N-substituted Amino Acids Research Articles

N-substituting perturbation on the interaction affinity and recognition specificity between rheumatic immune-related Abl SH3 domain and its peptoid ligands

Abl is a nonreceptor tyrosine kinase involved in a variety of disease pathways such as rheumatic immune. Full-length Abl protein consists of a catalytic tyrosine kinase (TK) domain as well as two regulatory Src homology domains 2 and 3 (SH2 and SH3, respectively); the latter recognizes and binds to those natural proline-rich peptide segments containing a PxxP motif on the protein surface of its interacting partners. However, natural peptides cannot bind effectively to the modular domain in high affinity and strong selectivity due to their small size and broad specificity. Here, a synthetic proline-rich peptide p41 was used as template; its structural diversity was extended by combinationally replacing the Pro0 and Pro+3 residues with a number of N-substituted amino acids. Consequently, peptide affinity change upon the replacement was derived to create a systematic N-substituting perturbation profile, from which we identified several N-substitution combinations at the Pro0 and Pro+3 residues of p41 PxxP motif that may moderately or significantly improve the peptide binding potency to Abl; they represent potent peptoid binders of Abl SH3 domain, with affinity improved considerably relative to p41. More significantly, the designed potent peptoids were also found to exhibit a good SH3-selectivity for their cognate Abl over other noncognate nonreceptor tyrosine kinases, with S = 9.7-fold.

Computational Analysis and Rational Design of N-Substituting Perturbation on the Affinity and Specificity of Pediatric Fyn SH3–Peptoid Interaction

Human nonreceptor tyrosine kinase (TK) Fyn is implicated in various cellular processes and has been exploited as a sophisticated druggable target of pediatric T-cell lymphoma by blocking its kinase domain with small-molecule inhibitors or disrupting its regulatory SH2 and SH3 domains with peptidic inhibitors. In this study, the proline-heavy 9P1Y-peptide was found as a good binder of Fyn SH3 domain, of which the chemical diversity space was extended by replacing its proline residues with a variety of N-substituted amino acids, since N-substitution can mimic the side-chain location and shape of proline. A systematic single-point N-substituting perturbation profile (SSNPP) for 9P1Y-peptide was created theoretically, from which a systematic combinatorial peptoid library (SCPL) was then generated by introducing favorable N-substituted to different proline residue positions of the peptide. The computational design was further substantiated by fluorescence spectroscopy assays to identify five promising peptoid hits in the SCPL as potential Fyn SH3 binders. In particular, the designed peptoid Ptoid2 exhibited a good affinity to Fyn SH3 domain ([Formula: see text]M) and a moderate selectivity for Fyn over Lyn (3.8-fold), which are improved substantially from the natural 9P1Y-peptide. Molecular mechanism underlying the N-substituting perturbation effect was also examined in detail.

Pore size control of monodisperse mesoporous silica particles with alkyl imidazole ionic liquid templates for high performance liquid chromatography applications

The control of pore size in mesoporous silica particles is still a topic of considerable research attention for the preparation of chromatographic packing materials. In this study, three alkyl imidazole ionic liquid surfactants ([BRIJOn-MIM]Ts (n = 2, 10, 20)) were synthesized from polyoxyethylene oleyl ether (BRIJ™On, n = 2, 10, 20), 1-methylimidazole, and p-toluenesulfonyl chloride. [BRIJOn-MIM]Ts (n = 2, 10, 20) exhibited different template behaviors in the preparation of mesoporous silica, and hollow silica with worm-like vesicle structures, spherical silica, and gel were obtained, respectively. Monodisperse sub-3 µm core-shell silica particles (CSSPs) with narrow pore size distribution were successfully prepared by the ultrasound-assisted sol-gel method using [BRIJO10-MIM]Ts as the template. A shell thickness of up to 300 nm was obtained, and the pore size was expanded from 4.7 to 7.3 nm by adjusting the reaction parameters, overcoming the limitation of pore size to less than 4 nm when using organic alkyl quaternary ammonium or alkyl imidazolium surfactants as the templates. The synthesized 2.7 µm CSSPs were functionalized with tert-butylcarbamoylated quinine and used as an anion exchange chiral stationary phase, exhibiting excellent separation performance for N-substituted amino acid enantiomers.

