Phenylalanine Analogues Research Articles

Phenylalanine and Phenylglycine Analogues as Arginine Mimetics in Dengue Protease Inhibitors.

Dengue virus is an increasingly global pathogen. One of the promising targets for antiviral drug discovery against dengue and related flaviviruses such as West Nile virus is the viral serine protease NS2B-NS3. We here report the synthesis and in vitro characterization of potent peptidic inhibitors of dengue virus protease that incorporate phenylalanine and phenylglycine derivatives as arginine-mimicking groups with modulated basicity. The most promising compounds were (4-amidino)-L-phenylalanine-containing inhibitors, which reached nanomolar affinities against dengue virus protease. The type and position of the substituents on the phenylglycine and phenylalanine side chains has a significant effect on the inhibitory activity against dengue virus protease and selectivity against other proteases. In addition, the non-natural, basic amino acids described here may have relevance for the development of other peptidic and peptidomimetic drugs such as inhibitors of the blood clotting cascade.

Exploring the biological consequences of conformational changes in aspartame models containing constrained analogues of phenylalanine

The dipeptide aspartame (Asp-Phe-OMe) is a sweetener widely used in replacement of sucrose by food industry. 2′,6′-Dimethyltyrosine (DMT) and 2′,6′-dimethylphenylalanine (DMP) are two synthetic phenylalanine-constrained analogues, with a limited freedom in χ-space due to the presence of methyl groups in position 2′,6′ of the aromatic ring. These residues have shown to increase the activity of opioid peptides, such as endomorphins improving the binding to the opioid receptors. In this work, DMT and DMP have been synthesized following a diketopiperazine-mediated route and the corresponding aspartame derivatives (Asp-DMT-OMe and Asp-DMP-OMe) have been evaluated in vivo and in silico for their activity as synthetic sweeteners.

The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers.

Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.

Site Selective Azo Coupling for Peptide Cyclization and Affinity Labeling of an SH3 Protein.

A key challenge in bioconjugation is to control the site selectivity of the reaction. Chemical reagents often react with proteineous chemical groups without showing preference to their location or microenvironment in the protein; to confine the reaction to an amino acid at a specific site, one needs to distinguish this residue from others despite their identical chemical properties. Here, we report a strategy that utilizes proximity-driven reactivity to achieve site selective azo coupling between tyrosine and aryldiazonium. A phenylalanine analogue with an aryldiazonium moiety at its side chain was incorporated into a synthetic peptide and was found to react only with tyrosine in its vicinity but also to remain inert to others that are not immediately adjacent, a property attained by fine regulation of the electronic effect of the substituent on the aryl ring. Proximity-driven intramolecular azo coupling was showcased in cyclization of a β-hairpin peptide, structural features of the azo linked cyclic peptide was elucidated by NMR, and intermolecular azo coupling was achieved between an SH3 protein Abl-SH3 and its polyproline peptide ligands at specific tyrosine residues. This approach is generally applicable to develop covalent affinity labels for SH3 proteins because of the high occurrence rate of tyrosine at the peptide-binding site of SH3 proteins.

Structures and Energetics of Protonated Clusters of Methylamine with Phenylalanine Analogs, Characterized by Infrared Multiple Photon Dissociation Spectroscopy and Electronic Structure Calculations.

Gas-phase clusters of protonated methylamine and phenylalanine (Phe) derivatives have been studied using infrared multiple photon dissociation (IRMPD) spectroscopy in combination with electronic structure calculations at the MP2/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. Experiments were performed on several Phe derivatives including 4-chloro-l-phenylalanine (4Chloro-Phe), 4-nitro-l-phenylalanine (4Nitro-Phe), 3-cyano-l-phenylalanine (3Cyano-Phe), and 3-trifluoromethyl-l-phenylalanine (3CF3-Phe). Through comparisons between experimental IRMPD spectra and stimulated spectra obtained by electronic structure calculations, charge-solvated structures were found to be prevalent in both 4Chloro-Phe and 4Nitro-Phe, whereas 3Cyano-Phe favored zwitterionic structures and 3-CF3-Phe likely have both zwitterionic and charge-solvated structures present.

