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Amino acid synthesis from formaldehyde and hydroxylamine
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
86,
115-130 (1959)
Amino Acid Synthesis from Formaldehyde and Hydroxylaminels 2 J. Oro’, A. Kimball, R. Fritz and F. Master From the Department
of Chemistry,
University of Houston,
Houston,
Texas
Received March 30, 1959
The synthesis of amino acids, in one-step processes, by reaction of simple compounds of carbon (C, and C,) with simple nitrogen compounds has been accomplished by several investigators using highly activating forms of energy such as electric discharges, ultraviolet light, and ionizing radiation (l-23). In particular, Loeb (l), Miller (2-6), Abelson (7, S), and Heyns et al. (9) used the action of silent and spark discharges upon aqueous mixtures of totally reduced or partially oxidized carbon and nitrogen compounds. Photochemical syntheses of amino acids in aqueous systems were claimed some time ago by Baudisch (10) from potassium nitrite, carbon monoxide, and ferric chloride, by Baly et al. (11) from formhydroxamic acid and formaldehyde, and by Dhar and Mukherjee (12) from nitrates, simple carbon compounds such as glycol, and titanium dioxide. More recently, Bahadur and his associates (13-16) have observed the formation of amino acids by the action of light upon aqueous solutions of potassium nitrate, paraformaldehyde, and ferric chloride, and upon aqueous mixtures of paraformaldehyde and molybdic oxide in the presence of air. Also, recently Groth and von Weyssenhoff (17, 18) and Miller (6) have reported amino acid syntheses by irradiating with ultraviolet light aqueous mixtures containing ammonia as a nitrogen source, and ethane and methane, respectively, as a carbon source. The formation of amino acids by the action of ionizing radiations upon similar simple mixtures has been shown by Hasselstrom et al. (19), Paschke et al. (20), and Dose et al. (21, 22) using P-rays, y-rays and x-rays, respectively. A common feature of all these experiments is the employment of highly 1Work supported in part by a grant from the Robert A. Welch Foundation. 2 A preliminary report appearedin Am. Chem. Sot., Div. of Biol. Chem., Abstracts, p. 16C. 130th Meeting, Atlantic City, 1956. 115
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activating forms of energy. In the experiments of Miller, part of the energy of the electric discharges is used to oxidize methane to formaldehyde; and in the first experiments of Bahadur, part of the light energy is used in the reduction of nitrates. Thus, it was of interest to find out if certain C1 and N1 compounds of intermediate degrees of oxidation would be sufficiently active to yield amino acids even in the absence of radiant energy or electric discharges. This has been found to be so, as it has now been possible to synthesize appreciable amounts of glycine and lesser amounts of glycinamide and certain amino acids among other compounds by heating aqueous solutions of paraformaldehyde and hydroxylamine hydrochloride at moderate temperature. A study of the conditions of this reaction followed by an interpretation of the sequence of reactions involved in the over-all amino acid synthesis is presented. The significance of these experiments in relation to the amino acid syntheses obtained by other investigators is discussed. EXPERIMENTAL
Conditions of the Reaction In order to establish the optimal conditions of amino acid synthesis, several experiments were carried out in which the effect of several physical and chemical variables was tested. In a typical experiment, ten bml. samples of a 0.25 M solution of paraformaldehyde and hydroxylamine hydrochloride were placed in Pyrex test tubes. The tubes were closed with screw caps and then heated in an oven at 100”. The progress of the reaction was followed by analyzing the amino acid content of the tubes at different periods of time until the reaction ended. In some instances, amino acid amides, ammonia, and other products were also analyzed. The effect of temperature on the reaction rate was determined by observing the time needed for completion of the reaction at 80,90 and loo’, respectively, and calculating the corresponding &lo coefficient. The effect of pH on the amino acid yield was determined by measuring the total amount of amino acid synthesized in 0.25 M reaction mixtures which had different hydrogen-ion concentration. Hydrochloric acid or sodium hydroxide was used in adjusting the pH of these reaction mixtures. The role of inorganic ions as possible catalysts for the reaction was also tested. This was done by dissolving a small amount of the corresponding pure compound in the reaction mixture so as to form an ionic solution approximately IOW’ M and carrying out the reaction in the usual manner. The influence of other variables such as the concentration and the relative proportion of the reactants was also studied.
Determination
of Amino Acid and Amino Acid Amide Concentration
For the determination of the amino acid concentration in the large number of samples analyzed, the method of Awapara and Sato (24) was followed in part. Aliquots in 1 ml. of the reaction product were passed through small columns (1 X 5cm.) of Dowex 2 (8X, 200-400 mesh, OH- form). The columns were washed with 10 ml. water. These water eluates were left aside for the determination of amino acid amides
AMINO
ACID
SYNTHESIS
117
and other products. The amino acids were subsequently eluted with about 10 ml. of 4 N acetic acid and brought to an exact volume of 10 ml. with distilled water. Aliquots of 5 ml. of the acetic acid eluates were evaporated to dryness in a steam bath,3 and the residue was redissolved in 2 ml. of 0.25 N hydrochloric acid and analyzed by the method of Troll and Cannan (25) using glycine as standard. The amino acid concentration was expressed in millimoles per liter. For the determination of the total amino acid amide concentration, 5-ml. aliquots of the water eluates were subjected to hydrolysis in 0.5 N sodium hydroxide at 100” for 3 hr. The hydrolyzed samples were then evaporated under vacuum to about 1 ml. in order to remove the ammonia, neutralized with hydrochloric acid, and the amino acids were analyzed by the method previously described.
Determination
of Other Products
Occasionally, the water eluates were also analyzed for other bases, such as ammonia and hydroxylamine. In order to prevent any loss of ammonia or hydroxylamine by volatilization during the elution of the small Dowex 2 columns with water, the tips of the chromatographic columns were immersed into 10 ml. of a 0.1 N hydrochloric acid solution which had been previously placed in the collecting beakers. In this way, the eluted bases were transformed quantitatively into their respective chlorides. The ammonia and the hydroxylamine of these eluates were analyzed by nesslerization (26) and by the method of Csaky (27)) respectively. The hydrogen cyanide formed during the course of the reaction was analyzed directly in the reaction mixtures by the method of Schilt (28).
