Studies in prebiotic synthesis

J. Mol. Biol. (1968) 38, 121-128

Studies in Prebiotic Synthesis IV.7

Conversion

of 4-Aminoimidazole-5-carbonitrile

Derivatives

ROBERT A. SANCHEZ, JAMES P. FERRIS AND LESLIE

to Purines

E. OROEL

The Salk In.stitute for Biologicul Studies San Diego, California 92112, U.S.A. (Received27 June 1968) The synthesis of 4-aminoimidazole-Lcarbonitrile and its derivatives from HCN has been discussed in a preceding paper. It is shown here that these imidazoles are readily cyclized to mono- and di-substituted purines. Since the photochemical synthesis of 4-aminoimidazole&carbonitrile from HCN is considered to be a particularly plausible prebiotic pathway, the conversion of 4-aminoimidazole6carbonitrile and of its hydrolysis product 4-aminoimidazole-5carboxamide to adenine and guanine are examined in some detail.

1. Introduction The remarkable discovery that adenine is produced in concentrated ammoniacal solutions of hydrogen cyanide (Oro & Kimball, 1962) has subsequently been exploited both as a potentially useful organic synthesis (Yamada, Kumashiro & Taker&hi, 1967) and as a model of prebiological purine synthesis (Palm & Calvin, 1962; Kliss & Matthews, 1962; Lowe, Rees & Markham, 1963; Oro, 1965; Ponnamperuma, 1965; Sanchez, Ferris & Orgel, 1967). The following imidazoles have been identified by Oro & Kimball (1962) and Ferris & Orgel (1966) from aqueous ammonium cyanide. NH II HsN-C H,N x

NFC

N ’

N H

\ /

AICAI &Aminoimidazole5-carboxamidine

N

HzN x

’

N

H2N-C \ /

0 II

H,N x

H AICN 4-Aminoimidazole5-carbonitrile

N ’

N H

\ /

AICA 4-Aminoimidazole5-carboxamide

Since they are believed to be precursors of the purines in this system, the conditions favoring their synthesis have been studied in detail (Sanchez et al., 1967). It was concluded that AICN and AICAI are products of the reaction between HCN tetramer and formamidine and that they react with cyanide or formamidine to give adenine, These syntheses are limited primarily by the low yields of formamidine obtained from ammonium cyanide and by its instability in aqueous solution. The photot P&per III in this series is Ferris,

Sanchez

& Orgel,

121

1968.

122

R. A. SANCHEZ,

J. P. FERRIS

AND

L. E. ORGEL

chemical synthesis of AICN from HCN tetramer offers fewer difficulties. We have, therefore, concentrated on the synthesis of purines from AICN and from its hydra. lysis product AICA.

2. Materials

and Methods

General AICN p-toluenesulfonate was prepared as describedpreviously (Ferris & Orgel, 1966). AICA hydrochloride was purchased from Calbiochem (A grade) or prepared by the procedure of Montgomery, Hewson, Struck & Shealy (1959). The free bases were obtained by ion exchange chromatography over Dowex-1. Other compounds were synthesized as described previously (Sanchez et al., 1967) or were the best commercially available

products. The paper chromatographic methods of analyses are those already described (Sanchez et al., 1967). Unless otherwise specified, the solvent systems used were BW (n-butanol saturated with water), BAW (n-butanol-acetic acid-water, 4: 1: 1) and PA (n-propanol1 N-NH,OH, 3: 1).

3. Results (a) Hydrolysis of AICN and AICA 0.020 M solutions of AICN and AICA in 2% pHydrion buffers were prepared at pH values of 7-0, 8-O (potassium and sodium phosphate), 9.0 (potassium phosphate and sodium borate) and 10.0 (sodium borate and sodium carbonate). These were distributed into small ampules, purged with nitrogen, sealed off and placed in a thermostatically regulated water bath at 60°C or in a steam bath at 100°C.Solutions were analyzed periodically by diluting 250.fold into 0.20 M-phosphate buffer (pH 6.8) and measuring the U.V. spectra. Calibration curves were prepared using solutions containing mixtures of the two imidazoles. The results of the analysesare summarized in Table 1. TABLE

1

Hydrolysis of 4.aminoimidazde-5.curbonitde and 4.amin&midazole-5.curboxamide Temperature “C 100 60 30s op

t+ (d, days; y, years) pH 8.0 pH 9.0

pH 7.0 AICNt

AICA$

AICN

AICA

AICN

8*73d 220d -

114d -

2.19d 130d 3oy 2oooy

108d

0.64d

t X max 246 rnp, B max 11,000 at pH 6.8. $ h max 288 rnp, E max 10,000 at pH 6.8. 0 Estimated from Arrhmiua plot extrapolations

-

40d 5Y 5ooy

pH 10.0

AICA

AICN

AICA

66d

0.36d

29d

-

15d 2Y

1ooy

1Y -

-

and therefore very approximate.

