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Abiotic synthesis of amino groups
ARCHIVES
OF BIOCHEMISTRY
AND
Abiotic
BIOPHYSICS
106,303-307
Synthesis
of Amino
G. D. STEINMAX From
the Department
o.f Biochemistry,
Received
(1%)
Groups
H. A. LILLEVIK
AND
Michigan
State
October
University,
E. Lansing,
Michigan
9, 1963
An attempt was made to synthesize amino groups under nonreducing conditions from nitrogen. An aqueous solution of acetic acid was electrically sparked and yielded amino acid-like substances. The sparking of a solution of glycerol gave a product containing reducing amino compounds, some of which had the or-amino-aldehyde grouping. In both cases, molecular nitrogen extracted from the gaseous phase appeared to be the source from which amino nitrogen resulted.
Berthelot was one of the first to study the effects of silent electrical discharge on various materials in the presence of nitrogen (1). He noted that small amounts of nitrogen became fixed to the substrates in the process. Later, Cavendish observed that when an electric spark passed through a mixture of oxygen and nitrogen, nitric acid was synthesized (2). Irradiation of glycine yields acetic acid (3). Therefore, in order to investigate the possibility of synthesizing amino groups in the form of amino acids by sparking, acetic acid was chosen as a reactant in the present experiments. Likewise, it has already been shown that irradiation of alcohols yields reducing substances (4). Therefore, since amino group production was being studied, electric sparking of alcohols was explored for possible synthesis of aminocarbonyl cornpounds. The scope of the synthetic methods employed was defined by the intention to work under possible primordial conditions of simple reactants and high energy stimulators. The means for the prebiotic appearance of the reagents used have been proposed by Oparin (5). Nitrogen, in the presence of oxygen, was utilized in order to demonstrate the plausibility of amino group synthesis at that stage of the Earth’s development when the atmosphere contained an appreciable amount of oxygen. Likewise, the proposed
synthetic method would suggest a means by which an amino group could be added to certain substances while in solution. EXPERIMENTAL SPARKING IRRADIATION
APPARATUS
AND
PROCEDURE
The reagent solution to be sparked was introduced into a specially constructed vessel (Fig. one platinum electrode was 1) where (4 submerged within the solut’ion and the other (B) was suspended about 5 mm. above the liquid surface (C). The vessel was designed to contain a lo-ml. solution within the chamber. The vessel was then closed and remained open only to ventilation ports (I>) at the top which brought in and released acid- and thymol-filtered air under pressure. This arrangement removed unde,sired gases resulting from the sparking process. Filtration of the air before entry enhanced its microbiological purity. The spark was supplied from a high voltage source such as an induction coil, a transformer, or a trio of Tesla coils directly connected to t,he chamber’s electrodes. In this manner. a visible spark passed continuously between t,he upper electrode and the solution surface. The usual sparking period lasted two hours. il magnetic stirrer mixed the solution continuously. To minimize the possibility of contamination, high purity reagents and double distilled water were used in preparing solutions to be sparked. The glass reaction vessel was cleansed with chromic acid solution each time before use. Then, a lo<;,, (v/v) aqueous solution of reagent was sparked. Standard microbiological analyses in303
m
302
STEINMAN
AND
D
B --mmI I IC
nI
A I
FIG. 1. Schematic drawing illustrating vessel with submerged electrode (A), electrode (B), liquid surface (C), and ports (D).
sparking suspended ventilation
dicated solutions, effects.
in product nonspecific
no appreciable thus tending
AMINO
contamination to rule out
ACID SYNTHESIS
Paper Chromatography A 10% (v/v) aqueous solution of acetic acid (Baker Analyzed Reagent) was sparked for two hours under filtered air. Then, the product was separated with the following solvent systems in single dimension descending chromatography on Whatman No. 1 and No. 4 filter papers: (a) n-butanol:pyridine:water (6:6:6); (b) n-butanol: acetic acid:water (4:1:5); and (c) phenol:water (88:12). On the chromatograms, the sparked acetic acid product yielded a spot in all three solvent systems which favorably matched a glycine standard run in parallel when sprayed with a O.2’+& (w/v) solution of ninhydrin in Rbutanol or acetone. When an aqueous solution of acetic acid was sparked under an all nitrogen atmosphere, the product also gave a ninhydrin spot by paper chromatography which matched the glycine standard. Another, much less concentrated spot on the chromatogram appeared to be aspartic acid. Two other spots remained unidentified since the purpose of the procedure was merely to observe at least one or two representatives of the general class of amino acids.
