N-methylated Amino Acid Residues Research Articles

N-Methylated Analogs of hIAPP Fragments 18-22, 23-27, 33-37 Inhibit Aggregation of the Amyloidogenic Core of the Hormone.

Amylin (hIAPP) aggregation leads to the formation of insoluble deposits and is one of the factors in the development of type II diabetes. The aim of this research was to find N-methylated analogs of the aggregating amylin fragments 18-22, 23-27, and 33-37, which would not themselves be susceptible to aggregation and would inhibit the aggregation of the amyloidogenic cores of the hormone. None of the analogs of fragment 18-22 containing one or two N-methylated amino acid residues showed any tendency to aggregate. Only the peptide H-F(N-Me)GA(N-Me) IL-OH (6) derived from the 23-27 hIAPP hot spot did not form fibrous structures. All analogs of the 33-37 amylin fragment were characterized by the ability to form aggregates, despite the presence of N-methylated amino acids in their structures. N-Methylated peptides 1-5 demonstrated inhibitory properties against the aggregation of fragment 18-22. Aggregation of the amyloidogenic core of 23-27 was significantly inhibited by N-methylated peptides 1-3 derived from the (18-22) H-HSSNN-OH fragment and by the H-F(N-Me)GA(N-Me)IL-OH (6) fragment derived from the 23-27 amylin hot spot. Fragment (33-37) H-GSNTY-NH2 was found to be inhibited in the presence of N-methylated peptides 1-3 derived from the 18-22 fragment and by the double methylated peptide H-F(N-Me)GA(N-Me)IL-OH (6). Research on the possibility of using N-methylated analogs of amyloidogenic amylin cores as inhibitors of hormone aggregation is ongoing, with a focus on finding the minimum concentration of N-methylated peptides capable of inhibiting the aggregation of hIAPP hot spots.

Insulin Hot-Spot Analogs Formed with N-Methylated Amino Acid Residues Inhibit Aggregation of Native Hormone.

In this study, N-methylated analogs of hot-spots of insulin were designed and synthesized, in the expectation that they would inhibit the aggregation of both insulin hot-spots and the entire hormone. Synthesis of insulin “amyloidogenic” analogs containing N-methylated amino acid residues was performed by microwave-assisted solid phase according to the Fmoc/tert-Bu strategy. As a coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TosO-) was used. Three independent methods were applied in aggregation studies of the complexes of insulin with its N-methylated peptides. Additionally, circular dichroism (CD) measurements were used to confirm that aggregation processes did not occur in the presence of the N-methylated analogs of hot-spot insulin fragments, and that insulin retains its native conformation. Of the seven N-methylated analogs of the A- and B-chain hot-spots of insulin, six inhibited insulin aggregation (peptides 1 and 3–7). All tested peptides were found to have a lower ability to inhibit the aggregation of insulin hot-spots compared to the capability to inhibit native hormone aggregation.

Heterologous Production of a Novel Cyclic Peptide Compound, KK-1, in Aspergillus oryzae.

A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.

Cyclic mu-opioid receptor ligands containing multiple N-methylated amino acid residues

In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis.

Synthesis of linear and cyclic opioid-based peptide analogs containing multiple N-methylated amino acid residues.

A series of six novel opioid peptide analogs containing one to three N-methylamino acid residues, and six cyclic counterparts of these peptides were prepared by the solid-phase method. Introduction of two consecutive N-methylated amino acids, as well as cyclization of such conformationally constrained sequences, turned out to be challenging. The use of a recently reported triazine-based coupling reagent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate, enabled the synthesis and cyclization of the designed analogs in acceptable yields and with a lesser amount of by-products than observed with the standard coupling reagents such as TBTU or HATU.

Suppression of peptide ion dissociation under electron capture: role of backbone amide hydrogen.

The electron capture dissociation (ECD) of proteins/peptides is affected by the nature and sequence of amino acid residues. Electron capture/transfer with no dissociation is an intriguing phenomenon that has occasionally been observed. We have previously identified that diarginated peptides enriched with glutamic acid residues were found to show suppression of backbone fragmentation. In this paper, we report the effect of geometrical parameters of a peptide, including chain length, conformation and amide hydrogen, on the suppression of ECD fragmentation using synthetic model peptides. Glycine containing model polypeptides were used to probe the mechanism. Molecular-mechanics was used to obtain the conformation of the precursor ions. The ECD experiments were performed on a Bruker APEX III 4.7 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Significant decreases in the intensities of the fragment ions were observed for the 23-mer polypeptide with only one E residue. This implied that the E:R ratio was no longer the sole determining factor for the occurrence of suppression effects. Results of conformational searches showed that there was a hydrogen-bonding 'ladder' formed in the 23-mer polypeptide, which was not found in the 15-mer peptide. Substituting the normal amino acid residues by the corresponding N-methylated amino acid residues in the model peptide, the suppression effect disappeared. Our results indicate that survival of the intact reduced peptide ion after electron capture depends also on the length of the peptide. The amide hydrogen was critical in forming the resonance structure that suppressed the ECD fragmentation.

