INTRODUCTION:

The significant questions, how, when and where first speck of life begin and how far back chemical events in remote inanimate era gave rise to fundamental biomolecules have been engaging the minds of great thinkers, scientists and philosophers^1-7 since antiquity. The ancient sages of India held the view that five fundamental elements{Kshiti, Jal, Pavak, Gagan and Sameera} were responsible for the evolution of life and finding of past more than half a century are leading to us the same conclusion.

According to our present understanding ,the universe is 10 to 15 billion years old and there has been wide spread organic material present in the Milky Way Galaxy and in the solar system which was evolved by the collapse of the interstellar cloud about 4.6 billion years ago⁸. Atmospheric components like H₂, NH₃, H₂O and CH₄ are believed to have been formed under the action of UV light ,lightening, shock waves, heat and other forms of energy^9-12.

The primordial biomolecules like amino acids, purine, pyrimidines, sugars etc. and their precursors have been detected in meteorites and in the interstellar space. Hydrogen cyanide is considered to be a major precursor of nitrogenous organic molecules leading to the formation of cyanamide, cyanoacetylene and nitriles^13-14.

Hydrogen cyanide, acetonitrile, cyanoacetylene and amino nitriles have been reported as reactive intermediates during discharge experiments^15-16.Formation of acetonitrile by photolysis of acetylene and ammonia has been reported by Ferris and Ishikava¹⁷

CH≡CH +NH₃ → CH₃C≡ N

Kotake et.al¹⁸ reported that thermal energy produces hydrogen cyanide in high yield from methane and ammonia in the presence of silica or aluminium oxide.

CH₄ +NH₃ → HCN +3H₂+60 k.cal

C₂H₂ +2NH₃ → 2HCN +4H₂ +63 k.cal

CO + NH₃ → HCN +H₂O +10 k.cal

Presence of acetonitrile has also been detected in comets, in interstellar medium and is formed in Fischer-Tropsch Type (FTT) reaction from CO, NH₃ and H₂ as reported by Hoyatsu et.al.¹⁹.

Thus the making and breaking processes resulted in the formation of a number of simple molecules either in interstellar medium, comets and meteorites or in hydrothermal system of primitive earth or transported to them after being formed elsewhere on earth.

Hydrogen cyanide has been proposed as one of the key component in prebiological synthesis. The cyanohydrin mechanism has been suggested by Milller²⁰ to explain the formation of α-amino acids.

RCHO +NH₃+HCN → RCH(NH₂) CN +H₂O

RCH (NH₂) CN +2H₂O → R.CH (NH₂) COOH +H₂O

Oro and Kamat²¹, Low et. al.²², Ferris et. al.²³, have successfully synthesized amino acids, amino acid polymers, fatty acids and purines from aqueous solution of acetonitrile. Therefore, attempts have been made here to synthesise amino acids from aqueous solution of acetonitrile in presence and absence of silica and alumina under prebiotic wetting and drying cycles of primitive sea beach environment that may throw some light on the process of chemical evolution leading to the evolution of life on earth.

EXPERIMENTAL PROCEDURE:

Preparation of solution:

All the investigations were carried out in aqueous medium. Sterilized double distilled water was used as the solvent in every experiment wherever necessary the vapor of the double distilled water was allowed to pass through the reaction vessels. Every care was taken to ensure the purity of the samples employed.

Experimental solution (5 ml each) with pH 9.0 ± 0.5 were heated in borosil glass reaction vessels- Kjeldhal flasks (100 ml) fitted with air condensers on hot plates at a temperature of 90 ± 5⁰C in the presence and absence of metal oxides (silica, alumina). Samples of reaction concentrates were analyzed for the possible formation of amino acids using chromatographic techniques on Whatman No- 01 paper both by uni- and two dimensional chromatography using butanol- acetic acid- water (4:1:1 v/v, 4:1:5 v/v upper layer), butanol- acetic acid- pyridine- water (15:3:10:12 v/v) and phenol- water (80:20 v/v). Amino acid spots were visualized with ninhydrin, identified with isatin and also by comparison of their Rf values with authentic amino acids as well as of their DNP derivatives. For the separation of DNP amino acids the solvent system used was n- butanol saturated with water. Colorimetric estimation of amino acids was carried out by comparison of color intensity of the unknown compound with that of a standard solution employing photochemical colorimeter MK III.

Ultraviolet spectra:

Ultraviolet absorption spectra of the various reaction mixtures or elutes of some products were determined in aqueous solution using Jasco V- series spectrophotometer.

Infra red spectroscopy:

IR spectra of reaction concentrates were recorded in Perkin Elmer 881 (4000-6000 cm^-1) spectrophotometer.

