TRANSFORMATION
OF SOME
TO OTHER
HYDROXY
AMINO
AMINO
ACIDS
ACIDS
A. S. U. CHOUGHULEY, A. S. SUBBARAMAN, Z. A. KAZI, and M. S. CHADHA Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India
(Received 16 April, 1975) Abstract. It has been observed that fl-hydroxy-a-amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehydereactions, glycine also reacts with acetone to give fl-hydroxyvalineunder prebiologically feasible conditions. It is suggested, therefore, that the formation of fl-hydroxy-c~-aminoacids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions. 1. Introduction
The formation of serine and threonine by the reaction of formaldehyde and acetaldehyde respectively with a glycine-copper complex has been described by various authors (Akabori et al., 1959; Otani and Winitz, 1960; Sato et aI., 1957; Kutani et al., 1959). Likewise, the synthesis of some other aliphatic fl-hydroxy-c~-amino acids, by the reaction of appropriate aldehyde and ketones with a glycine-copper complex has been reported by Herman Mix (1962). However, fl-phenylserine could be obtained more simply, by the reaction of benzaldehyde with glycine in alkaline solution (Shaw and Fox, 1953). Recently, we have shown that these fl-hydroxy-~-amino acids are also formed when glycine and appropriate aldehydes are heated in dilute ammoniacal solutions (Subbaraman et al., 1972). It was observed that if higher temperatures were used, or phosphorous acid, phosphoric acid and their salts were present in the reaction mixture, alanine was formed along with serine. Likewise, with threonine, c~-aminobutyric acid was obtained. It was, therefore, likely that in the presence of acids or their salts, reduced or alkyl amino acids were being formed. Since phosphorous acid, phosphoric acid and their salts have prebiotic relevance, these were used in the present studies. The formation of corresponding alkyl amino acids by heating these fl-hydroxy-c~amino acids with phosphorous acid, phosphoric acid or their ammonium salts is reported in this paper. The possible significance of these reactions in chemical evolution is also considered. 2. Materials and Methods
Serine and threonine used in the present work were suppplied by Sigma Chemical Co., U.S.A. fl-Phenylserine (Shaw and Fox, 1953) and fl-hydroxyvaline (Mix, 1962) were prepared by the methods reported in the literature. Phosphoric acid was obtained from Reidel De Halen Ag., and phosphorous acid was prepared according to the Origins of Life 6 (1975) 527-535. All Rights Reserved Copyright 9 1975 by D. Reidel Publishing Company, Dordreeht-Holland
528
A.S.U. CHOUGHULEY ET AL.
procedure of Voight and Gallais (1953). Diammonium hydrogen phosphate was procured from E. Merck, Germany. A Beckman Unichrom Amino Acid Analyzer was used for ion exchange chromatography of the amino acids formed in the reactions. Gas chromatography was carried out on Toshniwal Gas Chromatograph Type RL04, supplied by Toshniwal Bros., India. Labelled amino acids (-1-14C) were made available by the Isotope Division of Bhabha Atomic Research Centre. The N M R spectra were recorded on a Varian A60A Spectrometer. The experimental conditions are summarized in the tables. In a typical experiment, an aqueous solution of the hydroxy amino acid and phosphorous acid, phosphoric acid or the corresponding ammonium salt was heated in a sealed tube at the desired temperature, for an appropriate length of time. An aliquot of the reaction mixture was taken in citrate buffer and analyzed by ion exchange chromatography (IEC). Another aliquot was examined by two dimensional paper chromatography (descending, Whatman No. 1) using n-butanol-acetic acidwater (100:22: 50) and phenol-water-16N NH4OH (80:20: 1) as the solvent system. For further support of the tentative identifications made by the above procedures, co-chromatography on the analyzer column with standard amino acids was employed. For co-chromatography on paper, the individual spots from a preparative paper chromatogram were eluted with 2 ~ acetic acid, mixed with the appropriate standard compounds and rechromatographed. The solvent systems used in the two directions were (1) n-propanol-ethanol-water (70:20: 10) and (2) pyridine-water (65:35). For the identification of alanine, valine and phenylalanine, appropriate radioactive amino acid tracers (alanine- 1- ~4C, valine- 1-14C and phenylalanine- 1-14C respectively) were added to the eluted material before rechromatography. The compounds were then identified by the coincidence technique of chromatography-autoradiography (Ponnamperuma et al., 1964; Choughuley and Lemmon, 1966). Gas chromatography was employed for further characterization of the products. The TAB (N-trifluoroacetyl-n-butyl ester) derivatives of the amino acids from the reaction mixture as well as of individual amino acids of interest were prepared by the method of Roach and Gehrke (1969). The columns used were SE-30 (6' x 0.25", 5~ on 80/100 mesh, DMCS treated chromosorb G, glass)and EGS (6'• 0.85", 1~ on 80/100 mesh, DMCS treated chromosorb G, stainless steel). The retention times of the amino acid derivatives from the reaction mixture were the same as those of the expected individual amino acids. Co-chromatography with the derivatives of expected amino acids gave enhanced peaks in each case, thus confirming the homogeneity of the amino acids. The conversion of the CHzOH group of serine or the -CH(OH) group of threonine
I
to corresponding -CH3 or -CH2- groups to give alanine and ~-aminobutyric acid (~-ABA), respectively, was also confirmed by comparison of the N M R data: Alanine (experimental as well as standard sample): 6(D20), 1.48 (d, 3H, -CH-CH3), 3.78 (q, 1H,-CH-CH3), 4.63 (HDO).