Separation Procedure For preparative purposes 3 1. of 0.25 M solution of paraformaldehyde and hydroxylamine hydrochloride was refluxed in a 5-1. flask provided with a long water-cooled condenser. Normally, the solution was placed directly in the flask and refluxed for a period of 60 hr. Alternatively, the solution was allowed to drop slowly into the refluxing apparatus during a 30.hr. period, and the refluxing operation was continued for 60 hr. additional. The separation procedure adopted is a combination of ion-exchange chromatography and selective precipitation of glycine. The chromatographic procedure is similar to that of Miller (3) and is shown in Fig. 1. At the end of the reaction, 2 1. of the resulting solution was passed through a 750-ml. column of Dowex 50 (8X, 200-400 mesh, H+ form), and the column was washed with distilled water until the effluent was neutral. This acidic fraction which contained hydrochloric acid and organic acids was left aside for processing later. The amino acids and bases were then eluted from the Dowex 50 column with 2 N ammonium hydroxide. The ammonium hydroxide eluate was concentrated to a small volume,4 placed in a 750-ml. column of Dowex 2 (8X, 200-400 mesh, OH- form), and the column was first eluted with water, then with 1 N hydrochloric acid.The water eluate contained amino acid amides and other bases, and the acid eluate amino acids. 3 The evaporation of the acid eluates was found to be necessary in order to obt.ain completely reproducible values for the amino acid concentration. 4 All concentrating operations were performed at 40” in a vacuum rotating evaporator unless otherwise stated.
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SEPARATION
MIXTURE REACTlON
AND
SCHEME
OF PRODUCTS
ORGANIC ACIDS AND NONIONICS
AUPHATIC
MASTER
AMINO ACIDS AND BASES
ACIDS AND ~
HI0
NONIONICS
NONIONICS /“M$X;1”“-;
,
HYDROXY
ACIDS
SOLUTION
AMINO ACIDS
1 “++
;;;;fATE
GLYCINE
COMPLEX
\ SILICA
GEL
FLOREX
XXX
SILICAGEL
-
DOWEX 50
DOWEX 50
FIG. 1. Separation solution
scheme of the products formed by refluxing of paraformaldehyde and hydroxylamine hydrochloride.
HYDROLYSIS
a 0.25 equimolar
Separation of Amino Acids and Bases The eluate containing the amino acids was concentrated and treated according to the method of Selim et al. (29) with cupric picrate which selectively precipitates glycine in the form of a glycine cupric picrate complex. 5 The solution remaining after the separation of most of the glycine was freed from the excess of cupric picrate (31) and was concentrated. The amino acids present in this solution were chromatographically separated by the method of Stein and Moore (32) using a Dowex 50 column of 250 ml. The water eluate containing the amino acid amides and other bases was concentrated to a small volume and chromatographed in a Dowex 50 column (Na+ form).
Separation of Organic Acids The organic acid fraction was extracted with ether, and the remaining aqueous solution was concentrated to a small volume. The ether-soluble acids of the ether extract and the water-soluble acids of the aqueous solution were separated by column chromatography on silica gel according to the method of Bulen et al. (33). Moreover, this organic acid fraction which also contained nonionics was chromatographed in a column of Florex XxX.(34), and the eluant was analyzed for reducing organic compounds,by the method of Folin-Wu (35).
IdentiJication
of Products
The formation of hydrogen cyanide, as an intermediate in the reaction, was determined by the specific method of Schilt (28) and by precipitation with silver nitrate 6 The precipitation by cupric picrate cipitation by 5.nitronapthalene-1-sulfonic
was found to be more effective acid (30).
than the pre-
119
AMINO ACID SYNTHESIS (36). The amino acids were identified by the melting points fonates (37)) and glycinamide by its chromatographic behavior raphy of amino acids (39)) column and paper chromatography, reactions were also used in other instances.
of their p-toluenesul(38). Gas chromatogand specific chemical
RESULTS
Progress of the Reaction Figure 2 shows the formation of amino acids in a typical experiment (0.25 M, 100”). It can be seen that at approximately 60 hr. the concentration of amino acids reached a maximum value of 17.7 mmoles/l. and stayed constant from there on. Since in theory at least two moles of formaldehyde is needed to form one mole of glycine, the molar yield of amino acids, measured as glycine, was 14.1%. Figure 2 shows also the formation of amino acid amides. It can be seen that the concentration reached a maximum at about 24 hr. and from there on decreased to a practically zero value. If the amino acid synthesis occurred by way of the amino acid amides, an amino acid amide curve such as this
6
HYDROGEN
CYANIDE
4 2
TlME IN HOURS
FIG. 2. Synthesis
of amino acids, amino acid amides and other compounds. Closed test tubes containing 5 ml. of a 0.25 equimolar solution of paraformaldehyde and hy; droxylamine hydrochloride were heated at 100” and analyzed for the above compounds at different times.
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should be expected since this is the characteristic kinetic curve for the formation and disappearance of an intermediate (40). That amino acid amides are formed earlier than amino acids was also demonstrated in an experiment in which the reaction was carried out at lower temperature and in a less polar solvent (TO”, 90 % methanol). The first ninhydrin-positive compound formed under these conditions of slow hydrolysis was glycinamide. The appearance of glycinamide in the chromatograms was followed a little later by the appearance of glycine. In addition, Fig. 2 shows the formation of ammonia and hydrogen cyanide. These compounds were formed readily, and their formation followed a parallel course with the synthesis of amino acids. Hydrogen cyanide was derived directly from formaldoxime hydrochloride (formaldehyde and hydroxylamine hydrochloride), since we have observed that pure formaldoxime hydrochloride in the presence of traces of water decomposes into hydrogen cyanide simply by long standing at room temperature. Additional evidence may be found in the literature (41). Ammonia, in the form of ammonium chloride, was also derived from formaldoxime hydrochloride after its dehydration to hydrogen cyanide or rearrangement to formamide (41). These two compounds were progressively hydrolyzed under the conditions of the reaction into formic acid and ammonia. The formation of hydrogen cyanide and ammonia as well as amino acids was accompanied by the corresponding disappearance of hydroxylamine and formaldoxime. This was evidenced by the direct analysis of samples for hydroxylamine, and also by the decreased formation of triformoxime with time. The white solid trimer of formaldoxime was deposited on the walls of the condenser of the refluxing apparatus in diminishing amounts as the reaction was progressing. Effect of Several Variables Figure 3 shows the effect of temperature on the formation of amino acids. It can be seen that at 80, 90, and 100” the reaction was completed at about 240, 120, and 60 hr., respectively. The factor by which the velocity of the reaction was increased on raising the temperature lo”, or temperature coefficient Qlo , is thus approximately 2. By extrapolating at room temperature, the reaction should reach completion in 1 year. Figure 4 shows the effect of pH. The synthesis occurred under acidic as well as under basic conditions, but no measurable formation of amino acids takes place between pH 3 and 6. The synthesis was optimal at about pH 1, which is the actual pH of a 0.25 equimolar solution of reactants. Although the mechanism of synthesis under acidic and basic conditions may not necessarily be the same, hydrogen cyanide and ammonia were always formed in either case, and no hydrogen cyanide or ammonia was ever formed at any pH between 3 and 6.
