Aza-amino Acid Research Articles

Cyclic Aza-peptide Integrin Ligand Synthesis and Biological Activity

Aza-peptides are obtained by replacement of the α-C-atom of one or more amino acids by a nitrogen atom in a peptide sequence. Introduction of aza-residues into peptide sequences may result in unique structural and pharmacological properties, such that aza-scanning may be used to probe structure-activity relationships. In this study, a general approach for the synthesis of cyclic aza-peptides was developed by modification of strategies for linear aza-peptide synthesis and applied in the preparation of cyclic aza-pentapeptides containing the RGD (Arg-Gly-Asp) sequence. Aza-amino acid scanning was performed on the cyclic RGD-peptide Cilengitide, cyclo[R-G-D-f-N(Me)V] 1, and its parent peptide cyclo(R-G-D-f-V) 2, potent antagonists of the αvβ3, αvβ5, and α5β1 integrin receptors, which play important roles in human tumor metastasis and tumor-induced angiogenesis. Although incorporation of the aza-residues resulted generally in a loss of binding affinity, cyclic aza-peptides containing aza-glycine retained nanomolar activity toward the αvβ3 receptor.

Azapeptides and Their Therapeutic Potential

Azapeptides are peptide analogs in which one or more of the amino residues is replaced by a semicarbazide. This substitution of a nitrogen for the α-carbon center results in conformational restrictions, which bend the peptide about the aza-amino acid residue away from a linear geometry. The resulting azapeptide turn conformations have been observed by x-ray crystallography and spectroscopy, as well as predicted based on computational models. In biologically active peptide analogs, the aza-substitution has led to enhanced activity and selectivity as well as improved properties, such as prolonged duration of action and metabolic stability. In light of these characteristics, azapeptides have found important uses as receptor ligands, enzyme inhibitors, drugs, pro-drugs, probes and imaging agents. Recent improvements in synthetic methods for their procurement have ushered in a new era of azapeptide chemistry. This review aims to provide a historical look at the development of azapeptide science along with a focus on recent developments and perspectives on the future of this useful tool for medicinal chemistry.

Peptidomimetic inhibitors of bacterial peptide deformylase

A series of N-formyl hydroxylamine peptide deformylase inhibitors containing a cyclic azaamino acid moiety between the P1' and P3' substituents are presented. Selected compounds display antibacterial activity against pathogens associated with respiratory tract infections with representative compounds showing excellent MICs against Haemophilus influenzae.

Synthesis of N′-substituted Ddz-protected hydrazines and their application in solid phase synthesis of aza-peptides

Hydrazine derivatives are of considerable scientific and industrial value. Substituted hydrazines are precursors for many compounds of great interest and importance, among them aza-peptides. (Aza-peptides are peptide analogues in which one or more of the α-carbons, bearing the side chain residues, has been replaced by a nitrogen atom.) Aza-amino acid residues conserve the pharmacophores necessary for biological activity while inducing conformational changes and increased resistance to proteolytic degradation. These properties make aza-peptides attractive tools for structure–activity relationship studies and drug design. We describe the synthesis of N′-substituted 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz) protected hydrazines. A general approach for solid phase synthesis of aza-peptides has been developed based on the in-situ activation of the N-Ddz, N′-substituted hydrazines with phosgene, followed by introduction to the N-terminus of a resin-bound peptide. The Ddz-aza-amino building units include aliphatic, aromatic and functionalized side chains, protected for synthesis by the Fmoc strategy. Solid phase aza-peptide synthesis is demonstrated including selective mild deprotection of Ddz with Mg(ClO 4) 2 and coupling of the next amino acid with triphosgene. Ddz deprotection is orthogonal with the Fmoc and Boc protecting groups, making the solid phase Ddz-aza-peptide synthesis compatible with both the Fmoc and the Boc strategies. The Ddz-protected hydrazines have wide applications in the synthesis of substituted hydrazines and in the synthesis of aza containing peptidomimetics.