Novel N-Substituted Amino Acid Hydrazone-Isatin Derivatives: Synthesis, Antioxidant Activity, and Anticancer Activity in 2D and 3D Models In Vitro

A series of novel mono and bishydrazones each bearing a 2-oxindole moiety along with substituted phenylaminopropanamide, pyrrolidin-2-one, benzimidazole, diphenylmethane, or diphenylamine fragments were synthesized, and their anticancer activities were tested by MTT assay against human melanoma A375 and colon adenocarcinoma HT-29 cell lines. In general, the synthesized compounds were more cytotoxic against HT-29 than A375. 3-((4-Methoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-N′-(2-oxoindolin-3-ylidene)propanehydrazide and (N′,N‴)-1,1′-(methylenebis(4,1-phenylene))bis(5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide) were identified as the most active compounds against HT-29 in 2D and 3D cell cultures. The same compounds showed the highest antioxidant activity among the synthesized compounds screened by ferric reducing antioxidant power assay (FRAP). Their antioxidant activity is on par with that of a well-known antioxidant ascorbic acid.

Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices.

As the only ribosomally encoded N-substituted amino acid, proline promotes distinct secondary protein structures. The high proline content in collagen, the most abundant protein in the human body, is crucial to forming its hallmark structure: the triple-helix. For over five decades, proline has been considered compulsory for synthetic designs aimed at recapitulating collagen's structure and properties. Here we describe that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, enabling synthesis of stable triple-helical collagen mimetic peptides (CMPs) with unprecedented side chain diversity. Supported by atomic-resolution crystal structures as well as circular dichroism and computational characterizations spanning over 30 N-gly-containing CMPs, we discovered that N-glys stabilize the triple-helix primarily by sterically preorganizing individual chains into the polyproline-II helix. We demonstrated that N-glys with exotic side chains including a "click"-able alkyne and a photosensitive side chain enable CMPs for functional applications including the spatiotemporal control of cell adhesion and migration. The structural principles uncovered in this study open up opportunities for a new generation of collagen-mimetic therapeutics and materials.

Identification of Substituted Amino Acid Hydrazides as Novel Anti-Tubercular Agents, Using a Scaffold Hopping Approach.

Discovery and development of new therapeutic options for the treatment of Mycobacterium tuberculosis (Mtb) infection, particularly drug-resistant strains, are urgently required to tackle the global burden of this disease. Herein, we reported the synthesis of a novel series of N-substituted amino acid hydrazides, utilising a scaffold hopping approach within a library of anti-tubercular agents. Efficacy and selectivity were evaluated against three strains of Mtb (wild-type, isoniazid-resistant and rifampicin-resistant), and cytotoxicity against macrophages in vitro. The antibacterial activity and therapeutic index of these molecules were significantly affected by modifications with the N-substituents. Introduction of a 3,5-dinitroaryl moiety demonstrated enhanced antibacterial activity against all three strains of Mtb. In contrast, the inclusion of an imidazo [1,2-a]pyridine-3-carboxy moiety resulted in enhanced activity towards isoniazid mono-resistant Mtb relative to wild-type Mtb. Consequently, this scaffold hopping approach showed significant promise for exemplification of novel molecules with specific activity profiles against drug-resistant tuberculosis.

Diverse N-Substituted Azole-Containing Amino Acids as Building Blocks for Cyclopeptides.

The preparation of 16 oxazole- or thiazole-containing amino esters bearing a wide array of N-substitution is reported. These were accessed in 40–92% yield via an AgClO4-promoted substitution reaction between a primary amine and a chloromethyl-functionalized thiazole or oxazole. These new synthetic building blocks will be useful for the preparation of new cyclopeptide analogues bearing heterocyclic backbone modifications. Four macrocyclic N-substituted oligoamides that include thiazole or oxazole heterocycles were obtained, following cyclooligomerization reactions of azole-modified N-substituted amino acids.

Synthesis and Pancreatic Lipase Inhibitory Activity of Phenacyl Esters of N-Aroyl Amino Acids

Background:Obesity has become a major life-threatening disorder as it is associated with many risk factors such as coronary heart disease, hypertension, type II diabetes and certain forms of cancer. Pancreatic lipase inhibition is one of the alternate strategies to treat obesity. In our previous study, phenacyl ester of N-sulphonyl beta alanine was obtained as a hit molecule when pharmacophore- based virtual screening was carried out using reported pancreatic lipase inhibitors.Objective:In search of novel pancreatic lipase inhibitors, three series of phenacyl esters of Nsubstituted amino acids were synthesized and evaluated for their pancreatic lipase inhibitory activity.Methods:N-aroylation of amino acids and subsequent esterification with different phenacyl bromides in the presence of N,N-Dimethylformamide (DMF) afforded the corresponding phenacyl esters in quantitative yields.Results:The synthesized compounds were found to have good pancreatic lipase inhibitory activity and showed IC50 in the range of 0.036 and 84 μg/ml.Conclusion:All the synthesized compounds showed better pancreatic lipase inhibitory activity and it indicates that the N-aroylated phenacyl esters could be considered as lead moiety for further studies to develop novel anti-obesity potentials.

