RESUMEN
BACKGROUND: Epithelial ovarian cancer has a poor prognosis, mostly due to its late diagnosis and the development of drug resistance after a first platinum-based regimen. The presence of a specific population of "cancer stem cells" could be responsible of the relapse of the tumor and the development of resistance to therapy. For this reason, it would be important to specifically target this subpopulation of tumor cells in order to increase the response to therapy. METHOD: We screened a chemical compound library assembled during the COST CM1106 action to search for compound classes active in targeting ovarian stem cells. We here report the results of the high-throughput screening assay in two ovarian cancer stem cells and the differentiated cells derived from them. RESULTS AND CONCLUSION: Interestingly, there were compounds active only on stem cells, only on differentiated cells, and compounds active on both cell populations. Even if these data need to be validated in ad hoc dose response cytotoxic experiments, the ongoing analysis of the compound structures will open up to mechanistic drug studies to select compounds able to improve the prognosis of ovarian cancer patients.
Asunto(s)
Antineoplásicos/farmacología , Ensayos Analíticos de Alto Rendimiento , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Estructura Molecular , Neoplasias Ováricas/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans-2-aminocyclohexane carboxylic acid (Achc) or trans-2-aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three-dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6-31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR-based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides.
Asunto(s)
Ácidos Ciclohexanocarboxílicos/química , Ciclohexilaminas/química , Péptidos Cíclicos/química , Dicroismo Circular/métodos , Cicloleucina/análogos & derivados , Cicloleucina/química , Conformación Molecular , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , PeptidomiméticosRESUMEN
The capability of a Support Vector Machines QSAR model to predict the antiproliferative ability of small peptides was evaluated by screening a virtual library of enkephalin-like analogs modified by incorporation of the (R,S)-(1-adamantyl)glycine (Aaa) residue. From an initial set of 390 compounds, the peptides, Tyr-Aaa-Gly-Phe-Met (2), Tyr-Aaa-Gly-Phe-Phe (3), Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5) were selected, synthesized and their antitumor activity was tested and compared to that of Met-enkephalin (1). The antiproliferative activity correlated with the computational prediction and with the foldamer-forming ability of the studied peptides. The most active compounds were the hydrophobic peptides, Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5), having a greater propensity to adopt folded structures than the other peptides.
Asunto(s)
Antineoplásicos/síntesis química , Biología Computacional/métodos , Citostáticos/síntesis química , Diseño de Fármacos , Encefalina Metionina/análogos & derivados , Modelos Moleculares , Adamantano/análogos & derivados , Adamantano/química , Antineoplásicos/química , Antineoplásicos/farmacología , Inteligencia Artificial , Línea Celular Tumoral , Dicroismo Circular , Citostáticos/química , Citostáticos/farmacología , Bases de Datos Factuales , Encefalina Metionina/química , Encefalina Metionina/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacología , Análisis de Componente Principal , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Programas InformáticosRESUMEN
While numerous examples of beta-peptides--exclusively composed of beta-amino acids--have been investigated during the past decade, there are only few reports on the conformational preference of a single beta-amino acid when incorporated into a cyclopeptide. The conformational bias of beta-amino acids on the secondary structure of cyclopeptides has been investigated by NMR spectroscopy in combination with distance geometry (DG) and molecular dynamics (MD) calculations using experimental constraints. The atomic coordinate RMSD criterion usually employed for clustering of conformations after DG and MD calculations does not necessarily group similar peptide conformations, as there is an insufficient correlation between atomic coordinates and torsion angles. To improve on this shortcoming and to eliminate any arbitrary decisions during this process, a torsion angle clustering procedure has been implemented. For the cyclic pentapeptides cyclo-(-Val-beta-Hala-Phe-Leu-Ile-) 1 and cyclo-(-Ser-Pro-Leu-beta-Hasn-Asp-) 3, the beta-amino acid is found in the central position of an extended gamma-turn (pseudo gamma-turn, Psigamma-turn), while the beta-Hpro residue in the cyclic hexapeptide cyclo-(-Ser-beta-Hpro-Leu-Asn-Ile-Asp-) 5 preferentially occupies position i+1 of a pseudo beta-turn (Psibeta-turn). These results further corroborate the hypothesis of beta-amino acids being reliable inducers of secondary structure in cyclic penta- and hexapeptides. They can be employed in the de novo design of biologically active cyclopeptides in pharmaceutical research, since the three-dimensional presentation of pharmacophoric groups in the side chains can be tailored by incorporation of beta-amino acids in strategic sequential positions.
