Photomodulation of conformational states. II. Mono- and bicyclic peptides with (4-aminomethyl)phenylazobenzoic acid as backbone constituent

It has been reported that backbone cyclization of octapeptides with the photoresponsive (4-aminomethyl)phenylazobenzoic acid imparts sufficient restraints to induce and stabilize ordered conformations of the peptide backbone in both the cis- and trans-azo-isomers (L. Ulysse, J. Cubillos, and J. Chmielewski, Journal of the American Chemical Society, 1995, Vol. 117, pp. 8466-8467). Correspondingly, the active-site octapeptide fragment H-Ala-Cys-Ala-Thr-Cys-Asp-Gly-Phe-OH [134-141] of thioredoxin reductase, with its high preference for a 3(10)-helix turn conformation centered on the Thr-Cys sequence, was backbone cyclized with this azobenzene moiety in the attempt to design a photoresponsive system where the conformational states of the peptide backbone are dictated by the configuration of the azobenzene and can be further modulated by the disulfide bridge. Nuclear magnetic resonance conformational analysis of the monocyclic compound clearly revealed the presence of two conformational families in both the cis- and trans-azo configuration. Of the higher populated conformational families, the structure of the trans-isomer seems like a pretzel-like folding, while the cis-isomer relaxes into a significantly less defined conformational state that does not exhibit any regular structural elements. Further restrictions imparted by disulfide bridging of the peptide moiety leads to an even better defined conformation for the trans-azo-isomer, whereas the cis-isomer can be described as a frustrated system without pronounced energy minima and thus with little conformational preferences. Our findings would suggest that this photoresponsive peptide template may not be of general usefulness for light-induced conformational transitions between two well-defined conformational states at least under the experimental conditions employed, even in the bicyclic form. However, trans --> cis isomerization of the bicyclic peptide is accompanied by a switch from a well-defined conformation to an ensemble of possible conformations.     

Photomodulation of conformational states. Synthesis of cyclic peptides with backbone-azobenzene moieties.

The search for photoresponsive conformational transitions accompanied by changes in physicochemical and biological properties led us to the design of small cyclic peptides containing azobenzene moieties in the backbone. For this purpose, (4-aminomethyl)phenylazobenzoic acid (H-AMPB-OH) and (4-amino)phenylazobenzoic acid (H-APB-OH) were synthesized and used to cyclize a bis-cysteinyl-octapeptide giving monocyclic derivatives in which additional conformational restriction could be introduced by conversion to bicyclic structures with a disulphide bridge. While synthesis with H-AMPB-OH proceeded smoothly on a chlorotrityl-resin with Fmoc/tBu chemistry, the poor nucleophilicity of the arylamino group of H-APB-OH required special chemistry for satisfactory incorporation into the peptide chain. Additional difficulties were encountered in the reductive cleavage of the S-tert-butylthio group from the cysteine residues since concomitant reduction of the azobenzene moiety took place at competing rates. This difficulty was eventually bypassed by using the S-trityl protection. Side-chain cyclization of the APB-peptide proved to be difficult, suggesting that restricted conformational freedom was already present in the monocyclic form, a fact that was fully confirmed by NMR structural analysis. Conversely, the methylene spacer in the AMPB moiety introduced sufficient flexibility for facile and quantitative side-chain cyclization to the bicyclic form. Both of the monocyclic peptides and both of the bicyclic peptides are photoresponsive molecules which undergo cis/trans isomerization reversibly.     

Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.     

N-(3-(4-Hydroxyphenyl)-propenoyl)-amino acid tryptamides as SIRT2 inhibitors

A series of N-(3-(4-hydroxyphenyl)-propenoyl)-amino acid tryptamides was based on a previously reported new SIRT2 inhibitor from our group, and it was designed to study if the molecular size of the compound could be reduced. The most potent compounds, N-(3-(4-hydroxyphenyl)-propenoyl)-2-aminoisobutyric acid tryptamide and N-(3-(4-hydroxyphenyl)-propenoyl)-L-alanine tryptamide, were equipotent, 30% smaller in molecular weight, and slightly more selective (SIRT2/SIRT1) than the parent compound.     

