Studies in prebiotic synthesis

J. Mol. Biol. (1972) 67, 25-33

Studies in Prebiotic Synthesis VI.? Synthesis of Purine Nucleosides WILLIAM D. FULLER,ROBERT A. SANCHEZAND LESLIE E. ORGEL The Salk Institute for Biological Studies, San Diego, Calif. 92112, U.S.A. (Received 11 November 1971) The isomers of 6-ribosylaminopurine (N6-ribosyladenine) are formed in up to 7496 yield when a dry mixture of adenine and ribose is heated. Under these conditions ribose also reacts with the primary amine group of adenosine, guanosine and probably guanine to form the corresponding ribosylaminopurines, while hypoxanthina fails to react. In the presence of Mg2 + and inorganic polyphosphates the natural nucleosides are also formed. Maximum yields of /3-inosine, /3-adenosine and /?-guanosine were So/& 4% and 9%, respectively.

1. Introduction Most theories of the origins of life presuppose that amino acids and nucleotides were formed abiotically on the primitive earth. The amino acids, nucleotide bases and sugars can all be prepared in the laboratory under plausibly prebiotic conditions. The purine nucleosides are the only monomers that remain to be synthesized prebiotically (Kenyon & Steinman, 1969). We have previously reported a prebiotic synthesis of the naturally-occurring pyrimidine nucleosides utilizing D-ribose, cyanamide and cyano-acetylene. The overall yield was small (Sanchez & Orgel, 1970). Ponnamperuma, Sagan & Mariner (1963, and references therein) described the synthesis of adenosine in O.Olo/o yield by t$he action of ultraviolet light on dilute solutions of adenine and D-ribose containing either NaCN, H,PO, or ethyl metaphosphate. We have been unable to duplicate these results. Schramm (1964) demonstrated that adenosine and its phosphate esters are produced when adenine and ribose are heated with alkyl polyphosphate esters. Recent experiments suggest that related inorganic polyphosphate salts: unlike organic polyphosphate esters, could have accumulated in large amounts on the primitive earth (Osterberg & Orgel, manuscript in preparation). Here we describe experiments which show that nucleosides are formed when mixtures of ribose and purine bases are heated with inorganic polyphosphates.

2. Materials and Methods cr-Adenosine was a gift from Dr L. Pichat of the Centre d’Etudes Nucleaire de Saclay. 9.fl-n-Ribopyranosyladenine was prepared from the product obtained by fusing a mixture of adenine and 1, 2, 3, 4-tetra-0-acetyl D-ribopyranose with p-toluenesulfonic acid.

cc-Inosine and 9-p-D-ribopyranosylhypoxanthine

were prepared

t Paper V in this series is Sanchez & Orgel (1970). 26

by the action of nitrous

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acid on u-adenosine and 9-j3-D-ribopyranosyladeninne, respectively. All other reagents were purchased from commercial sources. The purity of the sugars, bases and nucleosides was checked by chromatography. Paper chromatography was performed by descending elution on Whatman 3MM paper with the following solvents: A, n-butyl alcohol saturated with water and B, n-butyl alcohol and 5 N-&C&iC acid (2:l). Paper electrophoresis was carried out at 4000 V on Whatman 2.7 with

3MM paper concentrated

in the following buffers: C, 0.05 N-formic acid adjusted to a pH of ammonia and D, 0.05 N-boric acid adjusted to a pH of 8.5 with

sodium hydroxide. The chrometographic and electrophoretic mobilities of various compounds are assembled in Table 1. The glass fiber paper used as a support in some of the reactions was grade GF82 from Whatmsn. TABLE

Chromatographic system

1

and electrophoretic -RF A

Adenine D-Ribose 6-Ribosylaminopurine (I)$ cc-Adenosine p-Adenosine 9-p-D-Ribopyranosyladenine 6-Ribosylamino-9-fl-n-ribofuranosyl purine (11)s Guanine 2-Ribosylaminohypoxanthine (III)$ ,%Guanosine 2-Ribosylamino-9-/?-D-ribofuranosylhypo xanthine (IV)$ Hypoxanthine a-Inosine fl-Inosine 9.fl-n-Ribopyranosylhypoxanthine Xanthine ,%Xanthosine