Asunto(s)
Aminoácidos/química , Modelos Químicos , Péptidos/química , Estructura Secundaria de Proteína , Termodinámica , Análisis por Conglomerados , Simulación por Computador , Ciclización , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Modelos Moleculares , Péptidos/síntesis química , Conformación Proteica , Estándares de ReferenciaRESUMEN
To be efficient in vivo antisense oligonucleotides must reach the targeted cells and then cross the cellular membrane. We propose a two step system where the oligonucleotide is first electrostatically bound to a peptide coupled to a ligand of a cellular receptor. A complex is formed which allows the oligonucleotide to be bound to the membrane of the targeted cells. These oligonucleotides are then delivered inside the cells by the subsequent use of a transfection agent. As a reductionist model of peptide coupled to a ligand we have used a lipopeptide and characterized by a filter elution assay the stoichiometry between the peptide and the oligonucleotide in the complexes. Using HeLa cultured cells we have shown that addition of these complexes to the cells triggers the oligonucleotide binding to the cell membrane. The subsequent addition of dendrimers allows these antisense oligonucleotides to inhibit a reporter gene inside the cells.
Asunto(s)
Lipoproteínas/farmacocinética , Oligodesoxirribonucleótidos Antisentido/farmacocinética , Secuencia de Bases , Transporte Biológico , Biotransformación , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Genes Reporteros , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Cinética , Lipoproteínas/toxicidad , Proteínas Luminiscentes/genética , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , ARN Mensajero/genética , Proteínas Recombinantes/metabolismo , Tionucleótidos/farmacocinética , TransfecciónRESUMEN
The correlation between the helicity (absolute conformation) of the O-heterocyclic ring of chiral 2,3-dihydrobenzo[b]furan (1) and chromane (2) derivatives and their (1)L(b) band CD was investigated. The same helicity rule was found for both unsubstituted chromophores: P/M helicity of the heterocyclic ring leads to a negative/positive CD within the (1)L(b) band. While the substitution of the fused benzene ring by achiral substituents does not change this helicity rule for the chromane chromophore, it leads to its inversion for the 2,3-dihydrobenzo[b]furan chromophores. On the basis of these observations, the published absolute configurations of natural flavonol and pterocarpan derivatives were confirmed and the configurational assignments of several natural neolignans revised.
Asunto(s)
Benzofuranos/química , Cromanos/química , Dicroismo Circular , Conformación Molecular , Estereoisomerismo , TermodinámicaRESUMEN
Previous NMR studies on surfactin proposed two gamma or beta-turn-containing conformers while recent CD studies described beta-sheets and alpha-helices in surfactin. Since these data were not obtained in the same conditions, the conformation of surfactin was reinvestigated by FTIR spectroscopy, a diagnostic method for beta-sheets. In trifluoroethanol, the FTIR spectra of surfactin and its diester are compatible with gamma and/or beta-turn(s) and the differences in their CD spectra show the importance of the Glu(1) and Asp(5) COOH groups in stabilizing the lipopeptide conformation. The calcium-induced spectral changes of both lipopeptides suggest a first binding of the divalent ions to the surfactin COOH groups (until calcium-lipopeptide mole ratio reached 1) followed by bulk conformational changes (at higher mole ratios). In Tris buffer at pH 8.5, the FTIR amide I band shape, without the typical 1610-1628 and 1675-1695 cm(-1) bands, ascertains the absence of beta-sheets.
Asunto(s)
Proteínas Bacterianas/efectos de los fármacos , Calcio/farmacología , Péptidos Cíclicos , Proteínas Bacterianas/química , Dicroismo Circular , Lipopéptidos , Conformación Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
This report provides a detailed analysis on the influence of phosholipid bilayers on the conformation of poly[Lys(X(i)-DL-Ala(m))] (XAK, where X = Ser, Orn, Glu, or AcGlu) type branched polypeptides and their peptide conjugates. CD spectra of polycationic (SAK, OAK), amphoteric (EAK), or polyanionic (Ac-EAK) polylysine derivatives were recorded in 0.25M acetate buffer at pH 7.4 as well as in the presence of DPPC or DPPC/PG (95/5, 80/20 mol/mol) liposomes. Based on these data, two groups of polypeptides are described. Group one contains polypeptides with significantly ordered conformation even in buffer solution (SAK, AcEAK), which is essentially not altered by phospholipids. Group two, branched polypeptides (OAK, EAK), with only partially ordered conformation in aqueous solution in the presence of phospholipid bilayers with high PG content, could adopt more (EAK) or less (OAK) ordered alpha-helical structure depending on their charge properties. In addition, we report on the synthesis of two new sets of oligopeptide-branched polypeptide conjugates. Studies with selected conjugates suggest that these compounds are highly ordered in buffer solution almost regardless from the helix-forming ability of the carrier (AK, SAK, EAK) and from the hydrophilic/hydrophobic character of peptides attached (AVKDEL vs FWRGDLVFDFQV). Addition of phospholipid bilayers with different composition essentially had no modifying effect on conformation of conjugates. From this we can conclude that the covalently coupled oligopeptides has a predominant effect of the conformational properties of conjugates.
