N-alkylated Amino Acids Research Articles

A Racemization-Free Coupling Method for Peptides Having N-Methylamino Acids at the Carboxy-Termini.

To search for recemization-free coupling conditions for peptides having N-alkylamino acids at the carboxy-termini, a model coupling using Boc-Phe-MeAla-OH (MeAla, N-methylalanine) and H-Phe-OBzl was studied. When benzotriazolyl-N-oxytris(dimethylamino)phosphonium hexafluorophosphate or O-(7-azabenzotriazol-1-yl)-1, 1, 3, 3, -tetramethyluronium hexafluorophosphate was employed as a coupling reagent, no additives so far examined could sufficiently suppress the racemization of the carboxy-terminal MeAla residue. Though N-hydroxy compounds were not satisfactorily effective also in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI)-mediated coupling, CuCl2 could eliminate the racemization of MeAla in the coupling with WSCI even at room temperature.

Equilibrium of thecis-trans isomerisation of the peptide bond with N-alkyl amino acids measured by 2D NMR

The conformational cis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that the cis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing the cis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on the cis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L- or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of the cis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bond undergoing the cis/trans isomerisation moves the equilibrium to a significant amount of the cis form.

Gas-phase fragmentation of protonated mono-N-methylated peptides. Analogy with solution-phase acid-catalyzed hydrolysis

Fragmentation of protonated peptides with a single N-methylated residue was studied by low-energy collision-induced dissociation (CID) and the effects of the N-alkylation on the fragmentation were evaluated. Peptides with an N-terminal N-alkylated amino acid behave similarly to regular peptides except for an increased stability of the protonated molecular ion due to the increased proton affinity. On the other hand, the N-alkylation of an internal amino acid residue has very distinct effect on fragmentation. It causes activation of the amide bond on the C-terminal side of the N-alkylated residue resulting in preferential cleavage of this bond and formation of corresponding b ions. This is in a sharp contrast to the effect of a natural N-alkylated amino acid, proline, which activates its N-terminal amide bond providing abundant y ions upon CID. Both peptides with Ac-N-methylamino acid N-terminus and with an internal N-methylated amino acid exhibit the former type of fragmentation. A fragmentation mechanism is proposed that explains the observed effects based on fragmentation of model peptide analogs and isotopically labeled peptides. It was found that an interaction between carbonyl groups of neighboring amide moieties is an important factor in the formation of the b ion with a structure of a protonated N-methyloxazolone. AM1 calculations suggest that the stability of this ion favors its formation in the CID of N-methylated peptides. It was also shown that an incorporation of a basic site outside the peptide backbone does not affect the course of fragmentation of the peptides with an internal N-alkylamino acid. These observations provide support for the ‘mobile proton’ model for the initial stage of protonated peptide fragmentation in the low-energy CID. The observed phenomenon is analogous to the solution-phase acid-catalyzed hydrolysis of these peptides. © 1998 John Wiley & Sons, Ltd.

PF1022A--a novel anthelmintic cyclooctadepsipeptide. Modification and exchange of the N-methyl leucine residues.

The first structure-activity relationships of the anthelmintic cyclooctadepsipeptide PF1022A have been established via a systematic exchange of the leucine residues by a series of related N-alkylated amino acids. The data presented strongly suggest that (L)-N-methyl-leucine is crucial for high in vivo activity.

Enantiospecific Synthesis of N-(9-Phenylfluoren-9-yl)-α-amino Ketones

Enantiomerically pure N-(9-phenylfluoren-9-yl)-α-amino ketones were prepared in excellent yields by acylation of organolithium reagents with N-(9-phenylfluoren-9-yl)-α-amino acid-derived oxazolidinones. The method is not applicable for the acylation of Grignard reagents as they attack the methylenic carbon of the oxazolidinone to give the corresponding N-alkylated amino acids 13 in excellent yields. The resulting N-(9-phenylfluoren-9-yl)-α-amino ketones 8 could be stereoselectively reduced to the corresponding syn- or anti-β-amino alcohols depending upon the nature of the reducing agent.