Ion Exchange Chromdography A 10% (v/v) was sparked for was then applied exchange column
aqueous solution of acetic acid four hours under purified air. It to a prepared Dowex 50-X4 ion and eluted with pH 3.52 citric
LILLEVIK acid buffer similar to the method of Moore and Stein (6). The elution was carried out at room temperature and followed with ninhydrin reagent. Several well defined peaks were observed. In particular, fraction # 6 (later found to be glycine) had a concentration of 2.98 X 1OP mM per liter. A sample of acetic acid sparked under nitrogen also gave a positive ninhydrin response when tested in solution by the Moore and Stein method, whereas the original acetic acid reagent exhibited a negative response to this test. Using the Moore and Stein ninhydrin determination on a quantitative basis, considerably more amino group production was noted spectrophotometrically when the sparking of acetic acid took place under nitrogen than under air.
Derivatives Both an aqueous acetic acid solution sparked under air and another sparked under an all nitrogen atmosphere gave positive Hinsberg reactions for primary amines. One of the fractions (No. 6) from the ion exchange separation of sparked acetic acid yielded a tosyl derivative which had the same melting point as the corresponding glytine derivative. The sparking procedure under air was carried out on a 0.01% aqueous solution of sodium acetate bearing a Cl4 label at the carboxyl carbon (New England Nuclear). The Hinsberg test was performed on the product where after acidification to precipitate the derivative, the mixture was filtered and rinsed on No. 42 paper. The filter paper was then washed with 2 N NaOH to solubilize the amides originally formed from any primary amines. Both the first and second filtrates indicated radioactivity by scintillation counting; the first was probably due to residual acetate and the second to primary amines in the sparked product. The same procedure was repeated but with sparking under an all nitrogen atmosphere rather than air. In this case, evidence for the production of primary amines was also observed.
Radiotracers A 0.01% aqueous solution of CY-labeled sodium acetate was sparked under nitrogen for 5.5 hours. Nonlabeled glycine was then added as a carrier to the sparked labeled product. An aliquot of the mixture was placed on Whatman No. 1 paper which was run as a two dimensional chromatogram. Butanol-acetic acid-water (4:1:5) solvent was used in the first dimension and butanol-pyridinewater (6:6:6) solvent in the second. The chromatogram was then dried and placed in juxtaposition with X-ray film for three days in the dark. The X-ray was then developed to observe the radio-
ABIOTIC active products and sprayed with ninhydrin standard. Comparison sprayed chromatogram one of the radioactive glyrine standard, both
AMINO-CARBONYL
SYNTHESIS
chromatogram was locate the glycine of the X-ray with the indicated a coincidence of products and the “cold” in location and shape.
OF
AMINO
the
to
COMPOUND
SYNTHESIS
Reducing Properties A 10% (v/v) aqueous solution of glycerol (Central Scientific) was sparked under air for 2 hours. The reducing properties of the product solution were noted with Fehlings reagent as well as the ferricyanide and 2,4-dinitrophenylhydrazine tests Several spots were observed when the sparked glycerol product was chromatographed on Whatman No. 1 paper with et,hyl acetate.pyridine: water tFO:25:20) solvent in the ascending direction and was then dipped in benzidine solution.
Carbohydrate-Like Properties The sparked glycerol product yielded positive responses to the orthoaminodiphenyl, Molisch, and benxidine-periodate procedures. In particular, the product solution also exhibited a positive Bial reaction.
Amino Groups A 1O76 (v/v) aqueous glycerol solution was sparked under air for four hours and then separated on a prepared Dowex 50-X4 ion exchange column with 0.3 -v HCI. The eluates were followed with the ferricyanide, Elson-Morgan, and MooreStein ninhydrin reactions. Several peaks were observed, some of which were positive to all three tests. Likewise, the original unfractionated product solution exhibited positive responses to these three tests. A 1O70 (w/v) aqueous solution of ribose (Nutritional Biochemicals) was sparked under air for two hours. It was then placed on Whatman No. 1 paper in parallel with an aliquot of the sparked glycerol solution and resolved with ethyl acetate: pyridine:water (60:25:20) solvent in the descending direction. The sparked ribose exhibited several ninhydrin-positive spots, two of which matched a pair from the sparked glycerol.