AMINO
ACID
SYNTHESIS
121
The effect of some 20 inorganic compounds, mostly salts of metals, was investigated. Only sodium molybdate and sodium vanadate were found to enhance appreciably the synthesis of amino acids under acidic and basic conditions, respectively. The per cent amino acid increase caused by different concentrations of sodium molybdate at pH’s between 1 and 1.6 is recorded in Fig. 5. The values are corrected for the change in pH caused by the addition of sodium molybdate. Other compounds such as the chlorides of iron and of other heavy metals caused a slight decrease in the total amount of amino acids synthesized. Usually no qualitative differences were observed in the chromatographic pattern of the amino acids formed in the presence of inorganic ions. However, the addition of sodium phosphate caused the appearance in the chromatograms of a ninhydrin-positive compound (possibly an amino acid amide) with a lower migration rate than glycine and glycinamide in butanol-acetic acid. The effect of augmenting the equimolar concentration of the reactants from 0.2 to 5 M resulted in a progressive decrease in the molar yield of
I
TIME
IN HOURS
3. Effect of temperature on the formation of amino acids. Three series of test tubes, prepared as described in Fig. 2, were heated at 80, 90, and loo”, respectively, and analyzed for amino acids during the course of the reaction. Appropriate time scales are shown for each experiment. FIG.
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amino acids. A slight diminution was also observed when either of the two reactants, paraformaldehyde or hydroxylamine hydrochloride, was used in excess of the other. In addition, no significant change was observed when paraformaldehyde was replaced by formaldehyde. The amino acid yield could be increased, however, significantly by allow-
FIG. 4. Effect of the pH on the synthesis of amino acids. The tubes were prepared as described in Fig. 2, but the solutions had been previously adjusted to different pH’s with HCl or NaOH. The tubes were heated at 100” for 60 hr. and analyzed for amino acids.
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ACID
SYNTHESIS
123
FIG. 5. Effect of sodium molybdate on the synthesis of amino acids. Closed test tubes prepared as described in Fig. 2 but in addition contained the given concentrations of sodium molybdate (Merck). The pH of these solutions was measured, and the test tubes were subsequently heated at 100” for 60 hr. and analyzed for amino acids. The recorded values show the per cent increase over the values obtained in the absence of molybdate at the same pH.
ing an equimolar solution of reactants to enter slowly into the refluxing reaction vessel during 30 hr. and then continuing the refluxing operation for a regular period of 60 hr. In this way, a 20 % molar yield of amino acids was obtained from a 0.25 M reaction mixture as compared to the 14% yield usually obtained when the solution was refluxed all at once. The progressive addition of reactants apparently favors the condensation reactions that yield amino acids over other side hydrolytic reactions. Products Formed An elution diagram of the organic acid fraction chromatographed on silica gel (33) is shown in Fig. 6. The ether-soluble and the water-soluble acids were chromatographed under identical conditions, and the diagrams were superimposed. Three peaks corresponding, by the position, to formic acid (ether extract) and lactic and glycolic acids (water solution) can be
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FIG. 6. Elution diagrams from a silica gel column of the organic acid fraction obtained from 166 ml. of reaction mixture. The acids were identified only by the posi. tion of standards. The compounds corresponding to the small peak between lactic and glycolic acids and to the shoulder on the glycolic acid peak were not identified.
seen among other components. The molar yield of formic acid as measured from the elution diagram was 4.4 %. The molar yield of the other acids was one-fifth or less than that of formic acid. The organic acid-nonionic fraction contained also several reducing compounds as evidenced by column chromatography on Florex XXX and analysis of the effluent by the Folin-Wu method. In addition, paper chromatography of this fraction in butanol-acetic acid followed by reaction with aniline hydrogen phthalate (42) revealed the formation of one brown and two yellow spots. Although this indicates the presence of sugars or other carbonyl compounds, they could not be identified because they were present in very small amounts. The amino acid fraction obtained from 2 1. of reaction mixture yielded 13.6 g. glycine-cupric picrate complex from which about 2.1 g. glycine was recovered. An elution diagram of the amino acids remaining after partial precipitation of glycine is given in Fig. 7. Peaks, C, D, E, and F correspond to glycine, alanine, P-alanine, and ammonia, respectively. The fnst three compounds were identified by paper chromatography and by the melting points of their corresponding p-toluenesulfonates. Glycine peak, m.p. 148-149’;‘j mixed melting point with an 6 All the melting points are corrected.
AMINO
ACID
SYNTHESIS
125
authentic sample of glycine p-toluenesulfonate 148-149” (37). Alanine peak, m.p. 139.0-140.5”; mixed melting point with an authentic sample of alanine p-toluenesulfonate, 138.5-140.0”. p-Alanine peak, m.p. 119.5-120.5”; mixed melting point with an authentic sample of p-alanine p-toluenesulfonate, 119-120’ (43). Ammonia was identified by a positive reaction with t,he Nessler’s reagent and by paper chromatography (44). Alanine was also identified by gas chromatography (39). Peak A was found to be composed of three amino acids which were tentatively identified as serine, aspartic acid, and threonine by paper chromatography in phenol (45) and in the solvent pair 2-butanol-formic acid and 2-butanol-ammonia (46). The Rf values of the compounds in the phenol solvent were 0.34, 0.18, and 0.48, respectively. Serine was present in much larger amounts than the other two. From the ion-exchange and paper chromatographic analysis, it was determined that the amounts of alanine, /?-alanine, and serine formed were about 0.15, 0.16, and 0.11 mmoles/l. of reaction mixture. Thus the formation of each of these amino acids was at least 100 times smaller than the formation of glycine.
FIG. 7. Elution diagram from a Dowex 50 column of the amino acid fraction obtained from 2 1. of reaction mixture. Most of the glycine had been previously removed by precipitation with cupric picrate. The amino acids were identified by the position of standards, by derivatives, and by paper chromatography.
