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Possible role of hydrogen cyanide in chemical evolution: The oligomerization and condensation of hydrogen cyanide
J. MOE. Biol. (1973) 74, 511-518
Possible Role of Hydrogen Cyanide in Chemical Evolution :-f-The Oligomerization and Condensation of Hydrogen Cyanide J.P.
FERRIS, D.B.
DONNERAXD
A.P.
LOBO
Department of Chemistry Polytechnic Institute Troy, N.P. 12181, U.X.A.
Rensselaer
(Received 11 August 1972, and &a revised form 5 December 1972) The rate of cyanide oligomerization is independent of the presence of added nucleophiles such as azide, monomethylamine or triethylamine and is dependent only on the pH of the reaction mixture. The products formed, with the exception of urea, are also independent of the nucleophiles used to initiate reaction. In the presence of monomethylamine, monomethylmea is the main neutral product instead of urea, suggesting the intermediacy of cyanate. Evidence is presented which suggests that cyanogen may be the precurser to both oyanate and oxalic aaid in the cyanide oligomerization.
1. Introduction Hydrogen cyanide is believed to have had a significant role in the prebiotic synthesis of amino acids and purines (Sanchez et al., 1967, and references therein). In previous papers we presented evidence which indicates that in basic solution hydrogen cyanide oligomerizes to form a complex mixture of low molecular weight compounds (Ferris et al., 1972a,b). We found no evidence for the presence of peptides within this reaction system (Dormer et al., 1970; Ferris et al., 1972b; Ferris et al., 1973). Additionally, we were unable to find any experimental verification of the claim that polymers are formed. Large amounts of urea and smaller amounts of oxalic acid were isolated from the cyanide condensation system and we confirmed that hydrolysis of the oligomerization mixture results in the formation of amino acids. An investigation of the mechanisms by which urea and oxalic acid are produced and by which hydrogen cyanide oligomerizes was undertaken with the aim of understanding better the alkaline solution chemistry of hydrogen cyanide and the possible role of hydrogen cyanide in chemical evolution.
2. Materials and Methodsg (a) The oligomerization
of cyanide
in
the presence
of
added rvucleophiles
Cyanide oligomerization reactions were conducted in the presence of nucleophiles other than ammoninm hydroxide. The following reactions were investigated: (1) The pH of a solution of 1 N-sodium cyanide was adjusted to 9.2 with cone. HCl. (2) The pH of t Chemical Evolution XII. The previous paper in this series is Ferris et al. (19 73). $ General experimental procedures are given in the previous paper in this series (Ferris et al., 1973) and in the Ph.D. Thesis of D. Dormer (1971). 34
511
612
J. P. FERRIS
ET
AL.
a solution 0.1 M in HCN was adjusted to 9.2 using triethylamine. Diaminomaleonitrile (103 mg) was added to increase the rate of oligomerization. (3) The pH of a solution to 9-3 using monomethylamine. (4) The pH of a solution O-1 M in HCN was adjusted O-1 M in HCN was adjusted to 9.9 with ammonium hydroxide. Phenol (9.3 g, 0.1 mol, pK, 9.8; Cram, 1965) and 103 mg of diaminomaleonitrile were then added to the reaction to 9.2 with ammonium solution. (5) The pH of a solution O-1 M in HCN was adjusted hydroxide. The solution was made O-1 M in azide by the addition of sodium azide. In each instance described above, reaction was permitted to continue for 2 months at room temp. The formation or hydrolysis of diaminomaleonitrile (III) was monitored by U.V. spectroscopy. The reactions were then subjected to Sephadex and ion exchange fractionation in the same m-er as those oligomerization reactions in which ammonium hydroxide was utilized as the added base (Ferris et al., 1973). The infrared spectra of the fractions thus obtained were identical with those obtained from oligomerizations conducted in the presence of ammonium hydroxide only. The presence of urea as well as at least 5 other compounds exhibiting positive color reactions with Ehrlich’s reagent was noted in each reaction. Urea was assayed by paper chromatography (solvents ( 1) , (2) and (5)) and by thin-layer chromatography using silica gel layers (solvents (3), (8) and (9)). The yield of urea was much greater in those systems to which ammonium hydroxide was added. In reaction (3) (monomethylamine), monomethylurea was identified (in amounts greater than the yield of urea), however, N,N’-dimethylurea was not detected (paper chromatography in solvents (l), (2) and (5)). Th e 1knits of detection of N,N’-diiethylurea were less than 20 pg; that is, less than 2 mg of this compound could have been detected in the 435 mg of oligomer B isolated. In each case, the presence of oxalic acid in oligomer A was demonstrated by gas chromatography of the di-m-butyl ester of the isolated oxalic acid (Ferris et al., 1973) or by thinlayer chromatography on silica gel (solvents (2), (3) and (7)), polyamide (solvents (l), (7) and (8)) or by paper chromatography (solvents (2) and (5)). In one instance, the oxalic acid was sublimed from the reaction mixture (15O”C, 5 Torr, 1 h) and its structure confirmed by infrared and mass spectral analysis. In all of the reactions described, the same ammo acids were released on acid hydrolysis of oligomer A as were obtained from ammonium hydroxide oligomerizations (Table 1). (b) Oligomerizatiolz (i) Oligomerization
of
hydrogen cyanide derivatives hydrogen cyanide tetramer in the presence of ammonium hydroxide of
Hydrogen cyanide tetramer (280 mg) was dissolved in 90 ml water and the pH of this solution was adjusted to 10.8 with cone. ammonium hydroxide. The volume of the solution was then adjusted to 11. The solution turned brown and an insoluble material precipitated after several days. The filtrate was fractionated by the same method used for the HCN oligomerizations. Infrared and U.V. spectra of the oligomers obtained in this manner were identical with those obtained from HCN oligomerizations. Oligomer A was found to contain oxalic acid as shown by thin-layer chromatography (polyamide, solvents (1) and (7)) and paper chromatography (solvent (6)). Oligomer B was found to contain urea (paper chromatography, solvents (1) and (6); thin-layer chromatography, solvents (3) and (8)) and there was an indication that oxamide (paper chromatography, solvents (1) and (6)) might be present. The same amino acids were released on acid hydrolysis of oligomer A as were obtained from a HCN oligomerization with ammonium hydroxide as added base (Table 1). (ii)
Oligomerization
of hydrogen
cyantie
tetramer
in
the absence
of
base
Hydrogen cyanide tetramer (10 mg) was dissolved in 100 ml distilled water and left for 2 months. At the end of this time no insoluble material had formed (as in oligomerizations conducted using very dilute HCN solutions) and the solution was pale yellow. The presence of urea in the solution was ascertained by chromatography (paper, solvents (l), (2) and (3); thin-layer chromatography, solvents (2) and (3)). Glycine was detected (paper chromatography, solvents (1) and (2); thin-layer chromatography, solvents (2) and (3)) but oxalic acid was not, either before or after acid hydrolysis of the reaction mixture.
