Farnesyl diphosphate analogues with omega-bioorthogonal azide and alkyne functional groups for protein farnesyl transferase-catalyzed ligation reactions

Eleven farnesyl diphosphate analogues, which contained omega-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated as substrates for the alkylation of peptide cosubstrates by yeast protein farnesyl transferase. Five of the diphosphates were good alternative substrates for farnesyl diphosphate (FPP). Steady-state kinetic constants were measured for the active compounds, and the products were characterized by HPLC and LC-MS. Two of the analogues gave steady-state kinetic parameters (kcat and Km) very similar to those of the natural substrate.     

NMR double resonance study of azide binding to cytochrome c

Equilibrium constants for the binding of azide to ferri-cytochrome c at temperature rangeof 305—325 K were determined at pH=7 by using ~1H double resonance method.Thermodynamicvalues(⊿H~o=-34.5 kJ/mol,⊿S~o=-100 J/mol)were obtained from van't Hoff's relation andwere compared with those for azide binding to other ferric hemeproteins.The reason of loweraffinity of cytochrome c for azide was discussed.     

Understanding rubredoxin redox sites by density functional theory studies of analogues

Determining the redox energetics of redox site analogues of metalloproteins is essential in unraveling the various contributions to electron transfer properties of these proteins. Since studies of the [4Fe-4S] analogues show that the energies are dependent on the ligand dihedral angles, broken symmetry density functional theory (BS-DFT) with the B3LYP functional and double-ζ basis sets calculations of optimized geometries and electron detachment energies of [1Fe] rubredoxin analogues are compared to crystal structures and gas-phase photoelectron spectroscopy data, respectively, for [Fe(SCH(3))(4)](0/1-/2-), [Fe(S(2)-o-xyl)(2)](0/1-/2-), and Na(+)[Fe(S(2)-o-xyl)(2)](1-/2-) in different conformations. In particular, the study of Na(+)[Fe(S(2)-o-xyl)(2)](1-/2-) is the only direct comparison of calculated and experimental gas phase detachment energies for the 1-/2- couple found in the rubredoxins. These results show that variations in the inner sphere energetics by up to ～0.4 eV can be caused by differences in the ligand dihedral angles in either or both redox states. Moreover, these results indicate that the protein stabilizes the conformation that favors reduction. In addition, the free energies and reorganization energies of oxidation and reduction as well as electrostatic potential charges are calculated, which can be used as estimates in continuum electrostatic calculations of electron transfer properties of [1Fe] proteins.     

Distal metal effects in cobalt porphyrins related to CcO

Cobalt(II) porphyrins were studied to determine the influence of distal site metalation and superstructure upon dioxygen reactivity in active site models of cytochrome c oxidase (CcO). Monometallic, Co(II)(P) complexes when ligated by an axial imidazole react with dioxygen to form reversible Co-superoxide adducts, which were characterized by EPR and resonance Raman (RR). Unexpectedly, certain Co porphyrins with Cu(I) metalated imidazole pickets do not form mu-peroxo Co(III)/Cu(II) products even though the calculated intermetallic distance suggests this is possible. Instead, cobalt-porphyrin-superoxide complexes are obtained with the distal copper remaining as Cu(I). Moreover, distal metals (Cu(I) or Zn(II)) greatly enhance the stability of the dioxygen adduct, such that Co superoxides of bimetallic complexes demonstrate minimal reversibility. The "trapping" of dioxygen by a second metal is attributed to structural and electrostatic changes within the distal pocket upon metalation. EPR evidence suggests that the terminal oxygen in these bimetallic Co-superoxide systems is H-bonded to the NH of an imidazole picket amide linker, which may contribute to enthalpic stabilization of the dioxygen adduct. Stabilization of the dioxygen adduct in these bimetallic systems suggests one possible role for the distal copper in the Fe/Cu bimetallic active site of terminal oxidases, which form a heme-superoxide/copper(I) adduct upon oxygenation.     