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The basic fraction showed the presence of several ninhydrin-positive compounds when it was analyzed by paper chromatography. One of the main compounds was identified as glycinamide by its typical yellow color given by reaction with ninhydrin (38) and by the Rf values in three solvents : n-butanol-acetic acid-water (4 : 1: 1), 0.24; n-propanol-formic acidwater (7.5: 1.5: 1)) 0.37; pyridine-water (6.5: 3.5)) 0.78. The compound was also identified by its position in the elution diagram of a 15-cm. Dowex 50 column eluted with sodium citrate buffer according to the method of Moore et al. (47). Glycinamide emerged in the 63 to 84 ml. of effluent. Synthesis of the Products Identified In these experiments the synthesis of glycinamide and glycine can be interpreted to occur in the following steps. In a first step, formaldehyde and hydroxylamine react to form formaldoxime. Secondly, formaldoxime undergoes either by acid or base catalysis a dehydration to hydrogen cyanide. Finally, in the third step, hydrogen cyanide, formaldehyde and ammonia undergo a Strecker condensation into glycine nitrile, which by progressive hydrolysis yields first glycinamide and then glycine. This may be graphically represented by the following scheme of reactions: HCHO
+ NH,OH CHzNOH
HCHO
ti
+ HCN
+ NH3 + HCN
CH2(NHz)CN CH~(NH~)CONHP
CHzNOH
+ Hz0
+ Hz0
+ Hz0 + CHx(NHt)CN
+ H20 --+ CHz(NHz)CONHt + Hz0 + CH2(NHz)COOH
(1)
(2) @a) (3b) (3c)
The ammonia needed for the Strecker condensation is derived by decomposition of formaldoxime according to a reaction previously described (41) which involves either the direct hydrolysis of hydrogen cyanide or the rearrangement of formaldoxime to formamide and subsequent hydrolysis to formic acid and ammonia. The formation of glycolic acid can be interpreted in an analogous manner to the formation of glycine. Hydrogen cyanide condenses with formaldehyde, and the hydroxyacetonitrile formed is subsequently hydrolyzed to glycolic acid. The synthesis of glycine and glycolic acid by a Strecker’s condensation is a common feature of the present experiments and of the classical experiments of Miller with electric discharges. In both cases, the formation of hydrogen cyanide appears to be the key reaction for the synthesis of these compounds. The formation of the other amino and hydroxy acids in these experiments
AMINO
ACID
SYNTHESIS
127
can be explained on the basis of condensation reactions involving essentially formaldehyde and amides. The condensation of formaldehyde with glycinamide probably yields serinamide, which in turn can be converted into alaninamide according to the observations of Chambers and Carpenter (48). Hydrolysis of these amides would result in the formation of serine and alanine. Evidence for the condensation of formaldehyde with glycinamide to form amino acid amides has been obtained recently in this laboratory. In addition, the likelihood of such condensation is supported by the fact that serine can be obtained by condensation of formaldehyde with polyglycine (49), and threonine by the condensation of acetaldehyde with copper glycinate (50). It appears evident that when the ionization of the carboxyl group of glycine is prevented by formation of an amide or a chelate, the methylene group can easily undergo condensation reactions with formaldehyde or other aldehydes. The same mechanism that applies to the synthesis of alanine can also be used to explain the synthesis of lactic acid through the intermediate formation of lactamide. The formation of p-alanine is not completely clear at present, but it may involve the dehydration of lactamide to acrylamide, conversion of this compound to p-amino propionamide, and hydrolysis of the latter compound. In line with this reasoning, when aqueous solutions of either lactonitrile or acrylonitrile and hydroxylamine are heated at 100” for 24 hr. the formation of both alanine and P-alanine has been demonstrated. DISCUSSION
The present experiments have demonstrated that amino acids and hydroxy acids can be synthesized from aqueous mixtures of formaldehyde and hydroxylamine under acid as well as under basic conditions by heating at 100” or lower temperatures. The evidence obtained indicates that even at room temperature the synthesis can proceed at a measurable rate. The initial reactions of this synthetic process are the formation of formaldoxime and the dehydration of formaldoxime to hydrogen cyanide by acid or base catalysis. After this compound is formed, condensations take place to yield successively nitriles, amides, and finally hydroxy and amino acids. As a consequence of these findings, any process that may give rise to the formation of formaldoxime under acid or basic conditions may be expected to be a source of amino acids. Thus, for instance, the formation of amino acids in the experiments of Baly et al. (11) by photochemical action upon aqueous solutions of formhydroxamic acid and formaldehyde can be explained by the mechanism presented here, once the hydroxylamine liberated by hydrolysis of formhydroxamic acid combines with formaldehyde. Photochemical syntheses of amino acids in aqueous ferric chloride solu-
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tions containing potassium nitrite and carbon monoxide (10) or potassium nitrate and formaldehyde (13) can likely be explained on the basis of a similar mechanism. The role of ferric chloride in these solutions may be twofold. On one hand it enhances the photochemical reduction of nitrates (51) and nitrites (52) to some nitrogen compound of a lower oxidation level. This is done at the expense of partial oxidation of the carbon compounds which are always present in excess in these experiments. On the other hand, the addition of ferric chloride maintains the pH low enough to make possible the dehydration of formaldoxime to hydrogen cyanide. That this may be the case is indicated by the experiments of Menaul (53) who found that the formation of hydrogen cyanide in formaldehyde-nitrate mixtures exposed to light is directly dependent on the acidity of the medium. Of course, other mechanisms of photochemical synthesis of amino acids from formaldehyde and nitrogen compounds cannot be excluded. Additional evidence for the role of formaldoxime in the formation of hydrogen cyanide has been reported in relation to the thermal decomposition of nitromethane (54) and nitrite esters (55), which occurs through the intermediate formation of nitrosomethane. Nitrosomethane rearranges to formaldoxime, and formaldoxime decomposes thermally into hydrogen cyanide and water (56). In conclusion, the experiments described are of significance in the interpretation of the mechanisms of synthesis of amino acids and other fundamental biochemical compounds under conditions which may have existed in the earth’s crust during its early development (57-60). SUMMa4RY
The experiments reported here have shown that by heating an aqueous mixture of formaldehyde and hydroxylamine hydrochloride, several amino acids, hydroxy acids, and other biochemical compounds are formed. The predominant amino acid formed is glycine. The conditions under which the reaction takes place have been studied in some detail. The mechanism of synthesis has also been studied. Formaldoxime, hydrogen cyanide, and ammonia are among the first products formed. Strecker and cyanohydrin condensations yield nitriles which are subsequently hydrolyzed to amides and finally to acids. Other condensation reactions are proposed to account for the formation of serine, alanine, ,B-alanine, and lactic acid. The interpretation of some previous photochemical syntheses of amino acids appears possible in the light of the present evidence. The experiments are of significance in relation to the formation of biochemical compounds, from simple compounds of carbon and nitrogen, under conditions assumed to have existed during the early development of the earth’s crust.
