\ 0 ‘P-OH

‘P-OH

’0

~

HO-P ‘ oy ‘OH XXIV

OH ‘OCH,Plr

\

z,o c

o 110 H

Base

OH

HO’

SSV

3-phosphates but no 5-phosphates. The fact that this is observed experimentally strongly suggests that the internucleotide linkages are of this type. It would appear that enzymic hydrolysis proceeds by a different mechanism, since, in this case, some 5-phosphates are formed. Various investigations have supplied ample experimental justification for the analogy drawn by Brown and Todd between simple hydroxylated dialkyl phosphoric esters and polynucleotides. With the object of applying classical methods of carbohydrate chemistry, yeast ribonucleic acid was rnethylated, and the carbohydrates obtained after hydrolysis were examined.200-201 n-Ribose and mono-0-methyl- and di-0-methyl-D-riboses were (200) A. S.Anderson, G. R. Barker and K. R. Farrar, Nature, 163, 445 (1949). (201) A. S.Anderson, G. R. Barker, J. M. Gulland and M. V. Lock, J . Chem. SOC., 369 (1962).

321

NUCLEIC ACIDS

"""2coB HO

HoHzcG

O ,

__*

HO-P 'oH2cqoyBa~e

HO

OH

HO

J

Base

HO

Base

,O

HO-P' OH

H O '

HoH2YY Base

0,

OH

'P-OH

' O H

OH

No

HoH HO

OH

detected, and although the exact nature of the methylated products was not determined, the results were considered to indicate a branched structure, some D-ribose residues being triply esterified (those yielding n-ribose) and end residues being singly esterified (those yielding di-0-methyl-Dribose). The subsequent exposition of the mechanism of the hydrolysis of ribopolynucleotides, however, made it clear that migration of phosphoryl residues is to be expected during methylation, and this has subsequently been shown to occur. Moreover, there is some dephosphorylation during such methylation, so that the results do not necessarily indicate a branched structure. However, it was found that alkaline hydrolysis of the methylated polynucleotide resulted in the rapid liberation of methanol, followed by ksion of internucleotide linkages much more slowly than with the unmethylated polynucleotide.68 Since the formation of cyclic phosphates is prevented by methylation, the great,er resistance to hydrolysis is in accord

322

G . R. BARKER

with the views of Brown and Todd. It is interesting to note in this connection that, many years ago, it was observed that a phosphoryl residue attached to a methylated methyl D-riboside is resistant to hydrolysis.202 By hydrolysis under very mild alkaline conditions (with a boiling suspension of barium carbonate), ribonucleic acids have been shown to yield small quantities of cyclic phosphates as well as the normal n~cleotides.9~ These materials were identical electrophoretically with synthetic cyclic phosphates and were readily hydrolyzed to mixtures of 2- and 3-phosphates. Their formation in this way constitutes strong support for Brown and Todd's theory. The precise way in which the alkaline hydrolysis of the polynucleotide occurs has been studied using isotopically labeled water, and the results are in agreement202& with the scheme outlined above. On the basis of this theory, it is not possible to decide the exact positions of the internucleotide linkages, but certain locations can be excluded as the sites of phosphoric ester groups. Thus, dinucleoside 5-phosphates such as XIV are not labile toward alkalis. Linkages between the hydroxyl groups at C5 and either C2 or C3, or those a t C2 and C2, C2 and C3, or C3 and C3, would, however, be expected to be unstable toward alkalis. The final choice between these possibilities cannot yet be made, but there is a strong suggestion that they consist of linkages between the hydroxyl groups at C3 and C5. Evidence has already been mentioned which indicates the presence of some ester groups at the 5-p0sition,4~+ lg6and it must be pointed out that, since no phosphoryl migration to or from the 5-position is expected to occur, these conclusions are still valid. Similar conclusions have been reached through studies of the nature of ribonuclease action. Ribonuclease degrades ribonucleic acids by hydrolysis of certain of the internucleotide linkages with the liberation of mononucleotides,2'J3the larger proportion of which are pyrimidine n~cleotides,2~~ particularly in the earlier stages of the reaction.206Degradation to nucleotides is not complete, however, and a non-dialyzable, enzyme-resistant residue is left which contains a high proportion of purines.2OBOxidation of the dialyzable fragments with lead tetraacetate indicates that some of these are nucleoside 5-phosphates.207 There seems to be some disagreement as to whether the enzyme(202) P. A. Levene and s. A. Harris, J. B i d . Chem., 98, 9 (1932). (202a) D. Lipkin, P. T. Talbert and M. Cohn, J . Am. Chem. Soc., 78, 2871 (1954). (203) H. S. Loring and F. H. Carpenter, J . Bid. Chem., 160, 381 (1943). (204) G . Schmidt, R . Cubiles, B. H. Swartz and S . J. Thannhauser, J . B i d . Chem., 170, 759 (1947). (205) J. E. Bacher and F. W. Allen, J . Bid. Chem., 183, 633 (1950). (206) H. S. Loring, F. H. Carpenter and P. M. Roll, J . Biol. Chem., 169,601 (1947). (207) R. A. Becker and F. W. Allen, J . B i d . Chem., 196, 429 (1952).