Asunto(s)
Péptidos/química , Secuencia de Aminoácidos , Dicroismo Circular , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Conformación Proteica , SolucionesRESUMEN
Circular dichroism and Fourier-transform infrared spectroscopies were used to compare the conformational mobility of 13-mer peptides covering the 317-329 region of the envelope protein hemagglutinin of human influenza A virus subtypes H1, H2 and H3 with that of their truncated deca- and nonapeptide analogs. These peptides were demonstrated to bind to the murine I-Ed major histocompatibility complex encoded class II and human HLA-B*2705 class I molecules. Despite the amino acid substitutions in the three 13-mer subtype sequences, no significant differences in the conformational properties could be shown. Deletion of the N-terminal three residues resulted in a shift to an increased alpha-helical conformer population in the 317-329 H1 peptide and the breakage of the 3(10) or weakly H-bonded (nascent) alpha-helix in the H2 and H3 peptides. The conformational change observed upon deletion did not influence the efficiency of I-Ed peptide interaction, however, the C-terminal Arg had a beneficial effect both on MHC class II and class I binding without causing any remarkable change in solution conformation.
Asunto(s)
Hemaglutininas/química , Linfocitos T/inmunología , Animales , Dicroismo Circular , Hemaglutininas/inmunología , Humanos , Ratones , Conformación Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
We have prepared two types of cyclopeptides containing the 281DPVG284 sequence from the 276-284 region of glycoprotein gD-1 of the Herpes simplex virus (HSV). The syntheses were performed by solid phase methodology using MBHA or BHA resin and orthogonal protection schemes. Head-to-side-chain cyclization included the N-terminal part of the epitope, while side-chain-to-side-chain lactam bridge formation resulted in a peptide containing a C-terminal cycle. Peptides elongated by Cys at the N-terminal of the sequence were also prepared. Boc chemistry using Fmoc and OFm orthogonal protection was applied for on-resin cyclization. Based on the orthogonality of Bzl and cHex esters under a 1 M TMSOTf-thioanisole/TFA cleavage condition, a new approach for the cyclization on BHA-resin has also been developed. Preliminary studies on solution conformation of the cyclic peptides by CD spectroscopy indicated the importance of the location and the size of the cycle within the epitope sequence.
Asunto(s)
Simplexvirus/química , Proteínas del Envoltorio Viral/síntesis química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Epítopos , Glicoproteínas/síntesis química , Lactamas/química , Espectrometría de Masas , Péptidos/síntesis química , Estructura Secundaria de ProteínaRESUMEN
Oxidation of lincomycin with dimethyldioxirane resulted in the sulfoxide-glycosides 3a and 3b, whose treatment with osmium tetraoxide and N-methylmorpholine-N-oxide afforded the same sulfone; 4. According to FAB-MS and CD investigations, the absolute configuration of the sulfur atom in 3a and 3b is R and S, respectively. The new, unsaturated antibiotic analog (6) derived from clindamycin exists in the 4C1 conformation. The antibiotic activities of the synthesized compounds were also studied.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Lincomicina/análogos & derivados , Sulfonas/química , Sulfonas/farmacología , Sulfóxidos/química , Sulfóxidos/farmacología , Antibacterianos/síntesis química , Bacterias/efectos de los fármacos , Clindamicina/química , Lincomicina/síntesis química , Lincomicina/química , Lincomicina/farmacología , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Oxidación-Reducción , Sulfonas/síntesis química , Sulfóxidos/síntesis químicaRESUMEN
To investigate the role of secondary structure in the substrate specificity of human 72 kDa type IV collagenase, we synthesised linear and cyclic collagen sequence analogs. As Ca2+ plays a crucial role in the enzyme activity, the CD and FTIR spectra of the peptides were also measured in the presence of Ca2+. Most of the linear, but none of the cyclic peptides form stable 1:1 Ca2+ complexes. The cyclic hexapeptides adopt significantly different backbone conformations comprising not only beta-turns but also the less frequent gamma-turns. Consequently, in the cyclopeptides the scissile Gly-Ile(Leu) bond is embedded into a different conformational environment, but in spite of that none of them is a substrate or an inhibitor of the enzyme. The best substrate Ac-Pro-Leu-Gly-Leu-Ala-Gly-D-Lys-OH binds Ca2+, but does not form a stable 1:1 Ca2+ complex, which suggests that instead of a folded structure an extended flexible conformation is preferred by the enzyme.
