Thioester Compounds Research Articles

Ferroelectric properties of achiral banana-shaped and calamitic-chiral thioesters

Two thioester compounds: (S)-(+)-4-(1-methylheptyloxy)biphenyl 4′-heptylthiobenzoate (in short: MHOBS7) having rode-like, calamitic-chiral molecules, and 1,3-phenylene bis{4-[(4-octyloxybenzoyl)-sulfanyl]benzoate} (in short: 8OSOR) built of banana like achiral molecules, have been studied by complementary methods to show differences in their linear dielectric as well as ferroelectric properties. To this end the following experimental methods were used: differential scanning calorimetry, polarizing microscopy, dielectric spectroscopy and reversal current method. The first compound shows a ferroelectric SmC* phase with temperature dependent Goldstone mode displaying negative activation energy. At the N*--SmC* transition spontaneous polarization changes discontinuously showing first order character of this transition. For 8OSOR compound with bent core molecules only B1 phase shows up, which does not switch. However, it was possible to record reversal current spectra but temperature dependence of spontaneous polarization was unusual suggesting antiferroelectric order of this phase.

Comparison of the Specificity of Interaction of Cellular and Viral Zinc-Binding Domains with 2-Mercaptobenzamide Thioesters

The interactions of two 2-mercaptobenzamide thioester compounds with six diverse zinc-binding domains (ZBDs) have been analyzed by UV/visible spectroscopy, NMR spectroscopy, and nucleic acid binding assays. These thioester compounds serve as useful tools for probing the intrinsic chemical stability of ZBDs that exist within a variety of cellular and viral proteins. In our studies, the classical (Cys(2)His(2)) zinc finger ZBDs, the interleaved RING like ZBDs of protein kinase C delta (Cys(2)HisCys and HisCys(3)), and the carboxyl-terminal (Cys(2)HisCys) ZBD of Mouse Mammary Tumor Virus nucleocapsid protein (MMTV NCp10) were resistant to reaction with the thioester compounds. In contrast, the thioester compounds were able to efficiently eject zinc from the amino-terminal (Cys(2)HisCys) ZBD of MMTV NCp10, a Cys(2)HisCys ZBD from Friend of GATA-1 (FOG-1), and from both Cys(4) ZBDs of GATA-1. In all cases, zinc ejection led to a loss of protein structure. Interestingly, GATA-1 was resistant to reaction with the thioester compounds when bound to its target DNA sequence. The electronic and steric screening was calculated for select ZBDs to further explore their reactivity. Based on these results, it appears that both first and second zinc-coordination shell interactions within ZBDs, as well as nucleic acid binding, play important roles in determining the chemical stability and reactivity of ZBDs. These studies not only provide information regarding the relative reactivity of cysteine residues within structural ZBDs but also are crucial for the design of future therapeutic agents that selectively target ZBDs, such as those that occur in the HIV-1 nucleocapsid protein.

Synthesis, structural and spectroscopic study of aromatic thioester compounds

Eight novel thioesters were obtained and characterized by elemental analysis, mass spectrometry, infrared and NMR spectroscopy. The data were analyzed on the basis of the relevant resonance structures. The X-ray single crystal structure of o-methoxythiophenyl 4-methylbenzoate is reported.

Studies on the Mechanism of Inactivation of the HIV-1 Nucleocapsid Protein NCp7 with 2-Mercaptobenzamide Thioesters