Quantitative Analysis By the orthoaminodiphenyl test using a glucose standard, the sparked glycerol product appeared to contain 2.387;, of reducing material. With the Elson-Morgan procedure employing a glucosamine standard, 0.0837, of the product bore the or-amino-
305
GROUPS TABLE
I
SUMMARY OFTHE&UALITATIVEANALYSISOFTHE SPARKED
GLYCEROL
PRODUCT Test
SSltIlple
Fehling
Ribose Glucosamine Glycerol Sparked glycerol”
glucosamine fraction #lb
ninhy- Elsondrin Morgan
+” + -
+ --++ -
+
-
-
+
+
+
+
+
-
ferricyanide
ninhydrin
ElsonM0rgall
+ +
+ +
+ +
Test
Pertinent specificity
(7) (8)
reducing sugars carbohydrates, except 2amino sugars pentose, 2-deoxypentose, hexuronic acid, triose a-amino groups a-amino-aldehyde grouping reducing sugars
Fehling Molisch Bial
AMolisch Bial
(7)
Ninhydrin Elson-Morgan
(6) (7)
Ferricyanide
(9)
” Total product solution D Major fraction from the ion exchange separation of the sparked glycerol on Dowex 50-X4 with 0.3 iV HCI. c + positive response; - negative response.
aldehyde grouping. material synthesized group adjacent to hours of sparking.
Thus, 3.4% of all reducing appeared to have an amino its reducing group. after 2
pH CHANGES The sparking filtered). of nitric
pH
of water dropped significantly with under an air atmosphere (not acidThis was possibly due to the formation acid as observed earlier by Cavendish.
ULTRAVIOLETIRRADIATION Ninhydrin-positive material was produced from an aqueous solution of acetic acid when an ultraviolet lamp replaced the spark as the energy source. The sample was irradiated in an open Petri dish under air for 45 hours at 4°C. with the ultraviolet lamp (Mineralight, model R5, IJV
306
STEINMAN
Products, solution Morgan
Inc.). A similarly of glycerol exhibited reaction.
treated a positive
AND aqueous Elson-
DISCUSSION
Amino groups, in the form of amino acids and amino-carbonyl compounds, were synthesized in the presence of oxygen. Atmospheric nitrogen was probably the source of the amine nitrogen in the products since the synthesis was enhanced under an all nitrogen atmosphere in contradistinction to air. The precautions taken in the selection and preparation of solvents and solutions most likely ruled out predissolved forms of nitrogen as being significant contributors. Molecular nitrogen is known to dissociate into its atoms in an electrical discharge (10). From there, it could have possibly interacted with hydrogen to form a reactive precursor to the amino group such as an amide radical, which is also a known species (11). Therefore, one possible sequence of steps in the observed amino acid synthesis may be the following :
LILLEVIK
the positive Molisch and Bial reactions noted for the whole sparked glycerol product were possibly not induced by the particular ion exchange fraction noted. In accordance with the objectives set up for the experiment, evidence for the production of amino groups from nitrogen by sparking an alcohol in the presence of oxy gen was observed and documented. At least some of the products obtained from sparked glycerol displayed an amino group adjacent to a reducing group. In conjunction with the sparking of glycerol, additional substances resulted which exhibited certain characteristics of pentoselike and other carbohydrate-like materials. An aminoribose type of substance may have been one of them. Note added in proof: Glucose in 0.01 N HCI, when sparked under nitrogen, exhibited positive ninhydrin and ElsonMorgan reactions, as well as the same chromatographic properties as glucosamine-HCl. ACKNOWLEDGMENTS
N2-+2N CH,COOH + CH,COOH + H N + 3HzO + HNO, + 2H2 + H N + 2H + NH2 CH,COOH + NH, --) NH,CH,COOH
Such a sequence would also account for the lowering of pH. A similar sequence would probably be the case in the sparking of glycerol except that the oxidations and exchanges taking place at appropriate carbons would have to be considered as well. Although it is difficult to establish if all the steps of the syntheses took place within an aqueous environment, it is of importance to note that in all cases, the source of carbon was originally dissolved in aqueous solution. It was observed that the sparking of glycerol gave a product containing fractions which had the properties of reducing groups, amino groups, and amino sugars together. Often a substance exhibit,ing a positive Molisch reaction will not appear positive to the ninhydrin test nor the Elson-Morgan test (12). The latter procedure is quite diagnostic for the Lu-amino-aldehydic grouping, assuming no interferences. Therefore,