Isolation and Synthetic Diversification of Jadomycin 4-Amino-l-phenylalanine.

Streptomyces venezuelae ISP5230 was grown in the presence of phenylalanine analogues to observe whether they could be incorporated into novel jadomycin structures. It was found that the bacteria successfully produced jadomycins incorporating 4-aminophenylalanine enantiomers. Upon isolation and characterization of jadomycin 4-amino-l-phenylalanine (1), it was synthetically derivatized, using activated succinimidyl esters, to yield a small jadomycin amide library. These are the first examples of oxazolone-ring-containing jadomycins that have incorporated an amino functionality subsequently used for derivatization.

Asymmetric synthesis of 3-azide-4-fluoro-l-phenylalanine.

The asymmetric synthesis of N-Fmoc-protected 3-azide-4-fluoro-l-phenylalanine as a photoactive phenylalanine analog has been achieved by Schöllkopf's alkylation.

Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2–P4 study

Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 μM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.

Tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of SARS-CoV spike protein.

The spike (S) protein of severe acute respiratory syndrome-associated CoV (SARS-CoV) mediates membrane fusion and viral entry. These events involve structural rearrangements, including heteromerization between two heptad repeats (HR1 and HR2) to form a trimer of dimers as a six-helix bundle (6-HB), a quaternary protein structure that brings two distant clusters of hydrophobic sequences into the proximity of each other, the internal fusion peptide (IFP) preceding HR1, and the highly conserved tryptophan (Trp)-rich membrane proximal external region (MPER) following HR2. Here, we show that MPER can undergo self-oligomerization and heteromerization with IFP, events that are Trp-dependent. To delineate the roles of Trp residues of MPER in forming these quaternary structures and interacting with membranes, we employed a panel of synthetic peptides: MPER peptide (M-wt) and its alanine (Ala) and phenylalanine (Phe) analogues. Ala substitutions of Trp inhibited its association with cellular membranes. Chemical cross-linking experiments showed that M-wt can self-interact to form oligomers and cross-interact with IFP23, a synthetic IFP peptide, to form a heterohexamer. In comparison, little high-order oligomer was formed between M-wt and fusion peptide. The specific interaction between M-wt and IFP23 was confirmed by immunofluorescence staining experiments. In aqueous solutions, both M-wt and IFP23 displayed random secondary structures that became helical in hydrophobic solvents. Triple-Ala substitutions of Trp in M-wt, but not the corresponding triple-Phe analogue, disrupted oligomerization of M-wt and hetero-oligomerization of M-wt with IFP23. Overall, our results show that Trp residues of MPER play a key role in maintaining the structure and functions of MPER, allowing it to interact with IFP to form a MPER-IFP heteromer, a putative quaternary structure extending from the 6-HB, and function in membrane fusion. Finally, we showed that a MPER peptide could serve as an inhibitor in the entry process.

Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris.

The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs - benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid - were determined by the molecular replacement method at resolutions of 1.57 Å and 1.62 Å to clarify the broad substrate specificity profile of the enzyme. The conservative Leu211 and Leu254 residues (also present in both carboxypeptidase A and carboxypeptidase B) were shown to be structural determinants for recognition of hydrophobic substrates, whereas Asp263 was for recognition of positively charged substrates. Mutations of these determinants modify the substrate profile: the CpT variant Leu211Gln acquires carboxypeptidase B-like properties, and the CpT variant Asp263Asn the carboxypeptidase A-like selectivity. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 was shown to be responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S1' subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis. This is a novel insight into the substrate selectivity of metallocarboxypeptidases that demonstrates the importance of interactions between the S1' subsite and the catalytic center.

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

Inhibitory potencies of 24 α-aminophosphonic acids against barley seeds (Hordeum vulgare L.) metallo-aminopeptidase have been determined to evaluate structural requirements of this enzyme. The enzyme was sensitive mostly to the influence of phosphonic acid analogues of phenylalanine and its homologues, thus showing narrow specificity if compared with porcine aminopeptidases M1 and M17 and with Plasmodium aminopeptidase M17.

