Development of a new family of conformationally restricted peptides as potent nucleators of beta-turns. Design, synthesis, structure, and biological evaluation of a beta-lactam peptide analogue of melanostatin

Novel enantiopure (i)-(beta-lactam)-(Gly)-(i+3) peptide models, defined by the presence of a central alpha-alkyl-alpha-amino-beta-lactam ring placed as the (i+1) residue, have been synthesized in a totally stereocontrolled way by alpha-alkylation of suitable N-[bis(trimethylsilyl)methyl]-beta-lactams. The structural properties of these beta-lactam pseudopeptides have been studied by X-ray crystallography, Molecular Dynamics simulation, and NOESY-restrained NMR simulated annealing techniques, showing a strong tendency to form stable type II or type II' beta-turns either in the solid state or in highly coordinating DMSO solutions. Tetrapeptide models containing syn- or anti-alpha,beta-dialkyl-alpha-amino-beta-lactam rings have also been synthesized and their conformations analyzed, revealing that alpha-alkyl substitution is essential for beta-turn stabilization. A beta-lactam analogue of melanostatin (PLG amide) has also been prepared, characterized as a type-II beta-turn in DMSO-d6 solution, and tested by competitive binding assay as a dopaminergic D2 modulator in rat neuron cultured cells, displaying moderate agonist activity in the micromolar concentration range. On the basis of these results, a novel peptidomimetic design concept, based on the separation of constraint and recognition elements, is proposed.     

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.     

Incorporating beta-turns and a turn mimetic out of context in loop 1 of the WW domain affords cooperatively folded beta-sheets.

To probe the conformational requirements of loop 1 in the Pin1 WW domain, the residues at the i + 2 and i + 3 positions of a beta-turn within this loop were replaced by dPro-Gly and Asn-Gly, which are known to prefer the conformations required at the i + 1 and i + 2 positions of type II' and type I' beta-turns. Conformational specificity or lack thereof was further examined by incorporating into the i + 2 and i + 3 positions a non-alpha-amino acid-based beta-turn mimetic (4-(2'-aminoethyl)-6-dibenzofuran propionic acid residue, 1), which was designed to replace the i + 1 and i + 2 positions of beta-turns. All these Pin WW variants are monomeric and folded as discerned by analytical ultracentrifugation, NMR, and CD. They exhibit cooperative two-state transitions and display thermodynamic stability within 0.5 kcal/mol of the wild-type WW domain, demonstrating that the acquisition of native structure and stability does not require a specific sequence and, by extension, conformation within loop 1. However, it could be that these loop 1 mutations alter the kinetics of antiparallel beta-sheet folding, which will be addressed by subsequent kinetic studies.     

Ni-to-Ni+ 3-ethylene-bridged partially modified retro-inverso tetrapeptide beta-turn mimetic: design,synthesis, and structural characterization

A 10-membered heterocyclic ring system 1,3,8-trisubstituted 2,5,7-trioxo-1,4,8-triazadecane that represents a Ni-to-Ni+ 3-ethylene-bridged partially modified retro-inverso tetrapeptide beta-turn mimetic (EBRIT-BTM) has been designed, synthesized, and structurally analyzed. These compounds utilize an ethylene bridge to replace the COi...HNi + 3 10-membered hydrogen bond of standard beta-turns. The N,N'-ethylene-bridged dimer was obtained in 90% yield by reductive alkylation of phenylalanylamide with a tert-butyl N-(9-fluorenylmethyloxycarbonyl),N-(2-formylmethyl)-glycinate. An orthogonal protection strategy and HATU-mediated couplings allowed efficient stepwise additions of monomeric building blocks leading to a N(i)-to-N(i+3)-ethylene-bridged linear precursor: Further elaboration of the linear precursor generated the ethylene-bridged model compounds (16) and (18) (g, gem-diaminoalkyl; m, malonyl; and r, direction-reversed amino acid residue) in 44 and 39% yields, respectively. The structural features of the two EBRIT-BTM compounds were determined using 1H NMR and extensive computer simulations. The results indicate that the 10-membered rings are conformationally constrained with well-defined structural features and that the three amide bonds in the ring are in the trans orientation. The topological arrangement of the residues in the ring system closely resembles a type II' beta-turn. Transformation of CONH(2) in the N-terminal amino acid residue of 16 into NHCOCH3 in 18 resulted in the formation of a hydrogen bond between the NH of gPhe-COCH3 and the C-terminal carboxyl of Gly, initiating an antiparallel beta-sheet. The formulation of the concept applying a minimalistic structural elaboration approach and the synthetic exploration, together with the conformational analysis, offer a new molecular scaffolding system and a true tetrapeptide secondary structure mimetic that can be used to generate peptidomimetics of biological interest.     