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ACID
SYNTHESIS
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DANIELS, F., “Chemical Kinetics,” p. 27. Cornell Univ. Press, Ithaca, 1938. DUNSTAN, W. R., AND BOSSI, A. L., J. Chem. Sot. 73,353 (1898). PARTRIDGE, S. M., Nature 164, 443 (1949). HOLLEY, R. W., AND HOLLEY, A. D., J. Am. Chem. Sot. 71, 2131 (1949). POLLARD, F. H., AND BANISTER, A. J., Anal. Chim. Acta 14, 70 (1956). BLOCK, R. J., DURRUM, E. L., AND ZWEIG, G., “A Manual of PaDer Chromatography and Paper Electrophoresis,” p. 77. Academic Press Inc., New York, 1955. HAUSMANN, W., J. Am. Chem. Sot. 74, 3181 (1952). MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185 (1958). CHAMBERS, R. W., AND CARPENTER, F. H., J. Am. Chem. Sot. 77, 1527 (1955). AKABORI, S., OKAWA, K., AND SATO, M., Bull. Chem. Sot. Japan 29, 608 (1956). SATO, M., OKAWA, K., AND AKABORI, S., Bull. Chem. Sot. Japan 30, 937 (1937). ANDERSON, W. T., J. Am. Chem. Sot. 46, 797 (1924). KRISHNAN, K. S., AND GUHA, A. C., Proc. Indian Acad. Sci. lA, 242 (1934), C. A. 29, 1326 (1935). CULTRERA, R., Ann. staz. sper. agrar. Modena [N.S.], 6, 163 (1936), C. A. 32, 3266 (1938). MENAUL, P., J. Biol. Chem. 46, 297 (1921). MAKOVKY, A., AND LENJI, L., Chem. Revs. 68, 627 (1958). LEVY, J. B., Ind. Eng. Chem. 48, 762 (1956). TAYLOR, H. A., AND BENDER, H., J. Chem. Phys. 9, 761 (1941). Academic Press Inc., New OPARIN, A. I., “The Origin of Life on the Earth.” York, 1957. UREY, H. C., Proc. Natl. Acad. Sci. U. S. 38, 35 (1962). Fox, S. W., J. Chem. Educ. 34, 472 (1957). LEDERBERG, J., AND COWIE, D. W., Science 137, 1473 (1958).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
86,
115-130 (1959)
Amino Acid Synthesis from Formaldehyde and Hydroxylaminels 2 J. Oro’, A. Kimball, R. Fritz and F. Master From the Department
of Chemistry,
University of Houston,
Houston,
Texas
Received March 30, 1959
The synthesis of amino acids, in one-step processes, by reaction of simple compounds of carbon (C, and C,) with simple nitrogen compounds has been accomplished by several investigators using highly activating forms of energy such as electric discharges, ultraviolet light, and ionizing radiation (l-23). In particular, Loeb (l), Miller (2-6), Abelson (7, S), and Heyns et al. (9) used the action of silent and spark discharges upon aqueous mixtures of totally reduced or partially oxidized carbon and nitrogen compounds. Photochemical syntheses of amino acids in aqueous systems were claimed some time ago by Baudisch (10) from potassium nitrite, carbon monoxide, and ferric chloride, by Baly et al. (11) from formhydroxamic acid and formaldehyde, and by Dhar and Mukherjee (12) from nitrates, simple carbon compounds such as glycol, and titanium dioxide. More recently, Bahadur and his associates (13-16) have observed the formation of amino acids by the action of light upon aqueous solutions of potassium nitrate, paraformaldehyde, and ferric chloride, and upon aqueous mixtures of paraformaldehyde and molybdic oxide in the presence of air. Also, recently Groth and von Weyssenhoff (17, 18) and Miller (6) have reported amino acid syntheses by irradiating with ultraviolet light aqueous mixtures containing ammonia as a nitrogen source, and ethane and methane, respectively, as a carbon source. The formation of amino acids by the action of ionizing radiations upon similar simple mixtures has been shown by Hasselstrom et al. (19), Paschke et al. (20), and Dose et al. (21, 22) using P-rays, y-rays and x-rays, respectively. A common feature of all these experiments is the employment of highly 1Work supported in part by a grant from the Robert A. Welch Foundation. 2 A preliminary report appearedin Am. Chem. Sot., Div. of Biol. Chem., Abstracts, p. 16C. 130th Meeting, Atlantic City, 1956. 115
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activating forms of energy. In the experiments of Miller, part of the energy of the electric discharges is used to oxidize methane to formaldehyde; and in the first experiments of Bahadur, part of the light energy is used in the reduction of nitrates. Thus, it was of interest to find out if certain C1 and N1 compounds of intermediate degrees of oxidation would be sufficiently active to yield amino acids even in the absence of radiant energy or electric discharges. This has been found to be so, as it has now been possible to synthesize appreciable amounts of glycine and lesser amounts of glycinamide and certain amino acids among other compounds by heating aqueous solutions of paraformaldehyde and hydroxylamine hydrochloride at moderate temperature. A study of the conditions of this reaction followed by an interpretation of the sequence of reactions involved in the over-all amino acid synthesis is presented. The significance of these experiments in relation to the amino acid syntheses obtained by other investigators is discussed. EXPERIMENTAL
Conditions of the Reaction In order to establish the optimal conditions of amino acid synthesis, several experiments were carried out in which the effect of several physical and chemical variables was tested. In a typical experiment, ten bml. samples of a 0.25 M solution of paraformaldehyde and hydroxylamine hydrochloride were placed in Pyrex test tubes. The tubes were closed with screw caps and then heated in an oven at 100”. The progress of the reaction was followed by analyzing the amino acid content of the tubes at different periods of time until the reaction ended. In some instances, amino acid amides, ammonia, and other products were also analyzed. The effect of temperature on the reaction rate was determined by observing the time needed for completion of the reaction at 80,90 and loo’, respectively, and calculating the corresponding &lo coefficient. The effect of pH on the amino acid yield was determined by measuring the total amount of amino acid synthesized in 0.25 M reaction mixtures which had different hydrogen-ion concentration. Hydrochloric acid or sodium hydroxide was used in adjusting the pH of these reaction mixtures. The role of inorganic ions as possible catalysts for the reaction was also tested. This was done by dissolving a small amount of the corresponding pure compound in the reaction mixture so as to form an ionic solution approximately IOW’ M and carrying out the reaction in the usual manner. The influence of other variables such as the concentration and the relative proportion of the reactants was also studied.
Determination
of Amino Acid and Amino Acid Amide Concentration
For the determination of the amino acid concentration in the large number of samples analyzed, the method of Awapara and Sato (24) was followed in part. Aliquots in 1 ml. of the reaction product were passed through small columns (1 X 5cm.) of Dowex 2 (8X, 200-400 mesh, OH- form). The columns were washed with 10 ml. water. These water eluates were left aside for the determination of amino acid amides
AMINO
ACID
SYNTHESIS
117
and other products. The amino acids were subsequently eluted with about 10 ml. of 4 N acetic acid and brought to an exact volume of 10 ml. with distilled water. Aliquots of 5 ml. of the acetic acid eluates were evaporated to dryness in a steam bath,3 and the residue was redissolved in 2 ml. of 0.25 N hydrochloric acid and analyzed by the method of Troll and Cannan (25) using glycine as standard. The amino acid concentration was expressed in millimoles per liter. For the determination of the total amino acid amide concentration, 5-ml. aliquots of the water eluates were subjected to hydrolysis in 0.5 N sodium hydroxide at 100” for 3 hr. The hydrolyzed samples were then evaporated under vacuum to about 1 ml. in order to remove the ammonia, neutralized with hydrochloric acid, and the amino acids were analyzed by the method previously described.
