Thiostrepton Research Articles

Molecular orbital constrained gas electron diffraction study of N-acetyl N′-methyl alanine amide

Gas electron diffraction (GED) data were recorded at about 510 K for , N-acetyl N′-methyl alanine amide (ALA) and analysed using the Molecular Orbital Constrained Electron Diffraction procedure. The HF/6-31G * geometries of seven conformations were optimized (C 7 eq ( 1), C 5 ( 2), β 2 ( 3), C 7 ax ( 4), α R ( 5), α L ( 6), and α′ ( 7) and used as constraints of the GED data analysis. HF/6-31G * force constants were determined for each conformation and used to calculate mean amplitudes of vibration and shrinkage corrections. It is found that, in the case of ALA, the customarily applied linear approximation for calculating shrinkages yields values which are unacceptably large. Therefore data analyses were performed both without shrinkage and with shrinkages calculated at a lower temperature. Subject to the selected constraints our interpretation of the GED data supports the following conclusions for vapors of ALA at about 500 K: ( 1) the compound exists in a conformational equilibrium with several significantly populated states; ( 2) the C 5 form is not less populated than the global energy minimum, C 7 eq: ( 3) the helical form, α R, is not a significantly populated conformer.

Chromatographic behaviour of opioid peptides containing beta-methylphenylalanine isomers.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed to obtain pure erythro[2S3S, 2R3R]- and threo[2S3R, 2R3S]-beta-methylphenylalanine. These amino acids were incorporated into an enkephalin, H-Tyr-D-Ala-Gly-beta-MePhe-Val-Val-Gly-NH2, and into a deltorphin C, H-Tyr-D-Ala-beta-MePhe-Asp-Val-Val-Gly-NH2, analogue, which yielded four diastereoisomers of the peptides. The diastereoisomers were separated on different columns and with different eluent systems. The sequence of elution of the peptide diastereoisomers was determined after hydrolysis of the peptides. For identification of the beta-methylphenylalanine enantiomers, enzymatic degradation and an RP-HPLC method were used, with application of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide as derivatizing reagent.

Monitoring of optical isomers of some conformationally constrained amino acids with tetrahydroisoquinoline or tetraline ring structures. Part II

Abstract Conformationally constrained amino acids were synthesized in chiral or racemic forms: d - amd l -1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), the erythro - d,l -4-methyl analogue, d - and l -7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, d - and l -7-hydroxy-6,8-diiodo-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, d,l -2-aminotetraline-2-carboxylic acid, d,l -6-hydroxy-2-aminotetraline-2-carboxylic acid and d,l -6-methoxy-2-aminotetraline-2-carboxylic acid. The optical isomers were characterized and identified by applying precolumn derivatization with chiral reagents (1-fluoro-2,4-dinitrophenyl-5- l -alanine amide and 2,3,4,5-tetra-O-acetyl-β- d -glucopyranosyl isothiocyanate) and enzymatic digestion with l -amino acid oxidase, carboxypeptidase A and α-chymotrypsin. The HPLC conditions (pH, eluent composition and different buffers) were varied to obtain optimum separations.

The preferred solid‐state conformation of (αMe)Trp peptides

The two Z-L-Ala-DL-(alpha Me)Trp-NH2 diastereomeric dipeptides were synthesized from (Z-L-Ala)2(O) and H-DL-(alpha Me)Trp-NH2. The latter racemate, prepared by phase-transfer catalyzed alkylation of the N alpha-benzylidene derivative of alanine amide followed by acidic hydrolysis of the resulting Schiff base, was characterized by X-ray diffraction. The molecular and crystal structure of Z-L-Ala-L-(alpha Me)Trp-NH2, separated from its diastereomer by silica-gel column chromatography, was determined by X-ray diffraction analysis. Both independent molecules in the asymmetric unit of the dipeptide adopt a type-II beta-bend conformation. However, only the more regularly folded conformation of molecule B is stabilized by a 1<--4 C = O...H--N intramolecular H bond. The present results indicate that: (i) the C alpha-methylated (alpha Me)Trp residue is a strong beta-bend and helix former, and (ii) the relationship between (alpha Me)Trp chirality and helix screw sense tends to be opposite to that of protein amino acids. The implications for the use of the (alpha Me)Trp residue in designing conformationally restricted analogs of bioactive peptides are briefly discussed.