HYDROGEN
CYANIDE
IN CHEMICAL
EVOLUTION
513
(iii) Oligomerization of aminomakmonitde toluenesulfonate in base Aminomalononitrile toluenesulfonate in aqueous solution was neutralized with sodium carbonate to pH 7. The solution was extracted with ether and the U.V. spectrum of this extract exhibited an absorbance maximum at 296 nm, indicating that diaminomaleonitrile was present in the solution. (iv) Reaction of aminomalononitrile toluenesulfonate with sodium cyanide Aminomalononitrile toluenesulfonate (Ferris et al., 1968) (30 g) in an ice-water slurry, was mixed with sodium cyanide (30 g) dissolved in a minimal amount of cold water. After 1 min of reaction, diaminomaleonitrile (III, 2-l g) precipitated from the reaction solution and was isolated by vacuum filtration. No urea, cyanamide or oxalic acid could be detected in the filtrate. Both glycine (paper chromatography, solvents (1) and (3); thin-layer chromatography, solvent (3) and electrophoresis, buffer (2)) and oxalic acid (thin-layer chromatography, solvent (1); paper solvent (2); gas chromatography of the di-n-butyl ester) were shown to be present after acid hydrolysis. The same amino acids were released on acid hydrolysis of oligomer A as were obtained from HCN oligomerizations conducted in the presence of ammonium hydroxide (Table 1). (c) Reactions of cyanamide, cyanate and cyanogen in alkaline medium (i) A&dine hydrolysis of cyanamide Ammonium hydroxide was added to 100 mg of cyanamide (Bernard & Chemin, 1965) in 10 ml water, thereby adjusting the pH of the solution to 9.2. A similar reaction was conducted using monomethylamine as the added base. A blank consisting of cyanamide in water (without added base) was also prepared. Each solution was permitted to react for 1 week. Thin-layer chromatography (solvents (3) and (10)) was used to monitor the hydrolysis of cyanamide and the possible appearance of urea and cyanoguanidine. While urea was present in every sample (it was a trace contaminant in the cyanamide which was used) the amount did not appear to increase during the course of the reaction. No monomethylurea was detected in the reaction in which monomethylamine was the added base. Cyanoguanidine formation was observed in the reaction mixtures to which ammonium hydroxide or monomethylamine were added. (ii) Reactions of potassium cyanate in alkaline solution Ammonium hydroxide was added to a solution of potassium cyanate (5 g) in 50 ml water, adjusting the pH of the solution to 10.4. Cont. HCl was then used to adjust the pH of the solution to 9.2. The same reaction mixture was made up a second time using monomethylamine as the added base. A blank consisting of potassium cyanate in water (pH 9) was also prepared. The solutions were permitted to react for 1 week. All of the reaction solutions were tested for the presence of urea and N-methylurea by direct comparison with authentic samples using thin-layer chromatography (solvents (3), (9) and (10)). Urea was detected in all 3 reaction mixtures but in significantly larger amounts in the reaction where ammonium hydroxide and oyanate were mixed. No urea was detected in the KCNO prior to reaction. N-methylurea was found to be the principal product in the monomethylamine-oyanate reaction mixture, although smaller amounts of urea were also identified. (iii) Rmticm of cyanogen with arnmon&m hydroxide Cyanogen (1 g) was added to 200 ml of pH 9.2 aqueous NH&l buffer (prepared by adjusting the pH of an aqueous solution to 10.5 with cone. ammonium hydroxide and then titrating to 9.2 with cont. hydrochloric acid). Urea was detected after the reaction had been allowed to proceed for 24 h (thin-layer chromatography, solvents (l), (3), (8) and (9)). A portion of the reaction mixture was concentrated to dryness and subjected to sublimation conditions (IOO’C, 12 h, high vacuum) to volatilize oxalic acid. An infrared spectrum of the sublimate failed to show the presence of oxalic acid. Thin-layer chromatography on polyamide layers also indicated that oxalic acid was not present. The material which failed to sublime was subject to acid hydrolysis and oxalic acid was detected in the hydrolyzate by thin-layer chromatography (polyamide, solvents (1) and (8)).
614
J. P. FERRIS TABLE
Amino
ET
AL.