(b) Reactionsof 4.aminoimidazole-5.carbonitrik and 4.aminoimidawk-5.curbozumide (i) With HCN Solutions of each imidazole (0.10 N) in 1-O N-Nam were adjusted to pH 9 to 10 with HCl and then heated at 100°Cin sealedampules for one day. The solutions were

PREBIOTIC

SYNTHESIS

OF

123

PURINES

2

TABLE

Reactions of imidazoles with 1-ON-sodium cyanide at 100°C Product

composition

(%)

Imidazole

AICN AICA

AICN

AICA

Adenine

Hypoxenthine

22 0

19

7

1

48

0

3

centrifuged and subjected to chromatography in PA alongside authentic standards. The major absorbing spots were eluted, rechromatogrammed in BAW, re-eluted and the ultraviolet spectra measured in acidic and in basic solutions. The yields of purines are given in Table 2. Decreased yields of purines were obtained when the reactions were carried out at initial pH values of 1 or 12. The effects of NH,CN concentration on adenine yields from AICN are summarized in Table 3. The identity of the materials and the yields were established by chromatography alongside authentic standards in three solvent systems, and by measurements of ultraviolet spectra of material eluted from the BAW chromatograms. TABLE

3

Reactions of cl-aminoimidazok&carbonitrile with NH,CN at 1OO”Cfor 4 days

NH&N (mole/l.)

0.010 0.030 0.10 0.30 1.0

Product

composition

(%)

AICN

AICA

Adenine

31 23 15 8 2

10 17 23 33 34

1 2 5 8

11

The synthesis of adenine from AICN and hydrogen cyanide at 30°C and the effect of potential catalysts on it were studied. Solutions of AICN (0.05 M) and hydrogen cyanide (050 M) were buffered to a pH of 8.5 with sodium phosphate. The catalysts were added and the reactions allowed to proceed at 30°C. Analyses were performed by chromatography in PA followed by rechromatography of the eluted adenine areas in BW. The ultraviolet spectra of the re-eluted adenine spots were measured in acid and in base and the yields estimated from the optical densities. Hydroxylamine and perhaps hydrogen peroxide accelerate somewhat the synthesis of adenine from AICN and HCN. The following substanceshave little or no effect on the adenine yields: KCNO, NH,NH,, NH,CNS, CH,O, CaCl,, NH,, CH,NH,, pyridine. After 22 months the yields of adenine varied from 6 to 11%; 7.7% was obtained in the absenceof any catalyst. Recoveries of AICN ranged from 20 to 30% and the yields of AICA were between 15 and 20% in all experiments.

0.02 O-025 0.02 o-02 o-02 0.01 0.01 0.01 o-01 0.01 0.01

NH3 NH3 NH3 N%HPO1 NasHPOI NJ& N&i N-6 NH3 NJ& NH3

0.02 o-1 0.1 O-1 O-5 0.01 0.1 1.0 o-01 o-02 O-02

Base t concn (mole/l.) 0.2 0.4 o-2 0.2 o-2 0.1 0.1 0.1 2 0.2 o-2

CaNa concn (mole/l.)

t Identities and yields of the major products were established and measurement of ultraviolet spectra of &ted materials. i Similer resctions that had been heated at 100°C for longer

AICN AICN AICN AICN AICN AICA AICA AICA AICA AICA AICA

Imidazole & concn (mole/l.)

4

periods

3hr 3hQ 20 hr 20 hr 20 hr 21 hr 21 hr 21 hr 1w 24 hr 24 hr

8180 gave

very

Time at 100°C

chromatography

of time

by paper

to 1 hr 0 2 hr 2hr 2 hr lmin 1 min lmin 0 10 min 6hr

5 min

Time at room temperature

yields

standards

of purines.

in three

20 0 31 15 9 0 0 0 0 0 0

trace 2 5 9 17 47 52 100 23 4 authentic

Yield, y0 Diaminopwine

AICA

low or undetectable

alongside

50 100 13 19 23 0 0 0 0 0 0

AICN

Reactions of 4-aminoim~azok-5-carbonitrile and 4-aminoimirEazole-5-carbo~m~e with cyarwgenf

