Phenylalanine Analogues Research Articles

Positional effects of monofluorinated phenylalanines on histone acetyltransferase stability and activity

To explore the impact of global incorporation of fluorinated aromatic amino acids on protein function, we investigated the effects of three monofluorinated phenylalanine analogs para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the stability and enzymatic activity of the histone acetyltransferase (HAT), tGCN5. We selected this set of fluorinated amino acids because they bear the same size and overall polarity but alter in side chain shape and dipole direction. Our experiments showed that among three fluorinated amino acids, the global incorporation of pFF affords the smallest perturbation to the structure and function of tGCN5.

Positional Effects of Monofluorinated Phenylalanines on Histone Acetyltransferase on Stability, Activity and Specificity

To explore the impact of globally incorporated fluorinated aromatic analogs on functional proteins, we investigate the effects of the monofluorinated phenylalanine analogs para‐fluorophenylalanine (pFF), meta‐fluorophenylalanine (mFF), and ortho‐fluorophenylalanine (oFF) on the stability, activity and specificity of the histone acetyltransferase (HAT) protein, tGCN5. We selected this set of fluorinated amino acids because each analog bears the same overall polarity and size while altering the direction of the dipole and side chain shape. Our experiments demonstrate a positional effect of single fluorine substitution on tGCN5 in which pFF provides minimal perturbance to structure and activity followed by mFF and oFF. Surprisingly, all monofluorophenylalanines lead to an enhanced selectivity for the target histone H3 relative to the non‐histone p53 substrate. Based on these results, tGCN5 labeled with pFF appears to maintain stability and function, while improving its specificity for its target histone H3 substrate.

Conformationally constrained opioid ligands: The Dmt-Aba and Dmt-Aia versus Dmt-Tic scaffold

Replacement of the constrained phenylalanine analogue 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the opioid Dmt-Tic-Gly-NH-Bn scaffold by the 4-amino-1,2,4,5-tetrahydro-indolo[2,3- c]azepin-3-one (Aia) and 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffolds has led to the discovery of novel potent μ-selective agonists (Structures 5 and 12) as well as potent and selective δ-opioid receptor antagonists (Structures 9 and 15). Both stereochemistry and N-terminal N, N-dimethylation proved to be crucial factors for opioid receptor selectivity and functional bioactivity in the investigated small peptidomimetic templates. In addition to the in vitro pharmacological evaluation, automated docking models of Dmt-Tic and Dmt-Aba analogues were constructed in order to rationalize the observed structure–activity data.

Protein tyrosine kinase p56 lck-deficiency confers hypersusceptibility to ρ-fluorophenylalanine (pFPhe)-induced apoptosis by augmenting mitochondrial apoptotic pathway in human Jurkat T cells

Phenylalanine analog, ρ-fluorophenylalanine (pFPhe)-mediated cytotoxicity and several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, Bid cleavage, degradation of PARP and PLCγ-1, and DNA fragmentation were more significant in p56 lck-deficient Jurkat T cells (JCaM1.6) than in wild-type Jurkat T cells (E6.1). The susceptibility of JCaM1.6 toward apoptogenic activity of pFPhe decreased after acquisition of p56 lck by transfection. The p56 lck kinase activity increased 1.6-fold at 15–30 min after pFPhe treatment. The pan-caspase inhibitor (z-VAD-fmk) completely blocked the pFPhe-mediated apoptotic changes except caspase-9 activation. The caspase-8 inhibitor (z-IETD-fmk), which failed to influence pFPhe-induced caspase-9 activation, completely blocked caspase-8 activation and PLCγ-1 degradation with a marked reduction in caspase-3 activation and PARP degradation, indicating pFPhe-induced caspase-8 activation as a downstream event of mitochondria-dependent activation of caspase-9. These results indicate that the deficiency of p56 lck augments pFPhe-induced mitochondrial cytochrome c release and resultant apoptotic cell death in Jurkat T cells.

Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

Synthesis of Amino Acid Analogues of 5H‐Dibenz[b,f]azepine and Evaluation of their Radical Scavenging Activity

A method for the synthesis of tyrosine, phenyl alanine, hydroxy proline and threonine free amino acid analogues of 5H‐dibenz[b,f]azepine is proposed. 5H‐dibenz[b,f]azepine was prepared by known method. The key intermediate 3‐chloro‐1‐(5H‐dibenz[b,f]azepine‐5‐yl)propan‐1‐one was obtained by N‐acylation of 5H‐dibenz[b,f]azepine with 3‐chloro propionyl chloride. Further coupling of respective free amino acid to produce 2‐(3‐(5H‐dibenz[b,f]azepine‐5‐yl)‐3‐oxopropylamino)3‐(4 hydroxyphenyl) propanoic acid, 2‐(3‐(5H‐dibenz[b,f]azepine‐5‐yl)‐3‐oxopropylamino)‐3‐phenyl propanoicacid,1‐(3‐(5H‐dibenz[b,f]azepine‐5‐yl)‐3‐oxopropyl)‐3‐hydroxypyrolidine‐2‐carboxylic acid and 2‐(3‐(5H‐dibenz[b,f] azepine‐yl)‐3‐oxopropyl amino)‐3‐hydroxy butanoic acid. The synthesized compounds were evaluated for their potential over 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH) free radical scavenging activity. Butylated hydroxy anisole (BHA) and ascorbic acid (AA) were used as the reference antioxidant compounds and also the comparative study with synthesized compounds was done. Under our experimental conditions tyrosine, hydroxy proline and threonine analogues possess a direct scavenging effect on trapping the stable free radical DPPH. Hydroxy proline analogues showed a significant radical scavenging activity among the synthesized analogues

Partitioning of amino-acid analogues in a five-slab membrane model

The positional preferences of the twenty amino-acid residues in a phospholipid bilayer are investigated by calculating the solvation free energy of the corresponding side chain analogues using a five-slab continuum electrostatic model. The side-chain analogues of the aromatic residues tryptophan and tyrosine are found to partition in the head-group region, due to compensation between the increase of the non-polar component of the solvation free energy at the boundary with the aqueous region and the decrease in the electrostatic component. The side chain analogue of phenylalanine differs from the other aromatic molecules by being able to partition in both the head-group region and the membrane core. This finding is consistent with experimental findings of the position of phenylalanine in membrane helices. Interestingly, the charged side-chain analogues of arginine and lysine are shown to prefer the head-group region in an orientation that allows the charged moiety to interact with the aqueous layer. The orientation adopted is similar to the “snorkelling” effect seen in lysine and arginine residues in membrane helices. In contrast, the preference of the charged side-chain analogues of histidine (protonated) and aspartate (deprotonated) for the aqueous layer is shown to be due to a steep decrease in the electrostatic component of the solvation free energy at the boundary to the aqueous region. The calculations allow an understanding of the origins of side chain positioning in membranes and are thus useful in understanding membrane–protein:lipid thermodynamics.

Designing of Substrates and Inhibitors of Bovine α-Chymotrypsin with Synthetic Phenylalanine Analogues in Position P1

The primary specificity residue of a substrate or an inhibitor, called the P(1) residue, is responsible for the proper recognition by the cognate enzyme. This residue enters the S(1) pocket of the enzyme and establishes contacts (up to 50%) inside the proteinase substrate cavity, strongly affecting its specificity. To analyze the influence on bovine alpha-chymotrypsin substrate activity, aromatic non-proteinogenic amino acid residues in position P(1) with the sequence Ac-Phe-Ala-Thr-X-Anb(5,2)-NH(2) were introduced: L-pyridyl alanine (Pal), 4-nitrophenylalanine - Phe(p-NO(2)), 4-aminophenylalanine - Phe(p-NH(2)), 4-carboxyphenylalanine Phe(p-COOH), 4-guanidine phenylalanine - Phe(p-guanidine), 4-methyloxycarbonyl-phenylalanine - Phe(p-COOMe), 4-cyanophenylalanine - Phe(p-CN), Phe, Tyr. The effect of the additional substituent at the phenyl ring of the Phe residue was investigated. All peptides contained an amide of 5-amino-2-nitrobenzoic acid, which served as a chromophore. Kinetic parameters (k(cat), K(M) and k(cat)/K(M)) of the peptides synthesized with bovine alpha-chymotrypsin were determined. The highest value of the specificity constant k(cat)/K(M), reaching 6.0 x 10(5) [M(-1)xs(-1)], was obtained for Ac-Phe-Ala-Thr-Phe(p-NO(2))-Anb(5,2)-NH(2). The replacement of the acetyl group with benzyloxycarbonyl moiety yielded a substrate with the value of k(cat) more than three times higher. Peptide aldehydes were synthesized with selected residues (Phe, Pal, Tyr, Phe(p-NO(2)) in position P(1) and potent chymotrypsin inhibitors were obtained. The dissociation constant (K(i)) with the experimental enzyme determined for the most active peptide, Tos-Phe-Ala-Thr-Phe(p-NO(2))-CHO, amounted to 1.12 x 10(-8) M.

