N-methyl Alanine Research Articles

Carboxylate-Directed Pd-Catalyzed β-C(sp3)-H Arylation of N-Methyl Alanine Derivatives for Diversification of Bioactive Peptides.

This study presents a Pd(II)-catalyzed method for the β-C(sp3)-H arylation of N-Cbz- or N-Fmoc-protected N-methyl alanines, providing ready access to building blocks for N-methylated peptide synthesis. For this transformation, the native carboxylate was exploited as the directing group, attributing its success to the use of a monoprotected amino-pyridine ligand. Its synthetic utility was demonstrated by facile generation of nine analogues of the naturally occurring N-methylated cyclic peptide cycloaspeptide A.

Metabolomic changes after DAAs therapy are related to the improvement of cirrhosis and inflammation in HIV/HCV-coinfected patients

BackgroundA better understanding of the evolution of cirrhosis after hepatitis C virus (HCV) clearance is essential since the reversal of liver injury may not happen. We aimed to assess the evolution of plasma metabolites after direct-acting antivirals (DAAs) therapy and their association with liver disease scores in HIV/HCV-coinfected patients with advanced HCV-related cirrhosis. MethodsWe performed a prospective study in 49 cirrhotic patients who started DAAs therapy. Data and samples were collected at baseline and 36 weeks after SVR. Metabolomics analysis was carried out using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Inflammation-related biomarkers were analyzed using ProcartaPlex Immunoassays. ResultsAt 36 weeks after SVR, patients experienced significant decrease in taurocholic acid, 2,3-butanediol, and LPC(18:0); while several phosphatidylcholines (LPC(16:1), LPC(18:1), LPC(20:4), and PC(16:0/9:0(CHO))/PC(16:0/9:0(COH)), 2-keto-n-caproic acid/2-keto-isocaproic acid and N-methyl alanine increased, compared to baseline. The plasma decrease in taurocholic acid was associated with a reduction in Child-Turcotte-Pugh (CTP) (AMR=3.39; q-value=0.006) and liver stiffness measurement (LSM) (AMR=1.06; q-value<0.001), the plasma increase in LPC(20:4) was related to a reduction in LSM (AMR=0.98; q-value=0.027), and the rise of plasma 2-keto-n-caproic acid/2-keto-isocaproic acid was associated with a reduction in CTP (AMR=0.35; q-value=0.004). Finally, plasma changes in taurocholic acid were directly associated with inflammation-related biomarkers, while changes in LPC(20:4) were inversely associated. ConclusionsPlasma metabolomic profile changed after HCV clearance with all oral-DAAs in HIV/HCV-coinfected with advanced HCV-related cirrhosis. Changes in plasma levels of LPC (20: 4), 2-keto-n-caproic acid/2-keto-isocaproic acid, and taurocholic acid were related to improvements in cirrhosis scores and inflammatory status of patients.

Investigating How Proline Linkers Enhance Hybrid Antimicrobial Peptide Activity

To combat increasing antibiotic resistance in bacteria, antimicrobial peptides (AMPs) have been proposed as alternatives to conventional antibiotic treatments. AMPs are naturally found in many organisms, including humans, as part of the innate immune response. One approach to designing novel AMPs is to form a hybrid AMP by connecting two individual AMPs, often with a linker consisting of one or more amino acids. Previous studies have shown that some hybrid AMPs have increased antibacterial activity and reduced cytotoxicity to eukaryotic cells. The amino acid linker and hybrid design used may impact antibacterial activity; however, these effects have not yet been systematically evaluated. This study examines hybrid AMPs formed from two translocating histone-derived antimicrobial peptides (HDAPs): the naturally occurring buforin II (BF2) and the synthesized peptide DesHDAP1. Previous work in our laboratory has shown that proline linkers enhance the activity of these BF2/DesHDAP1 hybrids. To investigate which structural feature of proline contributes to this observed increase in activity, additional BF2/DesHDAP1 hybrids were designed using glycine and N-methyl alanine linkers. Antibacterial activity and circular dichroism spectroscopy results suggest that proline's inability to hydrogen bond contributes more to antibacterial activity than physical rigidity. Molecular dynamics simulations were also performed to compare the hybrids’ structure and interactions with membranes on a molecular level. Ongoing work is investigating the activity of these BF2/DesHDAP1 hybrid AMPs against eukaryotic cells.

