INTRODUCTION:

Nucleic acids along-with proteins constitute the main matrix of living systems. The important purine and pyrimidine bases of nucleic acids might have synthesized from primordial gases and other related compounds at an early stage of chemical evolution.

Oparin^1,2 postulated that life originated in the water of seas and oceans of the primitive earth when the atmosphere was reducing. He also suggested that life on earth must have arisen from a preformed 'pool' of organic compounds. According to this theory^1,3. The spontaneous generation was indeed the original pathway of life, although its 'spontaneity' had to be stretched to a billion years or more. Most of the Carbon present on early earth was in the form of hydrocarbons, Nitrogen in the form of Ammonia and Oxygen in the form of Water along-with noble gases. The powerful argument in favour of reduced primitive atmosphere was further supported by Urey⁴, Kuhn⁵ and others^6,7.

In presence of high energy radiations such as X-rays, γ-rays, UV light electric discharge, thunderstorms and volcanic eruptions large amount of heat was generated and simple organic compounds of C,H,N and Owere formed under ozone free atmosphere. Gradually the earth cooled and the organic compounds formed relatively in high temperature came down to the surface of the earth along with rainwater and finally accumulated in oceans^8,9. Due to repeated evaporation and condensation of rain droplets. The temperature of the earth crust cooled to about 1000^oC or lower and the resulting highly reactive free radicals as •CH,•CH₂,•CH₃,•NH₂,•H,•OH etc formed from CH₄,NH₃,H₂O vapours on intense photolysis as well as thermal fragmentation could have recombined to form a varierty of saturated and unsaturated hydrocarbons as follows:

H─OH→H+OH

C─H• →•CH

•CH +•CH→CH₄

2CH₄→HC≡CH

•CH +•CH→HC≡CH

2CH₄→HC≡CH +3H₂

Metal carbides and steam react with each other to form hydrocarbons as outlined in the following reaction- CaC₂+H₂O→HC≡CH+ CaO Ferris¹⁰ has reviewed various chemical processes of prebiotic origin leading to the formation of precursors of proteins and nucleic acids. Edmond et al.¹¹ and Von domm et al.¹² proposed that the submarine hydrothermal fluids contained high concentrations of metal ions such as iron, mangnese, copper and zinc which may be able to catalyse organic synthesis reactions and which are essential to many present day biochemical processes. Egami¹³ emphasized the role of trace elements in chemical evolutions and suggested that a close correlation exists between the concentration of transition elements in contemporary sea water and their biological importance. Ammonia was formed by direct combination of nitrogen and hydrogen in the upper surface of the atmosphere of few earlier stage of the existence of the Earth.

N₂+3H₂→2NH₃

The entire prebiotic phenonmenon leading to the formation of biomolecules in early evolution of life was termed as 'biopoesis' given by Bernal⁶. A concise but chemically relevant review of the introductory findings connected with formation of biologically important molecules such as amino acids, nucleic acid constituents and other related compounds.

Formation of biologically significant biomolecules from primordial gases such as acetylene, ammonia and water under a wide variety of conditions using various energy sources such as UV-light, X-rays, electric discharge and thermal energy have been reported by many earlier workers^14-20. However, investigations concerning biological synthesis of biomolecular matrices from acetylene- ammonia and water vapour in presence and absence of metal ferrocyanides available near sea shores or sea beds have not been studied in detail. Therefore an attempt have been made for possible synthesis of biomolecules such as nucleic acid constituents (purines/ pyrimidine bases) from reaction mixture of acetylene- ammonia and water vapour in presence and absence of metal ferrocyanides [Ni₂Fe(CN)₆, Zn₂Fe(CN)₆, Mn₂Fe(CN)₆, Co₂Fe(CN)₆, Cu₂Fe(CN)₆,] under simulated primitive earth's ocean- beach wetting/drying conditions using heat as a source of energy. The presence of large amount of solid surfaces such as, metal ferrocyanides, metal oxides and clay minerals on the prebiotic earth crust after the formation of hydrosphere has been reported.^22-26 The possibility that whether these solid surface play a role in the origin and early evolution of life on Earth?. This possibility has received considerable attention in the past few decades.