Carbohydrate–protein recognition probed by density functional theory and ab initio calculations including dispersive interactions

Carbohydrate-protein recognition has been studied by electronic structure calculations of complexes of fucose and glucose with toluene, p-hydroxytoluene and 3-methylindole, the latter aromatic molecules being analogues of phenylalanine, tyrosine and tryptophan, respectively. We use mainly a density functional theory model with empirical corrections for the dispersion interactions (DFT-D), this method being validated by comparison with a limited number of high level ab initio calculations. We have calculated both binding energies of the complexes as well as their harmonic vibrational frequencies and proton NMR chemical shifts. We find a range of minimum energy structures in which the aromatic group can bind to either of the two faces of the carbohydrate, the binding being dominated by a combination of OH-pi and CH-pi dispersive interactions. For the fucose-toluene and alpha-methyl glucose-toluene complexes, the most stable structures involve OH-pi interactions, which are reflected in a red shift of the corresponding O-H stretching frequency, in good quantitative agreement with experimental data. For those structures where CH-pi interactions are found we predict a corresponding blue shift in the C-H frequency, which parallels the predicted proton NMR shift. We find that the interactions involving 3-methylindole are somewhat greater than those for toluene and p-hydroxytoluene.

1-Amino-2-Phenylcyclopentane-1-carboxylic Acid: A Conformationally Restricted Phenylalanine Analogue

DFT calculations at the B3LYP/6-311G(d,p) level have been used to investigate the intrinsic conformational preferences of 1-amino-2-phenylcyclopentane-1-carboxylic acid (c5Phe), a constrained analogue of phenylalanine in which the alpha and beta carbons are included in a cyclopentane ring. Specifically, the N-acetyl-N'-methylamide derivatives of the cis and trans stereoisomers, where cis and trans refer to the relative position between the amino group and the phenyl ring, have been calculated. Solvent effects have been examined using a self-consistent reaction field (SCRF) method. Results indicate that the conformational space of the cis stereoisomer is much more restricted than that of the trans derivative both in the gas phase and in solution.

C-terminal constrained phenylalanine as a pharmacophoric unit in peptide-based proteasome inhibitors

Here we report the synthesis and biological properties of peptide-based molecules bearing constrained analogues of phenylalanine at the C-terminal. Compounds were tested as proteasome subunits' inhibitors. Dehydro-peptides showed good inhibition, in particular against trypsin-like (T-L) proteasome activity while some C-terminal Tic-derivatives inhibit only caspase-like activity in enzymatic β1 subunits with a certain degree of efficacy. The best analogues of the series demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.

Use of the Novel Fluorescent Amino Acidp-Cyanophenylalanine Offers a Direct Probe of Hydrophobic Core Formation during the Folding of the N-Terminal Domain of the Ribosomal Protein L9 and Provides Evidence for Two-State Folding

Fluorescence-detected stopped flow measurements are the method of choice for studies of protein folding kinetics. However, the methodology suffers from the limitation that the protein of interest either must contain an intrinsic fluorophore or can tolerate its introduction by mutagenesis. Recently, the cyano (nitrile) analogue of phenylalanine has been proposed for use as a fluorescence analogue. Here we take advantage of this new methodology to monitor the formation of the hydrophobic core during the folding of the N-terminal domain of L9 (NTL9). Phenylalanine 5, which is completely buried in the folded state of NTL9, was replaced with p-cyanophenylalanine (p-cyano-Phe). This derivative reports on the formation of the hydrophobic core. The variant adopts the same fold as wild-type NTL9 and is slightly more stable. Refolding and unfolding were monitored using both guanidine HCl and urea jump experiments. In both cases, plots of the natural log of the observed relaxation rate versus denaturant concentration, so-called chevron plots, exhibited the characteristic V shape expected for two-state folding, and no hint of deviation from linearity was observed at low denaturant concentrations. The stability calculated from the measured folding and unfolding rates is in very good agreement with the value obtained from equilibrium measurements as is the m value. The relative compactness of the transition state for folding as defined by the Tanford beta parameter is identical to that of the wild type. The results illustrate the applicability of p-cyano-Phe analogues in protein folding studies and provide further evidence of two-state folding of NTL9.