Sameuramide A, a new cyclic depsipeptide isolated from an ascidian of the family Didemnidae

Sameuramide A (1), a new cyclic depsipeptide encompassing one each of alanine, N-methyl alanine, N-methyl dehydroalanine, N,O-dimethyl threonine, phenyllactic acid, three β-hydroxy leucines, and two propionates, was isolated from a didemnid ascidian collected at the northern part of Japan. The planar structure was established based on the interpretation of MS and NMR data. The absolute configuration of the subunits was determined by the advanced Marfey’s method and the chiral LC-MS analysis. Compound 1 exhibited the activity of maintaining colony formation of murine embryonic stem (mES) cells without leukemia inhibitory factor (LIF). Down regulation of the gene expression of Krüppel-like transcription factor 4 (Klf4) indicated that 1 itself was not able to maintain the undifferentiated state of the mES cells. However, the expression levels of the marker genes (Nestin, T, Sox17) for three germ layers were upregulated in embryoid bodies (EBs) after treatment of 1 together with LIF, suggesting that 1 plays a supportive role for LIF in maintaining the multipotency of mES cells.

Biologically active new metabolites from a Florida collection of Moorea producens

A bioassay-guided investigation (cancer cell cytotoxicity) of a Moorea producens collection from Key West, Florida, led to the discovery of two new bioactive natural products [(+)-malyngamide Y and a cyclic depsipeptide, (+)-floridamide]. Their planar structures were deduced through extensive analysis of 1D and 2D NMR spectroscopic data and supported by HRFAB mass spectrometry. The new cyclic depsipeptide contains four amino acids units, including N-methyl phenylalanine, proline, valine and alanine, beside the unique unit, 2,2-dimethyl-3-hydroxy-octanoic acid. In addition to the discovery of these two new compounds, two previously reported metabolites were also isolated and identified from this cyanobacterial collection; (−)-C-12 lyngbic acid and the antibacterial agent (−)-malyngolide.

Low energy CID and action IRMPD provide insights into a minor subpopulation of the gas-phase conformers of triply charged bradykinin

Unique fragmentation of the IMS-resolved conformers of triply charged bradykinin is shown to occur at very low collision energies by quadrupole CID. For the major C conformation of bradykinin, the unique fragmentation appears to result from a subpopulation that has a different average CCS than the dominant C population that has been previously identified as TTC (trans, trans, cis at the three Pro residues). The three major gas-phase isomers exhibit minimal differences in fragmentation at collision energies greater than 12eV, presumably because the amide bonds of the proline residues isomerize prior to dissociation at these higher energies. The differences in fragmentation observed in the low collision energy regime (<12eV) are attributed to the conformational differences of the fragmenting subpopulations of the mobility resolved populations associated with the cis or trans isomerization state of the three proline residues. Action IRMPD of the b2 ion supports a cis-Pro2 isomerization state for the subpopulation of conformation C fragmenting at low energies, assuming no isomerization was induced by the low energy CID. Substitution of N-methyl alanine, an acyclic proline mimic, independently, for each of the three prolines of bradykinin and low energy fragmentation of the minor subpopulation of conformation C allowed for tentative assignments of the isomerization state of two of the three proline resides of this subpopulation. Density functional theory calculations support the conclusion made from NMA-substitutions and together suggest that the subpopulation of conformation C is cis at Pro2 and trans at Pro7. It is not clear whether the minor subpopulation is a typical feature of triply charged bradykinin or whether unintended activation in the instrument used here produces this population. The results illustrate the utility of ion mobility coupled with low-energy CID for the determination of a minor subpopulation that was not visible by MS or IM alone.