NUCLEIC ACIDS

323

resistant residue is oxidized by periodate,208,20g but it should be possible to decide the point, since residues which are oxidizable by periodate must be terminal nucleotides phosphorylated a t the 5-position only, and these, according to Brown and Todd’s theory, will give rise to nucleosides on alkaline hydrolysis. By hydrolyzing ribonucleic acids with ribonuclease in low concentration, and removing the fission products continuously by dialysis, cyclic phosphates have been obtained.g4*96 Furthermore, ribonuclease, although having no action on cyclic phosphates of purine nucleosides, hydrolyzes pyrimidine nucleoside cyclicf phosphates to give exclusively the corresponding 3 - p h o ~ p h a t e *lo . ~ ~This ~ accounts satisfactorily for the fact that successive action of ribonuclease and phosphodiesterase produces, besides some 5-phosphates, considerable quantities of cytidine 3- and uridine 3-phosphatesh6 The significance of these observations in connection with the present argument is that, since cyclic phosphates are formed as intermediates both in chemical and enzymic degradations, the nature of the end products cannot allow of a distinction between 2- and 3-phosphate ester linkages in the original polynucleotide molecule. It is therefore necessary to consider fission products of complexity greater than that of mononucleotides. Hydrolysis of ribonucleic acids by snake venom was found to yield inorganic phosphate, nucleosides, and pyrimidine ribonucleoside diphosphate~.‘~’ These diphosphates were shown by their behavior toward various enzymes to be mixtures of 2,5- and 3,5-diphosphates, and it therefore seems likely that they were formed through intermediate, cyclic phosphates. Thus, although this evidence confirms the existence of 2(or 3) 4 5 linkages, it does not distinguish between the 2- and 3-positions, Alkaline hydrolysis has been reported to yield, besides mononucleotides, more complex fission products, but these materials have not yet been examined in detail.211 The enzyme-resista.nt residue left after ribonuclease action is not dialyzable and was believed to have a high molecular weight. This has now been shown to be due not to molecular size but to electrostatic effects, and in the presence of salts the enzyme-resistant core readily dialyzes.212Moreover, it consists of a mixture of relatively small oligonucleotides containing some di- and tri-nucle~tides.~~ Some of these oligonucleotides have been charac(208) (1951). (209) (210) (211) (212)

L. F. Cavalieri, S. E. Kerr and Alice Angelos, J . Am. Chem. SOC.,7 3 , 2567 E. Volkin and W. E. Cohn, J . Biol. Chem., 206, 767 (1953). D . M. Brown, C. A. Dekker and A. R. Todd, J. Chem. Soc., 2715 (1952). K. C. Smith and F. W. Allen, J . Am. Chem. Soe., 76, 2131 (1953). R . Markham and J. D. Smith, Biochem. J . (London), 62, 565 (1952).

