Cis Amide Bond Research Articles

Stereoisomers of 4-amino-3-hydroxy-1-cyclohexanecarboxylic acid and 4-amino-3-oxo-1-cyclohexanecarboxylic acid as mimetics of a twisted cis-amide bond

Stereoselective synthesis of the title compounds was performed. The relative configuration of methyl t-4-( tert-butoxycarbonylamino)-c-3-hydroxy-r-1-cyclohexanecarboxylate and methyl trans-4-( tert-butoxycarbonylamino)-3-oxo-r-1-cyclohexanecarboxylate was confirmed by X-ray diffraction methods. Analogues of cyclolinopeptide A (CLA) containing these twisted cis-amide bond mimetics were then synthesised.

3-(1-Aminoalkyl)isoxazole-4-carboxylic acids as peptide bond replacements

An orthogonally protected 3-(1-aminoalkyl)isoxazole-4-carboxylic acid has been prepared by 1,3-dipolar cycloaddition of a suitably protected α-aminonitrile oxide with an enaminoester dipolarophile; this protected amino acid has been deprotected and coupled independently at either the C- or N-terminus to produce pseudopeptide segments as peptide mimetics that contain a cis-amide bond replacement.

2,2,5,5,8,8-hexamethyl-2,3,5,6,7,8-hexahydroimidazo

The title compound, C12H19N3O2, is an unusual product of silica-catalyzed intermolecular condensation of α-amino­isobutyric acid. The mol­ecule has three types of C—N bonds: a double bond, a cis-amide bond and single bonds, two of which are typical and two having intermediate lengths due to π-electron delocalization between C=N and C=O groups. The cis-amide moieties interact to form dimers via hydrogen bonds which stack in parallel layers.

New pyrazole containing bicarboxylic α-amino acids: mimics of the cis amide bond

A series of novel optically active α-amino acids, containing a pyrazole ring, which can be regarded as building blocks for peptidomimetics, have been prepared starting from readily available α-amino acids. The synthetic strategy employed allows the regio- and stereoselective preparation of 1,3- or 1,5-substituted pyrazolyl rings. The pyrazole containing peptidomimetics can be considered as analogues of peptides with a cis amide bond.

Pseudo-prolines in cyclic peptides: Conformational stabilisation of cyclo[Pro-Thr(ψ Me,Mepro)-Pro

Linear peptide H-Pro-Thr(ψ Me,Mepro)-Pro-OH containing a preformed cis-Pro-Thr(ψ Me,Mepro) tertiary amide bond cyclises instantaneously and free of formation of oligomeric structures to the cyclic tripeptide cyclo-[Pro-Thr(ψ Me,Mepro)-Pro]. Even at concentrations up to 10 −1 M peptide, no oligomeric structures are detected by mass spectroscopy and HPLC. 2D 1H NMR studies of purified cyclotripeptide reveal the compound to exist in one single conformation with all peptide bonds in the cis conformation. These results indicate enhanced cyclisation tendencies of cis-amide bond containing peptides of short chain length.

Effect of the tetrazole cis-amide bond surrogate on the complexing ability of some enkephalin analogues toward Cu(II) ions

A study of the effect of the tetrazole moiety, a cis-amide bond surrogate, on the Cu(II) coordinating properties of oligopeptides is reported. The insertion of the tetrazole moiety ψ(CN 4) into the peptide sequence of [Leu 5]enkephalin considerably changes the coordination ability of the ligand. Potentiometric and spectroscopic results indicate that if the tetrazole moiety is in a suitable position in the peptide chain, i.e. if it follows the third residue, an unusual stable CuH −1L species involving 4N coordination is formed in the physiological pH region. The tetrazole ψ(CN 4) ring provides one of these nitrogens. The data indicate that Cu(II) ions are strongly trapped inside a bent peptide backbone. However, the coordination mode involving the tetrazole ring nitrogen does not prevent the hydrolysis process under strongly basic conditions.