Designed peptides with homochiral and heterochiral diproline templates as conformational constraints

Diproline segments have been advanced as templates for nucleation of folded structure in designed peptides. The conformational space available to homochiral and heterochiral diproline segments has been probed by crystallographic and NMR studies on model peptides containing L-Pro-L-Pro and D-Pro-L-Pro units. Four distinct classes of model peptides have been investigated: a) isolated D-Pro-L-Pro segments which form type II' beta-turn; b) D-Pro-L-Pro-L-Xxx sequences which form type II'-I (betaII'-I, consecutive beta-turns) turns; c) D-Pro-L-Pro-D-Xxx sequences; d) L-Pro-L-Pro-L-Xxx sequences. A total of 17 peptide crystal structures containing diproline segments are reported. Peptides of the type Piv-D-Pro-L-Pro-L-Xxx-NHMe are conformationally homogeneous, adopting consecutive beta-turn conformations. Peptides in the series Piv-D-Pro-L-Pro-D-Xxx-NHMe and Piv-L-Pro-L-Pro-L-Xxx-NHMe, display a heterogeneity of structures in crystals. A type VIa beta-turn conformation is characterized in Piv-L-Pro-L-Pro-L-Phe-OMe (18), while an example of a 5-->1 hydrogen bonded alpha-turn is observed in crystals of Piv-D-Pro-L-Pro-D-Ala-NHMe (11). An analysis of pyrrolidine conformations suggests a preferred proline puckering geometry is favored only in the case of heterochiral diproline segments. Solution NMR studies, reveal a strong conformational influence of the C-terminal Xxx residues on the structures of diproline segments. In L-Pro-L-Pro-L-Xxx sequences, the Xxx residues strongly determine the population of Pro-Pro cis conformers, with an overwhelming population of the trans form in L-Xxx=L-Ala (19).     

Beta-turn mimic in tripeptide with Phe(1)-Aib(2) as corner residues and beta-strand structure in an isomeric tripeptide: an X-ray diffraction study

A single crystal X-ray diffraction study of the tripeptide Boc-Phe-Aib-Leu-OMe (Aib = alpha-aminoisobutyric acid) reveals that it forms structurally one of the best type II beta-turns so far reported in tripeptides, stabilized by 10 atom intramolecular hydrogen bonding. In contrast, the isomeric tripeptide Boc-Phe-Leu-Aib-OMe adopts a beta-strand like conformation. Interestingly, a previously reported structure of another isomeric tripeptide, Boc-Leu-Aib-Phe-OMe, shows a double bend conformation without any intramolecular hydrogen bonding. These results demonstrate an example of the creation of structural diversities in the backbone of small peptides depending upon the co-operative steric interactions amongst the amino acid residues.     

Do benzodiazepines mimic reverse-turn structures?