324

G. R. BARKER

terized by their behavior on chromatography before and after hydrolysis, and all appear to consist of nucleosides joined through phosphoric acid at the 5- and 2(or 3)-positions. Some also contain cyclic-phosphate groupings. No positive evidence was adduced for the presence of 5-phosphate linkages, but if the formulation of the dinucleotides containing a cyclic-phosphate group as in XXVI is correct, it follows that the assumption of a 5-phosphate group is also correct. Again, this work in itself does not give any further

HoH2cGsI HoH Guanine

0, 0t

OH

0 0, I

Guanine

OH

Hb H2A Cytosine

/'\OH 0

tosine

OH

0

\

OR

o~pi::

' O H XXVI

XXVII

HoH2cc$ HoH2cYY Guanine

0

0, I O/P,OH

Guanine

0

OH

Cytosine

0,i /p'OH 0

Hako9 OHC

HO OH XXVIII

OH

Cytosine

CHO

XXIX

information concerning the internucleotide linkage. However, by the action of ribonuclease, XXVI is converted to XXVII, which is dephosphorylated by prostate phosphomonoesterase to a dinucleoside monophosphate

NUCLEIC ACIDS

325

(XXVIII).213 Compound XXVIII, on oxidation with sodium metaperiodate, gives the dialdehyde (XXIX) which, with Russell's viper venom a t pH 10, is split to give cytosine and gusnosine 3-phosphate. This confirms the structure XXVII for the original dinucleotide and suggests that at least some of the internucleotide linkages in the polynucleotide from which it was derived consist of phosphoester groupings linking the 3- and 5-positions. The same conclusion has also been reached by fission of the ribonucleaseresistant core by an extract of spleen, which yields only 3-phosphates without the observable formation of cyclic phosphates as intermediate^.^^^ Oligonucleotides have also been separated by ion-exchange chromatography of yeast ribonucleic acid treated either with acid216 or with ribonuclease~osAlkaline hydrolysis of the fission products obtained with the latter gives rise to pyrimidine nucleoside 3-phosphates and mixtures of purine nucleoside 2- and 3-phosphates. Bone phosphomonoesterase1g6 followed by alkaline hydrolysis gives pyrimidine nucleosides and purine

Pyrimidine where

&

@ = the phosphate group

nucleoside 2- and 3-phosphates. These facts are consistent with the formulation of the oligonucleotides with a pyrimidine nucleotide as the terminal residue, esterified at the 3-position. It cannot be concluded from this evidence, however, that all the nucleotides are esterified in the 3-position. The most convincing evidence in favor of a uniform 3,5-diester linkage between nucleotides has been obtained by the action of various enzymes on synthetic diesters of known 217 Ribonuclease and spleen extracts were found to act only on nucleoside 3-(benzyl hydrogen phosphates), but not on other isomers, to give nucleoside cyclic phosphates which are broken down further to give nucleoside 3-phosphates. It is concluded, by analogy, that polynucleotides, which are substrates for these enzymes, also possess ester groupings at the 3-positions, rather than a t the (213) P. R. Whitfield and R. Markham, Nature, 171, 1151 (1953). (214) L. A. Heppel, R . Mnrkham and R. J. Hilmoe, Nature, 171, 1152 (1953). (215) R. B. Merrifield and D. W. Woolley, J . Biol. Chem., 197, 521 (1952). (216) D. M. Brown and A. R. Todd, J. Chem. SOC.,2040 (1953). (217) D. M. Brown, L. A. Heppel and R . J. Hilmoe, J . Chem. Soc., 40 (1954).

326

G . R. BARKER

2-positions. Thus, there is strong justification for the formulation of ribopolynucleotides as chains of nucleosides joined through phosphoryl residues esterified alternately at the 3- and 5-positions. As yet, little evidence has been obtained concerning the order of attachment of the nucleotide residues. The arrangement of the mononucleot,ides is, however, most likely not random. It has been claimed that the liberation of nucleotides by alkaline hydrolysis of ribonucleic acids takes place in a regular manner?l8 and the oligonucleotides discussed above also appear to conform to some sort of pattern. It is interesting to note that this type of arrangement is in agreement with the views of Schmidt and his collaborators, who first suggested, from a study of ribonuclease action, that polynucleotides consist of alternating chains of purine and pyrimidine nucle0tides.2~~ A method has been developed which is designed to remove nucleotide residues singly from a polynucleotide chain, and it is anticipated that more precise information will shortly be forthcoming regarding the order in which the nucleotides are linked.220The method is based on the fact that esters of %ox0 alcohols are unstable toward alkali. In agreement with this, it is found that the products of the action of periodate on adenosine 5-phosphate (XXX) or adenosine 5-(benzyl hydrogen phosphate) are hydrolyzed under very mild conditions. Thus, after removal of terminal, singlyesterified phosphoryl residues from a polynucleotide chain, it is anticipated that periodate oxidation and hydrolysis will result in the removal of the