Molecular design and optimization of hepatic cancer SLP76-derived PLCγ1 SH3-binding peptide with the systematic N-substitution of peptide PXXP motif.

The phospholipase Cγ1 (PLCγ1) is essential for T-cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP-containing decapeptide segment (185 QPPVPPQRPM194 , termed as SLP76185-194 peptide) of adaptor protein SLP76 following T-cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76185-194 PXXP motif with nonnatural N-substituted amino acids, as the proline is the only endogenous N-substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate-sized N-substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N-substituted peptides as well as native SLP76185-194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence-based assays. Two N-substituted peptides, SLP76185-194 (N-Leu187/N-Gln190) and SLP76185-194 (N-Thr187/N-Gln190), are designed to have high potency (Kd =0.67±0.18 and 1.7±0.3μM, respectively), with affinity improvement by, respectively, 8.5-fold and 3.4-fold relative to native peptide (Kd =5.7±1.2μM).

Exploring the systematic effect of N-substituted PxxP motifs on peptoid affinity to ARHGEF5/TIM SH3 domain and its relationship with ARHGEF5/TIM activation.

The TIM protein is a short isoform of full-length Rho guanine nucleotide exchange factor 5 (ARHGEF5), which acts as a functional regulator of Rho-dependent signaling pathways by activating the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a proline-rich region 47 SSPRQPRKAL56 (termed as SSP peptide) between the putative helix and the DH domain. Previously, we demonstrate that the auto-inhibitory state of TIM protein can be relieved by targeting its SH3 domain with rationally designed peptide ligands. However, the designed natural peptides have only a moderately increased affinity (~2-fold) as compared to the cognate SH3-SSP interaction and are susceptible to protease degradation. Here, considering that proline is the only endogenous N-substituted amino acid that plays a critical role in SH3-peptide recognition, the two key proline residues Pro49 and Pro52 in the core 49 PxxP52 motif of SSP peptide are systematically replaced by 19 N-substituted amino acid types to derive a variety of nonnatural peptoid ligands for TIM SH3 domain. Dynamics and energetics analyses reveal that the replacement would impair the active polyproline II (PPII) helical conformation of SSP peptide due to lack of structural constraint introduced by the five-membered ring of native proline side-chains, thus increasing the peptide flexibility that could incur a large entropy penalty upon binding to the domain. However, the impairment is not very significant and the peptide affinity may also be restored and improved if the N-substituted motif of derived peptiod ligands can effectively interact with the PxxP-binding site of TIM SH3 domain. Consequently, a number of potent peptoids are successfully designed by fluorescence spectroscopy confirmation, in which three (ie, SSP[N-Ile49, N-Asn52], SSP[N-Phe49, N-Gln52], and SSP[N-Tyr49, N-Asn52]) exhibit considerably increased affinity (Kd = 0.09, 0.07, and 0.04 μM, respectively) relative to the native SSP peptide (Kd = 0.87 μM). In addition, guanine nucleotide exchange assays also substantiate that the designed SH3-targeted peptiods can effectively enhance TIM-catalyzed RhoA exchange activity (EA), which is observed to present an exponential relationship with the measured SH3-peptoid binding affinity (pKd ).

N-Substituted amino acid inhibitors of the phosphatase domain of the soluble epoxide hydrolase

The soluble epoxide hydrolase (sEH) is a bifunctional enzyme implicated in the regulation of inflammation. The N-terminal domain harbors a phosphatase activity (N-phos) with an affinity to lipid phosphomonoesters, and the C-terminal domain has an activity to hydrolyze anti-inflammatory lipid epoxides (C-EH). Although many potent inhibitors of C-EH have been discovered, little is known about inhibitors of N-phos. Here, we identify N-substituted amino acids as selective inhibitors of N-phos. Many of the N-substituted amino acids inhibited differently mouse and human N-phos; phenylalanine derivatives are relatively selective for mouse N-phos, whereas tyrosine derivatives are more selective for human N-phos. The best inhibitors, Fmoc-l-Phe(4-CN) (67) and Boc-l-Tyr(Bzl) (23), inhibited mouse and human N-phos competitively with KI in the low micromolar range. These compounds inhibit the N-phos activity 37- (67) and 137-folds (23) more potently than the C-EH. The differences in inhibitor structure activity suggest different active site structure between species, and thus, probably a divergent substrate preference between mouse and human N-phos.