1
acid analyses of oligomer A obtained from various oligomerization reactions Added base or chemical species
Amino acid
NH*OH/HCN
OH-/HCN
Et,N/HCN
PhONH,OH/HCN
NH,i&ICN$
Asp Thr Ser Glu
0.025 0,001 0.038 0.010
0.012 0.005 0.007 0.012
0.013 0.037 0.003 0.015
0.042 0.001 0.003 0.011
0.001 0.001 0.001 0.002
Et Ala val Ile Leu NH, LYS
0.763 0.006 0.001 0.002 4.511 0.003 -
0.292 0.010 0.002 0.002 0.003 7.381 0.003 trace
0.197 O-008 trace trace 0.001 3.621 0.002 trace
0.531 0.005 0.001 0.001 0.001 4.840 0.001 trace
0.027 0.001 trace trace trace 2.448
His
NH,OH/III Asp Thr Ser Glu VW+ GUY Ala Val Ile Lell NH. LYS Hi
o-019 0.005 0.011 0.019 0.250 0.017 0.010 0*008 0.011 4.736 0.010 0.004
N&N/II 0.014 0.001 trace 0.113 0.002
-
0.822 0.001 -
-
CH,NH,/HCN 0.016 0.004 0.016 0.010 0.195 0.012 0.003 0.002 0.007 4.573 0.006 0.007
Results are reported as ~01 of amino acid/mg of applied sample. All samples were subjected to sublimation at 100°C (1 Torr) for 24 h to remove NH&X and oxalic acid before ammo acid analysis. Results obtained on the Technicon Autoanalyzer. Substances with the same retention volume as the listed amino acids were found. This does not necessarily mean these ammo acids were actually present. t Glutamic acid and citrulline were not resolved in these analyses. $ Other products (salts itnd decomposition products of azide) were present in this sample.
3. Results and Discussion In our previous studies, the oligomerization of cyanide was initiated by adjusting the pH of 0.1 M-HCN to 9.2 with ammonium hydroxide (Ferris et al., 1973). In the present study, the oligomerization was carried out at pH 9.2 in the presence of a variety of other bases and nucleophiles to ascertain if these compounds altered the rate or manner of reaction. The rate of cyanide oligomerization to diaminomaleonitrile was not affected by the presence of OH-, CHaNH,, (CH,CH,),N, NaN, or phenoxide. The initial stages of the cyanide oligomerization are dependent only on the pH of the reaction mixture and are independent of the presence of other nucleophiles. The added base apparently takes
HPDISOGEN
CYANIDE
IN
CHEMICAL
515
EVOLUTION
no direct part in the reaction but serves merely to adjust the relative proportions of hydrogen cyanide and cyanide ion. The invariability in the rate of diaminomaleonitrile formation in the presence of a variety of nuoleophiles demonstrates that none of these compounds is able to compete effectively with cyanide ion in the oligomerization process. The iV-alkyl derivatives of (I) react rapidly with cyanide to yield the corresponding monoalkylamino derivatives of (III) (Ferris et al., 1972aJ). The reaction of aminomalononitrile (II) with cyanide to produce (III) is complete within a few minutes (Sanchez et al., 1967). However, under similar conditions the formation of (III) from cyanide requires several hours to a day. These data demonstrate that k1 is rate-limiting and that the other nucleophiles we added to the reaction mixture are unable to compete with cyanide ion in this step. These same nucleophiles must be unable to compete with cyanide in any subsequent steps, since the yield of diaminomaleonit&e was independent of the nucleophile that was added. In each of the above reactions, the condensation of cyanide was allowed to proceed for about two months and the tan product mixture was isolated and characterized. There were no significant. variations in the amino acids released on acid hydrolysis as observed using an amino acid analyzer (Table 1). Thus, none of the added nucleophiles is being incorporated into the product mixture. We also found that the same cyanide condensation products were observed if diaminomaleonitrile or aminomalononitrile and cyanide were used as the reactants. The same proportions of amino acids were released after acid hydrolysis of the tan reaction product. Since it is not possible to form higher oligomers by the addition of cyanide to diaminomaleonitrile (Ferris et al., 1972a,b), it would appear that the tan reaction product is being generatedfroman equilibirium mixtureof oligomersas shown in scheme 1. Any one of these oligomers may be used to generate the entire reaction system?. Hydrolytic and oxidation-reduction processes then account for the large number of compounds formed from this equilibrium mixture.
HCN
k,
k,
+HCN = -HCN
+HCN HN=CHCN
I
-3E
k3 NH&H(CN),
+HCN = -HCN
W
CN
H,N
CN
m
III
Hydrolysis and redox reactions
1
Urea, oxalic acid and unknown compounds which yield amino acids on further hydrolysis.
SCHEME1. Oxalic acid was isolated from each of the above reaction mixtures. The yield was about the same in each instance, indicating that the added nucleophiles do not affect the formation of this compound. t The generation of cyanide ion when dkminomaleonitrile further support for the proposed equilibria (Lotz, unpublished
ix dissolved results).
in water
provides
516
J. P. FERRIS
ET
AL.