Hydrolysis theory for cisplatin and its analogues based on density functional studies

Hydrolysis of cisplatin, the most widely used anticancer drug in the world, is believed to be the key activation step before the drug reaching its intracellular target DNA.To obtain an accurate hydrolysis theory for this important class of square-planar Pt(II) complexes, three typical reactions, i.e., the first and second hydrolyses of cisplatin and the hydrolysis of [Pt(dien)Cl](+) (dien = diethylenetriamine), were studied at the experimental temperature with the solvent effect using mPW1PW91/SDD from a comprehensive methodological study on the Hartree-Fock (HF) ab initio method, electron correlation methods, pure density functional theory (DFT) methods, and hybrid HF-DFT methods with several basis sets. The true five stationary states in the second-order nucleophilic substitution (S(N)2) pathway for the hydrolysis process, namely, reactant (R) --> intermediate 1 (I1) --> TS --> intermediate 2 (I2) --> product (P) were obtained and characterized theoretically for the first time. The most remarkable structural variations and the associated atomic charge variations in the hydrolysis process were found to occur in the equatorial plane of the five-coordinate trigonal-bipyramidal (TBP)-like structures of I1, TS, and I2. The reaction with the TS structure of smaller L-M-E angle and more lengthened M-L and M-E bonds was found to have a smaller Gibbs free energy change and accordingly the better hydrolysis yield. It is found that the sum of the three concentric angles in the TBP's equator is near 360 degrees in I1 and I2 and is almost 360 degrees in TS in each reaction. The associated energy profiles again demonstrated a typical S(N)2 reaction curve. The computed forward and backward reaction enthalpy (Delta H(++)) and reaction entropy (Delta S(++)) in the rate-determining step I1 --> TS --> I2 are in good agreement with the experiments. Natural bonding orbital population analysis shows that the charge-separating extent follows the same order of Delta G in studied reactions. Comparing with the computational results of gas-phase reactions, it can be concluded that the solvent effect should be considered to obtain an accurate hydrolysis picture. The most affected structural parameters after solvation are related to the equatorial plane of the TBP-like geometry. The results provide theoretical guidance on detailed understanding on the mechanism of the hydrolysis of cisplatin, which could be useful in the design of novel Pt-based anticancer agents.     

Virtual screening for binding of phenylalanine analogues to phenylalanyl-tRNA synthetase

Although incorporation of nonnatural amino acids provides a powerful means of controlling protein structure and function, experimental investigations of amino acid analogues for utilization by the protein biosynthetic machinery can be costly and time-consuming. In this paper, we describe a computational protocol (HierDock) for predicting the relative energies of binding of phenylalanine analogues to phenylalanyl-tRNA synthetase (PheRS). Starting with the crystal structure of Thermus thermophilus PheRS without bound ligand, HierDock predicts the binding site of phenylalanine (Phe) within 1.1 A of that revealed by the crystal structure of PheRS cocrystallized with Phe. The calculated binding energies of Phe analogues in PheRS, using HierDock, correlate well with the translational activities of the same analogues in Escherichia coli. HierDock identifies p-fluorophenylalanine and 3-thienylalanine as especially good substrates for PheRS, in agreement with experiment. These results suggest that the HierDock protocol may be useful for virtual screening of amino acid analogues prior to experiment.     

Synthetic studies of cyclosporin analogues

The syntheses of eleven analogues of Cyclosporin are described; the analogues which contain (Me)Thr, (Me)Ser, Hyp and Dab at position-1 and Abu, Nva, Nle and Thr at position-2 were prepared by stepwise assembly of the undecapeptide fragments followed by cyclisation with a variety of reagents. The highest yields were obtained using the Castro reagent, and in the best case, (Hyp¹ ,Abu²) cyclosporin, a yield of 65% was obtained.     