Investigations of the mechanism of the "proline effect" in tandem mass spectrometry experiments: the "pipecolic acid effect".

The fragmentation behavior of a set of model peptides containing proline, its four-membered ring analog azetidine-2-carboxylic acid (Aze), its six-membered ring analog pipecolic acid (Pip), an acyclic secondary amine residue N-methyl-alanine (NMeA), and the D stereoisomers of Pro and Pip has been determined using collision-induced dissociation in ESI-tandem mass spectrometers. Experimental results for AAXAA, AVXLG, AAAXA, AGXGA, and AXPAA peptides are presented, where X represents Pro, Aze, Pip, or NMeA. Aze- and Pro-containing peptides fragment according to the well-established "proline effect" through selective cleavage of the amide bond N-terminal to the Aze/Pro residue to give yn (+) ions. In contrast, Pip- and NMA-fragment through a different mechanism, the "pipecolic acid effect," selectively at the amide bond C-terminal to the Pip/NMA residue to give bn (+) ions. Calculations of the relative basicities of various sites in model peptide molecules containing Aze, Pro, Pip, or NMeA indicate that whereas the "proline effect' can in part be rationalized by the increased basicity of the prolyl-amide site, the "pipecolic acid effect" cannot be justified through the basicity of the residue. Rather, the increased flexibility of the Pip and NMeA residues allow for conformations of the peptide for which transfer of the mobile proton to the amide site C-terminal to the Pip/NMeA becomes energetically favorable. This argument is supported by the differing results obtained for AAPAA versus AA(D-Pro)AA, a result that can best be explained by steric effects. Fragmentation of pentapeptides containing both Pro and Pip indicate that the "pipecolic acid effect" is stronger than the "proline effect."

Evolved Diversification of a Modular Natural Product Pathway: Apratoxins F and G, Two Cytotoxic Cyclic Depsipeptides from a Palmyra Collection ofLyngbya bouillonii

A collection of Lyngbya bouillonii from Palmyra Atoll in the Central Pacific, a site several thousand kilometers distant from all previous collections of this chemically prolific species of cyanobacterium, was found to contain two new cancer cell cytotoxins of the apratoxin family. The structures of the new compounds, apratoxins F and G, were determined by 1D and 2D NMR techniques in combination with mass spectrometric methods. Stereochemistry was explored by using chromatographic analyses of the hydrolytically released fragments in combination with NMR and optical rotation comparisons with known members of the apratoxin family. Apratoxins F and G add fresh insights into the SAR of this family because they incorporate an N-methyl alanine residue at a position where all prior apratoxins have possessed a proline unit, yet they retain high potency as cytotoxins to H-460 cancer cells with IC(50) values of 2 and 14 nM, respectively. Additional assays using zone inhibition of cancer cells and clonogenic cells give a comparison of the activities of apratoxin F to apratoxin A. Additionally, the clonogenic studies in combination with maximum tolerated dose (MTD) studies provided insights as to dosing schedules that should be used for in vivo studies, and preliminary in vivo evaluation validated the predicted in vivo efficacy for apratoxin A. These new apratoxins are illustrative of a mechanism (the modification of an NRPS adenylation domain specificity pocket) for evolving a biosynthetic pathway so as to diversify the suite of expressed secondary metabolites.