The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu5]-enkephalin(1, H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE(2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3:2 where both conformers exhibit gamma and beta-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4. It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4, with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.

Non-proline Cis peptide bonds in proteins

In a non-redundant set of 571 proteins from the Brookhaven Protein Data Base, a total of 43 non-proline cis peptide bonds were identified. Average geometrical parameters of the well-defined cis peptide bonds in proteins determined at high resolution show that some parameters, most notably the bond angle at the amide bond nitrogen, deviate significantly from the corresponding one in the trans conformation. Since the same feature was observed in cis amide bonds in small molecule structures found in the Cambridge Structural Data Base, a new set of parameters for the refinement of protein structures containing non-Pro cis peptide bonds is proposed. A striking preference was observed for main-chain dihedral angles of the residues involved in cis peptide bonds. All residues N-terminal and most residues C-terminal to a non-Pro cis peptide bond (except Gly) are located in the β-region of a φ/ψ plot. Also, all of the few C-terminal residues (except Gly) located in the α-region of the φ/ψ plot constitute the start of an α-helix in the respective structure. In the majority of cases, an intimate side-chain/side-chain interaction was observed between the flanking residues, often involving aromatic side-chains. Interestingly, most of the cases found occur in functionally important regions such as close to the active site of proteins. It is intriguing that many of the proteins containing non-proline cis peptide bonds are carbohydrate-binding or processing proteins. The occurrence of these unusual peptide bonds is significantly more frequent in structures determined at high resolution than in structures determined at medium and low resolution, suggesting that these bonds may be more abundant than previously thought. On the basis of our experience with the structure determination of coagulation factor XIII, we developed an algorithm for the identification of possibly overlooked cis peptide bonds that exploits the deviations of geometrical parameters from ideality. A few likely candidates based on our algorithm have been identified and are discussed.

Synthesis and structure-activity relationships of 2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates with retinoidal activity.

The structure-activity relationships of two series of novel retinoids (2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates) have been investigated by evaluating their ability to induce differentiation in both human promyelocytic leukemia (HL60) cells and mouse embryonal carcinoma (P19) cells. The most active compound (ED50 = 8.3 x 10(-9) M) of the 2-pyrazinylcarboxamidobenzoates is 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethylquinoxalyl)carboxamido]benzoic acid (9u), while the most active analogue of the beta-ionylideneacetamidobenzoates is 4-[3-methyl-5-(2',6',6'-trimethyl-1'-cyclohexen-1'-yl)-(2E, 4E)-pentadienamido]benzoic acid (10a, ED50 = 3.2 x 10(-8) M). Our studies identify an absolute requirement for the carboxylic acid moiety on the aromatic ring to be para relative to the amide linkage for activity. Benzoate substitutions in the ortho position relative to the terminal carboxylate (9d,k,r) are well-tolerated; however, a methoxy substituent meta relative to the terminal carboxylate gives rise to only weakly active analogues (9x). Conformational studies (NMR, X-ray crystallography) of the 2-pyrazinylcarboxamidobenzoates indicate that the preferred conformation exhibits a trans-amide bond and an internal hydrogen bond between the quinoxaline N1 and HN amide which locks the torsional angle between C2 and CO in the s-trans conformation. N-Methylation (9y) results in loss of activity. Studies indicate that there is now a cis-amide bond present which redirects the carboxylate toward the pharmacophoric gem-dimethyl groups. The distance between the gem-dimethyl group and the terminal carboxylate appears to be too short to activate the retinoid receptor. N-Methylation in the beta-ionylideneacetamidobenzoate series (10c) also results in the formation of a cis-amide bond and loss of activity.

A new cyclolinopeptide containing nonproteinaceous amino acidN-methyl-4-aminoproline

We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established as cyclo(PPFFILLX), where X is a non-proteinaceous amino acid, N-methyl-4-aminoproline. This amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanar cis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysed cis/trans amide-Pro bond isomerisation.