The HIV-1 nucleocapsid protein (NCp7) is a small basic protein with two CysCysHisCys zinc-binding domains that specifically recognizes the Psi-site of the viral RNA. NCp7 plays a number of crucial roles in the viral lifecycle, including reverse transcription and RNA encapsidation. Several classes of potential anti-HIV compounds have been designed to inactivate NCp7 through zinc ejection, including a special class of thioester compounds. We have investigated the mechanism of action of two N-substituted-S-acyl-2-mercaptobenzamide compounds (compounds 1 and 2) that target NCp7. UV/Visible spectroscopy studies demonstrated that both thioesters were able to eject metal from NCp7. NMR and mass spectroscopy studies showed that the thioester compounds specifically ejected zinc from the carboxyl-terminal zinc-binding domain of NCp7 by covalent modification of Cys(39). Exposure of NCp7 to compounds 1 and 2 destroyed its ability to specifically bind RNA, whereas NCp7 already bound to RNA was protected from zinc ejection by the thioesters. The thiol component of the thioesters (compound 3, 2-mercaptobenzoyl-beta-alaninamide) did not eject zinc from NCp7, but when compound 3 was incubated with acetyl CoA prior to incubation with NCp7, we observed extensive metal ejection. Thus, the thiol released by the reaction of compounds 1 and 2 could be re-acylated in vivo by acyl CoA to form a new thioester compound that is able to react with NCp7. These studies provide a better understanding of the mechanism of action of thioester compounds, which is important for future design of anti-HIV-1 compounds that target NCp7.

Lysosomal ceroid depletion by drugs: Therapeutic implications for a hereditary neurodegenerative disease of childhood

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.

Inactivation of HIV-1 Nucleocapsid Protein P7 by Pyridinioalkanoyl Thioesters: CHARACTERIZATION OF REACTION PRODUCTS AND PROPOSED MECHANISM OF ACTION

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.

S-ethyl thiochloroformate, ClC(O)SCH 2CH 3: Unusual conformational properties?

The conformational equilibrium of S-ethyl thiochloroformate, ClC(O)SCH 2CH 3, was determined by vibrational spectroscopy (FTIR and FTRaman spectra), ab initio and density functional theory calculations (HF/3-21G*, HF/6-31G* and B3PW91/6-31G*). The evaluation of experimental and theoretical results suggests the existence of two conformers syn, gauche (CO double bond syn with respect to the S–C single bond and C–S single bond gauche with respect to the C–C single bond, respectively) and syn, anti, being the former most stable one. The existence of this rotational isomerism is rather unexpected for this thioester compound, owing to the known conformational equilibrium reported for some thioesters that depends on the ClCSC torsion angle. Photolysis products of the compound irradiated in adequate inert gas matrices were identified as CO, SCO, C 2H 5SCl and C 2H 5Cl.

Experimental and theoretical evidence of decrease of both the enthalpy difference and the isomerization barrier of some thioester compounds when the molecular ion is formed

The conformational properties of the molecular ions of some XC(O)SY compounds were studied with respect to the syn-anti isomerization process (syn and anti denote the position of the CO bond relative to the SY bond). Experimental investigations of both the neutral species and the molecular ion provide evidence that on ionization the enthalpy difference between the possible stable conformers decreases, as does the barrier to internal rotation around the CS single bond. This conclusion is based on the observation of fragment ions, which can only be interpreted as being derived from the less stable conformer. For these detected fragments to be formed, they must be derived from the less stable form, but with a lower energy difference compared with the predominant conformation of the molecular ion. Ab initio calculation at the (U)HF/6-31G * level of theory were carried out for the pairs FC(O)SNSO/FC(O)SNSO +, ClC(O)SNSO/ClC(O)SNSO + and FC(O)SCl/FC(O)SCl +. The theoretical results are in agreement with the experimental data.

Development of Surrogate Substrates for Juvenile Hormone Esterase

Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, juvenile hormone esterase (JHE). Substrates were designed that resembled the endogenous substrate juvenile hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman′s method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen β to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylesterase, and acetylcholinesterase were determined for the surrogate substrates. While JHE and porcine liver carboxylesterase hydrolyzed several of the substrates, acetylcholinesterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [ 3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher K m values but are more easily hydrolyzed ( V max) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [ 3H]JH III substrate.