Determination
of Other Products
Occasionally, the water eluates were also analyzed for other bases, such as ammonia and hydroxylamine. In order to prevent any loss of ammonia or hydroxylamine by volatilization during the elution of the small Dowex 2 columns with water, the tips of the chromatographic columns were immersed into 10 ml. of a 0.1 N hydrochloric acid solution which had been previously placed in the collecting beakers. In this way, the eluted bases were transformed quantitatively into their respective chlorides. The ammonia and the hydroxylamine of these eluates were analyzed by nesslerization (26) and by the method of Csaky (27)) respectively. The hydrogen cyanide formed during the course of the reaction was analyzed directly in the reaction mixtures by the method of Schilt (28).
Separation Procedure For preparative purposes 3 1. of 0.25 M solution of paraformaldehyde and hydroxylamine hydrochloride was refluxed in a 5-1. flask provided with a long water-cooled condenser. Normally, the solution was placed directly in the flask and refluxed for a period of 60 hr. Alternatively, the solution was allowed to drop slowly into the refluxing apparatus during a 30.hr. period, and the refluxing operation was continued for 60 hr. additional. The separation procedure adopted is a combination of ion-exchange chromatography and selective precipitation of glycine. The chromatographic procedure is similar to that of Miller (3) and is shown in Fig. 1. At the end of the reaction, 2 1. of the resulting solution was passed through a 750-ml. column of Dowex 50 (8X, 200-400 mesh, H+ form), and the column was washed with distilled water until the effluent was neutral. This acidic fraction which contained hydrochloric acid and organic acids was left aside for processing later. The amino acids and bases were then eluted from the Dowex 50 column with 2 N ammonium hydroxide. The ammonium hydroxide eluate was concentrated to a small volume,4 placed in a 750-ml. column of Dowex 2 (8X, 200-400 mesh, OH- form), and the column was first eluted with water, then with 1 N hydrochloric acid.The water eluate contained amino acid amides and other bases, and the acid eluate amino acids. 3 The evaporation of the acid eluates was found to be necessary in order to obt.ain completely reproducible values for the amino acid concentration. 4 All concentrating operations were performed at 40” in a vacuum rotating evaporator unless otherwise stated.
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SEPARATION
MIXTURE REACTlON
AND
SCHEME
OF PRODUCTS
ORGANIC ACIDS AND NONIONICS
AUPHATIC
MASTER
AMINO ACIDS AND BASES
ACIDS AND ~
HI0
NONIONICS
NONIONICS /“M$X;1”“-;
,
HYDROXY
ACIDS
SOLUTION
AMINO ACIDS
1 “++
;;;;fATE
GLYCINE
COMPLEX
\ SILICA
GEL
FLOREX
XXX
SILICAGEL
-
DOWEX 50
DOWEX 50
FIG. 1. Separation solution
scheme of the products formed by refluxing of paraformaldehyde and hydroxylamine hydrochloride.
HYDROLYSIS
a 0.25 equimolar
Separation of Amino Acids and Bases The eluate containing the amino acids was concentrated and treated according to the method of Selim et al. (29) with cupric picrate which selectively precipitates glycine in the form of a glycine cupric picrate complex. 5 The solution remaining after the separation of most of the glycine was freed from the excess of cupric picrate (31) and was concentrated. The amino acids present in this solution were chromatographically separated by the method of Stein and Moore (32) using a Dowex 50 column of 250 ml. The water eluate containing the amino acid amides and other bases was concentrated to a small volume and chromatographed in a Dowex 50 column (Na+ form).
Separation of Organic Acids The organic acid fraction was extracted with ether, and the remaining aqueous solution was concentrated to a small volume. The ether-soluble acids of the ether extract and the water-soluble acids of the aqueous solution were separated by column chromatography on silica gel according to the method of Bulen et al. (33). Moreover, this organic acid fraction which also contained nonionics was chromatographed in a column of Florex XxX.(34), and the eluant was analyzed for reducing organic compounds,by the method of Folin-Wu (35).
IdentiJication
of Products
The formation of hydrogen cyanide, as an intermediate in the reaction, was determined by the specific method of Schilt (28) and by precipitation with silver nitrate 6 The precipitation by cupric picrate cipitation by 5.nitronapthalene-1-sulfonic
was found to be more effective acid (30).
than the pre-
119
AMINO ACID SYNTHESIS (36). The amino acids were identified by the melting points fonates (37)) and glycinamide by its chromatographic behavior raphy of amino acids (39)) column and paper chromatography, reactions were also used in other instances.
of their p-toluenesul(38). Gas chromatogand specific chemical
RESULTS
Progress of the Reaction Figure 2 shows the formation of amino acids in a typical experiment (0.25 M, 100”). It can be seen that at approximately 60 hr. the concentration of amino acids reached a maximum value of 17.7 mmoles/l. and stayed constant from there on. Since in theory at least two moles of formaldehyde is needed to form one mole of glycine, the molar yield of amino acids, measured as glycine, was 14.1%. Figure 2 shows also the formation of amino acid amides. It can be seen that the concentration reached a maximum at about 24 hr. and from there on decreased to a practically zero value. If the amino acid synthesis occurred by way of the amino acid amides, an amino acid amide curve such as this
6
HYDROGEN
CYANIDE
4 2
TlME IN HOURS
FIG. 2. Synthesis
of amino acids, amino acid amides and other compounds. Closed test tubes containing 5 ml. of a 0.25 equimolar solution of paraformaldehyde and hy; droxylamine hydrochloride were heated at 100” and analyzed for the above compounds at different times.
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should be expected since this is the characteristic kinetic curve for the formation and disappearance of an intermediate (40). That amino acid amides are formed earlier than amino acids was also demonstrated in an experiment in which the reaction was carried out at lower temperature and in a less polar solvent (TO”, 90 % methanol). The first ninhydrin-positive compound formed under these conditions of slow hydrolysis was glycinamide. The appearance of glycinamide in the chromatograms was followed a little later by the appearance of glycine. In addition, Fig. 2 shows the formation of ammonia and hydrogen cyanide. These compounds were formed readily, and their formation followed a parallel course with the synthesis of amino acids. Hydrogen cyanide was derived directly from formaldoxime hydrochloride (formaldehyde and hydroxylamine hydrochloride), since we have observed that pure formaldoxime hydrochloride in the presence of traces of water decomposes into hydrogen cyanide simply by long standing at room temperature. Additional evidence may be found in the literature (41). Ammonia, in the form of ammonium chloride, was also derived from formaldoxime hydrochloride after its dehydration to hydrogen cyanide or rearrangement to formamide (41). These two compounds were progressively hydrolyzed under the conditions of the reaction into formic acid and ammonia. The formation of hydrogen cyanide and ammonia as well as amino acids was accompanied by the corresponding disappearance of hydroxylamine and formaldoxime. This was evidenced by the direct analysis of samples for hydroxylamine, and also by the decreased formation of triformoxime with time. The white solid trimer of formaldoxime was deposited on the walls of the condenser of the refluxing apparatus in diminishing amounts as the reaction was progressing. Effect of Several Variables Figure 3 shows the effect of temperature on the formation of amino acids. It can be seen that at 80, 90, and 100” the reaction was completed at about 240, 120, and 60 hr., respectively. The factor by which the velocity of the reaction was increased on raising the temperature lo”, or temperature coefficient Qlo , is thus approximately 2. By extrapolating at room temperature, the reaction should reach completion in 1 year. Figure 4 shows the effect of pH. The synthesis occurred under acidic as well as under basic conditions, but no measurable formation of amino acids takes place between pH 3 and 6. The synthesis was optimal at about pH 1, which is the actual pH of a 0.25 equimolar solution of reactants. Although the mechanism of synthesis under acidic and basic conditions may not necessarily be the same, hydrogen cyanide and ammonia were always formed in either case, and no hydrogen cyanide or ammonia was ever formed at any pH between 3 and 6.