Investigation of electron correlation effects on molecular geometries

A survey is presented as part of an ongoing program in which the effects of electron correlation on molecular structures are studied for the purpose of obtaining data that can be used in developing force field parameters for empirical molecular modeling. In our approach, molecular structures devoid of dispersion forces, i.e. optimized at the Hartree Fock (HF) level, are compared with the same structures optimized at the MP2 level, in which the dispersion forces are “switched on”. So far, the investigations have included hydrocarbons, to study aliphatic non-bonded interactions, systems with internal hydrogen bonding (ethylene glycol, glycerol, glycine and the model dipeptide N-formyl alanine amide), and silicates, which are studied in order to derive force field parameters applicable to modeling clays. In hydrocarbons, electron correlation effects lead to contractions in 1,5-non-bonded distances in all rotamers with gauche (G) torsions, particularly in GG sequences, in which small changes in torsional angles (< 5°) can cause large changes (a few tenths of an angstrom) in non-bonded distances in the attractive region of the van der Waals potential. In ethylene glycol and glycerol, hydrogen bonds in MP2 structures are 0.1 to 0.2 Å shorter than the same bonds in the corresponding HF structures. In silicates, significant correlation effects are found both in primary structural parameters and torsional angles, and again long-range non-bonded distances are contracted in structures in which correlation effects are active. In the context of the widely used single-point energy calculations at the correlation level (for example single-point MP2 energies calculated for HF geometries), we find that the errors connected with the lack of geometry optimization at the MP2 level increase with the size of a system and are larger for folded conformations than stretched ones.

Investigation of electron correlation effects on molecular geometries

A survey is presented as part of an ongoing program in which the effects of electron correlation on molecular structures are studied for the purpose of obtaining data that can be used in developing force field parameters for empirical molecular modeling. In our approach, molecular structures devoid of dispersion forces, i.e. optimized at the Hartree-Fock (HF) level, are compared with the same structures optimized at the MP2 level, in which the dispersion forces are “switched on”. So far, the investigations have included hydrocarbons, to study aliphatic non-bonded interactions, systems with internal hydrogen bonding (ethylene glycol, glycerol, glycine and the model dipeptide N-formyl alanine amide), and silicates, which are studied in order to derive force field parameters applicable to modeling clays. In hydrocarbons, electron correlation effects lead to contractions in 1,5-non-bonded distances in all rotamers with gauche (G) torsions, particularly in GG sequences, in which small changes in torsional angles (< 5°) can cause large changes (a few tenths of an angström) in non-bonded distances in the attractive region of the van der Waals potential. In ethylene glycol and glycerol, hydrogen bonds in MP2 structures are 0.1 to 0.2Å shorter than the same bonds in the corresponding HF structures. In silicates, significant correlation effects are found both in primary structural parameters and torsional angles, and again long-range non-bonded distances are contracted in structures in which correlation effects are active. In the context of the widely used single-point energy calculations at the correlation level (for example singlepoint MP2 energies calculated for HF geometries), we find that the errors connected with the lack of geometry optimization at the MP2 level increase with the size of a system and are larger for folded conformations than stretched ones.

Geometry optimization, energetics and solvation studies on four- and five-membered cyclic and disulfide-bridged peptides, using the programs quanta 3.3 and charmm 22

Force field parameters for peptides and proteins in the charmm 22 force field have been refined using the results of MP2/6-311G ∗∗ calculations on N-formyl alanine amide (Frey et al., J. Am. Chem. Soc., 114 (1992) 5369) and other data (Remek et al., J. Mol. Struct. (Theochem), 235 (1991) 1). Significant changes in the peptide backbone torsional energy terms in charmm 22 were made in order to be consistent with the MP2 results relative to previously fitted HF results. To test the new force field parameters against experimental data, molecular mechanics calculations were carried out on small four- and five-membered cyclic and disulfide-bridged peptides. It was found that in general these molecules are in their calculated minimum energy conformation or a low energy conformation in the X-ray structure. The average deviation of the dihedral angles calculated for the seven energy minimized vacuum structures from the X-ray values is 10°. The dihedral angle deviations of the minimized and dynamically averaged conformations are similar for studies which included explicit TIP3P solvent molecules. Upon solvation and minimization, internal vacuum energies increase by 4–7 kcal mol −1. In addition to calculated structural results, we report some of the force field parameters used for the calculations, energy decompositions, and harmonic vibrational free energies.

