Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

Formation of pyrophosphate,tripolyphosphate, and phosphorylimidazole with the thioester,N, S-diacetylcysteamine,as the condensing agent

Summary Reaction of 0.20M orthophosphate with 0.20M N,S-diacetylcysteamine in 0.40M imidazole at pH 7.0 or 8.0 under drying conditions at 50°C for 6 days yields pyrophosphate and tripolyphosphate in the presence and absence of 0.10M divalent metal ion. The efficiency of utilization of N,S-diacetylcysteamine in the formation of pyrophosphate linkages ranges from 3 – 8% under the above conditions. The thioester, N,S-diacetylcysteamine, and imidazole are required for phosphoanhydride formation.Reaction of 0.40M orthophosphate with 0.20M N, S-diacetylcysteamine in 0.40M imidazole at ambient temperature for 6 days yields phosphorylimidazole in the absence or presence of 0.05M MgCl₂. Phosphorylimidazole and pyrophosphate are formed in the presence of 0.05M CaCl₂; pyrophosphate and tripolyphosphate are formed with 0.15M CaCl₂. The efficiency of utilization of N,S-diacetylcysteamine in the formation of pyrophosphate linkages is roughly 7% at 6 days of reaction with 0.15M CaCl₂. The thioester, N,S-diacetylcysteamine and imidazole are required for the formation of phosphoanhydrides. The significance of these reactions to molecular evolution is discussed.Abbreviations P₁ orthophosphate - P₂ pyrophosphate - P₃ tripolyphosphate - ImP phosphorylimidazole - Ac-Csa(Ac) N, S-diacetylcysteamine - Im imidazole     

The effect of pyrophosphate, tripolyphosphate and ATP on the rate of the Fenton reaction

It has been recently reported that pyrophosphate, tri-polyphosphate, ATP and analogous ligands considerably decrease the yield of hydroxyl radicals by the Fenton reaction under conditions where [H₂O₂] > > [Fe(II)L_n]. It was suggested that this effect is due to the slowing down of the Fenton reaction by these ligands. This suggestion seemed surprising as polyphosphate ligands stabilize Fe(III). Indeed, a kinetic study points out that these ligands accelerate the rate of the Fenton reaction by several orders of magnitude. Thus it is suggested that the effect of the ligands on the yield of the hydroxyl radicals is due to the stabilization of the Fe(III) complexes which slows down, or inhibits, their reduction by the radicals formed in the system and thus decreases the overall yield of hydroxyl radicals.     

An efficient synthesis of GDP-hexanolamine,a key tool for the purification of fucosyltransferases

A new efficient synthesis of GDP-hexanolamine from hexanolamine is reported with an overall yield of 71%. The pyrophosphate formation, the key step of this preparation, was achieved through a sequential GMP activation procedure based on polytrifluoroacetylation of GMP followed by activation of the phosphate group by 1-methylimidazole.     

The formation of peptides from glycine thioesters

Summary The condensation products obtained from 0.01M S-glycyl-N-acetyl-cysteamine at different pH's were investigated. The highest yields of diketo-piperazine (approx. 50%) were observed in phosphate buffers between pH 7.5 and 8.5. The highest yields of diglycine (46%), triglycine (10%) and tetraglycine (2%) were observed in carbonate buffers at pH 9.5. At pH 8.0, over 90% of the glycyl residues of 0.15M S-glycyl-N-acetylcysteamine were incorporated into condensation products, mainly DKP (60–70%). The yields of products from the condensation of S-glycyl-ethanethiol under similar conditions closely re-sembled those obtained with S-glycyl-N-acetylcysteamine.Abbreviations Boc-gly N-tert-butyloxycarbonylglycine - Ac-cys N-acetylcysteine - csa cysteamine - Ac-csa N-acetylcysteamine - DKP diketopiperazine - (gly)₂ diglycine - (gly)₃ triglycine - (gly)₄ tetraglycine - glySEt S-glycyl-ethanethiol - glyS-(Ac-cys) S-glycyl-N-acetylcysteine - glyS-(Ac-csa) S-glycyl-N-acetylcysteamine - Boc-glyS-(Ac-cys) S-(Boc-glycyl)-N-acetylcysteine - Boc-glyS-(Ac-csa) S-(Boc-glycyl)-N-acetylcysteamine - Boc-glySEt S-(Boc-glycyl)-ethanethiol - gly-bydrox glycine hydroxamate     

Prebiotic synthesis of histidyl-histidine

Summary Histidyl-histidine (His-His) has been synthesized in a yield of up to 14.4% under plausible prebiotic conditions using histidine (His), cyanamide, and 4-amino-5-imidazole carboxamide. A trace amount of His trimer was also detected. Because the imidazole group of His is involved in a number of important enzymatic reactions, and His-His has been shown to catalyze the prebiotic synthesis of glycyl-glycine, we expect this work will stimulate further studies on the catalytic activities of simple His-containing peptides in prebiotic reactions.     

Pyrophosphate formation as the most efficient condensation reaction of activated nucleotides

Summary When an oligonucleotide primer pG₁₀ is incubated with the nucleotide analogue 9-[3-hydroxy-2-(hydroxymethyl)prop-1-yl] guanine diphosphate ( , I) in the presence of poly(C), addition of the monomer occurs almost exclusively at the 5′-terminal phosphate rather than the 3′-terminalcis-glycol. The implications of this finding in the context of prebiotic condensation reactions are discussed.     