AMINO
ACID
SYNTHESIS
121
The effect of some 20 inorganic compounds, mostly salts of metals, was investigated. Only sodium molybdate and sodium vanadate were found to enhance appreciably the synthesis of amino acids under acidic and basic conditions, respectively. The per cent amino acid increase caused by different concentrations of sodium molybdate at pH’s between 1 and 1.6 is recorded in Fig. 5. The values are corrected for the change in pH caused by the addition of sodium molybdate. Other compounds such as the chlorides of iron and of other heavy metals caused a slight decrease in the total amount of amino acids synthesized. Usually no qualitative differences were observed in the chromatographic pattern of the amino acids formed in the presence of inorganic ions. However, the addition of sodium phosphate caused the appearance in the chromatograms of a ninhydrin-positive compound (possibly an amino acid amide) with a lower migration rate than glycine and glycinamide in butanol-acetic acid. The effect of augmenting the equimolar concentration of the reactants from 0.2 to 5 M resulted in a progressive decrease in the molar yield of
I
TIME
IN HOURS
3. Effect of temperature on the formation of amino acids. Three series of test tubes, prepared as described in Fig. 2, were heated at 80, 90, and loo”, respectively, and analyzed for amino acids during the course of the reaction. Appropriate time scales are shown for each experiment. FIG.
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amino acids. A slight diminution was also observed when either of the two reactants, paraformaldehyde or hydroxylamine hydrochloride, was used in excess of the other. In addition, no significant change was observed when paraformaldehyde was replaced by formaldehyde. The amino acid yield could be increased, however, significantly by allow-
FIG. 4. Effect of the pH on the synthesis of amino acids. The tubes were prepared as described in Fig. 2, but the solutions had been previously adjusted to different pH’s with HCl or NaOH. The tubes were heated at 100” for 60 hr. and analyzed for amino acids.
AMINO
ACID
SYNTHESIS
123
FIG. 5. Effect of sodium molybdate on the synthesis of amino acids. Closed test tubes prepared as described in Fig. 2 but in addition contained the given concentrations of sodium molybdate (Merck). The pH of these solutions was measured, and the test tubes were subsequently heated at 100” for 60 hr. and analyzed for amino acids. The recorded values show the per cent increase over the values obtained in the absence of molybdate at the same pH.
ing an equimolar solution of reactants to enter slowly into the refluxing reaction vessel during 30 hr. and then continuing the refluxing operation for a regular period of 60 hr. In this way, a 20 % molar yield of amino acids was obtained from a 0.25 M reaction mixture as compared to the 14% yield usually obtained when the solution was refluxed all at once. The progressive addition of reactants apparently favors the condensation reactions that yield amino acids over other side hydrolytic reactions. Products Formed An elution diagram of the organic acid fraction chromatographed on silica gel (33) is shown in Fig. 6. The ether-soluble and the water-soluble acids were chromatographed under identical conditions, and the diagrams were superimposed. Three peaks corresponding, by the position, to formic acid (ether extract) and lactic and glycolic acids (water solution) can be
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FIG. 6. Elution diagrams from a silica gel column of the organic acid fraction obtained from 166 ml. of reaction mixture. The acids were identified only by the posi. tion of standards. The compounds corresponding to the small peak between lactic and glycolic acids and to the shoulder on the glycolic acid peak were not identified.
seen among other components. The molar yield of formic acid as measured from the elution diagram was 4.4 %. The molar yield of the other acids was one-fifth or less than that of formic acid. The organic acid-nonionic fraction contained also several reducing compounds as evidenced by column chromatography on Florex XXX and analysis of the effluent by the Folin-Wu method. In addition, paper chromatography of this fraction in butanol-acetic acid followed by reaction with aniline hydrogen phthalate (42) revealed the formation of one brown and two yellow spots. Although this indicates the presence of sugars or other carbonyl compounds, they could not be identified because they were present in very small amounts. The amino acid fraction obtained from 2 1. of reaction mixture yielded 13.6 g. glycine-cupric picrate complex from which about 2.1 g. glycine was recovered. An elution diagram of the amino acids remaining after partial precipitation of glycine is given in Fig. 7. Peaks, C, D, E, and F correspond to glycine, alanine, P-alanine, and ammonia, respectively. The fnst three compounds were identified by paper chromatography and by the melting points of their corresponding p-toluenesulfonates. Glycine peak, m.p. 148-149’;‘j mixed melting point with an 6 All the melting points are corrected.
AMINO
ACID
SYNTHESIS
125
authentic sample of glycine p-toluenesulfonate 148-149” (37). Alanine peak, m.p. 139.0-140.5”; mixed melting point with an authentic sample of alanine p-toluenesulfonate, 138.5-140.0”. p-Alanine peak, m.p. 119.5-120.5”; mixed melting point with an authentic sample of p-alanine p-toluenesulfonate, 119-120’ (43). Ammonia was identified by a positive reaction with t,he Nessler’s reagent and by paper chromatography (44). Alanine was also identified by gas chromatography (39). Peak A was found to be composed of three amino acids which were tentatively identified as serine, aspartic acid, and threonine by paper chromatography in phenol (45) and in the solvent pair 2-butanol-formic acid and 2-butanol-ammonia (46). The Rf values of the compounds in the phenol solvent were 0.34, 0.18, and 0.48, respectively. Serine was present in much larger amounts than the other two. From the ion-exchange and paper chromatographic analysis, it was determined that the amounts of alanine, /?-alanine, and serine formed were about 0.15, 0.16, and 0.11 mmoles/l. of reaction mixture. Thus the formation of each of these amino acids was at least 100 times smaller than the formation of glycine.
FIG. 7. Elution diagram from a Dowex 50 column of the amino acid fraction obtained from 2 1. of reaction mixture. Most of the glycine had been previously removed by precipitation with cupric picrate. The amino acids were identified by the position of standards, by derivatives, and by paper chromatography.