Conformational analysis and structural study by ab initio gradient geometry optimizations of the model tripeptide N-formyl L-alanyl L-alanine amide

The geometries of 15 conformations of the model tripeptide N-formyl l-alanyl l-alanine amide (Ala-Ala) were determined by ab initio gradient geometry refinements at the HF/4-21G level. The results can be compared with previous HF/4-21G calculations on the single residue, N-formyl alanine amide (Ala), and the model hexapeptide, N-formyl pentaalanine amide (Penta-Ala). Cooperative energy effects are found in Ala-Ala that are subtle in some (φ 1, ψ 1 and φ 2, ψ 2) conformations, such as (C7 eq C7 ax), (β, C7 eq) and (C7 ax, C7 ax), but more pronounced in bend forms, such as (C7 eq, α '), (C7 eq, α l ), and (C7 ax, 3 10 ). Optimization of α-helical structures leads to 3 10 -type helices, both in empirical charmm and ab initio HF/4-21G calculations, away from α R. This property of the calculated vacuum structures is in agreement with experimental investigations of short alanine based peptides in aqueous solution. The conformationally dependent structural trends found here for Ala-Ala are in agreement with the trends previously derived for Ala and Penta-Ala. They are particularly significant for the N-C(α)-C' angle. This parameter shows variations of up to 8° in the selected conformers and, regardless of how the residues are combined, its extension follows the approximate sequence β < C7 eq < C7 ax ≈ 3 10 -helix.

On the reaction of 2-aminopropionitrile in aqueous media

The reactions of 2- and 3-aminopropionitrile (APN), and 2,2′-iminodipropionitrile (IDPN) were carried out in aqueous ammoniacal media. 2-APN was found to give IDPN, N-(1-cyanoethyl)alanine amide, N-(1-cyanoethyl)alanine, N-(1-carbamoylethyl)alanine, 2,2′-iminodipropionic acid, alanine amide, and alanine. Compounds of biological significance such as peptides and amino acids other than alanine were not formed. The results were well consistent with those obtained for aminoacetonitrile. IDPN which can be formed easily from 2-APN in aqueous media, also yielded the same products as with 2-APN. On the other hand, 3-APN gave 3-alanine via 3-alanine amide under similar conditions. 3-APN was found to be more stable than 2-APN in aqueous media.

Determination of the chiral purity of benzylic amines using marfey's reagent

1-Fluoro-2,4-dinitrophenyl-5-(S)-alanine amide (Marfey's reagent) was used as a chiral derivatizing agent for benzylic amines in order to determine optical purities.

Structural Studies of Antarctic Fish Antifreeze Glycoproteins by One- and Two-Dimensional NMR Spectroscopy

Abstract The solution structure of antifreeze glycoproteins (AFGP's) of the polar fish Tetramatomus borchgrevinki has been investigated by 2D 1 H NMR spectroscopy as well as molecular modeling calculations (MM2). The simple glycotripeptide repeating structure in the shorter AFGP's (fractions 7 & 8) makes the structural analysis amenable. The resonance assignments of AFGP's 7 & 8 were determined by two-dimensional NMR techniques (COSY, Relayed-COSY, Phase Sensitive DQCOSY, NOESY). Information about the protein secondary structure was obtained by the coupling constants between the back-bone amide and α-carbon protons (obtained by phase sensitive COSY). Additional three dimensional constraints were obtained from NOESY through-space connectivities. The three dimensional solution structures of several AFGP's glycotripeptide fragments were based on MM2 calculations. The model structure was compared with the experimental data. Exchange rates of amide protons measured by dynamical spectroscopy show that the threonine and some of the alanine amide protons have two different and distinct exchange rates. GalNAc and the C-terminal Ala' amide protons appear to show relatively slow exchznge rates. The results suggets that the amide protons are not involved in any strong intramolecular hydrogen bonding.

Isolation and characterization of two novel peptide amides originating from myelin basic protein in bovine brain.

During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-amide and Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Ser-Ala-amide . These 12 and 16 residues peptides had the primary structure identical to the N-terminal fragment of myelin basic protein (MBP). The peptides were therefore designated myelin peptide amide-12 (MPA-12) and -16 (MPA-16). Unlike other amidated peptides, MPA might be generated from MBP by hydroxyl radicals produced via a Fenton reaction in situ. However, this unique amidation seems to occur exclusively to MBP in a site specific manner in the brain.

Cleavage kinetics and anchor linked intermediates in solid phase peptide amide synthesis

Kinetics and cleavage conditions of peptide amide synthesis were studied using the anchor molecules 5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid (4-ADPV-OH) and 5-(2'-aminomethyl-3'-5'-dimethoxyphenoxy) valeric acid (2-ADPV-OH). Unexpectedly the anchor amide alanyl-4-ADPV-NH2 was isolated and characterized as an intermediate during the cleavage with trifluoroacetic acid (TFA) of alanyl-4-ADPV-alanyl-aminomethyl-polystyrene to yield the alanine amide. As a matter of fact the NH--CH alpha bond of the alanyl spacer has to be cleaved to form this intermediate. Using TFA-dichloromethane (1:9) alanyl-4-ADPV-NH2 was obtained as a cleavage product in 50% yield within 60 min, whereas the isomeric alanyl-2-ADPV-NH2 was formed more slowly under these mild conditions. At high TFA concentration no difference between the 2- and 4-ADPV anchor was observed in the rate of formation of the free alanine amide. The presence of tryptophan amide in the cleavage mixture resulted in an anchor alkylated tryptophan amide, which remains stable in acidic solution but disappears rapidly in the presence of the resin. A low TFA/high TFA cleavage procedure is recommended for peptide amid synthesis applying the ADPV anchor.