Verrucamides A–D, Antibacterial Cyclopeptides from Myrothecium verrucaria

Four new cyclic tetradecapeptides, verrucamides A-D (1-4), have been isolated from the solid-substrate fermentation culture of the ascomycete fungus Myrothecium verrucaria. The structures of these compounds, each featuring six N-methylated amino acid residues, were elucidated primarily by NMR and MS methods. The absolute configurations of 1-4 were assigned by application of Marfey's method on their acid hydrolysates. Compounds 1-4 showed antimicrobial activity against the Gram-positive bacterium Staphylococcus aureus.

High-performance liquid chromatography and nuclear magnetic resonance study of linear tetrapeptides and octapeptides containing N-methylated amino acid residues

Chromatographic behavior of a series of N-methylated tetra and octapeptides on a reversed-phase sorbent was studied considering the information obtained on these compounds by NMR spectroscopy. The modified tetrapeptides were derived from GFFY-NH 2, GFFF-NH 2 and GFFH-NH 2 primary structures by N-methylation at various peptide bond positions. Similarly, the N-methylated octapeptides were based on TPK(Pac)T C-terminally elongated forms of GFFY and GFFF. It was found that many studied N-methylated peptides provide broad peaks as a consequence of cis/ trans isomerism of the R 1CO N(CH 3)R 2 peptide bond. The extent of the peak spreading depends on the following important factors: the nature of the surrounding amino acid residues, the location of the modified peptide bond within the peptide chain, temperature, and mobile phase flow-rate. All these aspects were critically evaluated. Nearly complete separation of the individual conformers of GF(NMe)FY-NH 2 was obtained applying fast chromatography on short column packed with 20–30 μm reversed-phase sorbent.

N-Methylation of Peptides on Selected Positions during the Elongation of the Peptide Chain in Solution Phase

[reactions: see text] An efficient and general solution-phase method for the site-specific N-methylation of peptides has been developed. This novel procedure involves synthesis of N-nosyl protected peptides and their subsequent N-methylation with diazomethane. Its efficiency was proved by the successful synthesis of various hindered oligopeptides containing N-methyl amino acid residues with excellent yield and purity. The method is particularly attractive in that the adopted conditions do not cause any detectable racemization of the peptide stereocenters and the process does not require chromatographic purification of the methylated products. A further advantage is the compatibility of this methodology with Fmoc solution-phase peptide synthesis.

Kendarimide A, a Novel Peptide Reversing P‐Glycoprotein‐Mediated Multidrug Resistance in Tumor Cells, from a Marine Sponge of Haliclona sp.

Abstract A novel modulator of multidrug resistance (MDR) in tumor cells, kendarimide A ( 1 ), was isolated from an Indonesian marine sponge of Haliclona sp. Compound 1 reversed MDR in KB-C2 cells mediated by P-glycoprotein (P-gp) at a 6 μM concentration, and the chemical structure of 1 was characterized to be a linear peptide composed of N-methylpyroglutamic acid (pyroMeGlu), N-methylated eight-membered cysteinyl-cysteine (ox-[MeCys-MeCys]) together with many N-methyl amino acid residues. The amino acid sequence of 1 was determined by 2D NMR and FAB MS analysis. The absolute configuration of the amino acid residues in 1 except for the MeCys part was determined to be l -form respectively, based on interpretation of the HPLC analysis of Marfey's derivatives of the hydrolysates of 1 and the synthetic method for the pyroMeGlu residue.

Kendarimide A, a novel peptide reversing P-glycoprotein-mediated multidrug resistance in tumor cells, from a marine sponge of Haliclona sp.

