Molecular Umbrella as a Nanocarrier for Antifungals.

A molecular umbrella composed of two O-sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a "trimethyl lock" (TML) or o-dithiobenzylcarbamoyl moiety as a cleavable linker. Three out of five conjugates demonstrated antifungal in vitro activity against C. albicans and C. glabrata but not against C. krusei, with MIC90 values in the 0.22-0.99 mM range and were not hemolytic. Antifungal activity of the most active conjugate 24c, containing the TML-pimelate linker, was comparable to that of intact cispentacin. A structural analogue of 24c, containing the Nap-NH2 fluorescent probe, was accumulated in Candida cells, and TML-containing conjugates were cleaved in cell-free extract of C. albicans cells. These results suggest that a molecular umbrella can be successfully applied as a nanocarrier for the construction of cleavable antifungal conjugates.

Towards Foldameric Miniproteins: A Helix-Turn-Helix Motif.

Numerous beta-amino acid containing peptides forming secondary structures have been already described, however the design of higher-order structures remains poorly explored. The methodology allowing construction of sequence patterns containing few rigid secondary element was proposed and experimentally validated. On the basis of 9/10/9/12-helix containing cis-2-aminocyclopentanecarboxylic acid (cis-ACPC) residues arranged in an ααßß sequence pattern, a conformationally stable helix-turn-helix structure was designed. The connection between two helices was also constructed using cis-ACPC residues. Five examples of designed peptides were obtained and analyzed using circular dichroism and nuclear magnetic resonance spectroscopy, which confirmed the assumed way of folding. The NMR structure was calculated for the peptide with the highest number of non-sequential contacts.

Systematic 'foldamerization' of peptide inhibiting p53-MDM2/X interactions by the incorporation of trans- or cis-2-aminocyclopentanecarboxylic acid residues.

A 'foldamerization' strategy for the discovery of biologically active peptide is evaluated using as an example the peptides that inhibit the p53-MDM2/X interactions. Application of a peptide scan with two constrained ß-residue of trans and cis stereochemistry indicated a substitution pattern that leads to active molecules with enhanced conformational stability and high resistance to proteolysis. This procedure led to the discovery of a peptide that showed subnanomolar inhibition of the p53-MDM2 interaction (Ki = 0.4 nM) with resistance to proteolysis enhanced by ca. two orders of magnitude. Crystallographic analysis and molecular modelling allowed for understanding of these peptide-protein interactions at the molecular level.

Synthesis, in vitro biological activity and docking of new analogs of BIM-23052 containing unnatural amino acids.

Somatostatin (SST) is an endogenous cyclic tetradecapeptide hormone that exerts multiple biological activities via a family of five receptors. BIM-23052 (DC-23-99) D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2 is a linear SST analog with established in vitro GH-inhibitory activity and high affinity to sstr5, sstr3 and sstr2. The different SSTR subtypes are expressed in different tissues and in some tumor cells. Based on this finding, a series of new analogs of BIM-23052 with expected antitumor activity have been synthesized. The Thr at position 6 in BIM-23052 was replaced by the conformationally hindered Tle, Aib, Ac5c and Ac6c of the new analogs. The peptides were synthesized by standard solid-phase peptide chemistry methods, Fmoc strategy. The cytotoxic effects of the compounds were tested in vitro against a panel of tumor cell lines: HT-29, MDA-MB-23, Hep-G2, HeLa and the normal human diploid cell line Lep-3. All five somatostatin receptor subtypes were modeled and docking was performed to determine the binding affinity of the analogs. The new peptides exhibited different concentration-dependent antiproliferative effect on the tumor cell lines after 24 h of treatment. The compound 3B (Aib6) demonstrated the most pronounced antiproliferative effects on HepG-2 cells with the IC50 = 0.01349 nM. Docking confirmed that all compounds bind well to SST receptors with preference to sstr3 and sstr5, which is most probably the reason for the observed biological effects.

Identification of the Amipurimycin Gene Cluster Yields Insight into the Biosynthesis of C9 Sugar Nucleoside Antibiotics.

Feeding studies indicate a possible synthetic pattern for the N-terminal cis-aminocyclopentane carboxylic acid (ACPC) and suggest an unusual source of the high-carbon sugar skeleton of amipurimycin (APM). The biosynthetic gene cluster of APM was identified and confirmed by in vivo experiments. A C9 core intermediate was discovered from null mutants of ACPC pathway, and an ATP-grasp enzyme (ApmA8) was reconstituted in vitro for ACPC loading. Our observations allow a first proposal of the APM biosynthetic pathway.

Insights into the structural features and stability of peptide nucleic acid with a D-prolyl-2-aminocyclopentane carboxylic acid backbone that binds to DNA and RNA.