IR spectroscopy of cationized aliphatic amino acids: Stability of charge-solvated structure increases with metal cation size

Gas-phase structures of alkali metal cationized (Li +, Na +, K +, Rb +, and Cs +) proline (Pro) and N-methyl alanine have been investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser and computational modeling. Measured IRMPD spectra are compared to spectra calculated at the B3LYP/6-311++G(2d,2p) level of theory to identify individual conformers. Calculations indicate that the stability of the salt bridge (SB; zwitterionic) conformer relative to the most stable canonical structure with a single formal charge site (charge solvation; CS) of aliphatic amino acids (e.g., Pro, N-methyl alanine, N-methyl glycine, and glycine) does not increase with size and polarizability of the alkali metal cations, in contrast to the trend commonly found for functionalized amino acids. In fact, the relative stability of SB over CS conformers reaches a maximum at [amino acid + Na] +. A uniform SB structure and two characteristic CS conformers are identified by theory to be relevant for alkali metalized Pro, N-methyl alanine, and N-methyl glycine. For CS structures, the alkali metal cation is either coordinated to the nitrogen and the carbonyl oxygen of the acid functionality (Li +, Na +) or is solely interacting with the carboxylic acid oxygens (K +, Rb +, and Cs +). The IRMPD spectra exhibit clearly distinguishable bands for the CO stretching modes of the carboxylic acid moiety in CS structures and for the carboxylate moiety in SB structures, allowing reliable structure assignments for all complexes investigated. The IRMPD spectra clearly exhibit the presence of mixed populations of SB and CS structures with the contribution of CS increasing toward the larger metal cations, in good agreement with the predictions from computational modeling. The special trend regarding formation and stability of individual gas-phase ion structures of aliphatic amino acids, lacking functionalized α-side chains, can be rationalized with the concept of hard and soft Lewis acids and bases. Furthermore, calculations show that the trends with metal cation size found for aliphatic amino acids with secondary amines are similar for ordinary aliphatic amino acids (Gly, Ala).

Three novel anabaenopeptins from the cyanobacterium Anabaena sp.

Three new cyclic peptides, anabaenopeptins NZ825, NZ841, and NZ857, were isolated from the hydrophilic extract of the cultured cyanobacterium Anabaena sp. The planar structure of the compounds was determined by homonuclear and inverse-heteronuclear 2D-NMR techniques as well as high-resolution mass spectrometry. The absolute configuration of the asymmetric centers was studied using Marfey's method for HPLC. This is the first report of anabaenopeptins that contain N-methyl glycine instead of the common N-methyl alanine. The incorporation of N-methyl glycine into the cyclic portion of the compounds results in their appearance as a mixture of two, equally stable, conformers, instead of the one distinct conformer in anabaenopeptins that contain N-methyl alanine or N-methyl homotyrosine. The three compounds were tested for inhibition of serine proteases and found to be not active.

Investigation of Gas Phase Ion Structure for Proline-Containing b 2 Ion

Unusual fragmentation was observed for doubly charged VPDPR in which cleavage C-terminal to proline and N-terminal to aspartic acid yielded b(2) (+ a(2))/y(3) complementary ions. This unique fragmentation is contradictory to trends previously established by statistical analysis of peptide tandem mass (MS/MS) spectra. Substitution of alanine for aspartic acid (i.e., VPAPR) did not change the fragmentation, indicating the cleavage was not directed by aspartic acid. Fragmentation patterns for VPAPR and V(NmA)APR (NmA = N-methyl alanine) were compared to determine whether conformational constraints from proline's cyclic side-chain contribute to b(2) ion formation. While both peptide sequences fragmented to yield b(2)/y(3) ions, only VPAPR produced a(2) ions, suggesting the VP b(2) ion is structurally different from the V(NmA) b(2) ion. Instead, the V(NmA) b(2) ion was accompanied by an ion corresponding to formal loss of 71. The suspected structural differences were confirmed by isolation and fragmentation of the respective b(2) ions (i.e., MS(3) spectra). Evidence supporting a diketopiperazine structure for the VP b(2) ion is reported. Fragmentation patterns for the VP b(2) ion and a synthetic VP diketopiperazine showed great similarity. N-terminal acetylation of VPAPR prevented the formation of the VP b(2) ion, presumably by blocking nucleophilic attack by the N-terminal amine on the carbonyl oxygen of the protonation site. Acetylation of the N-terminus for V(NmA)APR did not prevent the formation of the V(NmA) b(2) ion, indicating the V(NmA) b(2) ion has a structure, presumably that of an oxazolone, which requires no attack by the N-terminus for formation. Finally, high-resolution, accurate mass measurements determined that the V(NmA) (b(2)-71) ion results from losing a portion of valine from oxazolone V(NmA) b(2) ion, rather than cross-ring cleavage of the alternate diketopiperazine.