Template-directed synthesis of oligonucleotides under eutectic conditions

Summary One of the most important sets of model prebiotic experiments consists of reactions that synthesize complementary oligonucleotides from preformed templates under nonenzymatic conditions. Most of these experiments are conducted at 4°C using 0.01–0.1 M concentrations of activated nucleotide monomer and template (monomer equivalent). In an attempt to extend the conditions under which this type of reaction can occur, we have concentrated the reactants by freezing at –18°C, which is close to the NaCl–H₂O eutectic at –21°C.The results from this set of experiments suggest that successful syntheses can occur with poly(C) concentrations as low at 5×10^–4 M and 2MeImpG concentrations at 10^–3 M. It was also anticipated that this mechanism might allow the previously unsuccessful poly(A)-directed synthesis of oligo(U)s to occur. However, no template effect was seen with the poly(A) and ImpU system. The failure of these conditions to allow template-directed synthesis of oligo(U)s supports the previously proposed idea that pyrimidines may not have been part of the earliest genetic material.Because of the low concentrations of monomer and template that would be expected from prebiotic syntheses, this lower temperature could be considered a more plausible geologic setting for template-directed synthesis than the standard reaction conditions.     

Infrared spectral characterization of peptidic material produced by ionizing radiation in aqueous cyanides

Summary Oligomers formed by ionizing radiation in aqueous cyanide solutions, under various experimental conditions, have been characterized by infrared spectroscopy. IR bands appear in the region known to be characteristic for amides and peptides. The results are discussed in relation to radiation-induced formation of peptidic material and the potential role of ionizing radiation as an energy source for some processes in prebiotic molecular evolution.     

Prebiotic synthesis of histidine

Summary The prebiotic formation of histidine (His) has been accomplished experimentally by the reacton of erythrose with formamidine followed by a Strecker synthesis. In the first step of this reaction sequence, the formation of imidazole-4-acetaldehyde took place by the condensation of erythrose and formamidine, two compounds that are known to be formed under prebiotic conditions. In a second step, the imidazole-4-acetaldehyde was converted to His, without isolation of the reaction products by adding HCN and ammonia to the reaction mixture. LC, HPLC, thermospray liquid chromatography-mass spectrometry, and tandem mass spectrometry were used to identify the product, which was obtained in a yield of 3.5% based on the ratio of His/erythrose. This is a new chemical synthesis of one of the basic amino acids which has not been synthesized prebiotically until now.     

Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: Implications for the nature of the first genetic material

Summary Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system.Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template.Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3×10^–6 M), as well as the difficulty of prebiotic activation of phosphates.     

Acute bioenergetic insulin sensitivity of skeletal muscle cells: ATP-demand-provoked glycolysis contributes to stimulation of ATP supply

Skeletal muscle takes up glucose in an insulin-sensitive manner and is thus important for the maintenance of blood glucose homeostasis. Insulin resistance during development of type 2 diabetes is associated with decreased ATP synthesis, but the causality of this association is controversial. In this paper, we report real-time oxygen uptake and medium acidification data that we use to quantify acute insulin effects on intracellular ATP supply and ATP demand in rat and human skeletal muscle cells. We demonstrate that insulin increases overall cellular ATP supply by stimulating the rate of glycolytic ATP synthesis. Stimulation is immediate and achieved directly by increased glycolytic capacity, and indirectly by elevated ATP demand from protein synthesis. Raised glycolytic capacity does not result from augmented glucose uptake. Notably, insulin-sensitive glucose uptake is increased synergistically by nitrite. While nitrite has a similar stimulatory effect on glycolytic ATP supply as insulin, it does not amplify insulin stimulation. These data highlight the multifarious nature of acute bioenergetic insulin sensitivity of skeletal muscle cells, and are thus important for the interpretation of changes in energy metabolism that are seen in insulin-resistant muscle.     

Prebiotic syntheses of vitamin coenzymes: I. Cysteamine and 2-mercaptoethanesulfonic acid (coenzyme M)

Summary The reaction of NH₃ and SO _sup2– ^inf3 with ethylene sulfide is shown to be a prebiotic synthesis of cysteamine and 2-mercaptoethanesulfonic acid (coenzyme M). A similar reaction with ethylene imine would give cysteamine and taurine. Ethylene oxide would react with NH3 and N(CH₃)₃ to give the phospholipid components ethanolamine and choline. The prebiotic sources of ethylene sulfide, ethylene imine and ethylene oxide are discussed. Cysteamine itself is not a suitable thioester for metabolic processes because of acyl transfer to the amino group, but this can be prevented by using an amide of cysteamine. The use of cysteamine in coenzyme A may have been due to its prebiotic abundance. The facile prebiotic synthesis of both cysteamine and coenzyme M suggests that they were involved in very early metabolic pathways. Offprint requests to: S.L. Miller     

Cyanamide mediated synthesis under plausible primitive earth conditions

Summary The formation of glycerol occurs when a solution of DL-glyceraldehyde is heated in the presence of hydrogen sulfide at room temperature. DL-glyceraldehyde and dihydroxyacetone treated with hydrazine, as well as DL-glyceraldehyde incubated with formaldehyde are also partially converted to glycerol. The yields of the above reactions are from approximately 1% to about 3%. The formation of glycerophosphates occurs when glycerol is heated with ammonium dihydrogen phosphate and either urea or cyanamide. The yield of glycerophosphates is about 30%, most of which issn-glycero-1 (3)-phosphate. These findings indicate that glycerol andsn-glycero-3-phosphate, which are moieties of glycerolipids, could have been formed under conditions which may have prevailed on the primitive Earth.