TRANSFORMATION OF HYDROXY AMINO ACIDS
529
c~-Aminobutyric acid (experimental as well as standard sample): ~(D20), 0.97 (t, 3H,-CH2-CH3), 1.91 (m, 2H, CHE-CH3), 3.75 (t, 1H,-CH-CH2-), 4.58 (H_DO). For N M R spectra, the reaction mixtures containing the best yields of alanine and a-ABA were freed of salts and impurities by passing through Dowex-50H + (100-200 mesh), eluting with 1 N NH4OH, followed by freeze drying to obtain them as fine powders. As an analogy to the condensation of aldehydes with glycine (Subbaraman et al., 1972), the formation of fl-hydroxyvaline by the reaction of acetone with glycine was examined. Glycine (1 mmole) and acetone (0.3 ml) in ammoniacal solution (1 ml, pH 8.5) were heated at 100 ~ for 50 hr fl-Hydroxyvaline was formed in 5.7% yield. The comparison of its chromatographic and co-chromatographic behaviour on paper, by IEC and of its TAB derivative by GC was made with fl-hydroxyvaline (or its TAB derivative) prepared by the method of Herman Mix (1962). fl-Hydroxyvaline prepared by the latter method was used for transformation studies. 3. Results
The data in Table I indicate that alanine and glycine are formed when an aqueous solution of serine is heated with phosphorous acid, phosphoric acid, and their ammonium salts. The best yields of alanine were observed in experiments 2 and 9 ( ~ 22% and ~ 32% resp.). At lower temperatures, the formation of alanine was lower while more serine was recovered. The effect of the duration of heating (25 hr or 50 hr) was only marginal. In some cases longer heating periods seemed to give slightly reduced yields of the transformed products, although all the serine was degraded: At lower concentrations (10- 2 M ) of both the reactants, and at 120 ~ serine recovery was about 70%, with the formation of very small amounts of alanine and traces of glycine. At 140 ~ although most of the serine was degraded, the formation of alanine was around 2% and that of glycine, about 1%. With diammonium hydrogen phosphate (Expt. 11) alanine was formed in 7% yield, while in a control experiment (Expt. 12) in water (pH 8.5) it was obtained in less than 17ooyield. In the case of threonine (Table II), in addition to the expected e-aminobutyric acid and glycine, some formation of alanine was also observed. Serine was detected only when a 1 M solution of threonine, either with phosphorous acid or with phosphoric acid, was heated at 140 ~. Unlike serine and threonine, fi-phenylserine (Table III) gave the best yield of phenylalanine (3.5%) when a 1 M solution was heated with phosphorous acid at 120 ~ for 25 hr. Glycine formation was appreciable in all the experiments. fl-Hydroxyvaline with phosphorous acid in 1 M solution at 140 ~ for 50 hr gave glycine (47%), valine (0.39%) and recovered fl-hydroxyvaline (1.4%). When cysteine and cystine were heated with phosphoric acid in 1 M solution (pH 2, 140 ~ 50 hr), alanine and glycine were the only two products formed (21.8% and 30.0% alanine; and 0.9 and 0.7% glycine respectively). These experiments were carried out
~30
A.S.U. CHOUGHULEY
E T AL.