Rotational Barriers ofcis/transIsomerization of Proline Analogues and Their Catalysis by Cyclophilin

The rotational barriers for cis/trans isomerization of different proline analogues have been investigated by dynamic 1H NMR spectroscopy. To this end the analogues (S)-azetidine-2-carboxylic acid (Aze), (S)-piperidine-2-carboxylic acid (Pip), (R)-thiazolidine-4-carboxylic acid (4-Thz), (4R)-2-methylthiazolidine-4-carboxylic acid (2Me4-Thz), (R)-thiazolidine-2-carboxylic acid (2-Thz), (S)-oxazolidine-4-carboxylic acid (4-Oxa), (4S,5R)-5-methyloxazolidine-4-carboxylic acid (5Me4-Oxa), and (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (Hyp) and several N-alkylated amino acids were incorporated into the sequences Ala-Yaa-(4-)nitroanilide and Ala-Gly-Yaa-Phe-(4-)nitroanilide. NMR line-shape analyses of various cis and trans proton signals of these peptides were performed at different temperatures, and the rate constants of cis/trans isomerization were fitted to the Eyring equation. The rotational barriers of all cyclic proline analogues except hydroxyproline were found to be lower than that of proline by abou...

Pmc-protected amino acid esters as substrates in N-alkylamino acid synthesis

N-alkylamino acids may be synthesised via Mitsunobu reaction of N-(2,2,5,7,8-pentamethylchroman-6-sulphonyl-)-amino acid esters with various alcohols and subsequent deprotection.

Concerted base-promoted elimination in the decomposition ofN-halo amino acids

N-Chloroamino acids are unstable in aqueous solution and decompose through different pathways depending on the reaction conditions, yielding precursors of carcinogenic and/or mutagenic compounds. One of these pathways is a 1,2-elimination process, which has scarcely received any attention and for which no systematic analysis is available. The process is first order relative to the N-chloroamino acid and to that of hydroxide ion. The use of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoropropan-2-ol buffer solutions established that the process is general-base catalysed. The reaction rate is affected by the presence of a methyl group on the nitrogen atom and the nature of the leaving group. The results show an important steric effect due to the alkyl substituents on the α-carbon. With bulky alkyl substituents on the α-carbon, and in particular in the case of N-alkylamino acids, the catalytic effect increases as the base strength decreases. To characterize the transition state, Brønsted's β and βlg were used. A More O'Ferrall-Jencks diagram shows the transition state structure changing from carbanion-like to nitrenium-like, a large perpendicular effect being evident. The reaction proceeds through a concerted mechanism AxhDHDN instead of the stepwise AxhDH‡ + DN proposed earlier.

A new type of bradykinin B2 receptor antagonists: bradykinin analogs with N-alkyl amino acids at position 2

It is commonly assumed that bradykinin B2 receptor antagonists bind to a receptor site partially different from that for agonists. Thus, it is likely that there exists more than one key modification to convert bradykinin receptor agonists into antagonists. In this respect, [L-NMePhe2]-BK represents the basic structure of a new type of bradykinin B2 receptor antagonists without any replacement at position 7. This compound inhibits both in vitro bradykinin-induced contraction of the guinea pig lung strip and in vivo bradykinin-induced bronchoconstriction. Furthermore, this analog shows analgesic activity, blocks in a dose-dependent manner the bradykinin-induced Ca2+ release from macrophages and inhibits at a concentration of 10−13 M the bradykinin-induced cytokine release from mononuclear cells. Combinations with structural modifications previously performed for other B2 receptor antagonists rather reduce than enhance the potency.

Bradykinin Receptor Antagonists Containing N-Substituted Amino Acids: In Vitro and in Vivo B2 and B1 Receptor Antagonist Activity

We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.

STEREOCHEMISTRY OF COMPLEXES WITHN-ALKYLATED AMINO ACIDS. XI. CRYSTAL STRUCTURE AND MOLECULAR MECHANICS CALCULATIONS ON RACEMIC AQUABIS(L-N,N-DIMETHYLVALINATO)COPPER(II)

Abstract The structure of blue, triclinic (P 1) crystals of a 1:1 mixture of trans-aquabis(L-N,N-dimethylvalinato)copper(II) and its enantiomer was determined by X-ray diffraction methods and refined to R = 0.026. The structure consists of discrete molecules linked with OH–O hydrogen bonds. Coordination around copper(II) is distorted square-pyramidal with valine N and O atoms in the equatorial plane. The O-Cu-OW, O-Cu-N and N-Cu-OW valence angles range from 82.82(7) to 99.11(7)°. Values for O-Cu-O and N-Cu-N are 167.56(8) and 167.59(8)°, respectively. Cu-O [1.932(2), 1.929(2) A] and Cu-N [2.057(2), 2.043(2) A] bond lengths have values similar to those in other copper(II) complexes with N-alkylated amino acids, but the Cu-OW bond length is very short [2.312(2) A]. This was attributed to distortion of the coordination polyhedron, and the apical bond length was quite satisfactorily reproduced with a new theoretical method (N. Raos, J. Mol. Struct., in press.). The shape of the coordination polyhedron was rea...