Yk;> NH,

spoHzYo9

Ho\

HO O

OHC

xxx

CHO

terminal nucleoside residue only. Repetition of these processes should give a picture of the order of the nucleotides. It seems probable that the method which has been applied to oligonucleotides by Whitfield and Markham is (218) B. Magaiianik and E. Chargaff, Biochim. et Biophys. Acta, 7 , 396 (1951). (219) G . Schmidt, R. Cubiles, N. Zollner, Liselotte Hecht, Nancy Strickler, K. Seraidarian, Maria Seraidarian and S. J. Thannhauser, J . Biol. Chem., 192, 715 (1951). (220) D. M. Brown, M. Fried and A. R. Todd, Chemistry & Industry, 352 (1953); J . Chem. SOC.,2206 (1955).

327

NUCLEIC ACIDS

based on the same ~rinciple,213.~~O~ and this suggests that the method will prove successful. It has been pointed out221that, in a polynucleotide chain built up by esterification at the 3- and 5-positions, there are at least two possible ways in which the chain may be terminated with a singly-bound phosphoryl residue, as shown diagrammatically in XXXI and XXXII. According to

XXXI

XXXII

the theory of Brown and Toddlg2compound XXXII should, on hydrolysis by alkali, yield a nucleoside diphosphate and a nucleoside from the two end groups, whereas XXXI should yield only nucleoside 3- phosphates. It is found that nucleoside diphosphates and nucleosides are present to a considerable extent amongst the alkali-fission products of the ribonucleic acids of tobacco-mosaic virus and potato virus X, and it is concluded that these polynucleotides contain chains of the same type as depicted in It should be pointed out that both the above types of polynucleo-11. tide chain should be susceptible of degradation by the method of Brown, Fried and Todd. However, if, as has been suggested by Markham, Matthews and Srnith,s2lcyclic-phosphate end-groups are present, a slight modification of the technique may be necessary. As has been mentioned earlier1201branching at D-ribose centers in the polynuclcotide chain has been suggested, and on the basis of titration data, branching at phosphoryl groups has been both postulated and 225 Both these types of branching have been discussed by Volkin and Cohn.209 Evidence (for branching) derived from methylation studies so far made is now clearly inadmissible,68but branching a t D-ribose centers is not excluded on the basis of the new concepts regarding the properties of polynucleotides. On the other hand, since the dibenzyl and dimethyl esters of uridine 3-phosphate are unstable in aqueous solution over the whole range of pH, it appears very unlikely that triply esterified phosphate branch-points are present in the p ~ l y n u c l e o t i d e . ~ ~ ~ ~ (220a) P. R. Whitfield, Biochem. J. (London), 66, 390 (1954). (221) R. Markham, R. E. F. Matthews and J. D. Smith, Nature, 173, 537 (1954). (222) W. E. Fletcher, J. M. Gulland and D. 0. Jordan, J . Chem. SOC.,33 (1944). (223) L. Vandendriessche, Compt. rend. trav. lab. Carlsberg, Sbr. chim., 27, 341 (1951). (223a) D. M. Brown, D. I. Magrath and A. R. Todd, J . Chem. Soc., 4396 (1955).

