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     

Synthesis of homogeneous glycopeptides and their utility as DNA condensing agents

Two glycopeptides were synthesized by attaching purified glycosylamines (N-glycans) to a 20 amino acid peptide. Triantennary and Man9 Boc-tyrosinamide N-glycans were treated with trifluoroacetic acid to remove the Boc group and expose a tyrosinamide amine. The amine group was coupled with iodoacetic acid to produce N-iodoacetyl-oligosaccharides. These were reacted with the sulfhydryl group of a cysteine-containing peptide (CWK18), resulting in the formation of glycopeptides in good yield that were characterized by 1H NMR and ESIMS. Both glycopeptides were able to bind to plasmid DNA and form DNA condensates of approximately 110 nm mean diameter with zeta potential of +31 mV. The resulting homogeneous glycopeptide DNA condensates will be valuable as receptor-mediated gene-delivery agents.     

Model for prebiotic pyrophosphate formation: Condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents

Summary The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37°C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of³²PPi from³²Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.     

Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis

Prevention efforts and control of tuberculosis are seriously hampered by the appearance of multidrug-resistant strains of Mycobacterium tuberculosis, dictating new approaches to the treatment of the disease. Thiolactomycin (TLM) is a unique thiolactone that has been shown to exhibit anti-mycobacterial activity by specifically inhibiting fatty acid and mycolic acid biosynthesis. In this study, we present evidence that TLM targets two beta-ketoacyl-acyl-carrier protein synthases, KasA and KasB, consistent with the fact that both enzymes belong to the fatty-acid synthase type II system involved in fatty acid and mycolic acid biosynthesis. Overexpression of KasA, KasB, and KasAB in Mycobacterium bovis BCG increased in vivo and in vitro resistance against TLM. In addition, a multidrug-resistant clinical isolate was also found to be highly sensitive to TLM, indicating promise in counteracting multidrug-resistant strains of M. tuberculosis. The design and synthesis of several TLM derivatives have led to compounds more potent both in vitro against fatty acid and mycolic acid biosynthesis and in vivo against M. tuberculosis. Finally, a three-dimensional structural model of KasA has also been generated to improve understanding of the catalytic site of mycobacterial Kas proteins and to provide a more rational approach to the design of new drugs.     

The prebiotic synthesis and catalytic role of imidazoles and other condensing agents

In the past decade significant advances have been made in the synthesis of oligonucleotides and other polymers by means of imidazoles and other condensing agents. In spite of the current knowledge of the chemistry of imidazoles and their importance as prebiotic catalysts, their formation under primitive earth conditions has not been properly demonstrated. We have now been able to synthesize imidazole as well as its 2-methyl and 4-methyl derivatives under plausible prebiotic conditions. One method utilizes an aldehyde (formaldehyde or acetaldehyde), glyoxal and ammonia as the starting materials for the formation of imidazole and 2-methylimidazole. The other method uses a carbohydrate and ammonia as the key reagents for the synthesis of 4-methylimidazole. The importance of imidazole and related compounds (e.g., cyanamide) in the synthesis of oligonucleotides has been studied by us as well as others. Apparently the charge relay group (-N-C-N-) present in imidazoles, carbodiimides, cyanamide, or the histidine and arginine of enzyme active centers is essential for the synthesis of phosphodiester and pyrophosphate bonds.     

The prebiotic synthesis and catalytic role of imidazoles and other condensing agents

In the past decade significant advances have been made in the synthesis of oligonucleotides and other polymers by means of imidazoles and other condensing agents. In spite of the current knowledge of the chemistry of imidazoles and their importance as prebiotic catalysts, their formation under primitive earth conditions has not been properly demonstrated. We have now been able to synthesize imidazole as well as its 2-methyl and 4-methyl derivatives under plausible prebiotic conditions. One method utilizes an aldehyde (formaldehyde or acetaldehyde), glyoxal and ammonia as the starting materials for the formation of imidazole and 2-methylimidazole. The other method uses a carbohydrate and ammonia as the key reagents for the synthesis of 4-methylimidazole. The importance of imidazole and related compounds (e.g., cyanamide) in the synthesis of oligonucleotides has been studied by us as well as others. Apparently the charge relay group (–N–C–N–) present in imidazoles, carbodiimides, cyanamide, or the histidine and arginine of enzyme active centers is essential for the synthesis of phosphodiester and pyrophosphate bonds.     