The role of benzodiazepine derivatives (BZD) as a privileged scaffold that mimics beta-turn structures (Ripka et al. (1993) Tetrahedron 49:3593-3608) in peptide/protein recognition was reexamined in detail. Stable BZD ring conformers were determined with MM3, and experimental reverse-turn structures were extracted from the basis set of protein crystal structures previously defined by Ripka et al. Ideal beta-turns were also modeled and similarly compared with BZD conformers. Huge numbers of conformers were generated by systematically scanning the torsional degrees of freedom for BZDs, as well as those of ideal beta-turns for comparison. Using these structures, conformers of BZDs were fit to experimental structures as suggested by Ripka et al., or modeled classical beta-turn conformers, and the root-mean-square deviation (RMSD) values were calculated for each pairwise comparison. Pairs of conformers with the smallest RMSD values for overlap of the four alpha-beta side-chain orientations were selected. All overlaps of BZD conformers with experimental beta-turns yielded one or more comparisons where the least RMSD was significantly small, 0.48-0.86 angstroms, as previously suggested. Utilizing a different methodology, the overall conclusion that benzodiazepines could serve as reverse-turn mimetics of Ripka et al. is justified. The least RMSD values for the overlap of BZDs and modeled classical beta-turns were also less than 1 angstrom. When comparing BZDs with experimental or classical beta-turns, the set of experimental beta-turns selected by Ripka et al. fit the BZD scaffolds better than modeled classical beta-turns; however, all the experimental beta-turns did not fit a particular BZD scaffold better. A single BZD ring conformation, and/or chiral orientation, can mimic some, but not all, of the experimental beta-turn structures. BZD has two central ring conformations and one chiral center that explains why the four variations of the BZD scaffold can mimic all types of beta-turn structure examined. It was found, moreover, that the BZD scaffold also mimics each of the nine clusters of experimental orientations of side chains of reverse turns in the Protein Data Bank, when the new classification scheme for the four side-chain directions (the relative orientations of alpha-beta vectors of residues i through i+3) was considered (Tran et al. (2005) J Comput-Aided Mol Des 19:551-566).     

An unusual reverse turn structure adopted by a furanoid sugar amino acid incorporated in gramicidin S

A new reverse turn, replacing one of the native type II' beta-turns in the cyclic peptide antibiotic gramicidin S, induced by a furanoid sugar amino acid is revealed. The C3-hydroxyl function plays a pivotal role by acting as a H-bond acceptor, consequently flipping the amide bond between residues i and i + 1, as was established by NMR and X-ray crystallographic analysis.     

Spiro beta-lactams as beta-turn mimetics. Design, synthesis, and NMR conformational analysis

Molecular modeling calculations using high-level ab initio methods (MP2/6-31+G) of a new type of spiro beta-lactams predict that these systems could adopt a beta-turn secondary structure in solution. Strong intramolecular hydrogen bonds stabilize the beta-turn conformation with a geometry that is very close to the ideal type II beta-turns. The synthesis of the spiro beta-lactams is achieved by Staudinger reaction of a cyclic ketene derived from N-bencyloxycarbonyl-L-proline acid chloride with an imine. This reaction allows the formation of the spiranic backbone in a single-step with high diastereoselectivity and good yields. The new spiro beta-lactams obtained are the core for the preparation of different types of peptidomimetics using well-established peptide chemistry. The NMR conformational analysis shows that these compounds adopt beta-turn conformation as predicted by the theoretical studies.     

Water network dynamics at the critical moment of a peptide's beta-turn formation: a molecular dynamics study

All-atom molecular dynamics simulations for a single molecule of Leu-Enkephalin in aqueous solution have been used to study the role of the water network during the formation of beta-turns. We give a detailed account of the intramolecular hydrogen bonding, the water-peptide hydrogen bonding, and the orientation and residence times of water molecules focusing on the short critical periods of transition to the stable beta-turns. These studies suggest that, when intramolecular hydrogen bonding between the first and fourth residue of the beta-turn is not present, the disruption of the water network and the establishment of water bridges constitute decisive factors in the formation and stability of the beta-turn. Finally, we provide possible explanations and mechanisms for the formations of different kinds of beta-turns.     