Plasmid‐based, D‐aminopeptidase‐catalysed synthesis of (R)‐amino acids

AbstractThe gene for D‐aminopeptidase from Ochrobactrum anthropi SCRC Cl‐38 was cloned in Escherichia coli JM109. An expression plasmid pCl38DP (4.5 kb) was constructed. The amount of the enzyme in a cell‐free extract of E. coli JM109/pCl38DP was elevated up to 288000 units/liter culture, which is about 3600‐fold over that of O. anthropi SCRC Cl‐38. It was calculated that the enzyme comprised about 30% of the total extractable cellular protein. The intact cells of the E. coli transformant were used as a catalyst for (R)‐stereospecific hydrolysis of several racemic amino amides HCl. Complete hydrolysis of (R)‐alanine amide was achieved in a short time (4½h) from 5.0M racemic alanine amide HCl using cells of the E. coli transformant. The concentration of (R)‐alanine reached 220 g/l. The cells or the cell‐free extract catalyzed the synthesis of (R)‐2‐aminobutyric acid, (R)‐methionine, (R)‐norvaline and (R)‐norleucine from their amides in a similar manner.

Variable step molecular dynamics: An exploratory technique for peptides with fixed geometry

AbstractA new algorithm is presented for performing molecular dynamics simulations of peptides with fixed geometry, with the aim of simulating conformational changes and of exploring conformational space. The principle of the method is to expand the potential energy as a Taylor's series in the coordinates around the current point, retaining the force and its first two derivatives, and obtain a series solution of the resulting differential equations using a method due to Lyapunov. By choosing the time step so that the second term in the series is small compared to the first, the true solution can in principle be approximated to any desired degree of accuracy. The algorithm has been used to solve numerically Lagrange's equations of motion for N‐acetyl alanine amide and N‐acetyl methionide amide, regarded as fixed at their C‐termini, under the influence of the ECEPP/2 potential energy function, and time steps of 15–30 fsec have been achieved with little variation in the total energy. Possible directions for future development are discussed.

Pituitary thyroid function and prolactin release following intravenous administration of TRH and pyroglutamyl-histidyl-prolyl-β alanine amide to healthy volunteers.

The synthetic oligopeptide pyroglutamyl-histidyl-prolyl-β-alanine amide, structurally related to thyrotrophin releasing hormone (TRH), has been investigated in healthy male volunteers for its effect on pituitary thyroid function and on serum prolactin levels. In an open randomised 2-way crossover study the peptide has been compared with TRH on an equal dose basis following intravenous administration at 4 dose levels. The synthetic material had significantly lower thyrotrophin (TSH)-releasing activity than TRH while the prolactin releasing potencies of the 2 peptides were about equal.

A novel aminopeptidase from clostridium histolyticum

An aminopeptidase was found in the culture filtrate of Cl. histolyticum and purified to homogeneity (130 times) in a two-step procedure. All types of N-terminal amino acids, including proline and hydroxyproline are cleaved by the enzyme from small peptides and from polypeptides. A low rate of hydrolysis was observed for β-naphthylamides and for alanine amide; p-nitroanilides were not hydrolyzed. Kinetic parameters (Km and Vmax) for several tripeptides and the tetrapeptide Pro-Gly-Pro-Pro were determined. The enzyme has a pH optimum at 8.6. The presence of either Mn++ or Co++ is essential for its activity. Only slight activation was observed with Ni++ and Cd++, while Zn++ and Cu++ were inhibitory. The molecular weight of the native enzyme is about 340,000, and a molecular weight of about 60,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate (SDS).The culture filtrate of Cl. histolyticum has been shown to contain various proteolytic enzymes, in addition to collagenase1–5. In a search for enzymes acting on proline-rich peptides, we tested the crude filtrate with (Pro-Gly-Pro)n, (Pro-Gly-Pro)n-OMe, α,DNP-(Pro-Gly-Pro)n and poly-L-proline as substrates. Proline was formed only from (Pro-Gly-Pro)n and its methyl ester. This showed the presence in Cl. histolyticum filtrate of an aminopeptidase which cleaves N-terminal proline from polypeptides but not from polyproline. The purification and some of the properties of this clostridial aminopeptidase (CAP) are described in this communication.

Degradation of Thiostrepton.1 The Structure of Thiostreptine

<b>Degradation of Thiostrepton.</b>¹ <b>The Structure of Thiostreptine</b>