O
oo
o
[-,
co
9 o
Z O
t~ O
O'O
.,m o
T R A N S F O R M A T I O N OF H Y D R O X Y A M I N O ACIDS
IOO
I
J
I
l
IO
I
53 ]
[
G
O g~
O
O t~
OF SOME
TO OTHER
HYDROXY
AMINO
AMINO
ACIDS
ACIDS
A. S. U. CHOUGHULEY, A. S. SUBBARAMAN, Z. A. KAZI, and M. S. CHADHA Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India
(Received 16 April, 1975) Abstract. It has been observed that fl-hydroxy-a-amino acids are transformed into other amino acids, when heated in dilute solutions with phosphorous acid, phosphoric acid or their ammonium salts. It has been shown that as in the case of previously reported glycine-aldehydereactions, glycine also reacts with acetone to give fl-hydroxyvalineunder prebiologically feasible conditions. It is suggested, therefore, that the formation of fl-hydroxy-c~-aminoacids and their transformation to other amino acids may have been a pathway for the synthesis of amino acids under primitive earth conditions. 1. Introduction
The formation of serine and threonine by the reaction of formaldehyde and acetaldehyde respectively with a glycine-copper complex has been described by various authors (Akabori et al., 1959; Otani and Winitz, 1960; Sato et aI., 1957; Kutani et al., 1959). Likewise, the synthesis of some other aliphatic fl-hydroxy-c~-amino acids, by the reaction of appropriate aldehyde and ketones with a glycine-copper complex has been reported by Herman Mix (1962). However, fl-phenylserine could be obtained more simply, by the reaction of benzaldehyde with glycine in alkaline solution (Shaw and Fox, 1953). Recently, we have shown that these fl-hydroxy-~-amino acids are also formed when glycine and appropriate aldehydes are heated in dilute ammoniacal solutions (Subbaraman et al., 1972). It was observed that if higher temperatures were used, or phosphorous acid, phosphoric acid and their salts were present in the reaction mixture, alanine was formed along with serine. Likewise, with threonine, c~-aminobutyric acid was obtained. It was, therefore, likely that in the presence of acids or their salts, reduced or alkyl amino acids were being formed. Since phosphorous acid, phosphoric acid and their salts have prebiotic relevance, these were used in the present studies. The formation of corresponding alkyl amino acids by heating these fl-hydroxy-c~amino acids with phosphorous acid, phosphoric acid or their ammonium salts is reported in this paper. The possible significance of these reactions in chemical evolution is also considered. 2. Materials and Methods
Serine and threonine used in the present work were suppplied by Sigma Chemical Co., U.S.A. fl-Phenylserine (Shaw and Fox, 1953) and fl-hydroxyvaline (Mix, 1962) were prepared by the methods reported in the literature. Phosphoric acid was obtained from Reidel De Halen Ag., and phosphorous acid was prepared according to the Origins of Life 6 (1975) 527-535. All Rights Reserved Copyright 9 1975 by D. Reidel Publishing Company, Dordreeht-Holland
528
A.S.U. CHOUGHULEY ET AL.
procedure of Voight and Gallais (1953). Diammonium hydrogen phosphate was procured from E. Merck, Germany. A Beckman Unichrom Amino Acid Analyzer was used for ion exchange chromatography of the amino acids formed in the reactions. Gas chromatography was carried out on Toshniwal Gas Chromatograph Type RL04, supplied by Toshniwal Bros., India. Labelled amino acids (-1-14C) were made available by the Isotope Division of Bhabha Atomic Research Centre. The N M R spectra were recorded on a Varian A60A Spectrometer. The experimental conditions are summarized in the tables. In a typical experiment, an aqueous solution of the hydroxy amino acid and phosphorous acid, phosphoric acid or the corresponding ammonium salt was heated in a sealed tube at the desired temperature, for an appropriate length of time. An aliquot of the reaction mixture was taken in citrate buffer and analyzed by ion exchange chromatography (IEC). Another aliquot was examined by two dimensional paper chromatography (descending, Whatman No. 1) using n-butanol-acetic acidwater (100:22: 50) and phenol-water-16N NH4OH (80:20: 1) as the solvent system. For further support of the tentative identifications made by the above procedures, co-chromatography on the analyzer column with standard amino acids was employed. For co-chromatography on paper, the individual spots from a preparative paper chromatogram were eluted with 2 ~ acetic acid, mixed with the appropriate standard compounds and rechromatographed. The solvent systems used in the two directions were (1) n-propanol-ethanol-water (70:20: 10) and (2) pyridine-water (65:35). For the identification of alanine, valine and phenylalanine, appropriate radioactive amino acid tracers (alanine- 1- ~4C, valine- 1-14C and phenylalanine- 1-14C respectively) were added to the eluted material before rechromatography. The compounds were then identified by the coincidence technique of chromatography-autoradiography (Ponnamperuma et al., 1964; Choughuley and Lemmon, 1966). Gas chromatography was employed for further characterization of the products. The TAB (N-trifluoroacetyl-n-butyl ester) derivatives of the amino acids from the reaction mixture as well as of individual amino acids of interest were prepared by the method of Roach and Gehrke (1969). The columns used were SE-30 (6' x 0.25", 5~ on 80/100 mesh, DMCS treated chromosorb G, glass)and EGS (6'• 0.85", 1~ on 80/100 mesh, DMCS treated chromosorb G, stainless steel). The retention times of the amino acid derivatives from the reaction mixture were the same as those of the expected individual amino acids. Co-chromatography with the derivatives of expected amino acids gave enhanced peaks in each case, thus confirming the homogeneity of the amino acids. The conversion of the CHzOH group of serine or the -CH(OH) group of threonine
I
to corresponding -CH3 or -CH2- groups to give alanine and ~-aminobutyric acid (~-ABA), respectively, was also confirmed by comparison of the N M R data: Alanine (experimental as well as standard sample): 6(D20), 1.48 (d, 3H, -CH-CH3), 3.78 (q, 1H,-CH-CH3), 4.63 (HDO).
