The first step of the material characterization is to identify the purity of the crystalline complexes. Figure 1a-drepresentsMnHCCo, FeHCCo, NiHCCo and ZnHCCo complexes XRD patterns. JCPDS diffraction filesareused for analyzing theXRD pattern of MHCCo complexes. Diffraction peaks obtained are carefully matched with the relative intensities of the MHCCo complexes, JCPDS file no. for MnHCCo (22–1167), JCPDS file no. for FeHCCo (89-3736), JCPDS file no. for NiHCCo (22–1184) and JCPDS file no. for ZnHCCo (32–1468). The FT-IR spectrum of MHCCo complexes showed four significant peaks. In case of ZnHCCo, the band occurs at 2181 cm− 1 corresponds to strong CN stretching frequency, at 1607 cm− 1 peak represents the O–H bending of interstitial water molecules, 700 cm− 1 occurs due to bending of metal-carbon while band occurs at 451 cm− 1 represent the metal-cyanide bending. The bands occur due to otherMHCCocomplexes are indicated in Table 1.
The MHCCocomplexes were further characterized by TG/DT analysis. Figure 2a-d represents the obtained thermograms. With the help of TG curve of MHCCo complexes, degree of hydration was calculated. Figure 2a represent thermogram forMnHCCo complexes that indicated mass loss which corresponds to nearly two water molecules, FeHCCo complex showed a mass loss ofthree water molecules (Fig. 2b), NiHCCo complex showed a mass loss of three water molecules (Fig. 2c), ZnHCCo complex showed a mass loss of four water molecules (Fig. 2d). The percentage of C, H, N in the MHCCo complexes were analyzed by CHNS analysis and are depicted in Table 2. The experimental results obtained by elemental analysis matched with the theoretical value. The synthesized MHCCo complexes were analyzed by XRD, CHN analysis, TG/DTA, are as follows:
1. Mn3[Co(CN)6]2.2H2O (Brown) ;
2. Fe3[Co(CN)6]2.3H2O (Blue);
3. Ni3[Co(CN)6]2.3H2O (Sky Blue);
4. Zn3[Co(CN)6]2.4H2O (White)
The Fig. 3a, 3b, 3c and 3d show the FE-SEM images and EDX spectra of MnHCCo, FeHCCo, NiHCCo and ZnHCCo respectively. The structural morphology of FeHCCo, NiHCCo and ZnHCCo particles was observed to be spherical and even, whereas that of MnHCCo particles appeared to be polygon in shape (square shape was observed mostly). The particles size of FeHCCo, NiHCCo and ZnHCCo was found to be uniform suggesting a narrow size distribution, and that ofMnHCCo particles was found to be non-uniform suggesting a wide size distribution. The energy dispersive X-ray (EDX) spectra clearly indicate the presence of the corresponding metal in the MHCCo complexes.
To find the catalyzing properties of the MHCCo complexes, the oligomerization reaction of amino acid (glycine and alanine) were carried out at varies temperature i.e 60, 90, 120°C for 5 weeks (7, 14, 21, 28, 35 days)in the presence of MHCCo complexes. We studied the effects of temperature and time for the glycine and alanine oligomerization on MHCCo complexes. The catalytic efficiency of tested MHCCo complexes varies considerably with time and temperature are shown in Fig. 4(a-c) to 7(a-c). The yields of MHCCocatalyzedglycine and alanine oligomerization at different temperature 60, 90, 120°C on a completion of 5th week are summarized in Table 3 and Table 4.It is found that, product yield Vs time as functions of temperature behave linear relationship. The yields on MHCCocomplexes are much higher than those produced in blank experiment. On the completion of 5th week, diketopiperazine(DKG) of glycine and dimer of glycine, Glycyl-glycine (gly)2 was found in experiments of glycine without catalyst, while peptide formation was not obtained in experiment of alanine without catalyst. Formation of DKG(glycine), dimer of glycine and absence of condensation of alanine in the control experiment from glycine and alanine in absence ofMHCCo complexes, the results is in accord with the previous studies [39, 52].MHCCo complexes provide the surface for catalyzing the thermal condensation of glycine and alanine at relatively short time at temperature below 100°C. For identification and quantification of the obtained product in the reaction mixtures were analyzed by HPLC and ESI-MS techniques.The analysis of the reaction products by HPLC and ESI-MS revealed that peptides up to tetramer were formed from glycine while dimer in case of alanine. The Fig. 8(a-d) and 9(a-d) represent the HPLC chromatogram showing the separation of diketopiperazine, oligomers of glycine and alanine at optimum conditions. In the experiment, reaction was performed at varied temperature from 60 to 120°C for 5 weeks without applying dry/ wetting cycle. The reaction was monitored per week.