Mono- and bimetallic gold(I) and silver(I) pentafluoropropionates and related compounds

A series of eight new carboxylate complexes of the general type (L)_nMOC(O)R (L=PMe₃; n=1; M=Ag, Au; R=C₂F₅. L=PPh₃; n=1-3; M=Ag; R=C₂F₅, t-Bu) have been prepared in high yields. Crystal and molecular structures have been determined for three representative examples. The crystal structure of (Ph₃P)AgOC(O)C₂F₅ contains dimers in which the silver atoms are bridged by the carboxylate oxygen atoms. This bridging resembles the structural motif found in silver carboxylates without ligand support. Usage of the smaller phosphine PMe₃ leads to the formation of a polymeric chain structure in (Me₃P)AgOC(O)C₂F₅ with bridging carboxylate anions and short Ag-Ag contacts holding the monomers together. The reaction of (4-Me₂N-C₆H₄)Ph₂ PAuCl with two equivalents of C₂F₅CO₂Ag leads to the formation of a mixed metal product containing both gold and silver. The crystal structure analysis of this compound revealed a tetranuclear complex containing a central dimeric silver pentafluoropropionate unit which is chelated by the (triarylphosphine)gold(I) pentafluoropropionate molecules via Ag-Au metallophilic contacts and Ag-O donor/acceptor interactions.     

Synthesis of A 3-oxo-4(S)-amino acid analog of pepstatin. A new inhibitor of carboxyl (acid) proteases

A ketone analog of pepstatin, in which the 3S hydroxyl group is oxidized to a ketone group, has been synthesized and shown to be a potent inhibitor of pepsin. Kinetics of inhibition of pepsin provide evidence that the ketone pepstatin analog binds to pepsin differently than pepstatin. The relationship of these complexes to crystal complexes of pepstatin-carboxyl proteases is discussed.     

Role of amino acid properties to determine backbone tau(N-Calpha-C') stretching angle in peptides and proteins

The analysis of the basic geometry of amino acid residues of protein structures has demonstrated the invariability of all the bond lengths and bond angles except for tau, the backbone N-Calpha-C' angle. This angle can be widened or contracted significantly from the tetrahedral geometry to accommodate various other strains in the structure. In order to accurately determine the cause for this deviation, a survey is made for the tau angles using the peptide structures and the ultrahigh resolution protein structures. The average deviation of N-Calpha-C' angles from tetrahedral geometry for each amino acid in all the categories were calculated and then correlated with forty-eight physiochemical, energetic and conformational properties of amino acids. Linear and multiple regression analysis were carried out between the amino acid deviation and the 48 properties. This study confirms the deviation of tau angles in both the peptide and protein structures but similar forces do not influence them. The peptide structures are influenced by physical properties whereas as expected the conformational properties influence the protein structures. And it is not any single property that dominates the deviation but the combination of different factors contributes to the tau angle deviation.     

The (17)O-NMR shielding range and shielding time scale and detection of discrete hydrogen-bonded conformational states in peptides

The (17)O-NMR shielding range and shielding time scale due to hydrogen-bonding interactions in peptides are critically evaluated relative to those of (1)H-NMR. Furthermore, the assumptions and conclusions in previous (17)O-NMR studies on the detection of discrete conformational states in peptides (V. Tsikaris et al., Biopolymers, 2000, Vol. 53, pp. 135-139) are reconsidered. Consistent examination of the method demonstrates that although (17)O shieldings of peptide oxygens are very sensitive to hydrogen bonding interactions, the (17)O-NMR shielding time scale is not advantageous compared to that of (1)H-NMR, and thus it is not suitable for the detection of discrete hydrogen-bonded conformational states in peptides. (17)O-NMR spectroscopy is prone to interpretation errors due to the formation of (17)O-labeled impurities during the synthetic procedures (A. Steinschneider et al., International Journal of Peptide and Protein Research, 1981, Vol. 18, pp. 324-333).     

L-2-hydroxytetradecanoic acid as a constituent of Salmonella lipopolysaccharides (lipid A)

L-2-Hydroxytetradecanoic acid was recognized as a characteristic, although minor, constituent of the lipid A component of Salmonella lipopolysaccharides. The 2-hydroxy fatty acid was present in lipid A as an ester, probably bound to the hydroxyl group of some D-3-hydroxytetradecanoic acid residues. A survey of enterobacterial lipopolysaccharides showed that L-2-hydroxytetradecanoid acid was also present in Klebsiella and Serratia strains. It was absent, however, from lipopolysaccharides of other genera of the family including Escherichia, Shigella, Proteus, Enterobacter and Yersinia. This restricted distribution of the 2-hydroxy acid may be of significance for taxonomic studies of bacterial genera.     

Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid.

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.     

Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes

Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.     

Synthesis of novel protected Nalpha(omega-thioalkyl) amino acid building units and their incorporation in backbone cyclic disulfide and thioetheric bridged peptides.

General methods for the preparation of protected Nalpha(omega-thioalkyl) amino acids building units for backbone cyclization using reductive alkylation and on-resin preparation are described. The synthesis of non-Gly Fmoc-protected S-functionalized N-alkylated amino acids is based on the reaction of readily prepared protected omega-thio aldehyde with the appropriate amino acid. Preparation of Fmoc-protected S-functionalized N-alkylated Gly building units was carried out using two methods: reaction of glyoxylic acid with Acm-thioalkylamine and an on-resin reaction of bromoacetyl resin with Trt-thioalkylamines. Three model peptides were prepared using these building units. The GlyS2 building unit was incorporated into a backbone cyclic analog of somatostatin that contains a disulfide bridge. Formation of the disulfide bridge was performed by on-resin oxidation using 12 or Tl(CF3COO-)3. Both methods resulted in the desired product in a high degree of purity in the crude. The AspS3 building unit was also successfully incorporated into a model peptide. In addition, the in situ generation of sulfur containing Gly building units was demonstrated on a Substance P backbone cyclic analog containing a thioether bridge.     

Quinacrine and 9-amino acridine inhibit B-Z and B-H(l) form DNA conformational transitions

The interaction of quinacrine and 9-amino acridine with right-handed B-form, left-handed Z-form, and left-handed protonated (H(L))-form structures of polydG-me(5)dC was investigated by circular dichroism and absorption spectral analysis. Both the compounds bind strongly to the B-form structure and convert the Z-form and H(L)-form back to the bound right-handed form. Circular dichroic data revealed that the conformation at the binding site is right-handed even though adjacent regions of the polynucleotide may have left-handed conformation. The rate and extent of B-form-to-Z-form transition were decreased in the presence of these compounds. Scatchard analysis revealed that both quinacrine and 9-amino acridine bind strongly to the polynucleotide in the B-form in a noncooperative manner, in sharp contrast to the highly cooperative binding to the Z-form and H(L)-form. Results indicated that the cooperative binding of these drugs with the Z-form and the H(L)-forms was associated with a sequential conversion of the polynucleotide from a left-handed to a bound right-handed conformation. Experimental data enabled the calculation of the number of base pairs of Z-form (7-8 with quinacrine and 9-amino acridine) and H(L)-form (4 and 25, respectively, with quinacrine and 9-amino acridine) that adopt a right-handed conformation for each bound ligand. As these compounds are known to bind preferentially to alternating guanine--cytosine sequences, which are capable of easily undergoing the B-to-Z or B-to-H(L) transition, these effects may be important in understanding their biological activities.     

Interactions of d(10) metal ions with hippuric acid and cytosine. X-ray structure of the first cadmium (II)-amino acid derivative-nucleobase ternary compound.

The interactions of Zn(II), Cd(II) and Hg(II) with hippuric acid (hipH) were studied and several novel compounds were synthesized and studied by NMR. Some new metal-hippuric-cytosine ternary compounds were formed and the structure of the [Cd(hip)(2)(cyt)(H(2)O)](2) ternary complex resolved. Each cadmium (II) atom has a distorted trigonal bipyramid coordination which is linked to a water molecule, a cytosine via N(3), a carboxylic oxygen atom of a hippurate moiety and two bridging dicoordinated hippurates bound through the carboxylic oxygen atoms. To these five main bonds, two longer ancillary interactions can be observed: the second oxygen of the monocoordinated hippurate group and the carboxylic oxygen of the cytosine ligand. The compound is stabilized by an intramolecular stacking between the benzene and cytosine rings and by the hydrogen bonds between the coordinated water molecules and the ligands. This is, to our knowledge, the first structure of a cadmium-amino acid derivative-natural nucleobase compound described so far.