Calcitonin Gene-Related Peptide Analogues with Aza and Indolizidinone Amino Acid Residues Reveal Conformational Requirements for Antagonist Activity at the Human Calcitonin Gene-Related Peptide 1 Receptor

Calcitonin gene-related peptide antagonists have potential for the treatment and prevention of disease states such as non-insulin-dependent diabetes mellitus, migraine headache, pain, and inflammation. To gain insight into the spatial requirements for CGRP antagonism, three strategies were employed to restrict the conformation of the potent undecapeptide antagonist, [D31,P34,F35]CGRP27-37. First, aza-amino acid scanning was performed, and ten aza-peptide analogues were synthesized and examined for biological activity. Second, (3S,6S,9S)-2-oxo-3-amino-indolizidin-2-one amino acid (I2aa) and (2S,6S,8S)-9-oxo-8-amino-indolizidin-9-one amino acid (I9aa) both were introduced at positions 31-32, 32-33, 33-34, and 34-35, regions of the backbone expected to adopt turns. Finally, the conformation of the backbone and side-chain of the C-terminal residue, Phe35-Ala36-Phe37-NH2, was explored employing (2S,4R,6R,8S)-9-oxo-8-amino-4-phenyl-indolizidin-9-one amino acid (4-Ph-I9aa) as a constrained phenylalanine mimic. The structure-activity relationships exhibited by our 26 analogues illustrate conformational requirements important for designing CGRP antagonists and highlight the importance of beta-turns centered at Gly33-Pro34 for potency.

Aza‐scanning of the Potent Melanocortin Receptor Agonist Ac‐His‐d‐Phe‐Arg‐Trp‐NH2

The melanocortin pathway is an important participant in the regulation of skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. Melanocortin agonists contain the putative sequence 'His-Phe-Arg-Trp', which has been designated as the 'message' sequence for melanocortin peptides, and this sequence has been hypothesized to adopt a bioactive reverse turn conformation. Exploring the relationship between its structure and biological activity, we report the synthesis and evaluation of seven aza-analogs of the potent melanocortin receptor agonist Ac-His-D-Phe-Arg-Trp-NH2. Aza-amino acids, in which the alpha-carbon was replaced by nitrogen, were inserted along the peptide sequence to probe the importance of local configuration and turn conformation on the biology of this tetrapeptide. Although systematic substitution of aza-amino acids for the D-Phe and Arg residues led to a significant loss of activity relative to the parent peptide for all melanocortin receptor subtypes examined, substitution of aza-amino acids at the C-terminal Trp residue gave analogs equipotent to the parent peptide. In summary, the aza-scan has demonstrated that recognition of this tetrapeptide by the melanocortin receptors is particularly sensitive to modifications of configuration and conformation at the D-Phe and Arg residues versus the Trp amino acid. In light of aza-residues imparting resistance from enzymatic degradation, C-terminal aza-amino acid analogs may be used to design new peptide mimics with enhanced metabolic stability.

Interaction of Papain-like Cysteine Proteases with Dipeptide-Derived Nitriles

A series of 44 dipeptide nitriles with various amino acids at the P2 position and glycine nitrile at position P1 were prepared and evaluated as inhibitors of cysteine proteinases. With respect to the important contribution of the P2-S2 interaction to the formation of enzyme-inhibitor complexes, it was focused to introduce structural diversity into the P2 side chain. Nonproteinogenic amino acids were introduced, and systematic fluorine, bromine, and phenyl scans for phenylalanine in the P2 position were performed. Moreover, the N-terminal protection was varied. Kinetic investigations were carried out with cathepsin L, S, and K as well as papain. Changes in the backbone structure of the parent N-(tert-butoxycarbonyl)-phenylalanyl-glycine-nitrile (16), such as the introduction of an R-configured amino acid or an azaamino acid into P2 as well as methylation of the P1 nitrogen, resulted in a drastic loss of affinity. Exemplarily, the cyano group of 16 was replaced by an aldehyde or methyl ketone function. Structure-activity relationships were discussed with respect to the substrate specificity of the target enzymes.

Aza-Amino Acid Scanning of Secondary Structure Suited for Solid-Phase Peptide Synthesis with Fmoc Chemistry and Aza-Amino Acids with Heteroatomic Side Chains