The authors are indebted to Drs. J. R. Brunner, C. Ponnamperuma, and C. Sheba for their helpful suggestions. Portions of this work were carried out at Michigan State University, as well as Tel Hashomer Hospital, Tel Aviv, Israel and NASAAmes Research Center, Moffett Field, California. This project was supported in part by the NSF Undergraduate Research Program at Michigan State University. REFERENCES 1. GLOCKLER, G., AND LIND, S. C., “Electrochemistry of Gases and Other Dielectrics.” Wiley, New York, 1939. 2. BABOR, J. A., “General Chemistry.” Thomas Y. Crowell Co., New York, 1929. 3. MAXWELL, C. R., PETERSON, D. C., AND SHAWLESS, N. E., ~udiation Res. 1, 530 (1954). Chemistry of Water 4. ALLEN, A. O., “Radiation and Aqueous Solutions.” Van Nostrand, Princeton, New Jersey, 1961. 5. OPARIN, A. I., “Origin of Life.” Dover, New York, 1953. 6. MOORE, S., AND STEIN, W. H., J. Bid. Chem. 192, 663 (1951). Biochemistry.” 7. LITWACK, G., “Experimental Wiley, New York, 1960.
ABIOTIC
SYNTHESIS
8. MORROW, C. A., AND SANDSTROM, W. M., “Biochemical Laboratory Methods.” 2nd edition, Wiley, New York, 1927. 9. ASHWELL, G., Calorimetric Analysis of Sugars, in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.) Academic Press, New York, 1957. 10. HESLOP, It. B., AND ROBINSON, P. L., “In-
OF
AMINO organic
GROIJPS
305
Chemistry.”
Elsevier,
New
York,
1960. 11. GOCLD, Organic
E. S.,
“Mechanism
Chemistry.”
and Holt,
New
Structure York,
in 1962.
.12. KENT, P. W., AND WHITEHOUSE, M. W., “Biochemistry of the Amino Sugars.” Academic Press, New York, 1955.
OF BIOCHEMISTRY
AND
Abiotic
BIOPHYSICS
106,303-307
Synthesis
of Amino
G. D. STEINMAX From
the Department
o.f Biochemistry,
Received
(1%)
Groups
H. A. LILLEVIK
AND
Michigan
State
October
University,
E. Lansing,
Michigan
9, 1963
An attempt was made to synthesize amino groups under nonreducing conditions from nitrogen. An aqueous solution of acetic acid was electrically sparked and yielded amino acid-like substances. The sparking of a solution of glycerol gave a product containing reducing amino compounds, some of which had the or-amino-aldehyde grouping. In both cases, molecular nitrogen extracted from the gaseous phase appeared to be the source from which amino nitrogen resulted.
Berthelot was one of the first to study the effects of silent electrical discharge on various materials in the presence of nitrogen (1). He noted that small amounts of nitrogen became fixed to the substrates in the process. Later, Cavendish observed that when an electric spark passed through a mixture of oxygen and nitrogen, nitric acid was synthesized (2). Irradiation of glycine yields acetic acid (3). Therefore, in order to investigate the possibility of synthesizing amino groups in the form of amino acids by sparking, acetic acid was chosen as a reactant in the present experiments. Likewise, it has already been shown that irradiation of alcohols yields reducing substances (4). Therefore, since amino group production was being studied, electric sparking of alcohols was explored for possible synthesis of aminocarbonyl cornpounds. The scope of the synthetic methods employed was defined by the intention to work under possible primordial conditions of simple reactants and high energy stimulators. The means for the prebiotic appearance of the reagents used have been proposed by Oparin (5). Nitrogen, in the presence of oxygen, was utilized in order to demonstrate the plausibility of amino group synthesis at that stage of the Earth’s development when the atmosphere contained an appreciable amount of oxygen. Likewise, the proposed
synthetic method would suggest a means by which an amino group could be added to certain substances while in solution. EXPERIMENTAL SPARKING IRRADIATION
APPARATUS
AND
PROCEDURE
The reagent solution to be sparked was introduced into a specially constructed vessel (Fig. one platinum electrode was 1) where (4 submerged within the solut’ion and the other (B) was suspended about 5 mm. above the liquid surface (C). The vessel was designed to contain a lo-ml. solution within the chamber. The vessel was then closed and remained open only to ventilation ports (I>) at the top which brought in and released acid- and thymol-filtered air under pressure. This arrangement removed unde,sired gases resulting from the sparking process. Filtration of the air before entry enhanced its microbiological purity. The spark was supplied from a high voltage source such as an induction coil, a transformer, or a trio of Tesla coils directly connected to t,he chamber’s electrodes. In this manner. a visible spark passed continuously between t,he upper electrode and the solution surface. The usual sparking period lasted two hours. il magnetic stirrer mixed the solution continuously. To minimize the possibility of contamination, high purity reagents and double distilled water were used in preparing solutions to be sparked. The glass reaction vessel was cleansed with chromic acid solution each time before use. Then, a lo<;,, (v/v) aqueous solution of reagent was sparked. Standard microbiological analyses in303
m
302
STEINMAN
AND
D
B --mmI I IC
nI
A I
FIG. 1. Schematic drawing illustrating vessel with submerged electrode (A), electrode (B), liquid surface (C), and ports (D).
