Conformational preference and potential templates of N-methylated cyclic pentaalanine peptides

Systematic N-methylation of all peptide bonds in the cyclic pentapeptide cyclo(-D-Ala-Ala(4)-) has been performed yielding 30 different N-methylated derivatives, of which only seven displayed a single conformation on the NMR time scale. The conformation of these differentially N-methylated peptides was recently reported by us (J. Am. Chem. Soc. 2006, 128, 15 164-15 172). Here we present the conformational characterization of nine additional N-methylated peptides from the previous library which are not homogeneous but exist as a mixture in which at least one conformation is preferred by over 80 %. The structures of these peptides are investigated employing various 2D-NMR techniques, distance geometry calculations and further refined by molecular dynamics simulations in explicit DMSO. The comparison of the conformation of these nine peptides and the seven conformationally homogeneous peptides allow us to draw conclusions regarding the influence of N-methylation on the peptide backbone of cyclic pentapeptide of the class cyclo(-D-Ala-Ala(4)-). Here we present the different conformational classes of the peptides arising from the definitive pattern of N-methylation which can eventually serve as templates for the design of bioactive peptides.     

An ONIOM investigation of the effect of conformation on bond dissociation energies in peptides

In the present study, we use the ONIOM strategy of Morokuma and coworkers to examine the various C H bond dissociation energies (BDEs) of a small peptide (2ONW) and compare these with values obtained for its component individual amino acid residues. To evaluate suitable methods for ONIOM-based geometry optimizations, we test an “internal consistency” approach against full B3-LYP//B3-LYP results, and find B3-LYP/6-31G(d):AM1 to be appropriate. We find that the BDEs at the α-carbon in 2ONW are generally larger than the corresponding values for the individual residues on their own. This is attributed to the constraints of the peptide backbone leading to conformations that are not ideal for captodative stabilization of the resulting α-radicals. At the more flexible β- and γ-positions, the differences between the BDEs in 2ONW and the individual residues are smaller. Overall, the α-BDEs are smaller than the β- and γ-BDEs in most cases. Thus, to rationalize the inertness of peptide backbones with respect to α-hydrogen abstraction that is frequently found experimentally, it is necessary to consider alternative protection mechanisms such as the polar effect. © 2018 Wiley Periodicals, Inc.     

Synthesis and anticancer activities of proline-containing cyclic peptides and their linear analogs and congeners

A solution phase method was adopted for the synthesis of proline-containing cyclic pentapeptide 2 and total synthesis of naturally occurring cyclic heptapeptide Reniochalistatin B 3. For the synthesis of 3, both divergent and convergent strategies were used to improve the overall yield from 12 to 25%. Different N and C terminal modified linear analogs and congeners of 2 and 3 were synthesized. Both cyclic peptides 2 and 3 and their linear analogs/congeners were evaluated for anti-cancer activity against HeLa cell line, among which pentapeptide 2?h and hexapeptide 3n with N-terminal protected hexafluoroisopropyl carbamates (HFIPC) interestingly showed higher cytotoxicity with an IC₅₀ of 2.73 and 4.3?µM, respectively compared to their Boc-protected analogs 2a (IC₅₀ 20?µM) and 3c (IC₅₀ 38.51?µM) and cyclic peptides 2 (>100?µM) and 3 (47?µM). These results were further validated by biological experiments such as colony formation and wound healing assays.     

Two Cyclic Peptides Produced by the Endophytic Fungus # 2221 from Castaniopsis fissa on the South China Sea Coast

New cyclic peptides 1 and 2 were isolated from the endophytic fungus #2221 from Castaniopsisfissa on the south China sea coast. By 2D NMR methods and chiral HPLC technique, their structures were elucidated as cyclo (L-Val-L-Leu-L-Val-L-Leu) and cyclo(L-Leu-L-Ala-L-Leu-L-Ala), respectively.     