mobilitiest

-JLamosm-

valueB

(mobilities relative /3-adenosine C D

to

0.38 0.17 0.16, 0.22 0.18 0.21 0.14

0.50 0.28 0.28, 0‘34 0.35 0.37 0.32

1.5 0.75 1.1 1.0 1.0

0

0.08, 0.13 0.17 0.09 0.05

0.22, 0.26 0.29 0.16, 0.21 o-24

0.41 0.76 0.39

1.1, 1.8, 1.9 0.18 0.60, 1.3 1.2

0.04, 0.10 0.28 0.13 0.15 0.10 0.21 0.028

0.14, 0.11 0.37 0.23 0.24 0.21 0.29 0.22

0.40 0.38 0.35 0.30 0.30 0.16 0.26

1.2, 1.4, 1,s 0.56 1.4 1.4 0.74 145 2.2

o-17, o-33, i-2, 1.3 1 1 0.29

tSee text for details of solvent systems. fThe nature of the isomeric ribosides is discussed in the text.

3. Experimental and Results (a) Reactions in aqueous solutions Aqueous solutions containing adenine (10-l to 10T4 M) and n-ribose (1 to 10m2 M) maintained at a variety of temperatures (30 to 100°C) and pH values (2 to 11) failed to produce significant yields of any products containing adenine and a sugar. Attempts to bring about the synthesis of adenosine in aqueous solution with the help of ultraviolet light (257 nm and 350 nm), condensing agents (cyanate, cyanamide, urea, cyanoacetylene, cyanogen, polyphosphate, etc.) and heterogeneous catalysts (charcoal, clays and minerals) were all unsuccessful. In most cases adenosine would have been detected if the yield had been as great as O*lo/; in some experiments much smaller quant&ies could have been estimated.

SYNTHESIS

OF

PURINE

$17

NUCLEOSIDES

We conducted similar experiments, although fewer in number, with cytosine, guanine, hypoxanthine and uracil. In no case were we able to detect significant amounts of adducts containing both a base and a sugar. (b) Reactions in dry phases (i) Hypoxanthine

and inosine

Xo more than traces of products were detectable when aqueous solutions of hypoxanthine or inosine and n-ribose were evaporated to dryness on glass fiber paper and heated at 100°C. (ii) Adenine and its derivatives Adenine (2.70 g, 20 m-moles) was dissolved in 200 ml. of boiling water, n-ribose (6.00 g, 40 m-moles) was added and the solution evaporated to dryness in vacua on a steam cone. The residue was heated in vacua (20 mm Hg) at 100°C for 6 hours. The resulting yellow solid was extracted with small volumes of water, leaving a residue of 2.0 g (74% recovery) of adenine. The combined extracts were stirred with 8 g of Norit-A (M.C. & B) and then filtered. The Norit was washed with water to remove traces of ribose and then eluted with pyridine/water to remove the adenine-containing compounds. The latter eluate was evaporated to dryness and yielded about 1 g ( 19(y0 HIN,Ribose I

Ribose

H,Nl$jN)

H%:I&.:-5 N

R’,bose

Ribose III

kibose Iv

FIG. 1. Structures of the ribosides produced as major products adenine (I), adenosine (II), guanine (III) and guanosine (IV).

by the reaction

of n-ribose

with

yield) of crude product (I). Crystallization of a portion of this material from ethanol/ ether gave a white powder having an indefinite melting range near 220°C. Microanalysisgave: C, 44.71%; H, 510%; andN, 2552%. Calculated values for Cl,,H,,N,O, are: C, 44.94%; H, 4.90%; and N, 26.20%. Hydrolysis of this material in aqueous acid produced adenine and ribose (identified by paper chromatography in systems A and B) in essentially quantitative yields. The half-life for hydrolysis in 0.10 M-HCl at 100°C was about 10 minutes. Chromatography in systems A and B revealed the presence of at least two corn-