328

G . R. BARKER

b. DeoxyribonucleicAcids.-It has been mentioned earlier that deoxyribopolynucleotides, although being relatively stable toward alkaline reagents, are split enzymically to a mixture of nucleoside 5-phosphates. Furthermore , acid hydrolysis leads to the formation of pyrimidine nucleoside 3 ,5-diphosphates, and this strongly suggests that, in the polynucleotide chain, the nucleoside residues are joined by esterification with phosphoric acid at the 3- and 5-positions, as proposed long ago.‘ Evidence for this type of structure has also been obtained from studies of the degradation of deoxyribonucleic acids by the enzyme deoxyribonuclease. This enzyme initially brings about disaggregation of the material, and subsequently effects hydrolysis to a mixture of relatively small fragments, 226, 226 Although there is a suggestion that leaving a non-dialyzable 2-deoxy-5’-methylcytidine 5-phosphate is present in deoxyribonuclease the dialyzable fragments are believed to consist mainly of oligonucleotides.228s229 Certain of these have been dephosphorylated by prostate phosphomonoesterase with the formation of dinucleoside monophos231 The dinucleotide of adenine and cytosine was also found to be ~hates.2~~8 split by snake-venom diesterase to give a mixture of 2-deoxyadenosine 5phosphate and 2-deoxycytidine 5-phosphate. The same diesterase split the corresponding dinucleoside monophosphate to a mixture of 2-deoxycytidine and 2-deoxyadenosine 5-phosphate. Hence, the singly esterified phosphoryl group of the dinucleotide must have been attached to the 2-deoxycytidine Ho\ O=P-OH,C

HO

1Gsl

Cytosine

0 0, I

/p\OH

(224) S. Zamenhof and E. Chargaff, J . B i d . Chem., 178, 531 (1949). (225) W. G. Overend and M. Webb, J . Chem. Soc., 2746 (1950). (226) S. G . Laland, W. G. Overend andM. Webb, Acta Chem. Scand., 6,1545 (1952). (227) J. L. Potter, K . D. Brown and M. Laskowslti, Biochim. et Biophys. Acta, 9, 150 (1852). (228) M. Kunita, J . Gen. Physiol., 33, 363 (1950). (229) A. H. Gordon and P. Reichard, Biochem. J. (London), 48, 569 (1951). (230) J. D. Smith and R. Markham, Biochim. et Biophys. Acta, 8, 350 (1952). (231) J. D. Smith and R. Markham, Nature, 170, 120 (1952).

NUCLEIC ACIDS

329

residue, and the dinucleotide is therefore to be formulated as follows. It may be mentioned that the venom diesterase apparently breaks the 3-phosphate bond preferentially, and it is possible that this specificity accounts for the formation of only 5-0-phosphonucleosides by enzymic fission of deoxyribonucleic acids. Electrometric titration of the oligonucleotides produced by deoxyribonuclease action indicates that one secondary-phosphate dissociation is liberated per four phosphorus atoms. This suggests that the products consist, on the average, of four nucleotides joined together.232An extra enolic hydroxyl group can also be titrated, and it is suggested that every fourth or fifth internucleotide linkage may involve a hydroxyl group of one of the bases. An attempt is also made to explain the inability of deoxyribonuclease to split every internucleotide linkage-by assuming that it is this type of bond which is hydrolyzed by the enzyme. Further evidence that the polyniicleotide contains some type of bond which is absent from the products of deoxyribonuclease action is that, whereas the oligonucleotides produced by the enzyme are hydrolyzed by phosphodiesterases from various sources, the original sodium deoxyribonucleate is resistant to these enzymes.233 Linkages of an entirely different type have been postulated as subsidiary, internucleotide bonds. The deoxyribonucleic acid from herring roe, on digestion with deoxyribonuclease, became more Feulgen-positive during the ~~ initial stage of the reaction, when disaggregation is taking p l a ~ e . 2This was interpreted as indicating the breakage of labile phosphoric ester groups a t C1 of the 2-deoxy-~-riboseresidues. Linkages of this type cannot, however, be regarded as fully established, and in all probability the main internucleotide linkage in deoxyribonucleic acids is an ester bond between phosphoric acid and the 3- and 5-hydroxyl groups of adjacent nucleosides. This general conclusion is also in agreement with the results of the following studies on the products of mild, acid hydrolysis. Deoxyribonucleic acids readily undergo hydrolysis whereby purine bases are removed t o give a derived polynucleotide originally named thymic acid, but now often called apurinic acid. Hydrolysis may be carried out with dilute mineral acid, but recently apurjnic acids have been prepared by fission a t room temperature with an acidic, ion-exchange resin.236Under carefully controlled conditions, removal of the purines can be performed quantitatively without destroying the polynucleotide nature of the material and without altering the inter-pyrimidine ratios of the original (232) (233) (234) (235) (236)