Peptide nucleic acid (PNA) is a powerful biomolecule with a wide variety of important applications. In this work, the molecular structures and binding affinity of PNA with a D-prolyl-2-aminocyclopentane carboxylic acid backbone (acpcPNA) that binds to both DNA and RNA were studied using molecular dynamics simulations. The simulated structures of acpcPNA-DNA and acpcPNA-RNA duplexes more closely resembled the typical structures of B-DNA and A-RNA than the corresponding duplexes of aegPNA. The calculated binding free energies are in good agreement with the experimental results that the acpcPNA-DNA duplex is more stable than the acpcPNA-RNA duplex regardless of the base sequences. The results provide further insights in the relationship between structure and stability of this unique PNA system.

Environmental Effects Determine the Structure of Potential ß-Amino Acid Based Foldamers.

This work shows that hybrid peptides formed by alternating trans-2-aminocyclopentanecarboxylic acid (trans-ACPC) and trans-2-aminocyclohexanecarboxylic acid (trans-ACHC) do not fold in the solvents typically used in the study of their homo-oligomers. Only when the peptides are assayed in SDS micelles are the predicted helical structures obtained. This indicates that the environment could play an equally important role (as the backbone stereochemistry) in determining their fold, possibly by providing a sequestered environment.

Evaluation of ß-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure.

ß-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and ß residues have been investigated as subunits in protein-like molecules that adopt discrete and predictable conformations. Two classes of ß residue have been widely explored in the context of generating α-helix-like conformations: ß3 -amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five-membered ring, such the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their ß3  homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained ß residue, however, can offset the destabilizing effect of αâ†’ß substitution. Here we extend the study of αâ†’ß substitutions, involving both ß3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two-, three-, or four-residue ß-hairpin loop, and we evaluate αâ†’ß replacements at each loop position for each variant. By referral to the ϕ,ψ angles of the native structure, one can choose a stereochemically appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three ß-containing Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Development of N-(Functionalized benzoyl)-homocycloleucyl-glycinonitriles as Potent Cathepsin K Inhibitors.

Cathepsin K is a major drug target for osteoporosis and related-bone disorders. Using a combination of virtual combinatorial chemistry, QSAR modeling, and molecular docking studies, a series of cathepsin K inhibitors based on N-(functionalized benzoyl)-homocycloleucyl-glycinonitrile scaffold was developed. In order to avoid previous problems of cathepsin K inhibitors associated with lysosomotropism of compounds with basic character that resulted in off-target effects, a weakly- to nonbasic moiety was incorporated into the P3 position. Compounds 5, 6, and 9 were highly selective for cathepsin K when compared with cathepsins L and S, with the Ki values in the 10-30 nM range. The kinetic studies revealed that the new compounds exhibited reversible tight binding to cathepsin K, while the X-ray structural studies showed covalent and noncovalent binding between the nitrile group and the catalytic cysteine (Cys25) site.

Synthesis and in vitro antitumor activity of new octapeptide analogs of somatostatin containing unnatural amino acids.

Some modified octapeptide analogs of somatostatin with the following structure D-Phe-c(Cys-Phe-D-Trp-Xxx-Yyy-Cys)-Thr-NH2, where Xxx is Lys or Orn and Yyy is Aib (α-aminoisobutyric acid), Ac5c (1-aminocyclopentanecarboxylic acid) or Ac6c (1-aminocyclohexane carboxylic acid) have been synthesized. The peptides were prepared by standard Fmoc-solid phase peptide chemistry method. The direct disulphide bond formation has been employed on the solid phase by Tl(CF3CO2)3. The cytotoxic effects of the compounds were tested in vitro against a panel of tumor cell lines: HT-29 (human colorectal cancer cell line), MDA-MB-23 (human breast cancer cell line), Hep-G2 (human hepatocellular carcinoma cell line), HeLa (cervical cancer cell line) and normal human diploid cell line Lep-3. The new peptides exhibited different concentration-dependent antiproliferative effect against the tumor cell lines after 24 h treatment. The compounds were most effective to the HT-29 tumor cells. The compound 4C (Orn(5), Aib(6)) demonstrated the most pronounced antiproliferative effects on HT-29 cells with the IC50 = 0.0199 µM.

Investigation of the structure-selectivity relationships and van't Hoff analysis of chromatographic stereoisomer separations of unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids on Cinchona alkaloid-based chiral stationary phases.

The enantiomers of four unusual, rather rigid isoxazoline-fused 2-aminocyclopentanecarboxylic acids were directly separated on a quinine- or a quinidine-based zwitterionic ion-exchanger as chiral selector. The effects of the mobile phase composition, the structures of the analytes and temperature on the separations were investigated. Experiments were performed at constant mobile phase composition in the temperature range 10-50°C to study the effects of temperature, and thermodynamic parameters were calculated from plots of ln α versus 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantiomer separations were in most cases predominantly enthalpy-driven, but entropically-driven separations were also observed. The sequence of elution of the enantiomers was determined in all cases.

Induced folding of protein-sized foldameric ß-sandwich models with core ß-amino acid residues.

The mimicry of protein-sized ß-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin-14 ß-sheet has been used as a template, and αâ†’ß residue mutations were carried out in the hydrophobic core (positions 12 and 19). ß-Residues with diverse structural properties were utilized: Homologous ß(3) -amino acids, (1R,2S)-2-aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six α/ß-peptidic chains were constructed in both monomeric and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced ß-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the ß-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the ß-sandwich. The novel ß-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.