sparking suspended ventilation
dicated solutions, effects.
in product nonspecific
no appreciable thus tending
AMINO
contamination to rule out
ACID SYNTHESIS
Paper Chromatography A 10% (v/v) aqueous solution of acetic acid (Baker Analyzed Reagent) was sparked for two hours under filtered air. Then, the product was separated with the following solvent systems in single dimension descending chromatography on Whatman No. 1 and No. 4 filter papers: (a) n-butanol:pyridine:water (6:6:6); (b) n-butanol: acetic acid:water (4:1:5); and (c) phenol:water (88:12). On the chromatograms, the sparked acetic acid product yielded a spot in all three solvent systems which favorably matched a glycine standard run in parallel when sprayed with a O.2’+& (w/v) solution of ninhydrin in Rbutanol or acetone. When an aqueous solution of acetic acid was sparked under an all nitrogen atmosphere, the product also gave a ninhydrin spot by paper chromatography which matched the glycine standard. Another, much less concentrated spot on the chromatogram appeared to be aspartic acid. Two other spots remained unidentified since the purpose of the procedure was merely to observe at least one or two representatives of the general class of amino acids.
Ion Exchange Chromdography A 10% (v/v) was sparked for was then applied exchange column
aqueous solution of acetic acid four hours under purified air. It to a prepared Dowex 50-X4 ion and eluted with pH 3.52 citric
LILLEVIK acid buffer similar to the method of Moore and Stein (6). The elution was carried out at room temperature and followed with ninhydrin reagent. Several well defined peaks were observed. In particular, fraction # 6 (later found to be glycine) had a concentration of 2.98 X 1OP mM per liter. A sample of acetic acid sparked under nitrogen also gave a positive ninhydrin response when tested in solution by the Moore and Stein method, whereas the original acetic acid reagent exhibited a negative response to this test. Using the Moore and Stein ninhydrin determination on a quantitative basis, considerably more amino group production was noted spectrophotometrically when the sparking of acetic acid took place under nitrogen than under air.
Derivatives Both an aqueous acetic acid solution sparked under air and another sparked under an all nitrogen atmosphere gave positive Hinsberg reactions for primary amines. One of the fractions (No. 6) from the ion exchange separation of sparked acetic acid yielded a tosyl derivative which had the same melting point as the corresponding glytine derivative. The sparking procedure under air was carried out on a 0.01% aqueous solution of sodium acetate bearing a Cl4 label at the carboxyl carbon (New England Nuclear). The Hinsberg test was performed on the product where after acidification to precipitate the derivative, the mixture was filtered and rinsed on No. 42 paper. The filter paper was then washed with 2 N NaOH to solubilize the amides originally formed from any primary amines. Both the first and second filtrates indicated radioactivity by scintillation counting; the first was probably due to residual acetate and the second to primary amines in the sparked product. The same procedure was repeated but with sparking under an all nitrogen atmosphere rather than air. In this case, evidence for the production of primary amines was also observed.