Exploration of the P6/P7 Region of the Peptide-binding Site of the Human Class II Major Histocompatability Complex Protein HLA-DR1

Crystal structures of the class II major histocompatibilty complex (MHC) protein, HLA-DR1, generally show a tight fit between MHC and bound peptide except in the P6/P7 region of the peptide-binding site. In this region, there is a shallow water-filled pocket underneath the peptide and between the pockets that accommodate the P6 and P7 side chains. We investigated the properties of this pocket with the idea of engineering substitutions into the corresponding region of peptide antigens to increase their binding affinity for HLA-DR1. We investigated d-amino acids and N-alkyl modifications at both the P6 and P7 positions of the peptide and found that binding of peptides to HLA-DR1 could be increased by incorporating an N-methyl substitution at position 7 of the peptide. The crystal structure of HLA-DR1 bound to a peptide containing a P7 N-methyl alanine was determined. The N-methyl group orients in the P6/P7 pocket, displacing one of the waters usually bound in this pocket. The structure shows that the substitution does not alter the conformation of the bound peptide, which adopts the usual polyproline type II helix. An antigenic peptide carrying the N-methyl modification is taken up by antigen-presenting cells and loaded onto endogenous class II MHC molecules for presentation, and the resultant MHC-peptide complexes activate antigen-specific T-cells. These results suggest a possible strategy for increasing the affinity of weakly immunogenic peptides that might be applicable to the development of vaccines and diagnostic reagents.

Enantiomeric determination of amines by high-performance liquid chromatography using chiral fluorescent derivatization reagents.

4-(2-carboxypyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-Pro), 4-(2-carboxypyrrolidin-1-yl)-7-(N,N-dimethylamino-sulphonyl)-2,1,3 - benzoxadiazole DBD-Pro), 4-(N-1-carboxyethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole NBD-N-Me-Ala), 4-(N-1-carboxyethyl-N-methyl) amino-7-(N,N-dimethylamino-2,1,3-benzoxadiazole. (DBD-N-Me-Ala) have been synthesized for the resolution of enantiomers of amines by high performance liquid chromatography (HPLC). The reagents react with amino group at room temperature in the presence of activation agents, 2,2'-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) to produce the corresponding diastereomers. The derivatives were detected at lambda ex = 469, lambda em = 569 for DBD-moeity and lambda ex = 469, lambda em = 535 for NBD moeity. The resulting diastereomers were efficiently resolved using reversed-phase column with aqueous acetonitrile and aqueous methanol as the mobile phase. The elution order of the derivatives were D and L when proline was used as the chiral selector but the order was reversed when the diastereomers were prepared with the reagent containing N-methyl alanine as the chiral selector. DBD-Pro and NBD-Pro seem to give better separation as compared to DBD-N-Me-Ala and NBD-N-Me-Ala.

Enantiomeric determination of amines by high‐performance liquid chromatography using chiral fluorescent derivatization reagents