The yield of urea did show significant variation when formed in the presence of different nucleophiles. A much lower yield of urea was obtained in the absence of added ammonium hydroxide. When monomethylamine was used in place of ammonia, the major neutral product isolated wasmonomethylurea, althoughasmaller amountof urea was also observed. Similarly, small amounts of urea were obtained in the presence of sodium hydroxide or triethylamine. The formation of monomethylurea in the presence of addedmonomethylaminedemonstratesthatanoxidationproduct ofcyanide is produced, which in turn reacts with ammonia and amines to form the corresponding urea. We considered cyanate and cyanamide as precursors which might yield monomethylurea when reacted with monomethylamine at pH 9.2. The funding that cyanamide dimerizes to cyanoguanidine in alkaline solution and that it does not produce urea in the presence of ammonium hydroxide or monomethylurea in the presence of monomethylamine eliminated this species from consideration. The extensive conversion of cyanamide to urea reported by Lohrman & Orgel (1968) must be due to the presence of phosphate in their reaction mixtures. These workers noted that the yield of urea decreased markedly as the concentration of phosphate decreased from 1 M to 0-I M; presumably little or no urea would be produced in the absence of phosphate, as we observed. Cyanate reacts rapidly with ammonium hydroxide and monomethylamine at pH 9.2 to produce urea and monomethylurea, respectively. In addition, a low yield of urea was observed on reaction of cyanate at pH 9 or when cyanatewas reacted in the presence of monomethylamine. Urea was apparently formed by alkaline hydrolysis of cyanate. Ammonia liberated upon hydrolysis of cyanate accounts for the low yields of urea, since this would presumably react with unhydrolyzed cyanate to produce urea. Theseresults areincomplete accordwith those obtainedinthe cyanidereactionsystems, suggesting strongly that cyanate is produced in the oligomerization of HCN. Two routes might be envisaged for the formation of cyanate; the hydrolysis of cyanogen or the oxidation of cyanide. Base-catalyzed reaction of water with oyanogen produces cyanide while acid hydrolysis yields oxamide. We have conducted the hydrolysis of cyanogen at pH 9.2 in the presence of ammonium hydroxide and found that urea was among the reaction products. In addition, smaller amounts of oxalic acid were observed after acid hydrolysis of the reaction mixture. These findings indicate that near neutral pH one may be observing the reaction products of both acidic and basic hydrolysis of cyanogen, and that the ratio of reaction products is similar to the proportion of urea and oxalic acid isolated from alkaline cyanide oligomerization. An alternative source of cyanate might be the direct oxidation of cyanide. Latimer (1952) noted that this oxidation proceeds with ease when mild oxidants are used. The possibility exists that some of the cyanide oligomers formed during the course of the condensation are sufficiently electron poor to oxidize cyanide to cyanate. Alternatively, atmospheric oxygen might be responsible for the oxidation of cyanide to cyanate. This does not seem likely, since cyanide ion is stable in aqueous solutions and it has been reported that oxygen has no effect on the oligomeric reactions of HCN (Volker, 1960). Cyanogen may be formed via the oxidation of cyanide with cyanide oligomers. For example, cyanogen is produced when cyanide ion is added to tetracysnoethylene (Webster et al., 1962). Similar formation of oyanogen reaction (1 (a)), could account for the isolation of oxalic acid when cyanide ion is added to aminomalononitrile. Cyanogen
HYDROGEN
CYANIDE
IN
CHEMICAL
517
EVOLUTION
formation could also account for the fact that the yield of diaminomaleonitrile tion l(b)), was never greater than 70% (Sanchez et al., 1967).
(reac-
Hf
i 1 CN
NH,CH(CN&
-I- CN-
-
x
(CNj2
+
NH, CH2CN
(I(a))
NH2-A-N
&NLN
K
(I(b)) ,,,xCN NH2
CN
SCHEME 2.
4. Conclusions The formation of an oligometric mixture in cyanide condensation reactions involves the reaction of cyanide to produce an equilibrium mixture of dimer (I), trimer (II) and tetramer (IV). The tetramer is the thermodynamically most stable oligomer formed. All of these species may undergo hydrolytic and/or oxidation reduction reactions resulting in the formation of urea, oxalic acid and numerous other, as yet unidentified, products. None of these other products is a polymer and, furthermore, peptide units are not present in these compounds. Hydrogen cyanide still appears to be a likely starting point for the synthesis of purines and amino acids on the primitive earth. The present work has indicated that it is unlikely that biopolymers of any type will be formed from cyanide. Cyanide oligomerization does, however, seem to be a very efficient means of forming a large number of biomonomers which might serve as an initiation point for other chemical processes leading to more complex compounds. Recently, it has been reported that diaminomaleonitrile will effect the condensation of amino acids to peptides in low yield (Chang et al., 1969). It has been reported that cyanogen and cyanate will mediate the condensation of phosphate to pyrophosphate (Lohrman & Orgel, 1968; Miller & Parris, 1964). The direct cyanide-mediated formation of the peptide or pyrophosphate link remains to be demonstrated. This work was supported by grant no. GP19255 from the National Science Foundation, grant no. NGR 30018148 from the National Aeronautics and Space Administration and a Career Development Award (no. GM 6380) to one of us (J. P. F.) from the U.S. Public Health Service. REFERENCES Bernard, M. A. & Chemin, A. (1965). Bull. Sot. Chem. Fr. 1633. Chang, S., Flores, J. & Ponnamperuma, C. (1969). Proc. Nat. Acad. Sci., Wash. 64, 1011. Cram, D. J. (1965). In lf’undamentals of Carbanion Chemistry, p. 41. Academic Press, New York. Donner, D. B. (1971). Ph.D. Thesis, Rensselaer Polytechnic Institute, Troy, N.Y. Donner, D. B., Lobo, A. P., Wos, J. D. & Ferris, J. P. (1970). Biol. BUZZ. 139, 419. Ferris, J. P., Sanchez, R. A. & Manusoo, R. W. (1968). In Organic Syntheses, ed. by P. Yates, vol. 48, p. 1. John Wiley & Sons, Inc., New York. Ferris, J. P., Dormer, D. B. & Lotz, W. (1972a). J. Amer. Chem. Sot. 94, 6968. Ferris, J. P., Donner, D. B. & Lotz, W. (19723). Bio-organic Chem. 2, 95. Ferris, J. P., Dormer, D. B. & Lobo, A. P. (1973). J. Mol. Biol. 74, 499. Latimer, W. M. (1952). In Oxidation Potentials, p. 50. Prentice Hall, New York.
618
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ET
AL.
Lohrman, R. & Orgel, L. E. (1968). Science, 161, 64 Miller, S. L. & Parris, M. (1964). Nature, 204, 1248. Sanchez, R. A., Ferris, J. P. St Orgel, L. E. (1967). J. Mol. Biol. 30, 223. Volker, T. (1960). Angew. Chem. 72, 379. Webster, 0. E., Mahler, W. & Benson, R. E. (1962). J. Amer. Chem. Sot. 84, 3678.