MATERIALS AND METHODS:

All the studies were carried out in aqueous medium. Sterilized double distilled water was used as the solvent in every experiment. Gases such as acetylene and ammonia were freshly prepared by usual methods^14-16in the laboratory before use.

Acetylene:

Acetylene was prepared by adding water drop by drop to calcium carbide. The gas was passed through a number of gas washing traps containing separately a solution of copper acetate, dil H₂SO₄ and finally it was passed through water. The gas thus purified was allowed to bubble through the solutions contained in several quartz vessels.

Ammonia:

Ammonia was prepared by slowly heating liquor ammonia of qualigens (A.R. Grade) on sand bath. Every care was taken to ensure the purity of the gases. All these gases, when used in the experiments were washed by passing through traps containing separately a solution of copper acetate, dilute sulphuric acid and finally it was washed through water. The gas thus purified was allowed to bubble through the solutions contained in several glass reaction vessels.

Preparation of Metal Ferrocyanides:

All the metal ferrocyanides were prepared by using Kourims methods²⁷. Potassium ferrocyanide solution (167ml, 0.1m) was slowly added to solution of respective metal salts (500ml, 0.1 M) with constant stirring. To improve the process of coagulation an excess of metal salt was used. The reaction mixture was than heated at 60°C on the water bath for 2-3 hrs and kept as such for 24 hrs, at ambient temperature. The precipitate were filtered and washed thoroughly with double distilled water and dried in an oven at 60°C. The dried product was ground and sieved with 100 mesh size sieve. Therefore, metal ferrocyanide grains used for adsorption studies were comprised of particles upto100 mesh size. Purity of metal ferrocyanides were checked by comparing the X-ray diffraction data of the complexes from literature. The relative intensity data and interplanner spacing [d] were also compared with reported values.

Experimental solution (10ml each) pH= 9±0.5 maintained at a pressure of 20±5mm/Hg were heated in borosil glass reaction vessels- Kjeldal flasks (100ml) fitted with air condenser on the hot plates at a temperature of 90±5^oC in the presence and absence of different metal ferrocyanides.

The reaction concentrates were analyzed by chromatographic techniques^28,29 on Whatman no. 1 paper by using n-butanol: acetic acid: water 4:1:5 V/V(upper layer). The developed and dried papergram was first washed with ether and then the papergram was either sprayed with or immersed in a solution of 0.25 M-mercuric nitrate in 0.5 nitric acid and finally with water. After drying the papergram was treated with dilute solution of ammonium sulphide. Black spots due to mercuric sulphide were appeared on the chromatogram in the location of purine and pyrimidine bases.

Ultra-violet absorption spectra of the various reaction mixture or elutes of some resulting products were determined by using Jasco UV/VIS-550 series spectrophotometer. IR spectra of the reaction concentrates were recorded in Perkin Elmer 881(4000-600 cm^-1) spectrophotometer.

The reaction samples were further identified by High performance liquid chromatography³⁰ using Shimadzu LC2010 CHT Luna 5μNH₂ 100A C₁₈ 250X4.60nm column UV-visible detector. For the detection of nucleic acid bases the UV-detector was monitored at 256nm. Triple distilled water : methanol (80:20 v/v) was taken as mobile phase, flow rate 1.5ml/min. at 23^oC temperature. Results were compared with retention times of the standard nucleic acid bases run in the same column under similar conditions.

RESULTS:

Gaseous mixture of acetylene-ammonia (25mm/hg each) in presence of water (10ml) was heated for varying periods up to 200hrs under simulated drying/ wetting, day/ night conditions assumed to be available near lithosphere/ hydrosphere boundary of primitive sea. The reaction concentrate was analyzed periodically by high performance liquid chromatography (HPLC) and paper chromatography (PC) for the plausible formation of nucleic acid bases. Best formation was observed at 200 hrs. of heating and revealed Cytosine (I), Uracil (II), Adenine(III), Thymine(V) and Hypoxanthine (VI).