TRANSFORMATION OF HYDROXY AMINO ACIDS
529
c~-Aminobutyric acid (experimental as well as standard sample): ~(D20), 0.97 (t, 3H,-CH2-CH3), 1.91 (m, 2H, CHE-CH3), 3.75 (t, 1H,-CH-CH2-), 4.58 (H_DO). For N M R spectra, the reaction mixtures containing the best yields of alanine and a-ABA were freed of salts and impurities by passing through Dowex-50H + (100-200 mesh), eluting with 1 N NH4OH, followed by freeze drying to obtain them as fine powders. As an analogy to the condensation of aldehydes with glycine (Subbaraman et al., 1972), the formation of fl-hydroxyvaline by the reaction of acetone with glycine was examined. Glycine (1 mmole) and acetone (0.3 ml) in ammoniacal solution (1 ml, pH 8.5) were heated at 100 ~ for 50 hr fl-Hydroxyvaline was formed in 5.7% yield. The comparison of its chromatographic and co-chromatographic behaviour on paper, by IEC and of its TAB derivative by GC was made with fl-hydroxyvaline (or its TAB derivative) prepared by the method of Herman Mix (1962). fl-Hydroxyvaline prepared by the latter method was used for transformation studies. 3. Results
The data in Table I indicate that alanine and glycine are formed when an aqueous solution of serine is heated with phosphorous acid, phosphoric acid, and their ammonium salts. The best yields of alanine were observed in experiments 2 and 9 ( ~ 22% and ~ 32% resp.). At lower temperatures, the formation of alanine was lower while more serine was recovered. The effect of the duration of heating (25 hr or 50 hr) was only marginal. In some cases longer heating periods seemed to give slightly reduced yields of the transformed products, although all the serine was degraded: At lower concentrations (10- 2 M ) of both the reactants, and at 120 ~ serine recovery was about 70%, with the formation of very small amounts of alanine and traces of glycine. At 140 ~ although most of the serine was degraded, the formation of alanine was around 2% and that of glycine, about 1%. With diammonium hydrogen phosphate (Expt. 11) alanine was formed in 7% yield, while in a control experiment (Expt. 12) in water (pH 8.5) it was obtained in less than 17ooyield. In the case of threonine (Table II), in addition to the expected e-aminobutyric acid and glycine, some formation of alanine was also observed. Serine was detected only when a 1 M solution of threonine, either with phosphorous acid or with phosphoric acid, was heated at 140 ~. Unlike serine and threonine, fi-phenylserine (Table III) gave the best yield of phenylalanine (3.5%) when a 1 M solution was heated with phosphorous acid at 120 ~ for 25 hr. Glycine formation was appreciable in all the experiments. fl-Hydroxyvaline with phosphorous acid in 1 M solution at 140 ~ for 50 hr gave glycine (47%), valine (0.39%) and recovered fl-hydroxyvaline (1.4%). When cysteine and cystine were heated with phosphoric acid in 1 M solution (pH 2, 140 ~ 50 hr), alanine and glycine were the only two products formed (21.8% and 30.0% alanine; and 0.9 and 0.7% glycine respectively). These experiments were carried out
~30
A.S.U. CHOUGHULEY
E T AL.
O
oo
o
[-,
co
9 o
Z O
t~ O
O'O
.,m o
T R A N S F O R M A T I O N OF H Y D R O X Y A M I N O ACIDS
IOO
I
J
I
l
IO
I
53 ]
[
G
O g~
O
O t~