Binding of phosphonate chelating agents and pyrophosphate to apotransferrin.

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.     

Prebiotic condensation reactions in an aqueous medium: A review of condensing agents

Biopolymers are formed by dehydration-type condensation reactions. In aqueous solutions dehydration reactions are very unlikely to happen spontaneously. However, coupling of dehydration-condensation to the hydrolysis of condensing agents could facilitate the synthesis of biopolymers in an aqueous solution. The literature shows that the peptides, nucleosides, nucleotides and oligonucleotides can be formed in this way. A careful study of the literature pertaining to prebiotic condensing agents was conducted in order to determine the most plausible prebiotic synthesis of biopolymers. The condensing agents taken into consideration are cyanamide, carbodiimide, dicyanamide, dicyandiamide, hydrogec-cyanide-tetramer, cyanogen and the linear- and cyclic polyphosphates. From both a chemical as well as biological point of view the polyphosphates appear to be the most plausible general prebiotic condensing agent.     

Optimization of unique, uncharged thioesters as inhibitors of HIV replication

A combinatorial chemistry approach was employed to prepare a restricted library of N-substituted S-acyl-2-mercaptobenzamide thioesters. It was shown that many members of this chemotype display anti-HIV activity via their ability to interact with HIV-1, HIV-2, SIV-infected cells, cell-free virus, and chronically and latently infected cells in a manner consistent with targeting of the highly conserved HIV-1 NCp7 zinc fingers. Compounds were initially screened using two different in vitro antiviral assays and evaluated for stability in neutral buffer containing 10% pooled human serum using a spectrophotometric assay. These data revealed that there was no significant correlation between thioester stability and antiviral activity, however, a slight inverse correlation between serum stability and virucidal activity was noted. Based on the virucidal capability and the ability to select lead compounds to inhibit virus expression from latently infected TNF-induced U1 cells, we next determined if these compounds could prevent HIV cell-to-cell transmission. Several thioesters demonstrated potent inhibition of HIV cell-to-cell transmission with EC₅₀ values in the 80–100 nM range. Thus, we have optimized a series of restricted thioesters and provided evidence that serum stability is not required for antiviral activity. Moreover, selected compounds show potential for development as topical microbicides.     

Formation of pyrophosphate by soluble guanylate cyclase from rat lung.

The guanylate cyclase reaction was studied to determine the identity of the product(s) formed other than guanosine-3′,5′-monophosphate (cyclic GMP). Partially purified guanylate cyclase preparations from rat lung catalyzed the formation of nearly equal amounts of PP₁ and of cyclic GMP from GTP. Column chromatography of the enzyme preparation on DEAE-Sephadex or Bio-Gel A-5m failed to separate the enzyme(s) involved in formation of cyclic GMP and of PP₁. Nucleotide inhibitors of cyclic GMP formation also inhibited PP₁ formation, and Ca²⁺, a stimulant of cyclic GMP formation in the presence of Mn²⁺, also stimulated PP₁ formation. Detectable PP₁ formation was not observed when ATP was present instead of GTP.The results show that guanylate cyclase, in vitro, catalyzes the formation of pyrophosphate from GTP concomitant with the synthesis of cyclic GMP.     

Analogues of geranyl pyrophosphate as inhibitors of prenyltransferase

Six analogues of geranyl pyrophosphate (the monophosphates of geraniol and tetrahydrogeraniol, and the pyrophosphates of nerol, octan-1-ol, tetrahydrogeraniol and citronellol) were synthesized, and were found to be inhibitors of pig liver prenyl- (geranyl-)transferase. The effects of each analogue were analysed in kinetic experiments, which showed the pyrophosphates of citronellol, tetrahydrogeraniol and octan-1-ol to be the most potent inhibitors. The results are interpreted to support a previous hypothesis that the main forces in the binding of substrates to prenyltransferase are non-specific lipophilic forces and a pyrophosphate-binding force.     

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [3H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cation. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/3H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cation are present.