Quantitation of the nearest-neighbour effects of amino acid side-chains that restrict conformational freedom of the polypeptide chain using reversed-phase liquid chromatography of synthetic model peptides with L- and D-amino acid substitutions

Side-chain backbone interactions (or "effects") between nearest neighbours may severely restrict the conformations accessible to a polypeptide chain and thus represent the first step in protein folding. We have quantified nearest-neighbour effects (i to i+1) in peptides through reversed-phase liquid chromatography (RP-HPLC) of model synthetic peptides, where L- and D-amino acids were substituted at the N-terminal end of the peptide sequence, adjacent to a L-Leu residue. These nearest-neighbour effects (expressed as the difference in retention times of L- and D-peptide diastereomers at pHs 2 and 7) were frequently dramatic, depending on the type of side-chain adjacent to the L-Leu residue, albeit such effects were independent of mobile phase conditions. No nearest-neighbour effects were observed when residue i is adjacent to a Gly residue. Calculation of minimum energy conformations of selected peptides supported the view that, whether a L- or D-amino acid is substituted adjacent to L-Leu, its orientation relative to this bulky Leu side-chain represents the most energetically favourable configuration. We believe that such energetically favourable, and different, configurations of L- and D-peptide diastereomers affect their respective interactions with a hydrophobic stationary phase, which are thus quantified by different RP-HPLC retention times. Side-chain hydrophilicity/hydrophobicity coefficients were generated in the presence of these nearest-neighbour effects and, despite the relative difference in such coefficients generated from peptides substituted with L- or D-amino acids, the relative difference in hydrophilicity/hydrophobicity between different amino acids in the L- or D-series is maintained. Overall, our results demonstrate that such nearest-neighbour effects can clearly restrict conformational space of an amino acid side-chain in a polypeptide chain.     

A theoretical case study of type I and type II beta-turns

NMR chemical shielding anisotropy tensors have been computed by employing a medium size basis set and the GIAO-DFT(B3LYP) formalism of electronic structure theory for all of the atoms of type I and type II beta-turn models. The models contain all possible combinations of the amino acid residues Gly, Ala, Val, and Ser, with all possible side-chain orientations where applicable in a dipeptide. The several hundred structures investigated contain either constrained or optimized phi, psi, and chi dihedral angles. A statistical analysis of the resulting large database was performed and multidimensional (2D and 3D) chemical-shift/chemical-shift plots were generated. The (1)H(alpha-13)C(alpha), (13)C(alpha-1)H(alpha-13)C(beta), and (13)C(alpha-1)H(alpha-13)C' 2D and 3D plots have the notable feature that the conformers clearly cluster in distinct regions. This allows straightforward identification of the backbone and side-chain conformations of the residues forming beta-turns. Chemical shift calculations on larger For-(L-Ala)(n)-NH(2) (n=4, 6, 8) models, containing a single type I or type II beta-turn, prove that the simple models employed are adequate. A limited number of chemical shift calculations performed at the highly correlated CCSD(T) level prove the adequacy of the computational method chosen. For all nuclei, statistically averaged theoretical and experimental shifts taken from the BioMagnetic Resonance Bank (BMRB) exhibit good correlation. These results confirm and extend our previous findings that chemical shift information from selected multiple-pulse NMR experiments could be employed directly to extract folding information for polypeptides and proteins.     

Impact of azaproline on amide cis-trans isomerism: conformational analyses and NMR studies of model peptides including TRH analogues

The beta-turn is a well-studied motif in both proteins and peptides. Four residues, making almost a complete 180 degree-turn in the direction of the peptide chain, define the beta-turn. Several types of the beta-turn are defined according to Phi and Psi torsional angles of the backbone for residues i + 1 and i + 2. One special type of beta-turn, the type VI-turn, usually contains a proline with a cis-amide bond at residue i + 2. In an aza-amino acid, the alpha-carbon of the amino acid is changed to nitrogen. Peptides containing azaproline (azPro) have been shown to prefer the type VI beta-turn both in crystals and in organic solvents by NMR studies. MC/MD simulations using the GB/SA solvation model for water explored the conformational preferences of azPro-containing peptides in aqueous systems. An increase in the conformational preference for the cis-amide conformer of azPro was clearly seen, but the increased stability was relatively minor with respect to the trans-conformer as compared to previous suggestions. To test the validity of the calculations in view of the experimental data from crystal structures and NMR in organic solvents, [azPro(3)]-TRH and [Phe(2), azPro(3)]-TRH were synthesized, and their conformational preferences were determined by NMR in polar solvents as well as the impact of the azPro substitution on their biological activities.     