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ponents (Table 1). Electrophoresis in system D, however, shows four well-defined components. Two of these move rapidly and are presumably cc-and p-ribofuranosides, for ribofuranosides are known to form stable borate complexes. Two components move very slightly and are presumably u- and ,!I-ribopyranosides, since pyranosides are known to form weaker borate complexes (Khym, Doherty & Cohn, 1954). We isomerized /I-n-pseudouridine under conditions known to yield four isomers (a- and /?-anomers of the furanose and pyranose ring forms; Chambers & Kurkov, 1964) and found that this mixture of isomers and mixture I give almost identical electrophoretic patterns in system D. The four isomers of I eluted from the electrophoretogram have identical ultraviolet maxima: pH 2, 272 nm; pH 5 (H,O), 264 nm; pH 12, 272 nm. Upon heating in water at pH 8 and lOO”C, each compound is converted to the same equilibrium mixture of four isomers; the pyranose anomers are most abundant in the equilibrium mixture. The optical-rotatory dispersion spectra of the pyranose isomers at pH 7.0 are mirror-image curves having similar intensities. The furanose isomers interconvert too rapidly to allow measurement of their optical-rotatory dispersion spectra. We have studied the effect of pH on the condensation of adenine and ribose, and find that optimal yields of I are obtained under slightly acidic conditions. When a solution of adenine and n-ribose was adjusted to a pH of 45 with HCl and then evaporated to dryness and heated, a 74% yield of adduct I was obtained. o-Arabinose, n-lyxose and n-xylose reacted with adenine under these conditions and produced similar yields of adducts having the same ultraviolet spectra as I. 6-Methylaminopurine and 6-dimethylaminopurine gave little or no new products when heated with n-ribose. The following additional facts are consistent with our identification of the adeninen-ribose adduct as F-substituted glycoside. (a) The U.V. spectrum is similar to that reported for 6-methylaminopurine (pH 1, h,,, 267 nm (c = 14,900); pH 11, h,,, 272 nm (e = 15,300); Elion, 1962) and unlike those reported for l-, 3-, 7- and 9-alkyl and glycosyladenines. (b) The intraconversion of the four components of mixture I is (Ukita, Hamada & typical of the behavior of N-glycosides such as N-ribosylureas Yoshida, 1964) and N-ribosylanilines (Berger & Lee, 1946); the corresponding interconversions of tertiary N-glycosides, such as adenosine do not occur, and (c) many primary amines such as aniline are known to react with sugars to yield mixtures of isomeric N-glycosides (Berger & Lee, 1946). Adenosine (4.0 g, 15 m-moles) and n-ribose (4.5 g, 30 m-moles) were dissolved in 400 ml. of water and adjusted to a pH of 45 with HCl. The solution was evaporated to dryness in vacua on a steam cone and the residue was further heated in vacua for 4 hours. The yellow solid was dissolved in 25 ml. of water and applied to a column of Amberlite CG50 (H+). Elution with water removed n-ribose, adduct II (about 40% yield) and the adenosine, in that order. Adduct II was further purified by deposition from ethanol, The amorphous white powder had an indefinite melting point. The U.V. spectra in water showed: pH 2, X,,, 266 nm (c = 17,000); pH 7.7 (HsO), Amax 265 nm gave: C, 4552%; (6 = 18,800); pH 12, h,,, 264 nm (c = 19,600). Microanalysis H, 5.36%; N, 17.15%. Calculated values for C&,HZ1Nj08 are: C, 45.11%; H, 5.25%: N, 1754%. Aqueous hydrolysis of the diriboside II in neutral or alkaline solution produced adenosine in essentially quantitative yield. In acid there was also a slower subsequent