Cell-penetrating helical peptides having l-arginines and five-membered ring α,α-disubstituted α-amino acids.

Cell-penetrating peptides are powerful tools in the delivery of drugs, proteins, and nucleic acids into cells; therefore, focus has recently been placed on their development. In this study, we synthesized seven types of peptides possessing three l-arginines (l-Arg) and six l-leucines (l-Leu) and/or 1-aminocyclopentane-1-carboxylic acids (Ac5c), and investigated their secondary structures and cell-penetrating abilities. The peptide composed of an equal number of l-Arg, l-Leu, and Ac5c formed 310/α-helical structures in TFE solution and exhibited the highest cell-penetrating ability of all the peptides examined. Additional cellular uptake studies revealed that the incorporation of Ac5c into peptides led to improved tolerability against serum. The results of the present study will help in the design of novel cell-penetrating peptides.

Asymmetric syntheses of enantiopure C(5)-substituted transpentacins via diastereoselective Ireland-Claisen rearrangements.

Asymmetric syntheses of (S,S,S)-2-amino-5-methylcyclopentanecarboxylic acid and (S,S,S)-2-amino-5-phenylcyclopentanecarboxylic acid were achieved in 9 steps from commercially available starting materials via the Ireland-Claisen rearrangement of two enantiopure ß-amino allyl esters, followed by ring-closing metathesis, reduction and deprotection.

High-performance liquid chromatographic enantioseparation of isoxazoline-fused 2-aminocyclopentanecarboxylic acids on a chiral ligand-exchange stationary phase.

The application of a chiral ligand-exchange column for the direct high-performance liquid chromatographic enantioseparation of unusual ß-amino acids with a sodium N-((R)-2-hydroxy-1-phenylethyl)-N-undecylaminoacetate-Cu(II) complex as chiral selector is reported. The investigated amino acids were isoxazoline-fused 2-aminocyclopentanecarboxylic acid analogs. The chromatographic conditions were varied to achieve optimal separation. The effects of temperature were studied at constant mobile phase compositions in the temperature range 5-45°C, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Δ(ΔH°) ranged from -2.3 to 2.2 kJ/mol, Δ(ΔS°) from -3.0 to 7.8 J mol(-1) K(-1) and -Δ(ΔG°) from 0.1 to 1.7 kJ/mol, and both enthalpy- and entropy-controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. The sequence of elution of the enantiomers was determined in all cases.

Alkyne-azide cycloadditions with copper powder in a high-pressure continuous-flow reactor: high-temperature conditions versus the role of additives.

A safe and efficient flow-chemistry-based procedure is presented for 1,3-dipolar cycloaddition reactions between organic azides and acetylenes. This simple and inexpensive technique eliminates the need for costly special apparatus and utilizes Cu powder as a plausible Cu(I) source. To maximize the reaction rates, high-pressure/high-temperature conditions are utilized; alternatively, the harsh reaction conditions can be moderated at room temperature by the joint application of basic and acidic additives. A comparison of the performance of these two approaches in a series of model reactions has resulted in the formation of useful 1,4-disubstituted 1,2,3-triazoles in excellent yields. The risks that are associated with the handling of azides are lowered, thanks to the benefits of flow processing, and gram-scale production has been safely implemented. The synthetic capability of this continuous-flow technique is demonstrated by the efficient syntheses of some highly functionalized derivatives of the antifungal cispentacin.

A new access route to functionalized cispentacins from norbornene ß-amino acids.

An efficient and simple new stereocontrolled access route to novel disubstituted cispentacin derivatives with multiple stereogenic centers from norbornene ß-lactam has been developed. The synthesis involves olefinic bond functionalization by dihydroxylation followed by oxidative ring cleavage and transformation of the dialdehyde intermediate through a Wittig reaction.

Microtubes with rectangular cross-section by self-assembly of a short ß-peptide foldamer.

In nature, complex and well-defined structures are constructed by the self-assembly of biomolecules. It has been shown that ß-peptide foldamers can mimic natural peptides and self-assemble into three-dimensional molecular architectures thanks to their rigid and predictable helical conformation in solution. Using shorter foldamers, which can be prepared more easily than longer ones, to form such architectures is highly desirable, but shorter foldamers have been overlooked due to the seemingly inferior number of intramolecular hydrogen bonds to stabilize a folded state in solution. Here we report that a ß-peptide tetramer, although it lacks full helical propensity in solution, does self-assemble to form well-defined microtubes with rectangular cross-section by evaporation-induced self-assembly.

Synthesis of highly functionalized fluorinated cispentacin derivatives.

Fluorinated highly functionalized cispentacin derivatives were synthetised starting from an unsaturated bicyclic ß-lactam through C=C bond functionalization via the dipolar cycloaddition of a nitrile oxide, isoxazoline opening, and fluorination by OH/F exchange.