Quantum chemical study of cyclic dipeptides

The preferred conformations of cyclic dipeptides were first studied systemically using the density functional theory (DFT) B3LYP method at the 6‐31G(d) level. The structural characteristics of cyclic dipeptides were revealed, most of which have not been confirmed until now. Our studies showed that the six‐member main circles of cyclic dipeptides composed of natural L ‐amino acid residues appeared as boat conformations. The important factors that influence conformations of cyclic dipeptides, such as molecular total energy, nuclear repulsion energy, molecular orbit, spatial effects, and reactive mechanism, are discussed in detail. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007     

An efficient approach for the characterization of mucin-type glycopeptides: the effect of O-glycosylation on the conformation of synthetic mucin peptides

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.     

Synthetic and conformational studies on dehydrovaline-containing model peptides

To explore the conformational preferences of α?-dehydrovaline (ΔVal) residue, three model dipeptide N-methylamides containing ΔVal were synthesized. Conformational investigations using 300 MHz NMR spectroscopy were based on delineation of intramolecularly hydrogen-bonded NH groups and Nuclear Overhauser Effect (NOE) studies. Temperature and solvent dependence studies in (CD₃)₂SO and CDC1₃-(CD₃)₂SO mixtures showed the absence of any intramolecular hydrogen bonding which suggests that all the three peptides have an extended conformation in solution. Dilution studies in CHC1₃ conducted using IR spectroscopy further supported the above conclusions. NOE studies also ruled out the existence of any type of discernible secondary structure for these peptides. Conformational behaviour of these dehydrovaline peptides is in contrast with corresponding peptides containing Δ^zPhe and Δ^zLeu, both of which stabilize ?-turn (type-II) structure. These results highlight the importance of steric factors in deciding the conformational properties of dehydropeptides.     

Conformation-dependent racemization of aspartyl residues in peptides

Biologically uncommon D-aspartyl (D-Asp) residues have been detected in proteins of various tissues of elderly humans. The presence of D-Asp has been explained as a result of the racemization of L-Asp (denoted as Asp) in the protein of inert tissues. We have previously suggested that the racemization of Asp may depend on the conformation of the peptide chain. However, the nature of the peptide conformation that affects the D-Asp formation has not yet been examined. Here we report the kinetics of Asp racemization in two model peptides, (Asp-Leu)(15) and (Leu-Asp-Asp-Leu)(8)-Asp, which form beta-sheet structures and alpha-helical structures, respectively. For the beta-sheet structures, the activation energy of racemization of Asp residues was 27.3 kcal mol(-1), the racemization rate constant at 37 degrees C was 2.14x10(-2) per year and the time required to reach a D/L ratio of 0.99 at 37 degrees C was 122.6 years as estimated from the Arrhenius equation. For the alpha-helical structures, the activation energy of racemization was 18.4 kcal mol(-1), the racemization rate constant 20.02x10(-2) per year and the time 13.1 year. These results suggest that Asp residues inserted in alpha-helical peptides are more sensitive to racemization than Asp residues inserted in peptides adopting beta-sheet structures. The results clearly indicate that the racemization rate of Asp residues in peptides depends on the secondary structure of the host peptide.     

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).     

A new hybrid algorithm for finding the lowest minima of potential surfaces: approach and application to peptides

A new algorithm is presented for finding the global minimum, and other low-lying minima, of a potential energy surface (PES) of biological molecules. The algorithm synergetically combines three well-known global optimization methods: the diffusion equation method (DEM), which involves smoothing the PES; a simulated annealing (SA) algorithm; and evolutionary programming (EP), whose population-oriented approach allows for a parallel search over different regions of the PES. Tests on five peptides having between 6 and 9 residues show that the code implementing the new combined algorithm is efficient and is found to outperform the constituent methods, DEM and SA. Results of the algorithm, in the gas phase and with the GBSA implicit solvent model, are compared with crystallographic data for the test peptides; good accord is found in all cases. Also, for all but one of the examples, our hybrid algorithm finds a minimum deeper than those obtained by a very extensive scan. TINKERs implementation of the OPLS-AA force field is employed for the structure prediction. The results show that the new algorithm is a powerful structure predictor, when a reliable potential function is available. Our implementation of the algorithm is time-efficient, and requires only modest computational resources. Work is underway on applications of the new algorithm to structural prediction of proteins and other biological macro-molecules.     