Tam

solvent

systems,

41 16 15 0 30 43

Guanine

PREBIOTIC

SYNTHESIS

OF

PURINES

125

(ii) With CaN, Alkaline solutions (pH 8 to 10) of AICN and AICA (O-01 to O-05 M) react rapidly with cyanogen at room temperature. Dark solids precipitate and the imidazoles disappear from solution. The solids are heterogeneous and largely insoluble except in more strongly alkaline solutions (pH 11). Their ultraviolet spectra in water are ill-defined. When either the suspensionsor the isolated and resuspendedsolids are heated the formation of soluble well-defined intermediates is detectable before the purine basesappear as major products. The results of a number of typical reactions are summarized in Table 4. Studies of the temperature and pH dependencesof the conversion of the initial imidazole-cysnogen precipitates to the purines were carried out in 2% buffer solutions. The solutions were analyzed periodically by paper chromatography. The products were first eluted from BAW or PA developments and their ultraviolet spectra were then measured. The results are summarized in Table 5. The kinetic data from these heterogeneous reactions were erratic. TABLE

5

Temperature and pH dependencein the synthesisof purinee from the imidawle-cyanogen intermediates

PH

7.0 8-O 9.0 10.0

diaminopurine

30% >30 20 3 1

from 60%

Approximate AICN-CoN, 100°C

2 1

co.1 co.1

-


00 16


half-life.

days gutmine

30%

from

AICA-C2Na

60°C

100°C

2


1.5 1 1

The intermediate dark-colored solid that forms from AICA and cyanogen is converted by brief heating (10 min, lOO”C, pH 10) to a mixture containing a new compound with the following properties: RF values, O-21 (BW), 044 (BAW), O-38 (PA) ; U.V. spectrum, A max 262 rnp (pH 2), 283 rnp (pH 10); infrared spectrum, no -C&N frequency at 2200-2400 cm-‘; gives a purple color with dirtzotized sulfanilic acid. The compound may be 4-ureido-5Gnidazolecarboxamide, but it was not characterized further. Heating at pH 7 to 10 converts it cleanly to guanine, which we identified by its chromatographio properties end by its ultrrtviolet and infmred spectra. (iii) With cyunate The major products of reaation with cyanate are dieminopurine from AICN and guanine from AICA. Solutions O-10Y in AICN or AICA, and O-50M in KCNO (initial pH about 7) were heated at 100°C for 90 hours. The solutions were analyzed by paper chromatography in four solvent systems, alongside authentic standards. The results are given in Table 6. Two solutions O-01M each in AICA and NH, were prepared. To one cyanogen was added and to the other KCNO, to give O-1 M solutions, After hectting at 100°C for 19 hours the solutions were analyzed by paper ohromatography. Approximate yields were, respectively, 20% and 50% of AICA, 50% and 10% of guanine.

126

R.

A.

SANCHEZ,

J.

P.

FERRIS TABLE

Reactions

from from

AICN: AICA

of imidazoles

AICN

AICA

10 0

60 70

:

with 0.50 Approximate Diaminopurine

AND

L.

E.

ORGEL

6 M-potassizcm product

3 0

at 100°C

cyanate

composition Guanine

(%) Xanthine

Adenine

3 3

tra.ce 0

20

(iv) With cyanamide Low yields (1 to 5%) of diaminopurine from AICN and of guanine from AICA were detected when 0.01 M solutions of the imidazoles were heated with O-10 Mcyanamide at 100°C for 20 hours About 2% of the cyanamide remained after this time ; the imidazoles were recovered in high yields. (v) With urea Xanthine and guanine were the major products (5 to 10%) when solutions containing 0.10 M-AICN or AICA and 0.50 H-urea were heated at 100°C for 6 days. About 50% of the original imidazoles were recovered. (vi) With amnzonium carbonate The reaction of O-02 M-AICN with 1-O M-NH,HCO, at 100°C for three days produced AICA and xanthine (20 to 30% each) as the major products, in addition to recovered AICN (20 to 30%) and a small amount of diaminopurine. In a reaction with AICA under the same conditions almost all of the imidazole was recovered and only traces of other substances detected. (c) Reactions of 4-aminoimiduzole-5-carboxamidine AICAI was synthesized as described previously (Sanchez et al., 1967). Purification by column chromatography over Amberlite CG50, Cellulose and Dowex 50, in that 7

TBLE

Reactions of 4-aminoimidazole-5-carboxamidine with HCN at pH 9-O to 9.5 Reclction time 0.6 hr