SYNTHESIS

OF PURINE

NUCLEOSIDES

29

hydrolysis to adenine. The estimated half-lives for the hydrolysis at 100°C in buffered solution were 5 minutes at pH 3,3 hours at pH 5,6 hours at pH 7 and 1 hour at pH 9. The reaction of adenine with ribose produced in addition to the 6-ribosylemino purines discussed above, very low yields of adenosine. In further experiments designed to determine the yield of adenosine, 10 ml. of a solution containing adenine (0.05 M) and n-ribose (0.30 M) was adjusted to a pH of 4.5 with HCl and then distributed onto six 4 cm x 6 cm pads of glass fiber paper. The papers were dried and then heated at 100°C for 2.5 hours in open glass tubes. The papers were eluted with water and the eluate was divided into two equal portions. One was adjusted with ammonia to a concentration of 2.0 M and heated at 100°C for 2 hours; the other was left to stand at room temperature. 14C-labeled adenosine marker was added to the hydrolyzed and unhydrolyzed samples which were then concentrated. Chromatography or electrophoresis followed by elution of the areas corresponding to u- and /I-adenosine was carried out successively in systems B, A, C and D. The estimated yields of a- and /3-adenosine were O.O3o/oin the unhydrolyzed sample and O*2lo/o in the hydrolyzed sample. The ratio of a-adenosine to fl-adenosine was about 1: 2 in each case. The U.V. spectra of the products were the same as that of /Ladenosine. (iii) Guunine and guanosine Guanine reacted with n-ribose in a dry phase on glass fiber paper to produce a compound tentatively identified as 2-ribosylaminohypoxanthine (III, N2-ribosylguanine; Shapiro & Gordon, 1964). However, the yield was very low, probably because guanine is extremely insoluble. This reaction was not studied further. A mixture of guanosine and n-ribose in a molar ratio of 1: 2 was dissolved in a minimum volume of warm water and adjusted to a pH of 45. The solution was evaporated to dryness in vacua at lOO”C, and heating was continued in vacua for 4 hours. Crude product IV in a yield of about 35% was extracted from the mixture with hot ethanol. Further purification was achieved by elution with water through a column of Amberlite CGBO(H + ) followed by recrystallization from ethanol. The white crystalline solid had an indefinite melting range near 175°C. Chromatography in systems A and B showed that guanosine and ribose had been removed and that two new isomeric components were present. The U.V. spectra of an unresolved mixture of the components in water showed: pH 2, h,,, 258 nm (E = 14,300), h sh 275 nm; pH 5 VW), knm 255 nm (c = 14,700), X sh 270 nm; pH 12, X,,, 259 to 265 nm (broad), (E = 12,600). Microanalysis gave: C, 42.57%; H, 5.17%; N, 16.11%. Calculated values for C,,H,,N,O,+H,O are: C, 42.35%; H, 5.17%; N, 16.47%. The half-lives for the hydrolysis of this diriboside at 100°C in 0.1 N-HCl (pH l), H,O (pH 6) and 2 M-NH,OH(pH 12) were approximately 12 minutes, 2 hours and 20 minutes, respectively. Guanosine was produced quantitatively at pH values of 6 and 12. At pH 1 the subsequent hydrolysis of guanosine to guanine was also rapid. (c) Reactions in dry $KZ.S~Sin the presence of various salts

(i) Hypoxanthiue We prepared a solution containing hypoxanthine (0.01 M), n-ribose (0.15 M)l, magnesium chloride (0.125 M) and sodium trimetaphosphate (0.125 M). Samples of 1-Oml. were adjusted to pH values of 3*0,5*0,7*0 and 9.0, and applied to 3 cm x 4 cm pads of glass fiber paper. These pads were subsequently dried and heated at 100°C

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for 2 hours. The aqueous eluates from the papers all had a pH of 4 to 5 and all showed the same chromatographic pattern in system B. The two major bands corresponded in RF to hypoxanthine (approx. 85% recovery) and inosine (approx. 15% yield). A control reaction in which the magnesium and trimetaphosphate salts were omitted gave only the band corresponding to hypoxanthine. The eluted “inosine” band from a larger-scale synthesis (yield approx. 20%) was rechromatogrammed in system A. It was resolved into pure u-inosine (4%) yield, /3-inosine contaminated with other products, and a very low yield of a substance with the R, of 9-/3-n-ribopyranosylhypoxanthine. The crude /3-inosine was eluted, applied to a column of Dowex-1 (formate form) and developed with a formic acid/water gradient. This resulted in the separation of pure /3-inosine in 8% yield from an unidentified component. The chromatographic mobilities of M- and ,%inosine obtained in this way were indistinguishable from those of authentic samples in several systems in addition to those listed in Table 1. Ultraviolet spectra (acid and base) and optical-rotatory dispersion spectra (phosphate buffer, pH 7.0) of our products and authentic materials coincided exactly. A detailed study of this reaction revealed that the rates of synthesis and destruction of inosine, and the rate of destruction of ribose depended in a complicated way on the ratio of reactants. The experiment described above was carried out under conditions close to those that optimize the rate of synthesis and the yield of inosine. The yields decrease markedly when the ribose/hypoxanthine ratio is reduced. (ii) Adenine One ml. of a solution containing adenine (0.050 M), D-ribose (0.30 M), magnesium chloride (0.25 M) and sodium trimetaphosphate (0.25 M) was adjusted to a pH of 7.0 with NaOH, applied to a 3 cm x 3 cm square of glass fiber paper and then allowed to dry. The paper was heated for 2 hours at 100°C in an open tube and then eluted with water. The eluate was divided into two equal portions. One of these was kept at room temperature and the other was evaporated, redissolved in 1 ml. of 6 M-NH,OH and then heated at 100°C for 4 hours. Both portions were concentrated and chromatogrammed in system B, together with /%[8-14C] adenosine marker. The unhydrolyzed sample contained some recovered adenine and large amounts of products with R, values similar to those of I, II and adenosine. The hydrolyzed sample contained adenine and smaller amounts of products. The areas corresponding to u- and p-adenosine were eluted and rechromatogrammed in system C. The combined u- and /3-adenosine yields were 1.7% from the unhydrolyzed sample and 7.2% from the hydrolyzed sample. In each case the ratio of u-adenosine to fl-adenosine was about 1:l. The identity of the u- and /3-adenosine was confirmed by comparing material obtained from larger-scale reactions with authentic standards. Our products had the correct optical-rotatory dispersion spectra, U.V. spectra, chromatographic mobilities in seven systems including those listed in Table 1, and the correct rates of hydrolysis in acid. 9-n-Ribopyranosyladenine (presumably both u- and ,&anomer) is also formed in these reactions but in lower yields. Little or no adenosine was obtained when either the magnesium chloride or the sodium trimetaphosphate was omitted. The yield of adenosine was little changed when manganese chloride replaced magnesium chloride, but with calcium chloride the