When we compared the runs, on the completion of 5th week, at lower temperature i.e. 60°C, only trimer of glycine, Glycyl-glycyl-glycine (Gly3) were observed in the presence of Mn-, Zn-HCCoand dimer of alanine (Ala2) were observed in the presenceof all three MHCCo complexes except FeHCCo but with low yield (Fig. 4a-7a) and without the presence of localized heat sources. Thus, in the presence of MHCCo, abiotic peptide synthesis occurs at short interval of time. It was observed that when the reaction temperature was 90°C, peptide was formed after short reaction times i.e after 1week in both the glycine and alanine with comparable yield. It has been found that yield of peptides were maximum on the completion of 4th week (Fig. 4b-7b) and after thatit was constant even at high temperature (Fig. 4c-7c).Thus our result reveals that the favorable condition for the polymerization of amino acids on the surface of MHCCo complexes are 90°C temperature and duration is 4 weeks. But in case of formation ofDKP(Gly) and DKP(Ala) on MHCCo complexes, it was observed that formation was more favorable from thermodynamic and kinetic point of view. DKP(Gly) and DKP(Ala) showed high yields, may be due to the low concentration of aqua layer on the MHCCo surface as compared to surrounding temperature at 120°C which do not favor elongation instead supports removal of water molecules from the dimeric glycine and alanine. In this study we found that high temperature supports the formation of DKPs, previously reported studies also revealed the same results [24, 31, 40]. Under hydrothermal conditions, rate of reaction for the formation of dimer and DKPs was also studied by Kawamura and co-workers [53, 54]. Bujdak and Rojas  found that polymerization of glycine and alanine using three different form of alumina i.e., acidic, neutral and basic and observed when neutral alumina used as a catalyst shows maximum polymerization of glycine and alanine.Activated alumina has been used as energy source for peptide bond formation , and this activated alumina oligomerize glycine upto (gly)11 . Catalytic activity of Ferrihydrite is due to its nanoporous high surface which dimerize glycine and alanine . According to Ferris, oligomer upto 55 monomer long have been formed by using mineral surface  while glycine condensation upto 16 unit (poly-Gly) have been done on SiO2 and TiO2 surface . At low temperature (-20°C) by using carbon dots as a photocatalyst polymerize amino acids to protein . Glycine polymerization by thermal condensation occurs on SiO2  and metal ferrite surface .