1 day

10 days

50 days

Product

AICAI AICA A AICAI AICA A AICAI AICA A AICAI AICA A

30%

80 0 2 30 0 4

Yield, y0 60%

86 7 3 6 42 11 0 1 24

100% 94 4 3 1 90 7 0 0.2 9 0 0 9

PREBIOTIC

SYNTHESIS

OF PURINES

127

order, yielded the product as a dihydrochloride. Its paper chromatographic mobilities and ultraviolet spectra were the same as those reported by Oro & Kimball (1962) and by Shaw (1950). Small amounts of adenine and AICA were present as contaminants. A solution was prepared which was 0.050 M in AICAI.2HCI and 0.50 M in NaCN. Sampleswere adjusted to pH values of 7.5, 9.0 and 10.5 with 12 M-HCl, and placed in baths maintained at 30,60 and 100°C(each &3”C). Analyses were made by descending chromatography in n-propanol-conc.NH,OH water (5.5:1*0:3*5). The R, values were AICAI, 0.48 ; adenine, 0.56 ; AICA, 0.64. Recoveries and yields were calculated from the ultraviolet absorption of the eluted spots. The results at pH 9.0 to 9.5 are shown in Table 7. At pH 7.5 to 8.0 AICAI survived somewhat longer and AICA yields were lower but the adenine yields were about the same. At pH 10.5 the yields were generally similar to those at pH 9.0 to 9.5.

4. Discussion 4-Aminoimidazole-5carbonitrile and its derivatives react with hydrogen cyanide, formamidine, cyanogen, cyanate, urea and carbonate to give a variety of mono- and disubstituted purines. Here we discuss in detail only those reactions which yield adenine or guanine. Extrapolation of the yields of adenine obtained from AICN and HCN at 30°C to 100°C suggests that AICN would react with the concentrated cyanide solutions (1 M or higher) formed as eutectic phasesin the temperature range -20°C to 0°C to give at least 10% of adenine and about 50% of AICA. The time of reaction would be 1 to 10 years. Since tetramer is formed in reasonable yield (> 10%) under these same conditions and since it is isomerized very efficiently by sunlight to AICN it follows that adenine could be formed in yields of up to a few per cent directly from very dilute cyanide solutions by cooling and irradiation. It is quite possible that suitable conditions for these reactions existed on the primitive earth. The production of guanine would require, in addition to AICA formed by the hydrolysis of AICN (Table I), either cyanogen or cyanate. Cyanogen could be generated photochemically from cyanide (Airey & Dainton, 1966), thermally (Harada & Fox, 1965) or in electric discharges (Schavo & Winkler, 1959); cyanate would be obtained from it by hydrolysis. The data in Tables 5 to 7 suggestthat yields of guanine of 10% or more based on AICA could be obtained in at most a few years at -20°C to 0°C. Thus an over-all yield of a few per cent based on cyanide is not impossible. While we believe that these are the most plausible routes to the biologically important purines, they are not the only possible ones. The synthesis of adenine from concentrated ammonium cyanide (Oro, 1965; Yamada et al., 1967) almost certainly involves formamidine ; a number of different routes could contribute.

Tetramer

128

R. A. SANCHEZ,

J. P. FERRIS

AND

L. E. ORGEL

Our studies suggestthat the observed yields of up to 1% in relatively concentrated ammonia solutions can readily be accounted for by the four routes shown above. This work was supported by Grant The authors are grateful to Mr Robert

GB6303 from the National Science Foundation. Mancuso for his very capable assistance.

REFERENCES Airey, P. L. t Dainton, F. S. (1966). Proc. Roy. Sot. A, 291, 340. Ferris, J. P. & Orgel, L. E. (1966). J. Amer. Chem. Sot. 88, 3829. Ferris, J. P., Sanchez, R. A. & Orgel, L. E. (1968). J. Mol. Biol. 33, 693. Harada, K. & Fox, S. W. (1966). In The O*ina of Prebiologicd Systema, ed. by S. W. Fox, pp. 187-201. New York: Academic Press, Inc. Kliss, R. M. & Matthews, C. N. (1962). Proc. Nut. Acd Sci., Wuah. 48, 1300. Lowe, C. V., Rees, M. W. t Markham, R. (1963). Nature, 199, 222. Montgomery, J. A., Hewson, K., Struck, R. F. & Shealy, Y. F. (1969). J. Org. Chem. 24, 256. Oro, J. (1965). In The Or@ins of PrebioZogicd Systems, ed. by S. W. Fox, pp. 137-171. New York: Academic Press, Inc. Oro, J. & Kimball, A. P. (1962). Arch. Biochem. Biophye. 96, 293. Palm, C. & Calvin, M. (1962). J. Ama-. Chem. Sot. 84, 2115. Ponnamperuma, C. (1965). In The Origins of Prebiologicul Systems, ed. by S. W. Fox, pp. 221-242. New York: Academic Press, Inc. Sanchez, R. A., Ferris, J. P. & Orgel, L. E. (1967). J. Mol. Biol. 30, 223. Schavo, A. & Winkler, C. A. (1969). Canud. J. Chem. 37, 665. Shaw, E. (1950). J. Biol. C&m. 185, 439. Yamada, Y., Kumashiro, I. & Takenishi, T. (1967). J. Org. Chem. 33, 642.