Impact of azaproline on Peptide conformation

The amino acid analog azaproline (azPro) contains a nitrogen atom in place of the C(alpha) of proline. Peptides containing azPro were shown to stabilize the cis-amide conformer for the acyl-azPro bond and prefer type VI beta-turns both in crystals and in organic solvents by NMR. The increased stability for cis-amide conformers was relatively minor with respect to the trans-conformers. Further, their conformational preferences were depended on solvent. To elucidate the impact of azPro substitution on amide cis-trans isomerism and peptide conformation, this paper reports ab initio studies on azPro derivatives and a comparison with their cognate Pro derivatives: 1-acetyl-2-methyl pyrrolidine (1), 1-acetyl-2-methyl pyrazolidine (2), Ac-Pro-NHMe (3), Ac-azPro-NHMe (4), Ac-azPro-NMe(2) (5), Ac-azAzc-NHMe (6), and Ac-azPip-NHMe (7). Conformational preferences were explored at the MP2/6-31+G** level of theory in vacuo. Solvation effects for 1 and 2 were studied implicitly using the polarizable continuum model and explicitly represented by interactions with a single water molecule. An increase in the conformational preference for the cis-amide conformer of azPro was clearly seen. An intramolecular hydrogen bond occurred solely in the trans-amide conformer that reduced the preference for the cis-conformer by 2.2 kcal/mol. The larger ring homolog aza-pipecolic acid (azPip), in which this internal hydrogen bond was diminished, significantly augmented stabilization of the cis-amide conformer. In aqueous solution, the preference for the cis-amide conformers was greatly reduced, mainly as a result of interaction between water and the lone pair of the alpha-nitrogen in the trans-amide conformer that was 3.8 kcal/mol greater than that in the cis-conformer. In the azPro analog, the energy barrier for cis-trans amide isomerization was 6 kcal/mol less than that in the cognate Pro derivative. Because the azPro derivatives can stabilize the cis-amide bond and mimic a type VI beta-turn without incorporation of additional steric bulk, such a simple chemical modification of the peptide backbone provides a useful conformational constraint when incorporated into the structure of selected bioactive peptides. Such modifications can scan receptors for biological recognition of reverse turns containing cis-amide bonds by the incorporation of type VI beta-turn scaffolds with oriented appended side chains.     

Efficient synthesis of enantiopure pyrrolizidinone amino acid

Enantiopure (3S,5R,8S)-3-[N-(Boc)amino]-1-azabicyclo[3.3.0]octan-2-one 8-carboxylic acid (1) was synthesized in nine steps and 16% overall yield from aspartate beta-aldehyde 7. Carbene-catalyzed acyloin condensation of 7, followed by acetylation and samarium iodide reduction, gave linear precursor (2S,7S)-alpha,omega-diamino-4-oxosuberate 11, which was converted to N-(Boc)aminopyrrolizidin-2-one carboxylic acid 1 by a reductive amination/lactam cyclization sequence. X-ray analysis of (3S,5R,8S)-methyl N-(Boc)aminopyrrolizidin-2-one carboxylate 21 showed that its internal backbone dihedral angles (psi = -149 degrees, phi = -49 degrees ) were in good agreement with the ideal values for a type II' beta-turn. Proton NMR experiments on N'-methyl-N-(Boc)aminopyrrolizidin-2-one carboxamide 23 demonstrated significantly different NH chemical displacements and temperature coefficients suggestive of solvent shielded and exposed hydrogens indicative of a turn conformation. Because pyrrolizidinone amino acids can serve as conformationally rigid dipeptide surrogates, this synthesis should facilitate their application in the exploration of conformation-activity relationships of various biologically active peptides.     