Double metal cyanide (DMC), MaI[MII(CN)n]b.xH2O is aninorganic coordinated complexes with 3D network. In DMC, inner metal MII is allied with the external metal MI by number of cyano-bridges (MII-C ≡ N-MI) where MI=divalent metal ions (Zn2+, Fe2+, Cd2+, Co2+, Cu2+, Ni2+, Mn2+ etc.)andMII=transitional metal ions (Fe2+, Fe3+, Co2+, Ni2+, Cr3+,Mo4+etc.). In DMC, the active site is the external metal MIwhich supports the catalytic functions.It was examined that DMC having varies inner and outer spheremetalsexhibited different catalytic properties. Ifexternal metaliszincon the surface of octacyanomolybdate (IV), exhibited higher catalytic activity for the formation of oligomers of glycine and alanine, comparedwithoctacyanomolybdate (IV) having other metals such as Mn2+,Fe2+,, Co2+,, Ni2+,, Cu2+,, Cd2+ . Present results showed that hexacyanocobaltate complexes having zinc as an external metal also showed evidence of high catalytic activity towards glycine and alanine i.e formation of oligomers of glycine and alanine, compared to other metals in outer sphere ofhexacyanocobaltate complexes such as Mn2+,Fe2+, Ni2+. The Table 3and Table 4 shows the % yield of Cyclic (Gly)2, di-, tri-, tetramer of glycine and Cyclic(Ala)2, dimer of alanine on the surface of MHCCo complexes. It was also observed that ZnHCCo and MnHCCo oligomerize glycine upto tetramer, NiHCCo afforded glycine upto the trimer while the FeHCCo oligomerize upto the dimer. ZnHCCo complex formed (Gly)4 (0.36%), (Gly)3(1.19%), (Gly)2(11.97%), DKP(Gly)(15.73%) from glycine while (Ala)2(6.27%), Cyclic(Ala)2(8.31%) formed from alanine on the completion of 5th week (35 days) at 90°C temperature. From the present study we also revealed that yield of glycine oligomers in the presence of MHCCo complexes is much more than that of oligomer of alanine, due to high activation energy required for alanine oligomerization . Lower amount and efficiency of catalytic sites may be the other factor which can be responsible for lower oligomerization of alanine on MHCCocomplexes .Greenstein reported thatstability constants of co-ordination complexes with amino acids are higher than the peptides supports the formation of oligomers of glycine and alanine . This mechanism revealed that as the chain length of amino acid elongates, oligomers concentration decreases (Table 3 and Table 4). On the basis of % yield of glycine and alanine oligomers in the presence of MHCCo complexes shows following catalytic activity trend:
ZnHCCo > MnHCCo > NiHCCo > FeHCCo
Surface area of MHCCo complexes (Table 5) and the yield of peptide bond formation (Table 3 and Table 4), observed that surface area of MHCCo complexes plays an important parameter for the polymerization of amino acids. Among MHCCo, ZnHCCo has highest surface area (683 m2g− 1) showed high catalytic activity for the peptide bond formation while FeHCCo has lower surface area (S.A = 167 m2g− 1) exhibited minimum catalytic activity.
The ESI-MS technique provide the additional analytical technique for the detection of oligomers of glycine and alanine in terms of mass, m/z = (M + H)+ ions, M indicated the amino acid/oligomers to be analyzed. Figures 10 and 11 represents the ESI-MS spectrum of formation of oligomers of glycine and alanine respectively on the surface of ZnHCCo at optimum temperature on the completion of 4th week. The ESI-MS (Fig. 10) clearly supports the formation of DKP (glycine), dimer, trimer, tetramer of glycine.The76.1 mass of glycine obtained in ESI-MS represents [Gly + H]+, 115 for [CycGly2 + H]+, 132.9 for [Gly2 + H]+, 189.9 for [Gly3 + H]+, 246.6 for [Gly4 + H]+. Similarly in Fig. 11ESI-MS for formation of oligomers of alanine such as DKP (alanine), dimer of alanine at optimum temperature on the completion of 4th week at the surface of ZnHCCo are shown. The 90.1 mass of alanine observed in ESI-MS represents [Ala + H]+, mass value 143 and 160.9 corresponds to [CycAla2 + H]+ and [ala2 + H]+ respectively. The both ESI-MS and HPLC data matched with results obtained throughout the experiments.
Divalent transition metal hexacyanocobaltates(III), in which central metal atom and carbon of cyanide group bonded through coordinate bond. It is found that these porous, water insoluble, mixed valency octahedral coordinated complexes are a part of Fm3m andPm3m space group [47, 66] with reduced size and high surface area promoted high catalytic activity [47, 66–70]. In the present study, MHCCo complexes also showed high catalytic activity for the production of peptide bond formationsbecause of mixed valency and high surface area. The evidences summarized above suggestcatalytic activity of MHCCo complexes for the condensation of amino acids and thus supported the chemical evolution of life.