Possible Role of Hydrogen Cyanide in Chemical Evolution :-f-The Oligomerization and Condensation of Hydrogen Cyanide J.P.
FERRIS, D.B.
DONNERAXD
A.P.
LOBO
Department of Chemistry Polytechnic Institute Troy, N.P. 12181, U.X.A.
Rensselaer
(Received 11 August 1972, and &a revised form 5 December 1972) The rate of cyanide oligomerization is independent of the presence of added nucleophiles such as azide, monomethylamine or triethylamine and is dependent only on the pH of the reaction mixture. The products formed, with the exception of urea, are also independent of the nucleophiles used to initiate reaction. In the presence of monomethylamine, monomethylmea is the main neutral product instead of urea, suggesting the intermediacy of cyanate. Evidence is presented which suggests that cyanogen may be the precurser to both oyanate and oxalic aaid in the cyanide oligomerization.
1. Introduction Hydrogen cyanide is believed to have had a significant role in the prebiotic synthesis of amino acids and purines (Sanchez et al., 1967, and references therein). In previous papers we presented evidence which indicates that in basic solution hydrogen cyanide oligomerizes to form a complex mixture of low molecular weight compounds (Ferris et al., 1972a,b). We found no evidence for the presence of peptides within this reaction system (Dormer et al., 1970; Ferris et al., 1972b; Ferris et al., 1973). Additionally, we were unable to find any experimental verification of the claim that polymers are formed. Large amounts of urea and smaller amounts of oxalic acid were isolated from the cyanide condensation system and we confirmed that hydrolysis of the oligomerization mixture results in the formation of amino acids. An investigation of the mechanisms by which urea and oxalic acid are produced and by which hydrogen cyanide oligomerizes was undertaken with the aim of understanding better the alkaline solution chemistry of hydrogen cyanide and the possible role of hydrogen cyanide in chemical evolution.
2. Materials and Methodsg (a) The oligomerization
of cyanide
in
the presence
of
added rvucleophiles
Cyanide oligomerization reactions were conducted in the presence of nucleophiles other than ammoninm hydroxide. The following reactions were investigated: (1) The pH of a solution of 1 N-sodium cyanide was adjusted to 9.2 with cone. HCl. (2) The pH of t Chemical Evolution XII. The previous paper in this series is Ferris et al. (19 73). $ General experimental procedures are given in the previous paper in this series (Ferris et al., 1973) and in the Ph.D. Thesis of D. Dormer (1971). 34
511
612
J. P. FERRIS
ET
AL.
a solution 0.1 M in HCN was adjusted to 9.2 using triethylamine. Diaminomaleonitrile (103 mg) was added to increase the rate of oligomerization. (3) The pH of a solution to 9-3 using monomethylamine. (4) The pH of a solution O-1 M in HCN was adjusted O-1 M in HCN was adjusted to 9.9 with ammonium hydroxide. Phenol (9.3 g, 0.1 mol, pK, 9.8; Cram, 1965) and 103 mg of diaminomaleonitrile were then added to the reaction to 9.2 with ammonium solution. (5) The pH of a solution O-1 M in HCN was adjusted hydroxide. The solution was made O-1 M in azide by the addition of sodium azide. In each instance described above, reaction was permitted to continue for 2 months at room temp. The formation or hydrolysis of diaminomaleonitrile (III) was monitored by U.V. spectroscopy. The reactions were then subjected to Sephadex and ion exchange fractionation in the same m-er as those oligomerization reactions in which ammonium hydroxide was utilized as the added base (Ferris et al., 1973). The infrared spectra of the fractions thus obtained were identical with those obtained from oligomerizations conducted in the presence of ammonium hydroxide only. The presence of urea as well as at least 5 other compounds exhibiting positive color reactions with Ehrlich’s reagent was noted in each reaction. Urea was assayed by paper chromatography (solvents ( 1) , (2) and (5)) and by thin-layer chromatography using silica gel layers (solvents (3), (8) and (9)). The yield of urea was much greater in those systems to which ammonium hydroxide was added. In reaction (3) (monomethylamine), monomethylurea was identified (in amounts greater than the yield of urea), however, N,N’-dimethylurea was not detected (paper chromatography in solvents (l), (2) and (5)). Th e 1knits of detection of N,N’-diiethylurea were less than 20 pg; that is, less than 2 mg of this compound could have been detected in the 435 mg of oligomer B isolated. In each case, the presence of oxalic acid in oligomer A was demonstrated by gas chromatography of the di-m-butyl ester of the isolated oxalic acid (Ferris et al., 1973) or by thinlayer chromatography on silica gel (solvents (2), (3) and (7)), polyamide (solvents (l), (7) and (8)) or by paper chromatography (solvents (2) and (5)). In one instance, the oxalic acid was sublimed from the reaction mixture (15O”C, 5 Torr, 1 h) and its structure confirmed by infrared and mass spectral analysis. In all of the reactions described, the same ammo acids were released on acid hydrolysis of oligomer A as were obtained from ammonium hydroxide oligomerizations (Table 1). (b) Oligomerizatiolz (i) Oligomerization
of
hydrogen cyanide derivatives hydrogen cyanide tetramer in the presence of ammonium hydroxide of
Hydrogen cyanide tetramer (280 mg) was dissolved in 90 ml water and the pH of this solution was adjusted to 10.8 with cone. ammonium hydroxide. The volume of the solution was then adjusted to 11. The solution turned brown and an insoluble material precipitated after several days. The filtrate was fractionated by the same method used for the HCN oligomerizations. Infrared and U.V. spectra of the oligomers obtained in this manner were identical with those obtained from HCN oligomerizations. Oligomer A was found to contain oxalic acid as shown by thin-layer chromatography (polyamide, solvents (1) and (7)) and paper chromatography (solvent (6)). Oligomer B was found to contain urea (paper chromatography, solvents (1) and (6); thin-layer chromatography, solvents (3) and (8)) and there was an indication that oxamide (paper chromatography, solvents (1) and (6)) might be present. The same amino acids were released on acid hydrolysis of oligomer A as were obtained from a HCN oligomerization with ammonium hydroxide as added base (Table 1). (ii)
Oligomerization
of hydrogen
cyantie
tetramer
in
the absence
of
base
Hydrogen cyanide tetramer (10 mg) was dissolved in 100 ml distilled water and left for 2 months. At the end of this time no insoluble material had formed (as in oligomerizations conducted using very dilute HCN solutions) and the solution was pale yellow. The presence of urea in the solution was ascertained by chromatography (paper, solvents (l), (2) and (3); thin-layer chromatography, solvents (2) and (3)). Glycine was detected (paper chromatography, solvents (1) and (2); thin-layer chromatography, solvents (2) and (3)) but oxalic acid was not, either before or after acid hydrolysis of the reaction mixture.