J. A. Little and G. C. Butler, J . Biol. Chem., 188, 695 (1951). R. 0. Hurst, J. A. Little and G. C. Butler, J . Biol. Chem., 188, 705 (1951). W. G . Overend, M. Stacey and M. Webb, J. Chem. SOC., 2450 (1951). S. G. Laland, Acta Chem. Scand., 8 , 449 (1954). C. Tamm, M. E. Hodes and E. Chargaff, J . Biol. Chem., 196, 49 (1952).

330

G. R. BARKER

The material is of high molecular weight, is electrophoretically homogeneous, and contains free, carbohydrate, reducing gr0ups.2~'It is not oxidized by periodate, which fact suggests that, in the polynucleotide chain, carbohydrate residues are doubly esterified. This conclusion is justifiable, since it appears that in the apurinic acids the reducing carbohydrate residues are in the aldehydo form, which means that in them the hydroxyl group a t C4 is free.2asThus, only if both the 3- and 5-hydroxyl groups are esterified would the material be expected to be resistant to periodate. Just as in the degradation of ribonucleic acids by ribonuclease, deoxyribonucleic acids are split by deoxyribonuclease in such a way that the dialyzable fragments contain proportions of bases different from those of the non-dialyzable core.239 This is believed to indicate dissymmetry in the arrangement of the nucleotides in the polynucleotide chain. From a knowledge of the structure of the dinucleotides formed from deoxyribonucleic acids by enzymic degradation, some information can be obtained concernit has been pointed ing the sequence of n u c l e o t i d e ~231 . ~However, ~~~ that conclusions from enzymic degradation may be unreliable, since there is the possibility of occurrence of exchange reactions similar to those observed with ribonu~leotides.~~~ Nevertheless, some evidence has been obtained by chemical degradation, and this is probably more reliable. It has been noted above that the non-glycosidic carbohydrate residues of apurinic acids are in the open-chain form. It is clear, therefore, that a free, or potentially free, hydroxyl group is situated adjacent b0t.h to 3- and to 5-phospho ester groups in the molecule, and such groupings are labile toward alkalis. In consequence, apurinic acids are partially degraded by sodium hydroxide, and the resistant residue is found to contain 85 % of the pyrimidine nucleotides and only 40 % of the non-glycosidic 2-deoxy-~-ribose therefore, that in the residues of the original apurinic a ~ i d . 2It~appears, ~ original polynucleotide, certain parts of the chain are made up principally of pyrimidine nucleotides. A similar conclusion has also been reached by alkaline degradation of an apurinic acid in which the reducing 2-deoxy-Dribose residues had been mercaptalated in order to ensure that they were in the aldehydo form.240Treatment of deoxyribonucleic acid with thioglycolic acid in the presence of zinc chloride plus sodium sulfate simultaneously effects removal of the purines and mercaptalation, to give a prod(237) C. Tamm and E. Chargaff, J . Biol. Chem., 203,689 (1953). (238) J. A. Lucy and P. W. Kent, Research (London), 6,495 (1953). (239) S. Zamenhof and E. Chargaff, J . Biol. Chem., 187, 1 (1950). (240) A. S. Jones and D. S. Letham, Biochim. et. Biophys. Acta, 14, 438 (1954). (241) L. A. Heppel, P. R. Whitfield and R. Markham, Biochem. J. (London), 66, iii (1954). (242) C. Tamm, H. S. Shapiro, Rakoma Lipshitz and E. Chargaff, J . Biol. Chem., 203, 673 (1953).