S-homoadenosyl-L-cysteine and S-homoadenosyl-L-homocysteine. Synthesis and binding studies of hon-hydrolyzed substrate analogues with S-adenosyl-L-homocysteine hydrolase

Treatment of homoadenosine [9-(5-deoxy-beta-D-ribo-hexofuranosyl)adenine] with thionyl chloride and pyridine in acetonitrile gave 6'-chloro-6'-deoxyhomoadenosine, which underwent nucleophilic displacement with L-cysteine or L-homocysteine to give homologated analogues of S-adenosyl-L-homocysteine. Each amino acid in aqueous sodium hydroxide at 60 degrees C gave excellent conversion from the chloronucleoside, and adsorption on Amberlite XAD-4 resin provided more convenient isolation than prior methods. Weak binding of these non-hydrolyzed analogues to S-adenosyl-L-homocysteine hydrolase was observed.     

Structural basis of thiamine pyrophosphate analogues binding to the eukaryotic riboswitch

The thiamine pyrophosphate (TPP)-sensing riboswitch is the only riboswitch found in eukaryotes. In plants, TPP regulates its own production by binding to the 3' untranslated region of the mRNA encoding ThiC, a critical enzyme in thiamine biosynthesis, which promotes the formation of an unstable splicing variant. In order to better understand the molecular basis of TPP-analogue binding to the eukaryotic TPP-responsive riboswitch, we have determined the crystal structures of the Arabidopsis thaliana TPP-riboswitch in complex with oxythiamine pyrophosphate (OTPP) and with the antimicrobial compound pyrithiamine pyrophosphate (PTPP). The OTPP-riboswitch complex reveals that the pyrimidine ring of OTPP is stabilized in its enol form in order to retain key interactions with guanosine 28 of the riboswitch previously observed in the TPP complex. The structure of PTPP in complex with the riboswitch shows that the base moiety of guanosine 60 undergoes a conformational change to cradle the pyridine ring of the PTPP. Structural information from these complexes has implications for the design of novel antimicrobials targeting TPP-sensing riboswitches.     

Density functional study of molecular properties of hydrazoic acid and methyl azide

Density functional calculations for hydrazoic acid HN₃ and methyl azide CH₃N₃ and for the respective singly ionized structures HN⁺₃ and CH₃N⁺₃ are reported. An analysis of the electrostatic solvent effects, based on the self-consistent reaction field approach, on the molecular properties and conformational equilibrium of CH₃N₃ is also reported. The results are sensitive to the basis set quality and show some dependence on the different representations for the exchange-correlation functions. For HN₃ very good agreement with experiment is observed for several properties, such as the geometry, dipole moment, vibrational frequencies and for the adiabatic first ionization energy. For CH₃N₃ the energy difference between eclipsed (ec) and staggered (st) conformers (δ_ec-st) is 2.5 kJ mol⁻¹, in good agreement with the experimental value (2.9 kJ mol⁻¹). However, for CH₃N⁺₃, δ_ec-st is −3.2 kJ mol⁻, reflecting a significant modification of the methyl group rotational potential after ionization. Solvent effects on the molecular properties of CH₃N₃ are important when it is solvated in a polar medium. The most significant modifications concern the dipole moment and the frequencies related to the CH₃ symmetric stretch and torsion vibrational modes.     

Molecular dynamics and density functional studies of substrate binding and catalysis of arginine deiminase

The active-site dynamics of arginine deiminase (ADI) complexed with the arginine substrate are investigated with ns molecular dynamics for the wildtype ADI and several mutants. It is shown that the substrate is held in the active site by an extensive hydrogen bond network, which may be weakened by substitution of active-site residues. In addition, the initial step of the catalysis is explored in several truncated active-site models with density functional theory. Evidence is presented in support of the hypothesis that the nucleophilic attack of the ADI Cys thiol at the guanidino carbon of the substrate is initiated by substrate-mediated proton transfer to a His residue in the catalytic triad (Cys-His-Glu). In addition, the active-site residues are found to strongly influence the reaction profile, consistent with their important role in catalysis.     

Density functional theory study of the intramolecular [2 + 3] cycloaddition of azide to nitriles

Density functional theory calculations using the hybrid functional B3LYP have been performed to study tetrazole formation by intramolecular [2 + 3] dipolar cycloaddition of organic azides and nitriles. Experimental reactivity trends are explained and rationalized in terms of a number of parameters, such as strain, tether length, and solvation and entropy effects. Interestingly, no correlation was found between the overall free energies and the free energies of activation of the reactions, due to the significant difference in strain and geometry between the transition states and products.     