Synthesis of Chiral, Monodentate Aminophosphane and Phosphoramidite Ligands Derived from Amino Acid Esters: Application in Rh‐Catalysed Asymmetric Olefin Hydrogenation Reactions

AbstractSix chiral monodentate ligands combining a 1,3‐dioxa‐2‐phosphacycloheptadinaphthyl moiety [(R)‐ or (S)‐binoP] either with a phenylalanine‐ or with an alanine‐derived fragment were synthesised. The new phosphoramidites are all relatively air stable. Related compounds in which the binoP moiety was replaced by a diphenylphosphanyl group were also prepared for comparison. The X‐ray structures of two phosphoramidite complexes, cis‐PtCl2[(R)‐binoP–NMeR]2 [5, R = (R)‐CH(CH2Ph)(CO2Me); 6, R = (S)‐CH(CH2Ph)(CO2Me)], were determined by single X‐ray analysis. In the solid state, both structures are nearly C2 symmetric, and the nitrogen atoms lies out of the coordination plane. Owing to the particular orientation of the benzyl groups in 6, the environment of the coordination sites occupied in this complex by the chlorine atoms is sterically more crowded than in 5. In the hydrogenation of 2‐(acetylamino)‐3‐(aryl)propenoic methyl esters (aryl = 4‐X‐C6H4, X = H, F, Cl; aryl = 3,4‐Cl2–C6H3), the alanine‐derived phosphoramidites turned out to be ca. twice as active as the corresponding phenylalanine analogues. The highest ee's were observed with the phenylalanine derivatives, for example 92 % in the hydrogenation of 2‐(acetylamino)‐3‐(phenyl)propenoic methyl ester, by using (R)‐binoP–NMe‐(R)‐CH(CH2Ph)(CO2Me). (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Use of Constrained Synthetic Amino Acids in β-Helix Proteins for Conformational Control

A highly constrained amino acid has been introduced in the turn region of a beta-helix to increase the conformational stability of the native fold for nanotechnological purposes. The influence of this specific amino acid replacement in the final organization of beta-helix motifs has been evaluated by combining ab initio first-principles calculations on model systems and molecular dynamics simulations of entire peptide segments. The former methodology, which has been applied to a sequence containing three amino acids, has been used to develop adjusted templates. Calculations indicated that 1-amino-2,2-diphenylcyclopropanecarboxylic acid, a constrained cyclopropane analogue of phenylalanine, exhibits a strong tendency to form and promote folded conformations. On the other hand, molecular dynamics simulations are employed to probe the ability of such a synthetic amino acid to enhance the conformational stability of the beta-helix motif, which is the first requirement for further protein nanoengineering. A highly regular segment from a naturally occurring beta-helix protein was selected as a potential nanoconstruct module. Simulations of wild type and mutated segments revealed that the ability of the phenylalanine analogue to nucleate turn conformations enhances the conformational stability of the beta-helix motif in isolated peptide segments.

The Thylakoid Proton Gradient Promotes an Advanced Stage of Signal Peptide Binding Deep within the Tat Pathway Receptor Complex

Tat (twin arginine translocation) systems transport folded proteins across the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria. Tat precursors are targeted by hydrophobic cleavable signal peptides with twin arginine (RR) motifs. Bacterial precursors possess an extended consensus, (S/T)RRXFLK, of which the two arginines and the phenylalanine are essential for efficient transport. Thylakoid Tat precursors possess twin arginines but lack the consensus phenylalanine. Here, we have characterized two stages of precursor binding to the thylakoid Tat signal peptide receptor, the 700-kDa cpTatC-Hcf106 complex. The OE17 precursor tOE17 binds to the receptor by RR-dependant electrostatic interactions and partially dissociates during blue native gel electrophoresis. In addition, the signal peptide of thylakoid-bound tOE17 is highly exposed to the membrane surface, as judged by accessibility to factor Xa of cleavage sites engineered into signal peptide flanking regions. By contrast, tOE17 containing a consensus phenylalanine in place of Val(-20) (V - 20F) binds the receptor more strongly and is completely stable during blue native gel electrophoresis. Thylakoid bound V - 20F is also completely protected from factor Xa at the identical sites. This suggests that the signal peptide is buried deeply in the cpTatC-Hcf106 binding site. We further provide evidence that the proton gradient, which is required for translocation, induces a tighter interaction between tOE17 and the cpTat machinery, similar to that exhibited by V - 20F. This implies that translocation involves a very intimate association of the signal peptide with the receptor complex binding site.