Electron capture dissociation initiates a free radical reaction cascade

For small cyclic peptides, one electron capture by the [M + 2H](2+) ion generates numerous fragments corresponding to amino acid losses, side-chain losses, and losses of some low molecular weight species such as H(2)O, CH(3)(*), C(3)H(6), and (*)CONH(2). As predicted, the side-chain cleavages are amplified relative to linear peptides of similar size, but the amino acid losses were unexpected because they require that one electron capture cause more than one backbone cleavage, a phenomenon which necessitates further refinement or reinterpretation of current ECD mechanisms. A modified mechanism is postulated in which nonergodic electron capture fragmentation generates an alpha-carbon radical species that then propagates along the protein backbone. This radical migration initiates multiple free radical rearrangements, which cause both multiple backbone cleavages and additional side-chain cleavages.     

Intrinsic folding of small peptide chains: spectroscopic evidence for the formation of beta-turns in the gas phase

Laser desorption of model peptides coupled to laser spectroscopic techniques enables the gas-phase observation of genuine secondary structures of biology. Spectroscopic evidence for the formation of beta-turns in gas-phase peptide chains containing glycine and phenylalanine residues establishes the intrinsic stability of these forms and their ability to compete with other stable structures. The precise characterization of local minima on the potential energy surface from IR spectroscopy constitutes an acute assessment for the state-of-the-art quantum mechanical calculations also presented. The observation of different types of beta-turns depending upon the residue order within the sequence is found to be consistent with the residue propensities in beta-turns of proteins, which suggests that the prevalence of glycine in type II and II' turns stems essentially from an energetic origin, already at play under isolated conditions.     

Mobile and localized protons: a framework for understanding peptide dissociation

Protein identification and peptide sequencing by tandem mass spectrometry requires knowledge of how peptides fragment in the gas phase, specifically which bonds are broken and where the charge(s) resides in the products. For many peptides, cleavage at the amide bonds dominate, producing a series of ions that are designated b and y. For other peptides, enhanced cleavage occurs at just one or two amino acid residues. Surface-induced dissociation, along with gas-phase collision-induced dissociation performed under a variety of conditions, has been used to refine the general 'mobile proton' model and to determine how and why enhanced cleavages occur at aspartic acid residues and protonated histidine residues. Enhanced cleavage at acidic residues occurs when the charge is unavailable to the peptide backbone or the acidic side-chain. The acidic H of the side-chain then serves to initiate cleavage at the amide bond immediately C-terminal to Asp (or Glu), producing an anhydride. In contrast, enhanced cleavage occurs at His when the His side-chain is protonated, turning His into a weak acid that can initiate backbone cleavage by transferring a proton to the backbone. This allows the nucleophilic nitrogen of the His side-chain to attack and form a cyclic structure that is different from the 'typical' backbone cleavage structures.     

The Conformations of Amino Acids on a Gold(111) Surface

The interactions of amino acids with inorganic surfaces are of interest for biologists and biotechnologists alike. However, the structural determinants of peptide–surface interactions have remained elusive, but are important for a structural understanding of the interactions of biomolecules with gold surfaces. Molecular dynamics simulations are a tool to analyze structures of amino acids on surfaces. However, such an approach is challenging due to lacking parameterization for many surfaces and the polarizability of metal surfaces. Herein, we report DFT calculations of amino acid fragments in vacuo and molecular dynamics simulations of the interaction of all amino acids with a gold(111) surface in explicit solvent, using the recently introduced polarizable gold force field GolP. We describe preferred orientations of the amino acids on the metal surface. We find that all amino acids preferably interact with the gold surface at least partially with their backbone, underlining an unfolding propensity of gold surfaces.