4-(2-carboxypyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-Pro), 4-(2-carboxypyrrolidin-1-yl)-7-(N,N-dimethylamino-sulphonyl)-2,1,3-benzoxadiazole DBD-Pro), 4-(N-1-carboxyethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole NBD-N-Me-Ala), 4-(N-1-carboxyethyl-N-methyl) amino-7-(N,N-dimethylamino-2,1,3-benzoxadiazole. (DBD-N-Me-Ala) have been synthesized for the resolution of enantiomers of amines by high performance liquid chromatography (HPLC). The reagents react with amino group at room temperature in the presence of activation agents, 2,2′-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) to produce the corresponding diastereomers. The derivatives were detected at λ ex = 469, λ em = 569 for DBD-moeity and λ ex = 469, λ em = 535 for NBD moeity. The resulting diastereomers were efficiently resolved using reversed-phase column with aqueous acetonitrile and aqueous methanol as the mobile phase. The elution order of the derivatives were DL when proline was used as the chiral selector but the order was reversed when the diastereomers were prepared with the reagent containing N-methyl alanine as the chiral selector. DBD-Pro and NBD-Pro seem to give better separation as compared to DBD-N-Me-Ala and NBD-N-Me-Ala. © 1998 John Wiley & Sons, Ltd.

Inhibition of sodium-dependent l-leucine uptake in rat brain synaptosomes

Synaptosomes isolated from adult rat cerebral cortices were used for studying the uptake of l-leucine by the Na +-dependent route. Three non-metabolizable amino acid analogues, which had been used previously to discriminate the Na +-dependent A-type uptake system of animal cells, were employed in this study. It was found that Na +-dependent uptake of leucine was insensitive to inhibition by 2-aminoisobutyric acid (AIB) and N-methylaminoisobutyric acid (MeAIB) whereas N-methylalanine (NMA) was markedly inhibitory. Inhibition by NMA was stereospecific—only the l-isomer had a pronounced effect. Na +-dependent uptake of leucine as well as its inhibition by l-NMA were rather insensitive to changes in pH from 6 to 9. Kinetic analysis of inhibition by l-NMA of Na +-dependent uptake revealed a non-competitive type of inhibition with a K i value of approximately 0.5 mM.

Excess enthalpies and apparent molar volumes of some N-methyl substituted amino acids in aqueous solutions

The excess enthalpies and limiting partial molar volumes at 25°C for aqueous solutions of N-methyl glycine, N-methyl alanine, and N-methyl serine are reported and compared with the same properties for the parent amino acids. For each N-methyl derivative the enthalpic contribution to the pairwise interaction is less favorable than that for the parent amino acid. The contribution of the N-methyl substituent to V20 is similar for each amino acid, and is about 2 cm3-mol−1 greater than for a methyl substituent on the α-carbon. These observations have been rationalized in terms of the likely solute-solvent interactions. In addition a rigid particle model is used with the volume data to examine solute-solvent interactions.

The potential of mean force surface for the alanine dipeptide in aqueous solution: a theoretical approach

Results of an application of integral equation theory to the determination of the intramolecular potential of mean force for the alanine dipeptide. N-methyl alanine acetamide, in aqueous solution are presented. The calculations are based on Ornstein—Zernike-like equations for polar systems with an intramolecular superposition approximation. The solvated free energy surface for the dipeptide as a function of the dihedral angles φ and ψ (Ramachandran plot) is determined and compared with the vaccum surface calculations. Conformations that are essentially forbidden in vaccum are found to be significant in aqueous solution. The solvent contributions to the free energy surface are decomposed into enthalpic and entropic terms. Possible applications and extensions of the method are outlined.

THE OCCURRENCE AND OTHER ASPECTS OF N-METHYL ALANINE (NMA) AND N-METHYL SERINE (NMS) INDICHAPETALUM CYMOSUM(ENGL.) (GIFBLAAR) AND OTHER MEMBERS OF THE DICHAPETALACEAE

Summary The isolation and characterization of two novel plant amino acids N-methyl alanine and N-methyl serine are described briefly. It is shown that these amino acids are not toxic and do not form part of Gifblaar protein. The concentration of these amino acids and fluoroacetate was determined in different parts of Gifblaar. In an examination of the amino acid composition of 16 other members of the Dichapetalaceae it was found that N-methyl alanine occurs in six and N-methyl serine in three other species of Dichapetalum.