Synthesis of Novel Exocyclic Allenes with a Norbornene Fragment

N-Substituted amino acids with a norbornene fragment were synthesized by the direct fusion of amino acids with endic anhydride at 150°C. The synthesized compounds were used for one-pot synthesis of novel stable allenes with an exocyclic cumulene group derived from l-phenyl-3-(triphenylphosphoranylidene)-pyrrolidine-2,5-dione has been carried out.

Synthesis of Phthaloylglycyl Hydrazide Derivatives: Selective Protection of Phthalimide Group from Hydrazinolysis

Background: N-phthalimide amino acid hydrazide is a class of compounds that have the potential therapeutic use. In general, hydrazinolysis of N-substituted amino acid(s) ester removes the ester group and yields the corresponding hydrazide. However, in case if N-substitution group is phthalimide, phthalimide group is cleaved and not the ester group. The resulted compound, therefore, is amino acid ester rather than Nphthalimide amino acid hydrazide. The above class of compounds, because of susceptibility of phthalimide group to hydrazinolysis, has previously been synthesized by a lengthy three-step procedure. Objective: N-phthaloylglycyl hydrazide was synthesized by using new efficient, simplified, one step process. Hydrazone derivatives from substituted benzaldehydes, and substituted furaldehyde were also synthesized. Method: N-phthaloylglycyl hydrazide was synthesized from the corresponding carboxylic acid using 1-Ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC) as a coupling agent and hydroxybenzotriazole (HOBt) as an activator. Hydrazone derivatives were synthesized by condensation of N-phthaloylglycyl hydrazide with substituted benzaldehyde/substituted furaldehydes. All the compounds were characterized by IR, 1H-NMR, 13CNMR, mass spectroscopy and elemental analysis. Results: The presence of EDC/HOBt resulted in hydrazinolysis of the carboxylic acid group and not the phthalimide group. N-phthaloylglycyl hydrazide was synthesized in good yield. Conclusion: We report the improved process of the synthesis of N-phthaloylglycyl hydrazide. This is the first report where stability of phthaloyl amino acid compound to hydrazine is demonstrated. The reaction may be explored for the reaction schemes where stability of phthalimide group to hydrazinolysis is required.

Solid-Phase Synthesis of Oligopeptides Containing Sterically Hindered Amino Acids on Nonswellable Resin Using 3-Nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium Hexafluorophosphate (PyNTP) as the Condensing Reagent.

Peptides are still difficult to synthesize when they contain sterically hindered amino acids, such as α,α-disubstituted amino acids and N-substituted amino acids. In this study, solid-phase syntheses of oligopeptides containing multiple α-aminoisobutyric acid (Aib) residues were performed in high yields by using a nonswellable resin as the solid-support and 3-nitro-1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyNTP) as the condensing reagent.

QUANTUM-CHEMICAL CALCULATION METHODS TO DETERMINE THE IONIZATION CONSTANTS OF N-SUBSTITUTED AMINO ACID DERIVATIVES

A theoretical study was conducted to determine the ionization constants of N-substituted amino acid derivatives using HF, DFT, and MP2 calculation methods. The extent of compatibility of these methods was determined by discussing the theoretical variables calculated in the three methods mentioned above, the relationship between the calculated physical variables have been found to be theoretical and to determine their nature. These variables were then correlated with the known chemical values of amino acids as pK a ionization parameters. The results obtained by this relationship were found to be good. This is indicated by the results of the statistical analysis across the correlation coefficient values. The theory that gave the best agreement between the values of the theoretical and the experimental ionization parameters were the MP2 method with good correlation coefficient (0.997) and standard error (0.162). As well as the large overlap between pK a values calculated theoretically with practical values where the difference (0.008) gives the opportunity to apply these variables in other studies.