Asunto(s)
Calcio/farmacología , Colágeno/química , Gelatinasas/metabolismo , Metaloendopeptidasas/metabolismo , Oligopéptidos/metabolismo , Estructura Secundaria de Proteína/efectos de los fármacos , Secuencia de Aminoácidos , Dicroismo Circular , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Humanos , Cinética , Metaloproteinasa 2 de la Matriz , Oligopéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Proteínas Recombinantes/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Especificidad por SustratoRESUMEN
An A/PR/8/34 (IHN1) influenza virus hemagglutinin (HA)-specific monoclonal antibody (Z38) was found to react with the solid phase adsorbed influenza virus expressing uncleaved (HA0) molecules but not to bind to virus particles bearing enzymatically cleaved hemagglutinin. Synthetic peptides corresponding to the uncleaved HA0 317-341 intersubunit region of subtypes H1-H3 (IP1-IP3) or comprising either the C-terminal 317-329 amino acids of HA1 (CP1) or the N-terminal 330-341 of HA2 (fusion peptide, FP) subunits of cleaved HA were used to characterize the fine specificity of Z38 mAb. Circular dichroism and Fourier-transform infrared spectroscopy showed that, compared to IP2 and IP3 comprising the H2 and H3 subtype fragments of the intact intersubunit region, IP1 has relatively low helicity but a tendency to adopt beta-turns in trifluoroethanol. The immunological and conformational properties of multiple antigenic peptides (MAPs) containing four copies of CP1 were also studied. Based on the appearance of an infrared component band at 1637 cm-1 (beta-turn band), the CP1 arms of MAPs also contain repeats of beta-turns. However, it is only the MAP1-FP construct comprising also the fusion peptide, which binds Z38 mAb as strongly as IP1 does. This puts emphasis on the role of the fusion region in modifying conformation and consequently the ability of peptides to elicit an antibody response. The results obtained for peptide conformation also suggests a beta-turn(s)/beta-sheet/beta-turn/beta-sheet conformational motif in the recognition by the hemagglutinin subtype-specific Z38 monoclonal antibody or by peptide-induced polyclonal antibodies.
Asunto(s)
Hemaglutininas Virales/química , Hemaglutininas Virales/genética , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Dicroismo Circular , Datos de Secuencia Molecular , Mapeo Peptídico , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Aluminium has been recognized to be a neurotoxic agent and a risk factor in Alzheimer's disease and other neuronal dysfunctions. CD spectroscopic studies on two synthetic fragments of the human neurofilament protein midsized subunit (NF-M), and their alanine-for-serine-substituted and/or serine-phosphorylated derivatives showed the formation of stable, citric acid resistant complexes of Al3+ with peptide ligands [M. Hollósi, Z. M. Shen, A. Perczel, and G. D. Fasman (1994) Proc. Natl. Acad. Sci. USA, vol. 9, pp. 4902-4906]. In the case of Ser-phosphorylated fragments, a beta-sheet inducing effect of Ca2+ and Al3+ ions was observed. However, the serine-containing parent peptides, NF-M13 (KSPVPKSPVEEKG) and NF-M17 (EEKGKSPVPKSPVEEKG), failed to show CD spectral changes reflecting beta-sheet formation upon addition of Al3+ ions. On the basis of the amide I region of the Fourier transform ir spectra, in trifluoroethanol, the peptide backbone of NF-M17 and NF-M17 (A6A11) shows marked changes in the presence of Al3+. The most significant spectral differences are seen in the carboxyl region (> 1700 cm-1). The high-frequency component bands above 1760 cm-1 in both spectra belong to the C = O of undissociated CF3COOH. Another strong band at 1710 cm-1 which appears only in the spectrum of NF-M17 (A6A11)(NF-M17 with Ser6 and Ser11 replaced by Ala) can be assigned to the side chain or C-terminal COOH groups. The differential protonation state of the carboxyl groups in the two peptides suggests the formation of Al3+ complexes of different structure and stability.