HYDROGEN
CYANIDE
IN CHEMICAL
EVOLUTION
513
(iii) Oligomerization of aminomakmonitde toluenesulfonate in base Aminomalononitrile toluenesulfonate in aqueous solution was neutralized with sodium carbonate to pH 7. The solution was extracted with ether and the U.V. spectrum of this extract exhibited an absorbance maximum at 296 nm, indicating that diaminomaleonitrile was present in the solution. (iv) Reaction of aminomalononitrile toluenesulfonate with sodium cyanide Aminomalononitrile toluenesulfonate (Ferris et al., 1968) (30 g) in an ice-water slurry, was mixed with sodium cyanide (30 g) dissolved in a minimal amount of cold water. After 1 min of reaction, diaminomaleonitrile (III, 2-l g) precipitated from the reaction solution and was isolated by vacuum filtration. No urea, cyanamide or oxalic acid could be detected in the filtrate. Both glycine (paper chromatography, solvents (1) and (3); thin-layer chromatography, solvent (3) and electrophoresis, buffer (2)) and oxalic acid (thin-layer chromatography, solvent (1); paper solvent (2); gas chromatography of the di-n-butyl ester) were shown to be present after acid hydrolysis. The same amino acids were released on acid hydrolysis of oligomer A as were obtained from HCN oligomerizations conducted in the presence of ammonium hydroxide (Table 1). (c) Reactions of cyanamide, cyanate and cyanogen in alkaline medium (i) A&dine hydrolysis of cyanamide Ammonium hydroxide was added to 100 mg of cyanamide (Bernard & Chemin, 1965) in 10 ml water, thereby adjusting the pH of the solution to 9.2. A similar reaction was conducted using monomethylamine as the added base. A blank consisting of cyanamide in water (without added base) was also prepared. Each solution was permitted to react for 1 week. Thin-layer chromatography (solvents (3) and (10)) was used to monitor the hydrolysis of cyanamide and the possible appearance of urea and cyanoguanidine. While urea was present in every sample (it was a trace contaminant in the cyanamide which was used) the amount did not appear to increase during the course of the reaction. No monomethylurea was detected in the reaction in which monomethylamine was the added base. Cyanoguanidine formation was observed in the reaction mixtures to which ammonium hydroxide or monomethylamine were added. (ii) Reactions of potassium cyanate in alkaline solution Ammonium hydroxide was added to a solution of potassium cyanate (5 g) in 50 ml water, adjusting the pH of the solution to 10.4. Cont. HCl was then used to adjust the pH of the solution to 9.2. The same reaction mixture was made up a second time using monomethylamine as the added base. A blank consisting of potassium cyanate in water (pH 9) was also prepared. The solutions were permitted to react for 1 week. All of the reaction solutions were tested for the presence of urea and N-methylurea by direct comparison with authentic samples using thin-layer chromatography (solvents (3), (9) and (10)). Urea was detected in all 3 reaction mixtures but in significantly larger amounts in the reaction where ammonium hydroxide and oyanate were mixed. No urea was detected in the KCNO prior to reaction. N-methylurea was found to be the principal product in the monomethylamine-oyanate reaction mixture, although smaller amounts of urea were also identified. (iii) Rmticm of cyanogen with arnmon&m hydroxide Cyanogen (1 g) was added to 200 ml of pH 9.2 aqueous NH&l buffer (prepared by adjusting the pH of an aqueous solution to 10.5 with cone. ammonium hydroxide and then titrating to 9.2 with cont. hydrochloric acid). Urea was detected after the reaction had been allowed to proceed for 24 h (thin-layer chromatography, solvents (l), (3), (8) and (9)). A portion of the reaction mixture was concentrated to dryness and subjected to sublimation conditions (IOO’C, 12 h, high vacuum) to volatilize oxalic acid. An infrared spectrum of the sublimate failed to show the presence of oxalic acid. Thin-layer chromatography on polyamide layers also indicated that oxalic acid was not present. The material which failed to sublime was subject to acid hydrolysis and oxalic acid was detected in the hydrolyzate by thin-layer chromatography (polyamide, solvents (1) and (8)).
614
J. P. FERRIS TABLE
Amino
ET
AL.