Pyrazoles, 1,2,4-triazoles, and tetrazoles as surrogates for cis-amide bonds in boronate ester thrombin inhibitors.

Substituted pyrazoles, 1,2,4-triazoles, and tetrazoles are good surrogates for cis-amide bonds in a series of boronate ester thrombin inhibitors.

A backbone-cyclic, receptor 5-selective somatostatin analogue: synthesis, bioactivity, and nuclear magnetic resonance conformational analysis.

Cyclo(PheN2-Tyr-D-Trp-Lys-Val-PheC3)-Thr-NH2 (PTR 3046), a backbone-cyclic somatostatin analogue, was synthesized by solid-phase methodology. The binding characteristics of PTR 3046 to the different somatostatin receptors, expressed in CHO cells, indicate high selectivity to the SSTR5 receptor. PTR 3046 is highly stable against enzymatic degradation as determined in vitro by incubation with rat renal homogenate and human serum. The biological activity of PTR 3046 in vivo was determined in rats. PTR 3046 inhibits bombesin- and caerulein-induced amylase and lipase release from the pancreas without inhibiting growth hormone or glucagon release. The major conformation of PTR 3046 in CD3OH, as determined by NMR, is defined by a type II' beta-turn at D-Trp-Lys and a cis amide bond at Val-PheC3.

Conformation of the sebacyl beta1Lys82-beta2Lys82 crosslink in T-state human hemoglobin.

The crystal structure of human T state hemoglobin crosslinked with bis(3,5-dibromo-salicyl) sebacate has been determined at 1.9 A resolution. The final crystallographic R factor is 0.168 with root-mean-square deviations (RMSD) from ideal bond distance of 0.018 A. The 10-carbon sebacyl residue found in the beta cleft covalently links the two betaLys82 residues. The sebacyl residue assumes a zigzag conformation with cis amide bonds formed by the NZ atoms of betaLys82's and the sebacyl carbonyl oxygens. The atoms of the crosslink have an occupancy factor of 1.0 with an average temperature factor for all atoms of 34 A2. An RMSD of 0.27 for all CA's of the tetramer is observed when the crosslinked deoxyhemoglobin is compared with deoxyhemoglobin refined by using a similar protocol, 2HHD [Fronticelli et al. J. Biol. Chem. 269: 23965-23969, 1994]. Thus, no significant perturbations in the tertiary or quaternary structure are introduced by the presence of the sebacyl residue. However, the sebacyl residue does displace seven water molecules in the beta cleft and the conformations of the beta1Lys82 and beta2Lys82 are altered because of the crosslinking. The carbonyl oxygen that is part of the amide bond formed with the NZ of beta2Lys82 forms a hydrogen bond with side chain of beta2Asn139 that is in turn hydrogen-bonded to the side chain of beta2Arg104. A comparison of the observed conformation with that modeled [Bucci et al. Biochemistry 35:3418-3425, 1996] shows significant differences. The differences in the structures can be rationalized in terms of compensating changes in the estimated free-energy balance, based on differences in exposed surface areas and the observed shift in the side-chain hydrogen-bonding pattern involving beta2Arg104, beta2Asn139, and the associated sebacyl carbonyl group.

Design, construction and properties of peptide N-terminal cap templates devised to initiate α-helices. Part 1. Caps derived from N-(4-chlorobutyryl)-(2S )-Pro-(2S )-Pro-(2S )-Ala-OMe and N-[(2S )-2-chloropropionyl]-(2S )-Pro-(2S )-Pro-(2S,4S )-4-hydroxyPro-OMe