NUCLEIC ACIDS

33 1

SCH,C02H SCH,CO,H

0

0, I

/ ' \ O H

uct containing the above type of structure. Alkaline hydrolysis yielded, amongst other products, a trinucleotide linked to a mercaptalated 2-deoxyD-ribose phosphate residue. Complete identification of the fission products should enable much concerning the sequence of the nucleotides to be determined, but it is already apparent that a t least parts of the polynucleotide molecule consist of chains of pyrimidine nucleotides linked directly together. c. Macromolecular Structure.-In recent years, a considerable volume of work has been directed toward a study of both types of nucleic acid by physical methods. Some of these investigations have been referred to above. The majority have, however, been concerned with the macromolecular structure of polynucleotides. Most aspects of this work have been surveyed in informal discussions of the Faraday Society243and will not be discussed here, but it is desirable to summarize two lines of investigation briefly. As has been emphasized previously, whereas some early measurements indicated a molecular weight of the order of 1000 for the ribonucleic acid of yeast, it is now known that the molecule is considerably larger than this. To give accurate values for the molecular weights of ribonucleic acids is however, difficult, since not only do the estimates vary with the method of preparation, but also with the technique used for making the measure(243) Various authors, Trans. F a ~ a d a ySOC.,46,790 (1950);60, 290 (1954).

332

G . R. BARKER

ment. Recorded values lieZ44between 6.0 X los and 2.9 X lo6, but determinations based on sedimentation and diffusion are probably liable to considerable error. Values found for the molecular weight of deoxyribonucleic acids also vary considerably, but probably lie between 1.0 X lo6 and 4.4 X lo6. Various difficulties encountered in making such measurements have been discussed by J0rdan,2~4and it is probable that more reliable information will be obtained only when the behavior of polyelectrolytes in general is better understood. Certain of the techniques used are useful in detecting differences between different nucleic-acid preparations, but the discrepancies between the values given by different methods of measurement appear to vary with the degree of polym8rizatior1.2~~ Probably the most exact information concerning the molecular architecture of the nucleic acids has resulted from x-ray studies. On the assumption that deoxyri bonucleic acids consist of 2-deoxyribonucleosides joined, via esterification, a t the 3- and 5-hydroxyl groups with phosphoric acid, a model has been devised which is compatible with the x-ray data for sodium deo~yribonucleate.~~6-~~9 Two right-handed, helical, polynucleotide chains are arranged with the phosphoryl residues on the outside and the bases directed toward their common axis. The structure repeats at every ten residues in each chain, and the two helices are held together by hydrogen bonding between pairs of adjacent bases. Owing t o the dimensions of the model, one of such an adjacent pair must be a purine and the other a pyrimidine, and if it is assumed that the bases are present, in the keto form, adenine and thymine must be paired together, and similarly guanine and cytosine. It is pointed out that this pairing could account for the presence of adenine and thymine, or guanine and cytosine, in equimolar proportions as observed by Chargaff,IT2and earlier workers.' A variation of the helical structure originally proposed has been suggested; it would allow of the two helices to be separated longitudinally.2498 Such a structure cannot be constructed for a ribopolynucleotide, since the extra oxygen atom would result in too close a van der Waals contact. However, x-ray photographs of ribonucleic acids from various sources are found t o be identical, which suggests that, irrespective of differences in composition, there is an underlying configuration which is common to all (244) D. 0. Jordan, Ann. Rev. Biochem., al, 209 (1952). (245) M. Goldstein and M. E. Reichmann, J . Am. Chem. Soc., 76, 3337 (19.54). (246) J. D. Watson and F. H. C. Crick, Nature, 171, 737 (1953). (247) M. H. F. Wilkins, A. R. Stokes and H. R. Wilson, Nature, 171, 738 (1953). (248) Rosalind E. Franklin and R. G . Gosling, Nature, 171, 740 (1953). (249) M. H. F. Wilkins, W. E. Seeds, A. R. Stokes and H. R. Wilson, Nature, 172, 759 (1953). (249a) H. Linser, Biochim. et Biophz~s.Acta, 16,295 (1955).

NUCLEIC ACIDS

333

specimens.2b0The patterns are different from those given by deoxyribonucleic acids and, moreover, can be altered reversibly by variation of the water content of the material. From certain samples it has also been possible to prepare fibers which show birefringence similar to that exhibited by deoxyribonucleic acid. The physical structures of the two types of nucleic acid therefore appear to have some features in common, and it is possible that this follows from a similarity in the mode of union of the nucleotides. (250) A. Rich and

J. D. Watson, Nature, 173, 995 (1954).