Aza-peptides, peptide analogues in which the alpha-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit a propensity for adopting beta-turn conformations. A general Fmoc-protection protocol for the stepwise solid-phase synthesis of aza-peptides has now been developed based on the activation of N'-alkyl fluoren-9-ylmethyl carbazates with phosgene for coupling the aza-amino acid residues. This method has proven effective for introducing aza-amino acid residues with aliphatic (Ala, Leu, Val, and Gly) and aromatic (Phe, Tyr, and Trp) side chains. Acid promoted loss of aromatic side chains was noted with aza-Trp and aza-Tyr residues during peptide cleavage and suppressed by temperature control in the case of the latter. In addition, aza-peptides with heteroatomic side chain residues (Lys, Orn, Arg, and Asp) were conveniently synthesized using this protocol. Partial aza-amino acid scans were performed on three biologically active peptides: the potent tetrapeptide melanocortin receptor agonist, Ac-His-d-Phe-Arg-Trp-NH2; the growth hormone secretagogue hexapeptide, GHRP-6, His-d-Trp-Ala-Trp-d-Phe-Lys-NH2; and the human calcitonin gene-related peptide (hCGRP) antagonist, FVPTDVGPFAF-NH2. This practical procedure for aza-amino acid scanning using Fmoc-based solid-phase synthesis should find general utility for probing the existence and importance of beta-turn conformations in bioactive peptides.

A general strategy for the synthesis of azapeptidomimetic lactams

A selection of azapeptidomimetics containing constraining lactam rings have been prepared by Mitsunobu cyclization of serine/homologated serine-azaalanine derivatives. These include sterically-congested β-lactams, as well as γ-butyrolactam and δ-valerolactam analogs. A novel azaamino acid acylation method was developed to prepare the sterically demanding α-benzyl-serine-azaalanine precursor. In all cases, the Mitsunobu conditions were highly efficient in forming the desired azapeptidomimetic lactams. The reported process represents a general strategy for the synthesis of peptidomimetic structures with a constraining lactam ring.

Columns: Polymer Supports in Synthesis

The focus of this month's column is on a general protocol for the solid phase synthesis of azapeptides without racimization, using a polymer support, where the aza-amino acid residue is introduced as an N-Boc-aza-dipeptide. This development was recently reported by Melendez and Lubell at the University of Montreal (JACS, 2004, 126, 6759–64). However, before summarizing the synthetic pathway, azapeptides will be introduced.Azapetides are peptide analogs where the α-carbon of one or more of the amino acid residues has been replaced by a nitrogen atom. A normal dipeptide portion of a peptide is compared to an azapeptide in structures 1 and 2, respectively. Natural peptides are widely investigated as drugs, but they are frequently rapidly metabolized.

Aza-amino acid scan for rapid identification of secondary structure based on the application of N-Boc-aza(1)-dipeptides in peptide synthesis.

Azapeptides, peptide analogues in which the alpha-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit propensity for adopting beta-turn conformations. A general protocol for the synthesis of azapeptides without racemization on solid phase has now been developed by introducing the aza-amino acid residue as an N-Boc-aza(1)-dipeptide. This approach has been validated by the synthesis of six N-Boc-aza(1)-dipeptides and their subsequent introduction into analogues of the C-terminal peptide fragment of the human calcitonin gene-related peptide (hCGRP). By performing an aza-amino acid scan of such antagonist peptides, a set of aza-hCGRP analogues was synthesized to examine the relationship between turn secondary structure and biological activity.

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.

A theoretical study of conformational properties of N-methyl azapeptide derivatives.

The conformational properties of azapeptide derivatives, Ac-azaGly-NHMe (1), Ac-azaAla-NHMe (2), Ac-NMe-azaGly-NHMe (3), Ac-NMe-azaAla-NHMe (4), Ac-azaGly-NMe(2) (5), Ac-azaAla-NMe(2) (6), Ac-NMe-azaGly-NMe(2) (7), and Ac-NMe-azaAla-NMe(2) (8), were systematically examined by using ab initio MO and DFT methods. Structural perturbations in azapeptides resulting from cyclic substitution of a methyl group at three N-positions of an azaamino acid were studied on the basis of the structure of the simplest model azapeptide, 1. Potential energy surfaces were generated at the HF/6-31G level for 1-4 and at the HF/6-31G//HF/3-21G level for 5-8 by rotating two key dihedral angles (phi, psi) in increments of 30 degrees. The backbone (phi, psi) angles of the minima for 1-4 are observed at the i + 2 position to form the betaI(I')-, betaII(II')-, betaVI-turns or the polyproline II structure according to the orientation of the acetyl group and the positions of the N-methyl groups. Compounds 5-8 coupled to a secondary amine were found to preferentially adopt polyproline II, betaI(III)-turn, or alpha-helical structure or even extended conformations depending on the orientation of the acetyl group and the positions of the N-methyl groups. Furthermore, N-methyl groups, depending on their positions, were found to affect the orientation of the amide group in the lowest energy conformations, the pyramidality of the N2 atom, and the bond length in azapeptide derivatives. These unique theoretical conformations of N-methyl azapeptide derivatives could be utilized in the definite design of secondary structure for peptides and proteins, and in the development of new drugs and molecular machines.