SYNTHESIS

OF

PURINE

NUCLEOSIDES

31

yield was reduced by almost 50%. Ferrous and cuprous chlorides gave little or no adenosine. Higher polyphosphates (tetrametaphosphate, hexametaphosphate, etc.) were about as effective as trimetaphosphate. Tripolyphosphate, pyrophosphate and orthophosphate were decreasingly effective. When the initial product formed in the presence of orthophosphate was hydrolyzed, adenosine was obtained in less tha,n 05% yield. Adenosine was not obtained in yields greater than 0.1% in the presence of the following salts: NaCN, HC02Mg, KCNO, Ca(OH),, CaCO,, ZnCl,, NaHSO,, Na,SO,, Na,B,O,, FeSO1, Fe,(SO,),. The yield in reactions involving magnesium and polyphosphates is optimum when the solution is initially adjusted to a pH of about 7.0. If the dry reaction product is redissolved, the pH of the solution obtained is in the range 4-O to 4.5. This is also the optimum pH range for the dry phase synthesis of adducts I and II from adenine and adenosine, respectively. We have made some efforts to discover the mechanism of this adenosine synthesis, but without success. Ribose is partially phosphorylated under our reaction conditions. However, none of the phosphate-containing products gave adenosine when heated separately with adenine, magnesium chloride and sodium trimetaphosphate. tl- and /3-n-ribofuranose-l-phosphate were equally ineffective in the synthesis. We could not detect any phosphorylated derivatives of adenine. (iii) Guunine The reaction of guanine with ribose had to be studied under somewhat different conditions, since guanine is only very slightly soluble in water. A solution containing [2-14C] guanine (5 x low4 M, 10 pCi/ml.), n-ribose (0.15 M), magnesium chloride (0.125 M) and sodium trimetaphosphate (O-125 M) was adjusted to a pH of 7.0. A sample was applied to glass fiber paper, dried under an infrared lamp and then heated at 100°C for 2 hours. The paper was eluted with 6 M-NH,OH (15% of the original radioactivity remained on the paper) and the eluate was heated at 100°C for 4 hours. Non-radioactive guanosine carrier was added, then the guanosine bands were purified by chromatography successively in systems B and A. Further chromatogrpahy on a column of Dowex 50(H+) with 0.1 M-HCl at 4°C gave coincident bands of radioactivity (9% of the original amount) and U.V. absorption. The radioactive product co-chromatograms with /I-guanosine in systems A, B and D. The half-lives for the acid hydrolysis of the radioactive product and /?-guanosine carrier were identical; guanine was the sole radioactive product of hydrolysis. (iv) Temperature

dependence of Nucleoside synthesis

The effects of temperature on the rates of synthesis and yields of adenosine and inosine were determined. A solution containing adenine (0.010 M), hypoxanthine (O*OlOM), n-ribose (O-20 M), MgCl, (0.10 M) and Na,P,Oa (0.10 M) was adjusted to a pH of 7.0 with NaOH. Samples of 1-Oml. were applied to 3 cm x 4 cm pads of glass fiber paper and allowed to dry. The papers were heated at 65°C 85°C and 100°C for various times. Then radioactive markers were added (/3-[S-14C]adenosine and /3-[8-14C] inosine) and the products eluted with 5 M-NH,OH. After heating at 100°C for 1 hour the analysis was carried out as described previously (Results section (c)(ii)). The results are illustrated in Figure 2.