Radiotracers A 0.01% aqueous solution of CY-labeled sodium acetate was sparked under nitrogen for 5.5 hours. Nonlabeled glycine was then added as a carrier to the sparked labeled product. An aliquot of the mixture was placed on Whatman No. 1 paper which was run as a two dimensional chromatogram. Butanol-acetic acid-water (4:1:5) solvent was used in the first dimension and butanol-pyridinewater (6:6:6) solvent in the second. The chromatogram was then dried and placed in juxtaposition with X-ray film for three days in the dark. The X-ray was then developed to observe the radio-
ABIOTIC active products and sprayed with ninhydrin standard. Comparison sprayed chromatogram one of the radioactive glyrine standard, both
AMINO-CARBONYL
SYNTHESIS
chromatogram was locate the glycine of the X-ray with the indicated a coincidence of products and the “cold” in location and shape.
OF
AMINO
the
to
COMPOUND
SYNTHESIS
Reducing Properties A 10% (v/v) aqueous solution of glycerol (Central Scientific) was sparked under air for 2 hours. The reducing properties of the product solution were noted with Fehlings reagent as well as the ferricyanide and 2,4-dinitrophenylhydrazine tests Several spots were observed when the sparked glycerol product was chromatographed on Whatman No. 1 paper with et,hyl acetate.pyridine: water tFO:25:20) solvent in the ascending direction and was then dipped in benzidine solution.
Carbohydrate-Like Properties The sparked glycerol product yielded positive responses to the orthoaminodiphenyl, Molisch, and benxidine-periodate procedures. In particular, the product solution also exhibited a positive Bial reaction.
Amino Groups A 1O76 (v/v) aqueous glycerol solution was sparked under air for four hours and then separated on a prepared Dowex 50-X4 ion exchange column with 0.3 -v HCI. The eluates were followed with the ferricyanide, Elson-Morgan, and MooreStein ninhydrin reactions. Several peaks were observed, some of which were positive to all three tests. Likewise, the original unfractionated product solution exhibited positive responses to these three tests. A 1O70 (w/v) aqueous solution of ribose (Nutritional Biochemicals) was sparked under air for two hours. It was then placed on Whatman No. 1 paper in parallel with an aliquot of the sparked glycerol solution and resolved with ethyl acetate: pyridine:water (60:25:20) solvent in the descending direction. The sparked ribose exhibited several ninhydrin-positive spots, two of which matched a pair from the sparked glycerol.
Quantitative Analysis By the orthoaminodiphenyl test using a glucose standard, the sparked glycerol product appeared to contain 2.387;, of reducing material. With the Elson-Morgan procedure employing a glucosamine standard, 0.0837, of the product bore the or-amino-
305
GROUPS TABLE
I
SUMMARY OFTHE&UALITATIVEANALYSISOFTHE SPARKED
GLYCEROL
PRODUCT Test
SSltIlple
Fehling
Ribose Glucosamine Glycerol Sparked glycerol”
glucosamine fraction #lb
ninhy- Elsondrin Morgan
+” + -
+ --++ -
+
-
-
+
+
+
+
+
-
ferricyanide
ninhydrin
ElsonM0rgall
+ +
+ +
+ +
Test
Pertinent specificity
(7) (8)
reducing sugars carbohydrates, except 2amino sugars pentose, 2-deoxypentose, hexuronic acid, triose a-amino groups a-amino-aldehyde grouping reducing sugars
Fehling Molisch Bial
AMolisch Bial
(7)
Ninhydrin Elson-Morgan
(6) (7)
Ferricyanide
(9)
” Total product solution D Major fraction from the ion exchange separation of the sparked glycerol on Dowex 50-X4 with 0.3 iV HCI. c + positive response; - negative response.
aldehyde grouping. material synthesized group adjacent to hours of sparking.
Thus, 3.4% of all reducing appeared to have an amino its reducing group. after 2
pH CHANGES The sparking filtered). of nitric
pH
of water dropped significantly with under an air atmosphere (not acidThis was possibly due to the formation acid as observed earlier by Cavendish.