Azapeptides as inhibitors of the hepatitis C virus NS3 serine protease

Truncation and substitution SAR studies of azapeptide-based inhibitors of the Hepatitis C virus (HCV) NS3 serine protease have been performed. These azapeptides were designed from the HCV polyprotein's NS5A-NS5B trans cleavage junction and contained an azaamino acid residue at the P1 position. These azapeptides exhibited predominantly non-acylating, competitive inhibition, contrary to classical azapeptides.

Utility of azapeptides as major histocompatibility complex class II protein ligands for T-cell activation.

Major histocompatibility complex class II (MHC II) protein binding and antigen specific activation of CD4+ "helper" T cells are demonstrated with peptides composed of the antigenic hen egg ovalbumin 325-339 peptide (OVA) substituted with azaamino acids. AzaAla and azaGly substitutions were made at 10 sequential peptide positions (326Ala-335Asn) that lie in the binding groove. The peptide positions substituted with azaamino acids encompass almost the entire binding groove, including positions where the identity of the amino acid side chain is known to have the most significant effect on MHC binding and the least effect on T-cell recognition. In addition, the T-cell contact 333Glu was substituted with azaGlu to generate a partial agonist ligand for the 3DO-54.8 T-cell hybridoma. Binding to MHC II protein was assayed by measuring the kinetic stability of complexes formed between detergent-solubilized MHC II I-A(d) protein and fluorescein-labeled OVA peptides using a fluorescence-HPLC assay. T-cell activation was also evaluated for aza-substituted peptides with azaamino acid substitutions at the peptide positions known to interact with the MHC II protein. All aza-substituted peptides showed detectable MHC binding, and some were found to show T-cell activation potency equal to the native peptide. Several of these were also found to be weak or partial agonists. Our results demonstrate that azaamino acids substituted into an antigenic peptide cause a subtle, global effect on peptide conformation that can be used to design altered peptide ligands (APL) as T-cell partial agonists. These may have potential as T-cell epitopes for synthetic vaccines and therapeutic agents for autoimmune diseases.

Torsion angle based design of peptidomimetics: A dipeptidic template adopting β-I Turn (Ac-Aib-AzGly–NH 2)

We have attempted to design a model dipeptide (acetyl dipeptide amide, Ac-CA1-CA2–NH 2) that can adopt specifically typical torsion angles of the β-I turn (φ i+1 , ψ i+1 , φ i+2 , ψ i+2 =−60°, −30°, −90°, 0°). The key of the design is the combination of constrained amino acids that prefer to adopt the desired torsion angles. We chose Aib (aminoisobutyric acid) as the first residue of which φ and ψ angles must be −60° and −30°, respectively. Then, we selected an azaamino acid as the second residue since previous studies have indicated that they prefer to adopt ±90° of φ angle and 0° or 180° of ψ angle. The conformational preference of the resulting Ac-Aib-AzGly–NH 2 is investigated using ab initio methods. The conformations implying β-I and β-I′ turns are energetically most favorable, as we expected. Thus, we synthesized the designed molecule on the solid phase considering the future generation of combinatorial libraries using an automatic peptide synthesizer. Then, NMR spectroscopy was carried out to confirm their conformational preference in solution was carried out. The results indicated that the Ac-Aib-AzGly–NH 2 adopt β-I or β-I′ turns in solution forming an intramolecular hydrogen bonding between Ac–C(O) and terminal NH 2. We believe that such a small peptidomimetic template is highly useful for the design of drug candidates and molecular devices.