In presence of nickel ferrocyanide and zinc ferrocyanide six distinct spots appeared on the papergram. Products I-VI were formed in lesser amount while other products V and VI were formed in relatively good amount. Presence of manganese ferrocyanide four products (I-III, V) were appeared on the papergram. Out of these, product III was formed in appreciable amount. In presence of copper ferrocyanide in total five products (I-III, V and VI) were formed. Out of these products I, III and VI were formed in appreciable amount, while product, II and V were formed in good amount. When cobalt ferrocyanide was used as sensitizer, five products (I,II, IV, V and VI) were appeared on the papergram. Of these, products I, II and IV were formed in appreciable amount. Products I, II, III, V and VI were identified as cytosine, uracil, adenine, thymine and hypoxanthine respectively, while product IV could not be identified. The quantity of resulting products (I, II, III and IV) were measured on the basis of optical density at their corresponding λ_max values.

Thus depending upon the resulting purine and pyrimidine bases the effect of metal ferrocyanides on the reaction system of acetylene, ammonia and water vapour under wetting and drying conditions was found as follows: Zn₂[Fe(CN)₆]> Ni₂[Fe(CN)₆]> Cu₂[Fe(CN)₆]> Co₂[Fe(CN)₆]> Mn₂[Fe(CN)₆].

From the above results it may be concluded that purine bases (adenine, guanine and hypoxanthine) were formed more easily than the pyrimidine (cytosine) bases from acetylene, ammonia and water vapour under drying/ wetting day/night conditions and metal ferrocyanides were found most effective catalysts for the formation of purine and pyrimidine bases. The stability and increase in optical density of the resulting nucleic acid bases ascertained due to ion- lone pair interaction between cations of different metals and nitrogen of purine/ pyrimidine bases.

The above reaction sample of acetylene - ammonia and water vapour heated upto 200 hrs. was further analyzed by HPLC. 10μl sample of reaction concentrate was injected in SHIMAZU LC 2010 CHT Luna 5μ NH₂ 100A C₁₈ 250 X 4.60 mm column monitored at 256 nm, mobile phase methanol: water (20: 80 V/V), temperature 23˚C, flow rate 1.5 ml/min showed the peaks corresponding to cytosine (1.814 min.), thymine (2.130 min.), hypoxanthine (2.465 min.) and adenine (2.920 min.) matched with standard nucleic acid bases run under identical conditions.

Results are illustrated in figures 1-6 and recorded in table 1 and 2.

Figure 1. Hour study of reaction system of C₂H₂-NH₃-H₂O(V)

Figure 2. HPLC of reaction system heated upto 200hrs

Figure 3. Chromatogram showing the formation of purine and pyrimidine bases

Figure 4. Bar diagram of products formed

Figure 5 UV-absorption spectra of adenine

Figure 6. IR spectra of Adenine

Table 1. Formation of purine and pyrimidine bases from reaction system of acetylene -ammonia- water vapour heated up to 200 hrs under primitive earth condition.

Composition of reaction system	Duration of heating	Optical density of products formed							fig. ref.
Composition of reaction system	Duration of heating	I	II	III	IV	V	VI	VII	fig. ref.
C₂H₂-NH₃-H₂O(V)	25hrs	-	-	-	-	-	-	-	(a)
	50hrs	0.128	0.131	0.109	-	-	-	-	(b)
	100hrs	0.268	0.201	0.251	-	-	-	-	(c)
	150hrs	0.329	0.108	0.209	-	0.219	0.119	-	(d)
	200hrs	0.139	0.11	0.265	0.112	0.206	0.265	0.131	(e)
	250hrs	0.132	0.142	-	0.201	-	-	-	(f)
Physico-chemical properties:
1.R_f(%) values using BAW (4:1:5 V/V upper layer)		17	28	39	43	49	54	60
2. Colour with a. Mercuric nitrate		Bl	Bl	Bl	Bl	Bl	Bl	Bl
b. Sodium bichromate		R	R	R	R	R	R	R
3.Fluorescence in UV-light (253 nm.)		BW	BW	BW	BW	BW	BW	BW
4.R_f (%) values of standard nucleic acid bases with: BAW (4:1:1 upper layer)		17.2	28.1	39	-	49.1	54	-
5. λ_max (nm) in acidic medium		276	259	262	-	265	250	-
6.Nucleic acid bases overlapped in co-chromatography		Cytosine	Uracil	Adenine	-	Thymine	Hypoxanthine	-
7.Products identified		Cytosine	Uracil	Adenine	-	Thymine	Hypoxanthine	-

BAW, n-butanol: acetic acid : water Not detected; Bl, black; R, red; BW- bluish white.