The structural features required of N-terminal helix caps which might display high α-helix-forming propensities (Zimm–Bragg σ-values close to unity) were considered and two systems were identified as initial candidates for synthesis and evaluation. The first system was an eleven-membered cyclic all-trans triamide derived from N-(4-chlorobutyryl)-(2S)-Pro-(2S)-Pro-(2S)-Ala-OMe in which it was intended to remove the only amide NH proton to generate a better nucleophile, and then ring-close by displacing the 4-halogen atom as halide. The precursor was prepared in moderate overall yield and displayed conformational isomerism in chloroform solution. Some of the conformers appeared to contain one or more cis amide bonds. No suitable conditions could be found to effect macrocyclisation. The second system was a twelve-membered cyclic all-trans triamide derived from N-[(2S)-2-chloropionyl]-(2S)-Pro-(2S)-Pro-(2S,4S)-4-hydroxyPro-OMe in which it was intended to generate an alkoxide anion and then ring-close by displacing halide ion from the 2-chloropropionyl moiety. The precursor was prepared in moderate overall yield and populated one all-trans rotameric form in chloroform solution. This system also failed to macrocyclise under a variety of conditions. The major reason for the lack of reactivity in each system was analysed and appeared to be the generation of large molecular dipoles in the transition state for the cyclisation, originating from the requirement for the alignment of the carboxamide carbonyl groups. Useful modifications to the systems described here that were designed to overcome or minimise the effect of the dipole in destabilising the required conformation for helix cap generation are presented in the following two articles.

Spiro-substituted piperidines as neurokinin receptor antagonists. III. Synthesis of (+/-)-N-[2-(3,4-dichlorophenyl)-4-(spiro-substituted piperidin-1'-yl)butyl]-N-methylbenzamides and evaluation of NK1-NK2 dual antagonistic activities.

To discover a novel NK1-NK2 dual antagonist, we have synthesized a series of spiro-substituted piperidines utilizing YM-35375 as a lead compound, and evaluated affinities for NK1 and NK2 receptors. In the N-methylbenzamide moiety, introduction of methoxy groups increased affinity for the NK1 receptor without a significant loss of affinity for the NK2 receptor. We also found that a conformation in which the phenyl groups of the N-methylbenzamide and 3,4-dichlorophenyl moieties are close to each other through a cis-amide bond, may be favorable for showing high affinity for the NK1 receptor and that a hydrogen bond-accepting group in the spiro-substituted piperidine moiety may be crucial for exhibiting high affinity for the NK2 receptor. Among the compounds prepared, YM-44778 (31) showed high and well-balanced affinity for NK1 and NK2 receptors (IC50 values of 18 and 16 nM, respectively). This compound also exhibited potent antagonistic activities against both NK1 and NK2 receptors (IC50 values of 82 and 62 nM, respectively) in isolated tissues.

1,2-dithia-5-azacyclooctan-6-one and related compounds: Synthesis and conformation

The first synthesis of 1,2-dithia-5-azacyclooctan-6-one 11, the parent member of a heterocyclic ring synthesis is reported. Compound 11 was found by nOe experiments to exist in a conformation with a trans-amide bond. The substituted derivatives 12 and 13 were also prepared and compound 12 was found to possess a trans-amide bond whilst for compound 13 three conformations were identified with the major conformer possessing a cis-amide bond.

3‐Aminoacetylthiazolidine‐4‐carboxylate esters and their l‐thia‐4‐azaspiro[4.5]decane‐3‐carboxylate derivatives. Synthesis and stereochemical properties

AbstractDimethyl thiazolidine‐2,4‐dicarboxylate 2 and ethyl l‐thia‐4‐azaspiro[4.5]decane‐3‐carboxylates 3‐5 were obtained as a diastereoisomeric mixture while their 3‐aminoacetyl derivatives were isolated in only one isomeric form. These reactions of N‐acylation were stereoselective, which can be explained by an interconversion of the diastereoisomers via a seco intermediate. The 1H nmr analysis of amides 6 and 11 exhibited the presence of both cis and trans amide bond conformations, whereas only one cis conformation was observed for spiro amides 8‐10 and 13‐15.