A novel modulator of multidrug resistance (MDR) in tumor cells, kendarimide A ( 1 ), was isolated from an Indonesian marine sponge of Haliclona sp. Compound 1 reversed MDR in KB-C2 cells mediated by P-glycoprotein (P-gp) at a 6 μM concentration, and the chemical structure of 1 was characterized to be a linear peptide composed of N-methylpyroglutamic acid (pyroMeGlu), N-methylated eight-membered cysteinyl-cysteine (ox-[MeCys-MeCys]) together with many N-methyl amino acid residues. The amino acid sequence of 1 was determined by 2D NMR and FAB MS analysis. The absolute configuration of the amino acid residues in 1 except for the MeCys part was determined to be l-form respectively, based on interpretation of the HPLC analysis of Marfey's derivatives of the hydrolysates of 1 and the synthetic method for the pyroMeGlu residue.

A novel synthesis of N-methyl asparagine, arginine, histidine, and tryptophan.

[reaction: see text] N-Methyl amino acid residues in peptides modify several pharmacologically useful parameters, but synthesis of alkylated peptides is hampered by unavailability of N-methylated monomers. The syntheses of four N-methyl amino acids with basic side chains are presented. The side chains of these basic amino acids needed to be specially protected or constructed. This completes the set of 20 common L-amino acid N-methyl derivatives prepared via 5-oxazolidinone intermediates by our group.

Highly Efficient Synthesis of Sterically Hindered Peptides Containing N-Methylated Amino Acid Residues using a Novel 1 H-Benzimidazolium Salt

Novel 1 H-benzimidazolium type peptide coupling reagent, CMBI, was designed, synthesized, and shown to be efficient in the promotion of the formation of sterically hindered amide and ester bonds. Its high efficiency was proved by model reaction tests and the successful synthesis of various hindered oligopeptides and peptide segments containing N-methyl amino acid residues with fast reaction speeds, low racemization and excellent yields. A mechanism for amide bond formation mediated by the reagent was proposed.

2-Bromo-1-ethyl Pyridinium Tetrafluoroborate (BEP): A Powerful Coupling Reagent for N-Methylated Peptide Synthesis

Abstract 2-Bromo-1-ethyl pyridinium tetrafluoroborate (BEP) was firstly utilized to the synthesis of peptides containing N-methyl amino acid residues both in solution and solid phase, and demonstrated high reactivity, low racemization and excellent yields, which were approved by the successful synthesis of a series of oligopeptides and hindered peptide fragments, such as the 8-11 segment of Cyclosporine A and the pentapeptide moiety of Dolastatin 15.

Structure-based design of irreversible, tripeptidyl human rhinovirus 3C protease inhibitors containing N-methyl amino acids

Tripeptide-derived molecules incorporating N-methyl amino acid residues and C-terminal Michael acceptor moieties were evaluated as irreversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). Such compounds displayed good 3CP inhibition activity ( k obs [I] up to 610,000 M −1s −1) and potent in vitro antiviral properties (EC 50 approaching 0.03 μM) when tested against HRV serotype-14.

New method for the synthesis of N-methyl amino acids containing peptides by reductive methylation of amino groups on the solid phase.

Primary amino groups on the model peptide Xaa-Ala-Pro-Lys(ClZ)-Tyr(2BrZ), synthesized on a p-methylbenzhydryl amine resin with conventional Boc/benzyl protective group strategy, were reacted with 4,4'-dimethoxydityl chloride in dichloromethane, resulting in the introduction of the dimethoxydityl group, which is an acid-labile N-alkyl type of protective group. The secondary amino groups thereby formed can be methylated by treating the peptide-resin with formaldehyde and sodium cyanoborohydride in N,N-dimethylformamide. After the removal of the dimethoxydityl group with trifluoroacetic acid, the resulting N-methylated amino acid residues with a free secondary amino groups are accessible for acylation with the next activated Boc amino acid. With this method majority of the 20 common amino acids can be monomethylated directly on the resin and, in most cases, with very low levels of the side reactions. In the cases where the complete methylation is difficult to achieve, the remaining primary amino groups can be selectively acylated in the presence of secondary amino groups with trimethylacetic acid 1-hydroxybenzotriazole ester. The method provides a convenient general route to synthesize N-methylated derivatives of most of the occurring and synthetic amino acids.

Studies on fungal products. XI. Isolation and structures of novel cyclic pentapeptides from Aspergillus sp. NE-45.

Three new compounds designated as cycloaspeptides A (1), B (2), and C (3) were isolated along with ergosterol from the mycelial chloroform extract of Aspergillus sp. NE-45. The structures of cycloaspeptides A. B, and C were established on the basis of chemical and spectroscopic evidence as 1, 2, and 3, respectively. Cycloaspeptides are new cyclic pentapeptides containing an anthranilic acid residue and two N-methyl amino acid residues.