Aromaticity and amyloid formation: Effect of π-electron distribution and aryl substituent geometry on the self-assembly of peptides derived from hIAPP22–29

A comprehensive investigation of peptides derived from the 22–29 region of human islet amyloid polypeptide (hIAPP) that contain phenylalanine analogs at position 23 with a variety of electron donating and withdrawing groups, along with heteroaromatic surrogates, has been employed to interrogate how π-electron distribution effects amyloid formation. Kinetic aggregation studies using turbidity measurements indicate that electron rich aromatic ring systems consistently abolish the amyloidogenic propensity of hIAPP22–29. Electron poor systems modulate the rate of aggregation. Raman and Fourier transform infrared spectroscopy confirm the parallel β-sheet secondary structure of aggregates derived from peptides containing electron poor phenylalanine analogs and provide direct evidence of ring stacking. Transmission electron microscopy confirms the presence of amyloid fibrils. The effect of aryl substituent geometry on peptide self-assembly reveals that the electronic nature of substituents and not their steric profile is responsible for failure of the electron donating group peptides to aggregate. Non-aggregating hIAPP22–29 peptides were found to inhibit the self-assembly of full-length hIAPP1–37. The most potent inhibitory peptides contain phenylalanine with the p-amino and p-formamido functionalities. These novel peptides may serve as leads for the development of future aggregation inhibitors. A potential mechanism for inhibition of amylin self-assembly by electron rich hIAPP22–29 peptides is proposed.

Tailoring the substrate specificity of yeast phenylalanyl-tRNA synthetase toward a phenylalanine analog using multiple-site-specific incorporation.

A yeast phenylalanyl-tRNA synthetase variant with T415G mutation (yPheRS (T415G)) was rationally designed to recognize various phenylalanine (Phe) analogs allowing site-specific incorporation into an amber site of a protein in E. coli. However, the relaxed substrate specificity of yPheRS (T415G) led to a significant tryptophan (Trp) misincorporation restricting the utility of yPheRS for biosynthesis of proteins containing a Phe analog. In order to obtain yPheRS variants with high substrate-specificity toward a Phe analog, we developed a general high-throughput screening method. This method uses fluorescence reduction of green fluorescence protein (GFP) upon efficient introduction of a Phe analog into multiple sites of GFP by breaking the degeneracy of the Phe codons. Combined use of positive and negative screenings of a yPheRS saturation library led to a yPheRS variant (yPheRS_naph) very selective toward 2-l-naphthylalanine (2Nal), a model Phe analog. The yPheRS_naph exhibited 6-fold higher relative activity toward 2Nal (vs Trp) in ATP-PPi exchange assays and led to high-fidelity incorporation of 2Nal into an amber site of murine dihydrofolate reductase in both minimal and rich media. These results successfully demonstrate that the high-throughput screening method developed can be used to evolve yPheRS to be very selective toward a Phe analog.

ChemInform Abstract: Synthesis of Novel Fluoranthene‐Based Conformationally Constrained α‐Amino Acid Derivatives and Polycyclic Aromatics via the Diels—Alder Reaction.

Conformationally constrained cyclic α-amino acid moieties have been fused to the fluoranthene system. Aminoindanecarboxylic acid (Aic) and 1,2,3,4-tetrahydroisoquinolinecarboxylic acid (Tic) derivatives were synthesized by an alkylation sequence, whereas a aminotetralincarboxylic acid (Atc) derivative was assembled using the Diels–Alder reaction as a key step. These α-amino acid derivatives are considered as constrained analogues of phenylalanine (Phe) and play an important role in the design and synthesis of bioactive peptides and some fluorescence chemosensor molecules. Moreover, this strategy has been extended to polycyclic aromatics via the Diels–Alder reaction and subsequent aromatization with DDQ.

Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnatural phenylalanine analogues

CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the ‘Phe43 cavity’ of HIV-1 gp120. The results show that the conserved hydrophobic pocket in gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2Å in length and 8.0Å in width. This result provides useful information for developing novel gp120 inhibitors or new HIV detection tools.