The quantitative “structure – antibacterial activity” relationships in a series of N-substituted amino acids

The presence of the antibacterial and antifungal activity in derivatives of amino acids is determined by different mechanisms; therefore, a promising field of research is to obtain new N-substituted amino acids and study their antibacterial action. Aim. To determine the quantitative “structure – antibacterial action” relationships in a series of N-substituted amino acids. Materials and methods. The quantitative dependencies of the antibacterial action of the compounds studied on AlogPs values were calculated using the STATISTIKA 8 program. Results and discussion. The satisfactory values of the levels of correlation of AlogPs parameters calculated with the experimental data of the antibacterial activity of N-substituted amino acids against S. aureus, E. coli, P. vulgaris, P. aeruginosa, B. subtilis, and Cl. perfringens are statistically significant. The absence of the relationship between the antibacterial effect against C. albicans and the structure of threonine derivatives may indicate a possible role of the latter in the metabolism of these fungi. Conclusions. The statistically significant correlation values of AlogPs with the values of the antibacterial action of N-substituted amino acids against the microorganisms studied have been determined, and it quantitatively confirms the earlier assumptions of the existence of the “structure – action” relationship in this series of compounds and the degree of its manifestation.

Facile radiosynthesis of 18F-labeled β-glutamic acid as a new PET tracer for imaging lung cancer

In this paper, N-(2-[18F]fluoropropionyl)-β-glutamic acid 8 ([18F]FP-β-Glu), a new N-substituted 18F-labeled amino acid tracer, was synthesized from the precursor 4 (diethyl 3-(2-bromopropanamido)pentanedioate) via a two-step reaction on the modified FDG synthesizer. The radiochemical yield was 20 ± 5% (n = 10, decay-corrected) from [18F]fluoride within 40 min, the radiochemical purity was 98%. Moreover, microPET studies showed that [18F]FP-β-Glu 8 exhibited rapid tumor uptake and good tumor-to-lung ratio in SPC-A-1 tumor-bearing mice. A high accumulation of radioactivity was found in the kidneys and bladder, which suggested that the tracer was mainly eliminated through the urinary system.

One-Pot Assembly of Amino Acid Bridged Hybrid Macromulticyclic Cages through Multiple Multicomponent Macrocyclizations.

An important development in the field of macrocyclization strategies towards molecular cages is described. The approach comprises the utilization of a double Ugi four-component macrocyclization for the assembly of macromulticycles with up to four different tethers, that is, hybrid cages. The innovation of this method rests on setting up the macromulticycle connectivities not through the tethers but through the bridgeheads, which in this case involve N-substituted amino acids. Both dilution and metal-template-driven macrocyclization conditions were implemented with success, enabling the one-pot formation of cryptands and cages including steroidal, polyether, heterocyclic, peptidic, and aryl tethers. This method demonstrates substantial complexity-generating character and is suitable for applications in molecular recognition and catalysis.

Variation of the Molecular Conformation, Shape, and Cavity Size in Dinuclear Metalla-Macrocycles Containing Hetero-Ditopic Dithiocarbamate-Carboxylate Ligands from a Homologous Series of N-Substituted Amino Acids.

A homologous series of dithiocarbamate ligands derived from N-substituted amino acids was reacted with different diorganotin dichlorides to give 18 diorganotin complexes. Spectroscopic and mass spectrometric analysis evidenced the formation of assemblies with six-coordinate tin atoms embedded in skewed-trapezoidal bipyramidal coordination environments of composition C2SnS2O2. Single-crystal X-ray diffraction analysis for three of the compounds revealed a one-dimensional polymeric structure for the complex with the ligand derived from 5-aminopentanoic acid, which through further intermolecular Sn···O interactions generated an overall two-dimensional coordination polymer containing 40-membered hexanuclear tin macrocycles. On the contrary, the ligands derived from 6-aminohexanoic and 8-aminooctanoic acid provided the expected 22- and 26-membered dinuclear macrocyclic structures. Density functional theory calculations for a representative series of macrocyclic complexes of composition [Me2SnLx]2 with Lx = ¯S2CN(Me)-(CH2)x-COO¯ (x = 3-12) enabled a detailed analysis of the variations in the molecular conformation, shape, and cavity size of the macrocycles in dependence of the aliphatic spacer. Because of odd-even effects, the difunctional ligands can adopt either a curved or a twisted-pincer shape, while the SnSxO4-x (x = 0-4) moieties can act either as linear or angular tectons with varying connectivity angles.

N-Substituted amino acids as anticorrosion additives. Part III: N-Acyl amino acids

N-Substituted amino acids as anticorrosion additives. Part III: N-Acyl amino acids