Chelates of copper(II) with N-alkylated amino acids: distortion of coordination polyhedron and apical bond length

The length of the bond between copper(II) and an apically situated water molecule varies for six N-alkylated α-amino acid chelates with distorted coordination geometry from 2.206 to 2.464 Å. This is attributed to differences in distortion of the copper(II) coordination polyhedron. Apical bond lengths of six distorted and one strictly planar copper(II) complexes (with proline, Cu OH 2 = 2.52 A ̊ ) were correlated with ten measures of distortion (various combinations of valence angles defining the copper coordination geometry, distortion vectors and their components in the (CuOH 2) direction) yielding an r value typically in the range 0.80–0.93; multiple correlation yielded an R value up to 0.96. The ability of molecular mechanics models to reproduce the apical bond length is briefly discussed and an approach based on the use of regression functions for estimation of CuOH 2 bond lengths from the calculated distortion of the coordination polyhedron is proposed.

Analysis of N-alkylated amino acids in human hemoglobin: evidence for elevated N-methylvaline levels in smokers.

To investigate the contribution of cigarette smoking to the levels of N-methylvaline and N-hydroxyethylvaline in hemoglobin we analyzed samples from 32 smokers and 37 nonsmokers. The average background levels of the nonsmoking individuals were determined to be 1175 +/- 176 pmol N-methylvaline/g globin, ranging from 722 to 1516 pmol/g globin, and 46 +/- 12 pmol N-hydroxyethylvaline/g globin, ranging from 19 to 64 pmol/g globin. A significant correlation (P < 0.001) was found between both amino acids and the amount of cigarettes smoked per day, with an increase of 42 pmol N-methylvaline/g globin/cigarette per day and 11 pmol N-hydroxyethylvaline/g globin/cigarette per day. No influence of age, sex, and occupational exposure was observed. Furthermore, the levels of N-hydroxyethylvaline and N-methylvaline correlated for smokers but not for nonsmokers, indicating cigarette smoking as a common source for both adducts. To our knowledge, this is the first time N-methylvaline levels are reported to differ significantly between smokers and nonsmokers in the general population. Especially the analysis of N-methylvaline following low-level exposures to methylating agents should therefore take into consideration the influence of individual smoking habits. Additionally, the results of our study confirm the reliability of N-hydroxyethylvaline as an indicator of individual cigarette consumption. We successfully applied a new calibration technique to the analysis of N-hydroxyethylvaline, introducing a commercially available and well-defined dipeptide standard. The observed levels of N-hydroxyethylvaline in the samples are in line with those reported in the literature and verify the applicability of our calibration method.

Synthesis of dipeptide 4-nitroanilides containing non-proteinogenic amino acids.

A series of tert-butyloxycarbonyl amino acid 4-nitroanilides, including N-alkylated amino acids and (R)-thiazolidine-4-carboxylic acid, (S)-oxazolidine-4-carboxylic acid. (4S,5S)-5-methyloxazolidine-4-carboxylic acid, (4S,5R)-5-methyloxazolidine-4-carboxylic acid, (S)-azetidine-2-carboxylic acid, (S)-pipecolic acid and (S)-3,4-dehydroproline, were prepared conveniently by the isocyanate method or the mixed anhydride procedure. The resulting amino acid 4-nitroanilides were extended to corresponding dipeptide 4-nitroanilides with tert-butyloxycarbonyl-(S)-alanine. In the case of sterically hindered amino acid 4-nitroanilides the mixed anhydride procedure with diphenylphosphinyl chloride was successful.

Novel degradation pathway of glycated amino acids into free fructosamine by a Pseudomonas sp. soil strain extract.