UV–UV hole burning and IR dip spectroscopy of homophenylalanine by laser desorption supersonic jet technique

Conformer selected electronic and vibrational spectra of homophenylalanine, phenylalanine analogue molecule, were measured by UV–UV hole burning and IR dip spectroscopy combined with laser desorption technique. 10 conformers were found by UV–UV hole burning spectroscopy and their structures were assigned by IR dip and UV absorption spectra with aid of quantum chemical calculations in both S0 and S1. This study shows that the combination of simulated IR and UV spectra is powerful to assign flexible molecules.

Novel series of phenylalanine analogs endowed with promising anti-inflammatory activity: synthesis, pharmacological evaluation, and computational insights

A novel series of methyl-2-(substituted benzylideneamino)-3-phenyl propionate (2a–j) derivatives have been synthesized. The title compounds (2a–j) were screened for in vivo acute anti-inflammatory and analgesic activities at a dose of 200 mg/kg b.w. Compound 2e exhibited the most promising and significant anti-inflammatory profile while compounds 2b, 2h, 2i, and 2j showed moderate to good inhibitory activity at 2nd and 4th h, respectively. These compounds were also found to have considerable analgesic activity (acetic acid-induced writhing model) and antipyretic activity (yeast-induced pyrexia model). In addition, the tested compounds were also found to possess less degree of ulcerogenic potential as compared to the standard NSAIDs. The synthesized compounds were further evaluated for their inhibitory activity against cyclooxygenase enzyme (COX-1/COX-2), by in vitro colorimetric COX (ovine) inhibitor screening assay method. The results revealed that the compounds 2b, 2e, 2h, 2i, and 2j exhibited selective and effective inhibition against COX-2. In an attempt to understand the ligand–protein interactions in terms of their binding affinity, docking studies were also performed using Molegro Virtual Docker (MVD-2013, 6.0) for the title compounds. It was observed that the binding affinities calculated were in agreement with the experimental IC50 values.

Oxovanadium(IV) Schiff Base Complex Derived From Phenylalanine Analogue Containing 2,3-Diaminopropionic Acid (DAP): Synthesis, Computational Study, and Biological Evaluation

Reduced Schiff base ligand by condensation of phenylalanine analogue containing 2,3-diaminopropionic acid (DAP) with salicylaldehyde and its oxovanadium(IV) complex have been prepared. Chemical and structural properties were fully characterized. Density functional theory was used to pin down the structure combined with IR. The natural bond orbital analysis and energy calculations indicate that tetracoordinated complex of R-type is the most probable structure where vanadium atom is coordinated to 2 × Ophenolate and 2 × Nimine of the ligand. By biological evaluation, the metal complex exhibits antibacterial activity against four bacterial strains and antitumor activity against two tumor cell lines.

Residue-specific incorporation of phenylalanine analogues into protein biosynthesis in silkworm cultured cells

Residue-specific incorporation of phenylalanine analogues into protein biosynthesis in silkworm cultured cells

Biosynthesis of 2-phenylethanol from glucose with genetically engineered Kluyveromyces marxianus

2-Phenylethanol (2-PE) is an aromatic alcohol with a rose scent, which is used in the cosmetics, fragrance and food industries. 2-PE is produced in a few yeast strains by Ehrlich pathway. In this study, Kluyveromyces marxianus was genetically engineered for overproduction of 2-PE from glucose. About 1.0g/L of 2-PE was produced by overexpressing phenylpyruvate decarboxylase (ARO10) and alcohol dehydrogenase (ADH2) genes of Saccharomyces cerevisiae. A similar level of 2-PE was also produced from evolved K. marxianus, which was resistant to the phenylalanine analog, p-fluorophenylalanine. aroGfbr from Klebsiella pneumoniae encoding a feedback resistant mutant of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DHAP) synthase was overexpressed in the evolved K. marxianus. Finally, 1.3g/L of 2-PE was produced from 20g/L glucose without addition of phenylalanine in the medium.