Determination of Peptide Amide Configuration in a Model Amyloid Fibril by Solid-State NMR

The ‚-amyloid proteins (A‚), especially the variant A‚1-42, are the primary constituent of the amyloid plaques which are characteristic of Alzheimer's disease (AD). The C-terminus of A ‚ has been identified as crucial for rapid amyloid formation. A 9-residue C-terminal fragment of A‚ (designated ‚34-42, with sequence LMVGGVVIA) forms a structured aggregate which is classified as an amyloid fibril based primarily on its morphological and solubility properties and the classic cross- ‚ fiber diffraction pattern it displays. We have adopted this aggregate as a model of the A‚-dominated amyloid plaques associated with AD. Previous results from a series of internuclear 13 C- 13 C distance measurements along the peptide backbone indicate that the ‚34-42 backbone conformation differs considerably from that of the classic ‚-sheet, possibly including an unusual cis amide bond in the peptide's central glycyl -glycyl region. Here we report evidence which demonstrates the presence of a trans configuration at the peptide linkage between these residues. The evidence derives from solid-state NMR static echo and n ) 2 rotational resonance experiments which probe the orientation of the 13 C carbonyl chemical shift anisotropy (CSA) tensor of the peptide bond with respect to the dipolar tensor connecting the carbonyl carbon to the 13 C-

Cyclic Heptapeptides Axinastatin 2, 3, and 4: Conformational analysis and evaluation of the biological potential

AbstractThe conformational analysis of naturally occurring cytostatic cyclic heptapeptides axinastatin 2, 3, and 4 was carried out by two‐dimensional NMR spectroscopy in combination with distance‐geometry (DG) and molecular‐dynamics (MD) calculations in explicit solvents. The synthesized secondary metabolites were examined in (D6)DMSO. Axinastatin 2 was also investigated in CD3OH. In all structures, Pro2 is in the i + 1 position of a βI turn and Pro6 occupies the i + 2 position of a βVIa turn about the cis amide bond between residue 5 and Pro6. In all peptides, a bifurcated H‐bond occurs between residue 4 CO and the amide protons of residue 1 and 7. For axinastatin 2 and 3, an Asn Ig turn was found about Asn1 and Pro2. We compared these structures with conformations of cyclic heptapeptides obtained by X‐ray and NMR studies. A β‐bulge motif with two β turns and one bifurcated H‐bond is found as the dominating backbone conformation of cyclic all‐L‐heptapeptides. Axinastatin 2, 3, and 4 can be characterized by six trans and one cis amide bond resulting in a β/βVI(a)‐turn motif, a conformation found for many cyclic heptapeptides. Detailed biological tests of the synthetic compounds in different human cancer cell lines indicates these axinastatins to be inactive or of low activity.

NMR structural characterization of oligo-N-substituted glycine lead compounds from a combinatorial library.

Synthesis and screening of combinatorial libraries for pharmaceutical lead discovery is a rapidly expanding field. Oligo-N-substituted glycines (NSGs) were one of the earliest sources of molecular diversity in combinatorial libraries. In one of the first demonstrations of the power of combinatorial chemistry, two NSG trimers, CHIR-2279 and CHIR-4531, were identified as nM ligands for two 7-transmembrane G-protein-coupled receptors. The NMR characterization of these two lead compounds was undertaken to verify covalent connectivity and to determine solution conformations, if any. The sequential chemical shift assignments were performed using a new strategy for assigning 1H and 13C resonances of NSGs. The conformational preferences were then determined in both an aqueous co-solvent system and an organic solvent to probe the effects of hydrophobic collapse. NSGs are expected to be more flexible than peptides due to the tertiary amide, with both cis and trans amide bond conformations being accessible. Solution NMR studies indicate that although CHIR-2279 and CHIR-4531 have identical backbones and termini, and very similar side chains, they do not display the same solution conformational characteristics.