A Pseudomonas sp. soil strain, selected for its ability to grow on epsilon-(1-deoxyfructosyl) aminocaproic acid, was induced to express a membrane-bound enzymatic activity which oxidatively degrades Amadori products into free fructosamine. Apparent Km values for fructosyl aminocaproate, epsilon-fructosyl lysine, fructosyl glycine, and ribated lysine were 0.21 mM, 2.73 mM, 3.52 mM, and 1.57 mM, respectively. The enzyme was also active against alpha-fructosyl lysine and borohydride-reduced Amadori product, weakly active with ribated and glycated polylysine, and inactive with reducing sugars, amino acids, and glycated proteins. The enzymatic activity was highest at pH 6.5 and 25 degrees C in 0.1 M sodium phosphate, while over 80% of the activity was lost above 65 degrees C. Complete inhibition was observed by HgCl2, NaN3, and NaCN suggesting a role for SH groups and copper in the enzymatic activity. The reaction products were characterized by 1H NMR, 13C NMR, and GC/MS and found to correspond to 1-deoxy-1-aminofructose, i.e. free "fructosamine," and adipic acid. Confirmation of the free fructosamine structure was based on the complete spectroscopic identity of the borohydride reduction product with commercially available glucamine (1-amino-1-deoxyglucitol). The new enzyme is provisorily classified as fructosyl N-alkyl amino acid oxidase (EC 1.5.3) (fructosyl-amino acid:oxygen oxidoreductase) and may thus belong to a novel class of "Amadoriases" which deglycate Amadori products oxidatively. In contrast, however, the new enzyme acts on the alkylamine bond rather than the ketoamine bond of the Amadori product.

REVIEW: THE CHEMISTRY OF CHELATES WITH N-ALKYLATED AMINO ACIDS

Abstract N-alkylated α-amino acids are compounds very suitable for studying steric effects in chelates with heavy metals, especially copper(II). These chelates are especially interesting because of pronounced stereoselective effects (enantioselectivity) as well as because of distortion of the copper coordination polyhedron. In this short review all the aspects of coordination chemistry of N-alkylated amino acids are presented and steric effects are discussed from the viewpoint of results obtained from X-ray analyses and molecular mechanics calculations.

Stereochemistry of Complexes with N-Alkylated Amino Acids. V. Crystal Structure, Molecular Mechanics Calculations and Conformational Analysis of Bis (L,N,N-Dimethyl-α-Alaninato) Aqua Copper(II)

Stereochemistry of Complexes with <i>N</i>-Alkylated Amino Acids. V. Crystal Structure, Molecular Mechanics Calculations and Conformational Analysis of Bis (<i>L</i>,<i>N</i>,<i>N</i>-Dimethyl-α-Alaninato) Aqua Copper(II)

Sequence determination of N-terminal and C-terminal blocked peptides containing N-alkylated amino acids and structure determination of these amino acid constituents by using fast-atom-bombardment/tandem mass spectrometry.

Peptides, blocked either at the N or C terminus, and thus unsuited for Edman degradation, and those containing N-alkylated amino acids, which are not detectable when using conventional amino acid analysis, can be easily sequenced by applying a method in which fast atom bombardment (FAB) is combined with tandem mass spectrometry (MSMS). Moreover, the structure of the N-alkylated amino acid constituents is provided by this approach. A widely applicable strategy will be presented, and to demonstrate its scope and limitations eighteen analogues of sequences related to the C terminus of substance P, a biologically active neuropeptide, were investigated. The power and reliability of the approach will be demonstrated by analyzing an 'unknown' peptide. Moreover, the detection and structure elucidation of N-alkylated amino acids which usually escape amino acid analysis will be described, as will be the unequivocal differentiation and identification of isomeric MeLeu/MeIle. The influence of the N-alkylation on the mass spectrometric fragmentation behaviour will be discussed. Furthermore, the sequencing of two adipokinetic hormones by using the combined FAB-MSMS approach is described. Analysis of peptides can be achieved with sample sizes less than 0.1 mumol and be completed within 2-4 h.

Potentiometric study of enantioselective effects in α-amino acid complexes of copper(II)

Using the MINIQUAD-75 program, stability constants of heteroligand (ternary) copper(II) complexes with N-alkylated derivatives of valine and proline or hydroxyproline were evaluated from potentiometric titration data. In the majority of combinations, heteroligand complexes appear to be the favoured species at equilibrium with the two corresponding homoligand complexes. The extent of this disproportion reaction depends strongly on the optical configuration of the constituent amino acid ligands, which implies that enantioselectivity is a common feature in copper complexes with N-alkylamino acids.

Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (1) as a coupling reagent for N-alkyl amino acids

Le reactif du titre permet la formation de liaisons N-alkyl peptidiques avec un minimum de racemisation; exemples de copulations de BocMeX−OH et de MeY−OBzl (X et Y=Leu ou Val)