High performance liquid chromatography:

The reaction products were further identified by High Performance Liquid Chromatography which were ascertained by Shimadzu SPD- 10 A UV visible detector with C₁₈ column using triple distilled water: methanol (80:20 v/v) and 0.1% H₃PO₄: acetonitrile (40:60 v/v) as mobile phase, flow rate 1.5 ml/min at pH 7.0, temperature 25⁰C and UV detector monitored at 197-210 nm for the detection of amino acids. Results were compared with retention times of the standard amino acids run in the same HPLC column under similar conditions.

Source and procedure of heating under wetting – drying conditions:

Hot plates and heating mental (Ambassdor, temperature, range 0-100^oC) were used for the reaction solution in pyrex/ borosil glass reaction vessels fitted with air condensers (120cm length plugged with surgical sterilized cotton). Heating was continued carefully till the last drop of solutions were dried and 5ml of deionised water was suspended for the new cycle to start. Thus heating was done for 8-10 hrs/day in each cycle under wetting-drying conditions.

Procedure for taking out experimental solution:

Portions of heated samples were taken out with the help of sterilized measuring cylinders of different measurements. For the chromatographic analysis, the heated samples were concentrated in vacuum evaporator. The concentrates of experimental samples were analysed by paper chromatography on Whatman No. 1 filter paper chromatographically and also by chemical methods

Solvent system for amino acids:

Solvent system used in analysing amino acids were:

i. n- butanol-acetic acid-water [4:1:1 v/v]

ii. n-butanol-acetic acid-water [4:1:5 v/v upper layer]

High performance liquid chromatography [hplc]:

The reaction products were further identified by high performance liquid chromatography using Shimadzu SPD-10A UV- visible detector with C₁₈ column using triple distilled water: methanol (80:20 v/v) and 0.1% H₃PO₄: acetonitrile (40:60 v/v) as mobile phase, flow rate 1.5 ml/min at pH 7.0, temperature 25⁰C and UV detector at 197-210 nm for the detection of amino acids. Results were compared with retention times of the standard

RESULTS:

Formation of amino acids from reaction system of acetonitrile-water vapor have been carried out in presence and absence of metal oxides believed to have existed near hydrosphere-lithosphere boundaries of the primitive sea. The presence of large amount of clay and metal oxides on the primitive earth has been reported and played an important role in the origin and early evolution of life.

The effect of heat on reaction system comprised of acetonitrile and water vapour in presence and absence of metal oxides (silica and alumina) has been investigated for the possible formation of amino acids in round bottom flasks kept on hot plates at 90±5 ⁰C under wetting and drying conditions. The pH was maintained at 8.5 ± 0.5. Heating was continued till the last drop of reaction concentrate was left. After completion of each cycle fresh double distilled water was added to the flask for the next cycle to start. Heated concentrate of acetonitrile and water vapour drawn out periodically after 10 hrs, 25 hrs, 50 hrs, and 100 hrs were subsequently analyzed by paper chromatography and HPLC for the formation of amino acids. The resulting products were further characterized by various physico -chemical methods and UV/IR spectral studies.

Paper chromatographic analysis of reaction concentrate heated for short period up to 10 hrs showed no ninhydrin positive spots on the papergram .Prolonging the duration of heating for 25 hrs showed four chromatographically separable ninhydrin positive products. Products glycine (II) and leucine (IX) were formed in appreciable amount. (fig.1). Heating the reaction system up to 50 hrs enhanced the amount of all these products along with the formation of three new products (fig.1). On extending the heating period up to 75 hrs, in all nine products appeared on the papergram. Lysine was formed in moderate amount while other products in good amount. On further prolonging the duration of heating for 100 hrs almost identical range of products were formed. Glycine (II)was formed in good amount while products lysine, glutamic acid, α-alanine valine and leucine were formed in moderate amount fig (1). However, identity of products V, VI and VIII could not be ascertained Fig (1).

The quantitative estimation and physico-chemical properties of the resulting thermal products have been recorded in table 01 and shown in figure 01.

Thus, time lapse studies have shown that formation of amino acids from aqueous solution of acetonitrile resulted in the formation of amino acids depending upon the duration of heating under wetting and drying conditions.

The UV-absorption spectra of the reaction concentrate of acetonitrile solution exposed to heat upto 100 hrs. showed a band at 203nm.The band corresponding to 203 nm may be due to the presence of a mixture of amino acids because their absorption lies in this region 193-220nm (Fig 02).

Table -01. Heat induced (90 ±5⁰C) synthesis of amino acids from reaction system of acetonitrile -water vapor under wetting –drying conditions of primitive earth upto 100 hrs.

Composition of reaction system

Duration of heating

No. of products formed in (Quantity mg/lit.)

Fig.

Ref.