The β-turn preferential solution conformation of a tetrapeptide containing an azaamino acid residue

The global minimum energy conformation of an azapeptide model, For-Ala-azaAla-NH 2 was predicted by ab initio calculation of the 3-21G and 6-31G ∗ levels. The backbone torsion angle ( φ 1, ψ 1, φ 2, ψ 2) of an azapeptide model appeared to be the β-turn conformation with a dihedral angle (−61°,131°,79°,15°). This indicates that azaamino acid induces the β-turn motif regardless of the change of chain length by the addition of an amino acid in azapeptide when compared with the minimum energy conformation of For-azaAla-NH 2. We designed and synthesized a tetrapeptide containing azaamino acid, Boc-Ala-Phe-azaLeu-Ala-OMe ( 1) to verify whether this β-turn conformation is still conserved in solution. The solution conformation of this azapeptide model was determined by using IR, NMR and molecular modeling techniques. The conformational behavior of this azapeptide was compared with that of the tetrapeptide, Boc-Ala-Phe-Leu-Ala-OMe ( 2), which was not associated with azaamino acid. The IR evidence of intramolecular H-bonding, the characteristic nuclear Overhauser enhancement (NOE) patterns, the temperature coefficients of amide protons and small solvent accessibility for the azapeptide 1 reveal that it favors the β-turn structure, whereas the peptide 2 forms extended structure in CDCl 3 solution. The average structure of azapeptide 1 from a restrained molecular dynamics simulation indicated that the dihedral angles [( φ 2, ψ 2), ( φ 3, ψ 3)] of Phe-azaLeu fragment in a model peptide, Boc-Ala-Phe-azaLeu-Ala-OMe were [(−60±8°,125±24°), (88±21°,1±4°)], and this implies that the intercalation of an azaamino acid residue in tetrapeptide induces the βII-turn conformation, and the increase of chain length by the addition of an amino acid in azapeptide constituents would not disrupt the backbone dihedral angle of β-turn conformation.

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.

Novel Solid-Phase Synthesis of Azapeptides and Azapeptoides via Fmoc-Strategy and Its Application in the Synthesis of RGD-Mimetics

The cell adhesion motif Arg-Gly-Asp (RGD) has been used as a starting point for the development of several antagonists for the αIIbβ3 and αvβ3 integrins, which are implicated in various pathological processes. In this paper, an efficient method for the solid-phase synthesis and biological evaluation of linear RGD-mimetics containing an azaamino acid instead of glycine are described. Activation of the Fmoc-protected hydrazines 1, 4, and 6 with a solution of phosgene in toluene provided Fmoc-protected activated azaglycine (2), azasarcosine (5), and azaalanine (7) in high yields. Six aza-RGD-mimetics have been synthesized on solid support using Fmoc peptide synthesis and individually optimized reaction conditions for the incorporation of activated azabuilding blocks. Due to orthogonal anchoring and side-chain protection, our strategy yielded TentaGel-bound RGD-mimetics, which meet all requirements of the one-bead−one-compound concept. We observed differing activity and selectivity in bioassays for the αIIbβ3- and αvβ3-integrin receptor depending on the substitution pattern of the azabuilding blocks. Our biological data suggest that azapeptides and azapeptoides can be employed as selectivity- and activity-inducing templates in pseudobiooligomers.

Submonomer Solution Synthesis of Hydrazinoazapeptoids, a New Class of Pseudopeptides.

The development of oligomeric peptidomimetics is actually the focus of increasing attention.1 This arises from the very low bioavailability of peptides, which seriously limited their therapeutical applications. Azapeptides,2 peptoids,3 and ureapeptoids4 belong to a new conceptual class of peptidomimetic in which the side chains are carried by nitrogen atoms. Moreover, the potentiality to automate the synthesis of such oligomers by iterative procedures in the solid phase enables the creation of libraries useful for lead-finding in drug discovery. Our present results come within that general framework. More precisely, we are interested in the synthesis of new kinds of pseudopeptides that we termed hydrazinoazapeptoids by analogy with peptoids. These “hybrid” peptidomimetics combine a C-terminal azaamino acid unit (aza) or an N-substituted azaglycine (Naza) with NRsubstituted hydrazinoglycines (NRh) (Figure 1). Although the chemistry of azaamino derivatives has been widely explored and is well documented,2 that of hydrazino acids has been the object of much less attention.5 Recently, some progress has been made in their preparation, although through laborious methods, in particular concerning the synthesis of optically enriched compounds.6 Nevertheless, only a limited number of side chains seem to be compatible with the chemical methods involved at that time. Moreover, due to the presence of the additional NR, difficulties sometimes occurred during the preparation of hydrazino acids,6b and their utilization in pseudopeptides design for which the coupling with amino acids is not always regioselective.5 This should be the reason why commercially available hydrazinoacids are NR protected.