Conformational studies of some carbocyclic nucleoside analogues

Proton NMR spectra of some carbocyclic nucleoside analogs of tuberculin have been analyzed at 100 MHz in DMSO-d₆. The spectral characteristics and nuclear Overhauser effects indicate a preferential syn conformation about the glycosidic bond when a hydroxyl group, oriented toward the base, is available for intramolecular hydrogen bond formation.     

Brownian dynamics simulations of binding mRNA cap analogues to eIF4E protein

The binding of five analogues of the 5'-end mRNA cap, differing in their electrostatic and hydrodynamic properties, to the eukaryotic initiation factor eIF4E was simulated by means of Brownian dynamics methods. Electrostatic and hydrodynamic models of eIF4E protein and the ligands were prepared using established molecular electrostatics and hydrodynamics simulation methods for predicting ionization states of titratable groups, adequate for given experimental conditions, and for computing their translational and rotational diffusion tensors, respectively. The diffusional encounter rate constants obtained from simulations are compared with bimolecular association rate constants resulting from stopped-flow spectrofluorimeter measurements. A very good agreement between simulations and experiments was achieved, which indicates that the kinetics of binding 5'-mRNA caps can be satisfactory explained by referring to the Brownian motion of the particles with the electrostatic steering of the ligands toward the eIF4E binding site and electrostatic desolvation contributions upon complex formation.     

MeOPEG-bounded azide cycloadditions to alkynyl dipolarophiles

The MeOPEG-supported azide 2 was reacted in the presence of a number of alkynyl dipolarophiles. The corresponding 1-MeOPEG-supported-1,2,3-triazoles were obtained in nearly quantitative yields. Acidic hydrolysis of the cycloadducts 5b and 6b caused the removal of the MeOPEG pendant giving 4-methoxycarbonyl-1,2,3-triazole 9 and 5-methoxycarbonyl-1,2,3-triazole 10, respectively.     

Vibrational frequencies of hydrazoic acid and methyl azide: density functional theory study

Harmonic vibrational frequencies of HN3 and CH3N3 molecules and their several isotopomers are calculated using HF, MP2 and five popular density functional theory (DFT) methods. On the basis of the comparison between calculated and experimental results, assignments of fundamental vibrational modes arc examined. HF and MP2 results are in bad agreement with experimental values. Of the five DFT methods, BLYP reproduces the observed fundamental frequencies the most satisfactorily. Two hybrid DFT methods are found to yield frequencies generally higher than the observed fundamental frequencies. The results indicate that BLYP calculation is a very promising approach for understanding the observed spectral features.     

Theoretical studies on the magnetic bistability of dinickel complex tuned by azide

The magnetic-structural correlation in magnetic switchable dinickel(II) complex [LNi2(N3)3] (L- is a pyrazolate-based compartmental ligand) has been investigated on the basis of various unrestricted density functional theory (UDFT) combined with the broken symmetry (BS) approach. The calculated exchange coupling constants were in good agreement with experimental result by using the PBE0 method with LANL2DZ basis set. The antiferromagnetic interaction between the Ni(II) ions is mainly due to the large energy difference of the singly occupied molecular orbitals (SOMOs), and the p orbital overlap for nitrogen atoms on azido and the pyrazolate bridge groups. The analysis of the spin density distribution reveals that both the spin polarization and spin delocalization contribute to the antiferromagnetic interaction. Furthermore, the bistable magnetic behavior for this system (strong antiferromagnetic interaction in low-temperature phase and the week antiferromagnetic in high-temperature phase) results from the change of the Ni-NNN-Ni dihedral angle (tau) in mu1,3-N3. The increase of tau is the key role in decreasing the SOMOs energy difference and weakening the antiferromagnetic interaction. Therefore, the abrupt modulation of the magnitude of M-NNN-M dihedral angle tau in the binuclear-azide complex by external perturbations provides new possibilities for the design of molecular magnetic switching devices.