Glutamine vinyl ester proteasome inhibitors selective for trypsin-like (β2) subunit

Here we report the study of a new series of peptide-based proteasome inhibitors with a vinyl ester moiety at C-terminal. The presence of Tic, a rigid analogue of phenylalanine, in the central portion of some derivatives is not favourable for the activity. The best analogue of the series shows a potent and selective inhibition for the β2 subunit and good enzymatic stability.

Synthesis of Enantiomerically Pure 1‐Amino‐2‐phenylcycloalkanecarboxylic Acids (cnPhe)

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Synthesis of a new π‐deficient phenylalanine derivative from a common 1,4‐diketone intermediate and study of the influence of aromatic density on prolyl amide isomer population

Enantiopure (2S)-N-(Boc)-3-(6-methylpyridazinyl)alanine (14) has been synthesized to serve as a phenylalanine analog lacking significant pi-donor capability. Two approaches were developed to furnish the target compound from L-aspartic acid as chiral educt in respectively six and nine steps and 13% and 12% yields. In both routes, a key homoallylic ketone intermediate was synthesized by a copper-catalyzed cascade addition of vinylmagnesium bromide to a carboxylic ester. Dipeptide models Ac-Xaa-Pro-NHMe (21a-c) were prepared and the relative populations of prolyl cis- and trans-amide isomers were measured in chloroform, dimethylsulfoxide, and water by proton NMR spectroscopy in order to assess the significance of the electron density of the neighboring aromatic residue on the prolyl amide geometry.

A Helical, Aromatic, Peptide Nanotube

[Structure: see text] The self-assembly in the crystal state of the terminally protected, linear dipeptide Boc-(S,S)c3diPhe-(R,R)c3diPhe-NHiPr (1) through intermolecular hydrogen bonds leads to the formation of a supramolecular helix of large diameter (18 A), internally decorated with phenyl rings. As a result, a hollow helical channel large enough to accommodate guest molecules is observed. This supramolecular structure differs from previous examples of peptide nanotubes. Compound 1 incorporates a highly restricted cyclopropane phenylalanine analogue (c3diPhe) with remarkable conformational properties.

In vivo PET evaluation in tumour-bearing rats of 2-[ 18F]fluoromethyl- l-phenylalanine as a new potential tracer for molecular imaging of brain and extra-cranial tumours in humans with PET

The Na +-independent l-type LAT1 amino acid transport system for large and neutral amino acids has been shown to be expressed higher in tumour tissue relative to normal tissue and has been regarded as a key point for the development of new amino acid based tumour tracers for molecular imaging. We developed a new fluorinated phenylalanine analogue, 2-[ 18F]fluoromethyl- l-phenylalanine, considering that the spatial volume of FCH 3 is comparable with that of the iodine atom in 2-I- l-phenylalanine, of which we have proven that it is taken up excellently in tumours by the LAT1 system. The substrate molecule for radiolabeling, Boc-2-bromomethyl- l-phenylalanine- tButylester, was prepared by radical bromination of Boc-2-methyl- l-phenylalanine- tButylester. [ 18F −] for bromine exchange is performed within 3 min in conditions comparable to the [ 18F]FDG synthesis with a radiochemical yield of at least 85%. After deprotection and semi-preparative HPLC purification, the 2-[ 18F]fluoromethyl- l-phenylalanine is recovered n.c.a. (57%) with a high purity and 3.7 MBq were injected into R1M rhabdomyosarcoma tumour-bearing rats. Imaging was performed with a human PET camera from 5 to 45 min p.i. The tumour/background and tumour/blood ratios obtained from PET acquisition were at least 2.5. DUR values for the tumours were at least about 5. Furthermore, a small tumour implanted near a kidney could be well visualized completely separated from this kidney. Moreover in all tumours the “active” tumour tissue can clearly be differentiated from less active tumour tissue. This proves that 2-[ 18F]fluoromethyl- l-phenylalanine has a great potential as a new tracer for specific tumour diagnosis with PET.