1
acid analyses of oligomer A obtained from various oligomerization reactions Added base or chemical species
Amino acid
NH*OH/HCN
OH-/HCN
Et,N/HCN
PhONH,OH/HCN
NH,i&ICN$
Asp Thr Ser Glu
0.025 0,001 0.038 0.010
0.012 0.005 0.007 0.012
0.013 0.037 0.003 0.015
0.042 0.001 0.003 0.011
0.001 0.001 0.001 0.002
Et Ala val Ile Leu NH, LYS
0.763 0.006 0.001 0.002 4.511 0.003 -
0.292 0.010 0.002 0.002 0.003 7.381 0.003 trace
0.197 O-008 trace trace 0.001 3.621 0.002 trace
0.531 0.005 0.001 0.001 0.001 4.840 0.001 trace
0.027 0.001 trace trace trace 2.448
His
NH,OH/III Asp Thr Ser Glu VW+ GUY Ala Val Ile Lell NH. LYS Hi
o-019 0.005 0.011 0.019 0.250 0.017 0.010 0*008 0.011 4.736 0.010 0.004
N&N/II 0.014 0.001 trace 0.113 0.002
-
0.822 0.001 -
-
CH,NH,/HCN 0.016 0.004 0.016 0.010 0.195 0.012 0.003 0.002 0.007 4.573 0.006 0.007
Results are reported as ~01 of amino acid/mg of applied sample. All samples were subjected to sublimation at 100°C (1 Torr) for 24 h to remove NH&X and oxalic acid before ammo acid analysis. Results obtained on the Technicon Autoanalyzer. Substances with the same retention volume as the listed amino acids were found. This does not necessarily mean these ammo acids were actually present. t Glutamic acid and citrulline were not resolved in these analyses. $ Other products (salts itnd decomposition products of azide) were present in this sample.
3. Results and Discussion In our previous studies, the oligomerization of cyanide was initiated by adjusting the pH of 0.1 M-HCN to 9.2 with ammonium hydroxide (Ferris et al., 1973). In the present study, the oligomerization was carried out at pH 9.2 in the presence of a variety of other bases and nucleophiles to ascertain if these compounds altered the rate or manner of reaction. The rate of cyanide oligomerization to diaminomaleonitrile was not affected by the presence of OH-, CHaNH,, (CH,CH,),N, NaN, or phenoxide. The initial stages of the cyanide oligomerization are dependent only on the pH of the reaction mixture and are independent of the presence of other nucleophiles. The added base apparently takes
HPDISOGEN
CYANIDE
IN
CHEMICAL
515
EVOLUTION
no direct part in the reaction but serves merely to adjust the relative proportions of hydrogen cyanide and cyanide ion. The invariability in the rate of diaminomaleonitrile formation in the presence of a variety of nuoleophiles demonstrates that none of these compounds is able to compete effectively with cyanide ion in the oligomerization process. The iV-alkyl derivatives of (I) react rapidly with cyanide to yield the corresponding monoalkylamino derivatives of (III) (Ferris et al., 1972aJ). The reaction of aminomalononitrile (II) with cyanide to produce (III) is complete within a few minutes (Sanchez et al., 1967). However, under similar conditions the formation of (III) from cyanide requires several hours to a day. These data demonstrate that k1 is rate-limiting and that the other nucleophiles we added to the reaction mixture are unable to compete with cyanide ion in this step. These same nucleophiles must be unable to compete with cyanide in any subsequent steps, since the yield of diaminomaleonit&e was independent of the nucleophile that was added. In each of the above reactions, the condensation of cyanide was allowed to proceed for about two months and the tan product mixture was isolated and characterized. There were no significant. variations in the amino acids released on acid hydrolysis as observed using an amino acid analyzer (Table 1). Thus, none of the added nucleophiles is being incorporated into the product mixture. We also found that the same cyanide condensation products were observed if diaminomaleonitrile or aminomalononitrile and cyanide were used as the reactants. The same proportions of amino acids were released after acid hydrolysis of the tan reaction product. Since it is not possible to form higher oligomers by the addition of cyanide to diaminomaleonitrile (Ferris et al., 1972a,b), it would appear that the tan reaction product is being generatedfroman equilibirium mixtureof oligomersas shown in scheme 1. Any one of these oligomers may be used to generate the entire reaction system?. Hydrolytic and oxidation-reduction processes then account for the large number of compounds formed from this equilibrium mixture.
HCN
k,
k,
+HCN = -HCN
+HCN HN=CHCN
I
-3E
k3 NH&H(CN),
+HCN = -HCN
W
CN
H,N
CN
m
III
Hydrolysis and redox reactions
1
Urea, oxalic acid and unknown compounds which yield amino acids on further hydrolysis.
SCHEME1. Oxalic acid was isolated from each of the above reaction mixtures. The yield was about the same in each instance, indicating that the added nucleophiles do not affect the formation of this compound. t The generation of cyanide ion when dkminomaleonitrile further support for the proposed equilibria (Lotz, unpublished
ix dissolved results).
in water
provides
516
J. P. FERRIS
ET
AL.