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aluminio/química , Proteínas de Neurofilamentos/química , Fragmentos de Péptidos/química , Péptidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Dicroismo Circular , Humanos , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) beta-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of beta-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the beta-turn band showed up between 1639 and 1633 cm-1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band was also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3(10)-helical polypeptides and proteins in D2O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508-512; H. H. Mantsch, A. Perczel, M. Hollósi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch, A. Perczel, M. Hollósi, and G. Fasman (1992), Biopolymers, Vol. 33, pp. 201-207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Todd, and G. L. Millhauser (1992), Nature, Vol. 359, pp. 653-655], suggest that the amide I band, with a major contribution from the acceptor C = O of the 1<--4 intramolecular H bond of beta-turns, appears near or below 1640 cm-1, rather than above 1660 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Péptidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Dicroismo Circular , Datos de Secuencia Molecular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Epitopic peptides representing the C-terminal (HA1) region of cleaved hemagglutinin of influenza virus from different serotypes were synthesized. Circular dichroism and Fourier-transform infrared spectroscopic data showed that peptides HS2 and HS3 have a predominantly alpha-helical conformation in trifluoroethanol. Recently a component band appearing between 1640 and 1635 cm-1 in the amide I region of the Fourier-transform infrared spectra of polypeptides has been correlated with strongly H-bonded beta-turns (Ref. 8). Using this assignment, HS1 was found to contain less alpha-helix but have tendency to adopt beta-turn(s). Interestingly, fragment HS2 with the highest alpha-helix content proved to be the poorest T-cell epitope among serotypes HS1-HS3.
Asunto(s)
Epítopos/química , Hemaglutininas Virales/química , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Linfocitos T/inmunología , Secuencia de Aminoácidos , Dicroismo Circular , Análisis de Fourier , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/inmunología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría Infrarroja/métodos , Proteínas del Envoltorio Viral/químicaRESUMEN
Mono- and dithionated N-acyl amino acid and dipeptide N'-methylamides were synthesized using Lawesson's reagent and S-thioacetyl thioglycolic acid. The conformation of the thionated models was characterized by IR, 13C, and 1H NMR spectroscopy, including NOE experiments. The formation of -C = S...H-N-C = X (X = O or S) intramolecular H-bonds of the type 2----2, 1----3 and 1----4 was evidenced by the characteristic shifts of the IR stretching frequencies of the NH group. Act-Pro-NHCH3(4) and Act-Prot-NHCH3(5) were found to be present as mixtures of rotational isomers about the CS-N bond. 13C chemical shifts of the gamma- and beta-carbons of the proline ring elucidated the conformation (Z or E) of the tertiary thioamide group. Our results suggest that the conformation of thiopeptides is determined by two factors: 1) the H-bond donating and accepting ability of the thioamide group and 2) the repulsion between the thiocarbonyl sulfur atom and the side chain groups of the neighbouring amino acid residues.
Asunto(s)
Dipéptidos/química , Tioamidas/química , Enlace de Hidrógeno , Isomerismo , Espectroscopía de Resonancia Magnética , Conformación ProteicaRESUMEN
High hydrophobicity of the second amino acid N-terminal to the scissile bond (P2 residue) is generally considered to be the major factor in the specificity of the substrates for cysteine proteases of the papain family. To examine the catalytic contribution of the S2P2 hydrogen bond apparent from X-ray crystallographic studies, the kinetics of Z-Phe-Gly-OEt and its thiono derivative were compared. The thiono compound contains a sulfur atom in place of the carbonyl oxygen of the phenylalanine residue. It was found that the specificity rate constants for the reactions of the thiono substrate with various cysteine proteases are lower by 2-3 orders of magnitude as compared to the corresponding rate constants for the oxo substrate. This remarkable effect is not expected in the light of previous studies indicating that the change from oxygen to sulfur in the P1 residue was without an appreciable effect. The results are interpreted in terms of a distorted binding of the thiono substrate.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Papaína/metabolismo , Enlace de Hidrógeno , Cinética , Unión Proteica , Especificidad por SustratoRESUMEN
[Lyst6]GHRP, the C-terminally thionated analog of the highly potent growth hormone releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 was prepared by using solid support. The success of the synthesis showed that Lawesson's reagent can be used for selective thionation of an amide group not only in solution but also on the surface of a resin. The C-terminal thioamide group proved to be stable under the conditions of the solid phase synthesis.