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(b)-

Time (days) (c) FIG. 2. Yields of fl-adenosine and fl-inosine obtained at various scales are different in the different diagrams. (See text for details.)

temperatures. Note that the (a) 100°C; (b) 85’C; (c) 65°C.

4. Discussion Prebiotic syntheses of purines and sugars have been known for some time. We have recently discovered that urea, under quite mild conditions, brings about the quantitative conversion of ammonium phosphate to inorganic polyphosphates (Osterberg & Orgel, manuscript in preparation). Under the same conditions, nucleosides are converted to nucleotides, nucleoside di- and triphosphates and short oligonucleotides (Osterberg, Lohrmann 8.r Orgel, manuscript in preparation). The demonstration that purine nucleosides are formed from purines and ribose in the presence of inorganic polyphosphates thus completes a prebiotic synthesis of purine nucleotides. It is a striking fact that molecules as complex as adenosine triphosphate or the trinucleotide ApApA could now be synthesized using no other reagents than hydrogen cyanide, formaldehyde, urea and ammonium phosphat,e. All reactions occur in aqueous solution within the pH range 7 to 9, or in the solid films obtained by evaporating such solutions at 60 to 80°C in open vessels. The best conversions of purines to the naturally-occurring purine nucleosides amounted to S%, 4% and 9% in the cases of /3-inosine, p-adenosine and /3-guanosine, respectively. However, in all cases, most of the purine was recovered unchanged at the end of the experiment, so yields based on the purine destroyed were probably greater than 50%. These efliciencies compare favorably with those obtained in other prebiotic reactions. In a number of potentially prebiotic, heterogeneous reactions the character of the products depends critically on the nature of the chemically inert supporting phase (Paecht-Horowitz, Berger & Katchalsky, 1970; Neuman, Neuman & Lane, 1970; Krane & Glimcher, 1962). The catalytic effect of inorganic phosphates does not seem to depend on the formation of phosphorylated derivatives of the purines or the sugars. If, as seems likely, the polyphosphates are acting as acid catalysts for reactions that resemble the classical fusion-syntheses of nucleosides, it is probable that more effective catalysts can be found. We are searching for such catalysts.

SYNTHESIS

OF PURINE

NUCLEOSIDES

This research was aided by grant no. GB 24837 from the National

33 Science Foundation.

REFERENCES Berger, L. & Lee, J. (1946). J. Org. Chem. 11, 75 (and subsequent papers). 3, 327. Chambers, R. W. & Kurkov, V. (1964). Biochemistry, Elion, G. (1962). J. Org. Chem. 27, 2478. Kenyon, D. H. & Steinman, G. (1969). Biochemical Prededination. New York: McGrawHill. Khym, J. X., Doherty, D. G. & Cohen, W. E. (1954). J. Amer. Chem. Sot. 76, 5523. Krane, S. M. & Glimcher, M. J. (1962). J. Bid. Chem. 237, 2991. Lohrmann, R. & Orgel, L. E. (1971). Science, 171, 490. Neuman, M. W., Neuman, W. F. & Lane, K. (1970). Currents in Modern Biology, 3, 253. Paecht-Horowitz, M., Berger, L. & Katchalsky, A. (1970). Nature, 228, 636. Ponnamperuma, C., Sagan, C. & Mariner, R. (1963). Nature, 199, 222. Sanchez, R. A. & Orgel, L. E. (1970). J. Mol. BioZ. 47, 531. Schramm, G. (1964). In The Origins of Prebiological Systems, ed. by S. W. Fox, p. 229. New York: Academic Press. Shapiro, R. & Gordon, C. N. (1964). Biochem. Biophys. Res. Comm. 17, 160. Ukita, T., Hamada, A. & Yoshida, M. (1964). Chem. Pharm. Bull. Japan, 12, 454.