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The basic fraction showed the presence of several ninhydrin-positive compounds when it was analyzed by paper chromatography. One of the main compounds was identified as glycinamide by its typical yellow color given by reaction with ninhydrin (38) and by the Rf values in three solvents : n-butanol-acetic acid-water (4 : 1: 1), 0.24; n-propanol-formic acidwater (7.5: 1.5: 1)) 0.37; pyridine-water (6.5: 3.5)) 0.78. The compound was also identified by its position in the elution diagram of a 15-cm. Dowex 50 column eluted with sodium citrate buffer according to the method of Moore et al. (47). Glycinamide emerged in the 63 to 84 ml. of effluent. Synthesis of the Products Identified In these experiments the synthesis of glycinamide and glycine can be interpreted to occur in the following steps. In a first step, formaldehyde and hydroxylamine react to form formaldoxime. Secondly, formaldoxime undergoes either by acid or base catalysis a dehydration to hydrogen cyanide. Finally, in the third step, hydrogen cyanide, formaldehyde and ammonia undergo a Strecker condensation into glycine nitrile, which by progressive hydrolysis yields first glycinamide and then glycine. This may be graphically represented by the following scheme of reactions: HCHO
+ NH,OH CHzNOH
HCHO
ti
+ HCN
+ NH3 + HCN
CH2(NHz)CN CH~(NH~)CONHP
CHzNOH
+ Hz0
+ Hz0
+ Hz0 + CHx(NHt)CN
+ H20 --+ CHz(NHz)CONHt + Hz0 + CH2(NHz)COOH
(1)
(2) @a) (3b) (3c)
The ammonia needed for the Strecker condensation is derived by decomposition of formaldoxime according to a reaction previously described (41) which involves either the direct hydrolysis of hydrogen cyanide or the rearrangement of formaldoxime to formamide and subsequent hydrolysis to formic acid and ammonia. The formation of glycolic acid can be interpreted in an analogous manner to the formation of glycine. Hydrogen cyanide condenses with formaldehyde, and the hydroxyacetonitrile formed is subsequently hydrolyzed to glycolic acid. The synthesis of glycine and glycolic acid by a Strecker’s condensation is a common feature of the present experiments and of the classical experiments of Miller with electric discharges. In both cases, the formation of hydrogen cyanide appears to be the key reaction for the synthesis of these compounds. The formation of the other amino and hydroxy acids in these experiments
AMINO
ACID
SYNTHESIS
127
can be explained on the basis of condensation reactions involving essentially formaldehyde and amides. The condensation of formaldehyde with glycinamide probably yields serinamide, which in turn can be converted into alaninamide according to the observations of Chambers and Carpenter (48). Hydrolysis of these amides would result in the formation of serine and alanine. Evidence for the condensation of formaldehyde with glycinamide to form amino acid amides has been obtained recently in this laboratory. In addition, the likelihood of such condensation is supported by the fact that serine can be obtained by condensation of formaldehyde with polyglycine (49), and threonine by the condensation of acetaldehyde with copper glycinate (50). It appears evident that when the ionization of the carboxyl group of glycine is prevented by formation of an amide or a chelate, the methylene group can easily undergo condensation reactions with formaldehyde or other aldehydes. The same mechanism that applies to the synthesis of alanine can also be used to explain the synthesis of lactic acid through the intermediate formation of lactamide. The formation of p-alanine is not completely clear at present, but it may involve the dehydration of lactamide to acrylamide, conversion of this compound to p-amino propionamide, and hydrolysis of the latter compound. In line with this reasoning, when aqueous solutions of either lactonitrile or acrylonitrile and hydroxylamine are heated at 100” for 24 hr. the formation of both alanine and P-alanine has been demonstrated. DISCUSSION
The present experiments have demonstrated that amino acids and hydroxy acids can be synthesized from aqueous mixtures of formaldehyde and hydroxylamine under acid as well as under basic conditions by heating at 100” or lower temperatures. The evidence obtained indicates that even at room temperature the synthesis can proceed at a measurable rate. The initial reactions of this synthetic process are the formation of formaldoxime and the dehydration of formaldoxime to hydrogen cyanide by acid or base catalysis. After this compound is formed, condensations take place to yield successively nitriles, amides, and finally hydroxy and amino acids. As a consequence of these findings, any process that may give rise to the formation of formaldoxime under acid or basic conditions may be expected to be a source of amino acids. Thus, for instance, the formation of amino acids in the experiments of Baly et al. (11) by photochemical action upon aqueous solutions of formhydroxamic acid and formaldehyde can be explained by the mechanism presented here, once the hydroxylamine liberated by hydrolysis of formhydroxamic acid combines with formaldehyde. Photochemical syntheses of amino acids in aqueous ferric chloride solu-
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tions containing potassium nitrite and carbon monoxide (10) or potassium nitrate and formaldehyde (13) can likely be explained on the basis of a similar mechanism. The role of ferric chloride in these solutions may be twofold. On one hand it enhances the photochemical reduction of nitrates (51) and nitrites (52) to some nitrogen compound of a lower oxidation level. This is done at the expense of partial oxidation of the carbon compounds which are always present in excess in these experiments. On the other hand, the addition of ferric chloride maintains the pH low enough to make possible the dehydration of formaldoxime to hydrogen cyanide. That this may be the case is indicated by the experiments of Menaul (53) who found that the formation of hydrogen cyanide in formaldehyde-nitrate mixtures exposed to light is directly dependent on the acidity of the medium. Of course, other mechanisms of photochemical synthesis of amino acids from formaldehyde and nitrogen compounds cannot be excluded. Additional evidence for the role of formaldoxime in the formation of hydrogen cyanide has been reported in relation to the thermal decomposition of nitromethane (54) and nitrite esters (55), which occurs through the intermediate formation of nitrosomethane. Nitrosomethane rearranges to formaldoxime, and formaldoxime decomposes thermally into hydrogen cyanide and water (56). In conclusion, the experiments described are of significance in the interpretation of the mechanisms of synthesis of amino acids and other fundamental biochemical compounds under conditions which may have existed in the earth’s crust during its early development (57-60). SUMMa4RY
The experiments reported here have shown that by heating an aqueous mixture of formaldehyde and hydroxylamine hydrochloride, several amino acids, hydroxy acids, and other biochemical compounds are formed. The predominant amino acid formed is glycine. The conditions under which the reaction takes place have been studied in some detail. The mechanism of synthesis has also been studied. Formaldoxime, hydrogen cyanide, and ammonia are among the first products formed. Strecker and cyanohydrin condensations yield nitriles which are subsequently hydrolyzed to amides and finally to acids. Other condensation reactions are proposed to account for the formation of serine, alanine, ,B-alanine, and lactic acid. The interpretation of some previous photochemical syntheses of amino acids appears possible in the light of the present evidence. The experiments are of significance in relation to the formation of biochemical compounds, from simple compounds of carbon and nitrogen, under conditions assumed to have existed during the early development of the earth’s crust.
AMINO
ACID
SYNTHESIS
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