The yield of urea did show significant variation when formed in the presence of different nucleophiles. A much lower yield of urea was obtained in the absence of added ammonium hydroxide. When monomethylamine was used in place of ammonia, the major neutral product isolated wasmonomethylurea, althoughasmaller amountof urea was also observed. Similarly, small amounts of urea were obtained in the presence of sodium hydroxide or triethylamine. The formation of monomethylurea in the presence of addedmonomethylaminedemonstratesthatanoxidationproduct ofcyanide is produced, which in turn reacts with ammonia and amines to form the corresponding urea. We considered cyanate and cyanamide as precursors which might yield monomethylurea when reacted with monomethylamine at pH 9.2. The funding that cyanamide dimerizes to cyanoguanidine in alkaline solution and that it does not produce urea in the presence of ammonium hydroxide or monomethylurea in the presence of monomethylamine eliminated this species from consideration. The extensive conversion of cyanamide to urea reported by Lohrman & Orgel (1968) must be due to the presence of phosphate in their reaction mixtures. These workers noted that the yield of urea decreased markedly as the concentration of phosphate decreased from 1 M to 0-I M; presumably little or no urea would be produced in the absence of phosphate, as we observed. Cyanate reacts rapidly with ammonium hydroxide and monomethylamine at pH 9.2 to produce urea and monomethylurea, respectively. In addition, a low yield of urea was observed on reaction of cyanate at pH 9 or when cyanatewas reacted in the presence of monomethylamine. Urea was apparently formed by alkaline hydrolysis of cyanate. Ammonia liberated upon hydrolysis of cyanate accounts for the low yields of urea, since this would presumably react with unhydrolyzed cyanate to produce urea. Theseresults areincomplete accordwith those obtainedinthe cyanidereactionsystems, suggesting strongly that cyanate is produced in the oligomerization of HCN. Two routes might be envisaged for the formation of cyanate; the hydrolysis of cyanogen or the oxidation of cyanide. Base-catalyzed reaction of water with oyanogen produces cyanide while acid hydrolysis yields oxamide. We have conducted the hydrolysis of cyanogen at pH 9.2 in the presence of ammonium hydroxide and found that urea was among the reaction products. In addition, smaller amounts of oxalic acid were observed after acid hydrolysis of the reaction mixture. These findings indicate that near neutral pH one may be observing the reaction products of both acidic and basic hydrolysis of cyanogen, and that the ratio of reaction products is similar to the proportion of urea and oxalic acid isolated from alkaline cyanide oligomerization. An alternative source of cyanate might be the direct oxidation of cyanide. Latimer (1952) noted that this oxidation proceeds with ease when mild oxidants are used. The possibility exists that some of the cyanide oligomers formed during the course of the condensation are sufficiently electron poor to oxidize cyanide to cyanate. Alternatively, atmospheric oxygen might be responsible for the oxidation of cyanide to cyanate. This does not seem likely, since cyanide ion is stable in aqueous solutions and it has been reported that oxygen has no effect on the oligomeric reactions of HCN (Volker, 1960). Cyanogen may be formed via the oxidation of cyanide with cyanide oligomers. For example, cyanogen is produced when cyanide ion is added to tetracysnoethylene (Webster et al., 1962). Similar formation of oyanogen reaction (1 (a)), could account for the isolation of oxalic acid when cyanide ion is added to aminomalononitrile. Cyanogen
HYDROGEN
CYANIDE
IN
CHEMICAL
517
EVOLUTION
formation could also account for the fact that the yield of diaminomaleonitrile tion l(b)), was never greater than 70% (Sanchez et al., 1967).
(reac-
Hf
i 1 CN
NH,CH(CN&
-I- CN-
-
x
(CNj2
+
NH, CH2CN
(I(a))
NH2-A-N
&NLN
K
(I(b)) ,,,xCN NH2
CN
SCHEME 2.
4. Conclusions The formation of an oligometric mixture in cyanide condensation reactions involves the reaction of cyanide to produce an equilibrium mixture of dimer (I), trimer (II) and tetramer (IV). The tetramer is the thermodynamically most stable oligomer formed. All of these species may undergo hydrolytic and/or oxidation reduction reactions resulting in the formation of urea, oxalic acid and numerous other, as yet unidentified, products. None of these other products is a polymer and, furthermore, peptide units are not present in these compounds. Hydrogen cyanide still appears to be a likely starting point for the synthesis of purines and amino acids on the primitive earth. The present work has indicated that it is unlikely that biopolymers of any type will be formed from cyanide. Cyanide oligomerization does, however, seem to be a very efficient means of forming a large number of biomonomers which might serve as an initiation point for other chemical processes leading to more complex compounds. Recently, it has been reported that diaminomaleonitrile will effect the condensation of amino acids to peptides in low yield (Chang et al., 1969). It has been reported that cyanogen and cyanate will mediate the condensation of phosphate to pyrophosphate (Lohrman & Orgel, 1968; Miller & Parris, 1964). The direct cyanide-mediated formation of the peptide or pyrophosphate link remains to be demonstrated. This work was supported by grant no. GP19255 from the National Science Foundation, grant no. NGR 30018148 from the National Aeronautics and Space Administration and a Career Development Award (no. GM 6380) to one of us (J. P. F.) from the U.S. Public Health Service. REFERENCES Bernard, M. A. & Chemin, A. (1965). Bull. Sot. Chem. Fr. 1633. Chang, S., Flores, J. & Ponnamperuma, C. (1969). Proc. Nat. Acad. Sci., Wash. 64, 1011. Cram, D. J. (1965). In lf’undamentals of Carbanion Chemistry, p. 41. Academic Press, New York. Donner, D. B. (1971). Ph.D. Thesis, Rensselaer Polytechnic Institute, Troy, N.Y. Donner, D. B., Lobo, A. P., Wos, J. D. & Ferris, J. P. (1970). Biol. BUZZ. 139, 419. Ferris, J. P., Sanchez, R. A. & Manusoo, R. W. (1968). In Organic Syntheses, ed. by P. Yates, vol. 48, p. 1. John Wiley & Sons, Inc., New York. Ferris, J. P., Dormer, D. B. & Lotz, W. (1972a). J. Amer. Chem. Sot. 94, 6968. Ferris, J. P., Donner, D. B. & Lotz, W. (19723). Bio-organic Chem. 2, 95. Ferris, J. P., Dormer, D. B. & Lobo, A. P. (1973). J. Mol. Biol. 74, 499. Latimer, W. M. (1952). In Oxidation Potentials, p. 50. Prentice Hall, New York.
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Lohrman, R. & Orgel, L. E. (1968). Science, 161, 64 Miller, S. L. & Parris, M. (1964). Nature, 204, 1248. Sanchez, R. A., Ferris, J. P. St Orgel, L. E. (1967). J. Mol. Biol. 30, 223. Volker, T. (1960). Angew. Chem. 72, 379. Webster, 0. E., Mahler, W. & Benson, R. E. (1962). J. Amer. Chem. Sot. 84, 3678.