Lipopeptaibol metabolites of tolypocladium geodes: total synthesis,preferred conformation,and membrane activity

We have synthesized by solution methods and characterized the lipopeptaibol metabolite LP237-F8 extracted from the fungus Tolypocladium geodes and five selected analogues with the Etn-->Aib or Etn-->Nva replacement at position 8 and/or a triple Gln-->Glu(OMe) replacement at positions 5, 6, and 9 (Etn=Calpha-ethylnorvaline, Aib=alpha-aminoisobutyric acid, Nva=norvaline). Conformation analysis, performed by FT-IR absorption, NMR, and CD techniques, strongly supports the view that the six terminally blocked decapeptides are highly helical in solution. Helix topology and amphiphilic character are responsible for their remarkable membrane activity. At position 8 the combination of high hydrophobicity and Calpha tetrasubstitution, as in the Etn-containing LP237-F8 metabolite, has a positive effect on membrane interaction.     

Total solid-phase synthesis of the azathiocoraline class of symmetric bicyclic peptides

Thiocoraline is a potent antitumor agent isolated from the marine organism Micromonospora sp. This symmetric bicyclic depsipeptide binds the minor groove of DNA. Here we report two solid-phase strategies for the syntheses of azathiocoraline and its analogues. The thioester linkage was replaced by an amide bond to improve the compound's pharmacokinetic properties. The first strategy is based on a convergent (4+4) approach, whilst the second is a stepwise synthesis, cyclizations in both approaches occurring on the solid support. These two strategies were designed to overcome problems caused by the presence of consecutive noncommercial N-methyl amino acids, to avoid epimerization during cyclization and/or fragment condensation, and to form the disulfide bridge under solid-phase conditions. The heterocyclic moiety was added in the last step of the synthesis to assist the preparation of libraries of new compounds with potential therapeutic applications.     

Solid-phase synthesis of homodetic cyclic peptides from Fmoc-MeDbz-resin

Cyclic homodetic peptides are very appealing for medicinal chemistry programs. In addition to the high efficiency and selectivity inherently associated with peptides, a cyclic structure totally formed by amide bonds increases their stability under physiological conditions. Here Fmoc-MeDbz-resin was studied for the preparation of these peptides. Our results demonstrate the usefulness of this strategy for the preparation of cyclic “head-to-side chain” peptides through cyclative cleavage (simultaneous cyclization and release from the resin). In contrast, for the synthesis of the “head-to-tail” counterparts, the cyclization-cleavage should be carried out in the presence of thiophenol.     

Acyclic and cyclic thioenamino peptides: solution- and solid-phase synthesis

Seven- and 10-membered cyclic thioenamino peptides, that is, 1,4-thiazepinone (11) and cyclic thioenamino peptide 9 (which represents a potential γ-turn mimetic), were synthesized, and the structure of 11 was secured by X-ray diffraction analysis of its TFA salt. The aforementioned compounds were prepared in solution and by solid-phase synthesis. Additionally, we have prepared thioenamino diketopiperazine synthon 16.     

Synthesis and biological evaluation of the osteoblast proliferating cyclic peptides dianthins G and H

The first synthesis and osteoblast proliferative activity of the naturally occurring cyclic peptides dianthins G and H is described. The greater potency of naturally occurring dianthin G over dianthin H at physiological concentrations mirrored the osteoblast proliferative activity observed for synthetic dianthins G and H. Six alanine-scan analogues of the more potent dianthin G were also synthesised and osteoblast assays revealed that four of the six residues can be further modified for improved activity. We also confirmed by variable temperature ¹H NMR spectroscopic analysis that the sets of major and minor signals observed for dianthins G and H in DMSO-d₆ are in fact due to cis–trans rotational isomers of the proline ring.