I II III IV V VI VII VIII IX

CH₃CN-H₂O (V)

2. R_f in:

B:A:W(4:1:1) V/V

3. Colour with

i. Ninhydrin

ii. Isatin

4. Solubility in

a. Ether

b. Water

5. UV- Fluorescence

6. R_f of standard amino

acids in B:A:W(4:1:1) V/V

7.Amino acids overlapped in co-

Chromatography

8. Amino acids identified

10 hrs. - - - - - - - - -

25 hrs. T 0.65 - 0.39 - - - - 0.52 (1)

50 hrs. T 0.34 T 0.32 - - - - 0.36 (1)

75 hrs. 0.22 0.40 0.19 0.41 - - 0.44 - 0.40 (1)

100 hrs. 0.30 0.51 0.26 0.45 - - 0.51 - 0.49 (1)

22 30 43 47 50 56 61 72 80

V R V V BV V Y V V V

PBr P PB BP RV P P PB BP

Ins ins ins ins ins ins ins ins ins

S S S S S S S S S

WB WB WB WB WB WB WB WB WB

12.1 17 24.9 28 36 40 43 50 52.2

Lys Gly Glu α- Ala - - Val - Leu

Table- 2. Quantity (mg/lit) and physico-chemical characteristics of products formed from reaction system of acetonitrile-water vapour heated up to 100 hrs with metal oxides under wetting-drying conditions.

Composition of Reaction system		No. of products formed
Composition of Reaction system		I	II	III	IV	V	VI	VII	VIII	IX	Fig. ref.
CH₃CN-H₂O(V)-Al₂O₃		T	0.36	0.51	0.42	T	0.65	T	0.86	T	(4)
CH₃CN-H₂O(V)-SiO₂		T	0.44	0.59	0.53	T	0.74	T	0.91	T	(4)
1	R_f (%) in : B:A:W 4:1:1V/V	29	32	38	42	49	53	57	65	69
2	Colour with 1.Ninhydrin 2.Isatin	V R	BV R	V R	RV R	V R	V R	Y R	V R	V R
3	Solubility a. Ether b. Water	ins S	ins S	ins S	Ins S	ins S	ins S	ins S	Ins S	Ins S
4	UV Fluorescence	WB	WB	WB	WB	WB	WB	WB	WB	WB
5	Amino Acid over lapped in co-chromatography	lys	asp	ser	Gly	glu	α-ala	-	Val	Leu
6	Amino acid identified	lys	asp	ser	Gly	glu	α-ala	-	Val	Leu

BAW 4:1:1 V/V n-butanol: acetic acid: water 4:1:1V/V; -,not detected; T- trace ; V- violet; B- blue; P- pink; W, white; R, red; ins- insoluble; s-soluble; lys , lysine; asp, aspartic acid; ser, serine: gly, glycine; glu,glutamic acid; α-ala, α-alanine; val , valine ; leu, leucine

The results were further confirmed by high performance liquid chromatography. 10μl sample of reaction concentrate of acetonitrile and water vapour heated up to 100 hrs was injected in Shimadzu SPD 10A UV–visible detector with C18 column monitored at 210 nm, mobile phase 1 % H₃PO₄ (pH 2.8), temperature 24⁰C, flow rate 1.0 ml/min showed peaks corresponding to glutamic acid (3.840 min.), glycine (4.023 min), α-alanine (4.421 min.), valine (5.088 min.), leucine (6.965min.) and lysine (10.001min.) matched with standard amino acids run under identical conditions.(Fig 03)

Heating the reaction system of acetonitrile and water vapour with silicon oxide for a period of 100 hrs under wetting and drying condition and on subsequent chromatographic analysis showed the formation of nine ninhydrin positive products on the papergram. Out of these ,products aspartic acid, serine, glycine , α-alanine and valine were formed in appreciable amount while other products in trace amount. Results are recorded in table 2 and illustrated in fig 04.

When the same reaction system was heated in the presence of alumina for 100 hrs under identical conditions, showed considerable increase in the intensity of all the reaction products.

A comparative study of the quantity and the nature of the products formed by heating the reaction system of acetonitrile and water vapor under wetting and drying condition with metel oxides, showed that the effect of sensitizers on heat induced synthesis of amino acids was in the following order:

Al₂O₃>SiO₂>CH₃CN-H₂O

FIG-01: Chromatogram showing the formation of amino acids from reaction system of acetonitrile- water vapor heated Upto 100 hrs under wetting and drying conditions.

Fig 02:UV absorption spectra of reaction concentrate of acetonitrile-water vapor heated upto 100 hrs.

Fig 03: HPLC of reaction concentrate of acetonitrile-water vapor heated upto 100 hrs.

Fig 04: Chromatogram showing the formation of amino acids from reaction system of acetonitrile- water vapor heated with metal oxides (silica &alumina) upto 100 hrs under wetting and drying conditions.

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Received on 31.12.2012 Modified on 17.01.2013

Asian J. Research Chem. 6(2): February 2013; Page 101-105