Determination of the conformation in thioester compounds containing the NSO group through pre-resonance Raman study: Spectroscopic properties of S( N-sulphinylimine)-fluorocarbonylsulphane, FC(O)SNSO

Starting from fluorocarbonylsulphenyl chloride, FC(O)SCl, and N-trimethylsilysulphinylimine, (CH 3) 3SiNSO, the compound S( N-sulphinylimine)-fluorocarbonylsulphane, FC(O)SNSO, has been prepared. The IR, pre-resonant Raman, 13C NMR, mass, and UV spectra have been obtained and interpreted. The two bands appearing in the CO stretching fundamental mode region of the vibrational spectra were specially investigated to probe the existence of conformational isomerism in the molecule. From the analysis of the bands and their contours in the vapour-IR spectra, and the depolarization ratios together with the pre-resonant behaviour in the Raman spectrum of the liquid (whose extension is also evaluated), two planar forms can be determined for the molecule. The study of the CO envelope in the IR spectrum of the vapour phase reveals syn conformation (with respect to the CO and SN bonds) as the most stable structure for the molecule, and the anti conformation as the second structure.

Towards the absolute determination of the conformation in thioester compounds through the first pre-resonance Raman study in X-NSO molecules. Preparation and properties of S( N-sulphinylimine) chlorocarbonylsulphane, ClC(O)SNSO: IR, pre-resonant Raman, 13C NMR, mass and UV spectroscopic and conformational study

Starting from chlorocarbonylsulphenyl chloride, ClC(O)SCl, and N-trimethylsilylsulphinylimine, (CH 3) 3SiNSO, the compound S( N-sulphinylimine)-chlorocarbonylsulphane, ClC(O)SNSO, has been prepared. The IR, pre-resonant Raman, 13C NMR, mass and UV spectra have been obtained and interpreted. The two bands appearing in the C  O stretching fundamental mode region were investigated in order to determine the existence of conformational isomerism in the molecule. Both Raman studies at different temperatures and those using different excitations revealed the existence of the molecule in only one conformation. From these studies, a pre-resonant effect has been determined for the first time for XNSO compounds. Its extension is associated with the planarity of the molecule. The study of the C  O envelope in the IR spectrum of the vapour phase reveals the syn conformation (with respect to the C  O and SN bonds) as the only structure for the molecule in appreciable concentration at room temperature.

Induction of hepatic gamma-glutamyl transpeptidase in rats by repeated administration of aminopyrine

Twenty-nine thioester compounds were synthesized to test their effectiveness as surrogate substrates for the insect enzyme, juvenile hormone esterase (JHE). Substrates were designed that resembled the endogenous substrate juvenile hormone (JH), with one common factor being a thioester instead of carboxyl ester found in JH. The principle of the spectrophotometric assay is based on a modification of Ellman′s method. Characterization of the substrates showed that replacement of the carbon atom by a sulfur or oxygen β to the carbonyl of the acyl group of the substrates resulted in an approximate five- to sixfold increase in the rate of hydrolysis by JHE. The specific activities of JHE, porcine liver carboxylesterase, and acetylcholinesterase were determined for the surrogate substrates. While JHE and porcine liver carboxylesterase hydrolyzed several of the substrates, acetylcholinesterase did not produce any detectable hydrolysis of the substrates. Michaelis-Menten kinetic parameters of the surrogate substrates when compared to a previously reported partition assay, utilizing radiolabeled [3H]JH III, indicated that the surrogate substrates have lower affinity as indicated by higher Km values but are more easily hydrolyzed (Vmax) by JHE. Furthermore, optimal reaction conditions for substrate hydrolysis and the spectrophotometric reaction were determined. In addition, first order rate constants for base hydrolysis and critical micelle concentrations were determined for several surrogate substrates. The spectrophotometric assay was also compared with a Vmax and research spectrophotometer, and these two instruments produced almost identical slopes. The relative potency of four transition state inhibitors of JHE was found to be similar with those of the surrogate substrates and the [3H]JH III substrate.