ULTRAVIOLETIRRADIATION Ninhydrin-positive material was produced from an aqueous solution of acetic acid when an ultraviolet lamp replaced the spark as the energy source. The sample was irradiated in an open Petri dish under air for 45 hours at 4°C. with the ultraviolet lamp (Mineralight, model R5, IJV
306
STEINMAN
Products, solution Morgan
Inc.). A similarly of glycerol exhibited reaction.
treated a positive
AND aqueous Elson-
DISCUSSION
Amino groups, in the form of amino acids and amino-carbonyl compounds, were synthesized in the presence of oxygen. Atmospheric nitrogen was probably the source of the amine nitrogen in the products since the synthesis was enhanced under an all nitrogen atmosphere in contradistinction to air. The precautions taken in the selection and preparation of solvents and solutions most likely ruled out predissolved forms of nitrogen as being significant contributors. Molecular nitrogen is known to dissociate into its atoms in an electrical discharge (10). From there, it could have possibly interacted with hydrogen to form a reactive precursor to the amino group such as an amide radical, which is also a known species (11). Therefore, one possible sequence of steps in the observed amino acid synthesis may be the following :
LILLEVIK
the positive Molisch and Bial reactions noted for the whole sparked glycerol product were possibly not induced by the particular ion exchange fraction noted. In accordance with the objectives set up for the experiment, evidence for the production of amino groups from nitrogen by sparking an alcohol in the presence of oxy gen was observed and documented. At least some of the products obtained from sparked glycerol displayed an amino group adjacent to a reducing group. In conjunction with the sparking of glycerol, additional substances resulted which exhibited certain characteristics of pentoselike and other carbohydrate-like materials. An aminoribose type of substance may have been one of them. Note added in proof: Glucose in 0.01 N HCI, when sparked under nitrogen, exhibited positive ninhydrin and ElsonMorgan reactions, as well as the same chromatographic properties as glucosamine-HCl. ACKNOWLEDGMENTS
N2-+2N CH,COOH + CH,COOH + H N + 3HzO + HNO, + 2H2 + H N + 2H + NH2 CH,COOH + NH, --) NH,CH,COOH
Such a sequence would also account for the lowering of pH. A similar sequence would probably be the case in the sparking of glycerol except that the oxidations and exchanges taking place at appropriate carbons would have to be considered as well. Although it is difficult to establish if all the steps of the syntheses took place within an aqueous environment, it is of importance to note that in all cases, the source of carbon was originally dissolved in aqueous solution. It was observed that the sparking of glycerol gave a product containing fractions which had the properties of reducing groups, amino groups, and amino sugars together. Often a substance exhibit,ing a positive Molisch reaction will not appear positive to the ninhydrin test nor the Elson-Morgan test (12). The latter procedure is quite diagnostic for the Lu-amino-aldehydic grouping, assuming no interferences. Therefore,
The authors are indebted to Drs. J. R. Brunner, C. Ponnamperuma, and C. Sheba for their helpful suggestions. Portions of this work were carried out at Michigan State University, as well as Tel Hashomer Hospital, Tel Aviv, Israel and NASAAmes Research Center, Moffett Field, California. This project was supported in part by the NSF Undergraduate Research Program at Michigan State University. REFERENCES 1. GLOCKLER, G., AND LIND, S. C., “Electrochemistry of Gases and Other Dielectrics.” Wiley, New York, 1939. 2. BABOR, J. A., “General Chemistry.” Thomas Y. Crowell Co., New York, 1929. 3. MAXWELL, C. R., PETERSON, D. C., AND SHAWLESS, N. E., ~udiation Res. 1, 530 (1954). Chemistry of Water 4. ALLEN, A. O., “Radiation and Aqueous Solutions.” Van Nostrand, Princeton, New Jersey, 1961. 5. OPARIN, A. I., “Origin of Life.” Dover, New York, 1953. 6. MOORE, S., AND STEIN, W. H., J. Bid. Chem. 192, 663 (1951). Biochemistry.” 7. LITWACK, G., “Experimental Wiley, New York, 1960.
ABIOTIC
SYNTHESIS
8. MORROW, C. A., AND SANDSTROM, W. M., “Biochemical Laboratory Methods.” 2nd edition, Wiley, New York, 1927. 9. ASHWELL, G., Calorimetric Analysis of Sugars, in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.) Academic Press, New York, 1957. 10. HESLOP, It. B., AND ROBINSON, P. L., “In-
OF
AMINO organic
GROIJPS
305
Chemistry.”
Elsevier,
New
York,
1960. 11. GOCLD, Organic
E. S.,
“Mechanism
Chemistry.”
and Holt,
New
Structure York,
in 1962.
.12. KENT, P. W., AND WHITEHOUSE, M. W., “Biochemistry of the Amino Sugars.” Academic Press, New York, 1955.