Table 2. Formation of purine and pyrimidine bases from reaction system of acetylene -ammonia and water vapour heated up to 200 hrs. in presence of metal ferrocyanides under prebiotic wetting and drying conditions.

Composition of reaction system	Optical density of products formed							fig.ref.
Composition of reaction system	I	II	III	IV	V	VI	VII	fig.ref.
C₂H₂-NH₃-H₂O(V)-[Ni₂Fe(CN)₆]	0.076	0.086	0.092	0.078	0.189	0.169	0.076	(b)
C₂H₂-NH₃-H₂O(V)-[Zn₂Fe(CN)₆]	0.089	0.068	0.098	0.079	0.198	0.160	0.089	(c)
C₂H₂-NH₃-H₂O(V)-[Mn₂ Fe(CN)₆]	0.192	0.109	0.637	-	0.192	-	0.192	(d)
C₂H₂-NH₃-H₂O(V)-[Cu₂ Fe(CN)₆]	0.629	0.239	0.642	-	0.212	0.625	0.629	(e)
C₂H₂-NH₃-H₂O(V)-[Co₂Fe(CN)₆]	0.608	0.421	-	0.629	0.098	0.121	0.608	(f)
Physico-chemical properties:
1.R_f(%) values using BAW (4:1:5 V/V upper layer)	16	27	38	44	48	55	16
2.colour with a. Mercuric nitrate	Bl	Bl	Bl	Bl	Bl	Bl	Bl
b.Sodium bichromate	R	R	R	R	R	R	R
3.Fluorescence in UV-light (253 nm.)	BW	BW	BW	BW	BW	BW	BW
4. λ_max (nm) in acidic medium	276	259	262	265	-	250	276
5. Products formed in co-chromatography	Cyto0.	Ura0.	Ade0.	-	Thy0.	Hypo	Cyto0.
6.Products identified	Cyto0.	Ura0.	Ade	0.-	Thy0.	Hypo	Cyto0.

BAW, n-butanol: acetic acid : water; - not detected; Bl, black; R, red;; BW- bluish white, Cyto; Cytosine, Ura; uracil, Ade; adenine, Thy; thymine, Hypo; Hypoxanthine

The UV- fluorescent spots corresponding to cytosine and adenine were confirmed by UV and IR which showed resemblance with authentic cytosine and adenine as follows-

(a) Cytosine: The IR spectra showed absorption frequency 3500-3300cm^-1 due to NH stretch of NH₂ group, 3300- 3000 cm^-1due to imino (=NH) group, 1690- 1640 cm^-1 due to >COgroup in pyrimidine ring 1650- 1450 cm^-1 (w)due to NH bending like that of authentic cytosine.

(b) Uracil: The UV-absorption band of the eluted product in water was found at λ_max 260.5nm. which was identical with reference uracil. IR spectra of this product was showed absorption frequency at 3.108.41 cm^-1 due to -NH stretching of imino group, 1734.13 cm^-1 due to C=O group in pyrimidine ring, 1662.20 cm^-1 due to NH bending like authentic uracil.

(c). Adenine: The UV- spectra of this product was perfectly matched with standard adenine λ_max 262 nm in acidic medium. The IR spectra of product III, which resembled with authentic adenine, showed absorption frequency at 3400- 3300 cm^-1 due to N-H stretch of NH₂ group and 1650-1450 cm^-1 due to N-H bending.

ACKNOWLEDGEMENT:

Authors are grateful to the Head of Chemistry and Physics department for providing necessary research facilities.

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Received on 05.12.2021 Modified on 22.01.2022

Asian J. Research Chem. 2022; 15(2):133-137.

DOI: 10.52711/0974-4150.2022.00021