Phenylalanine-containing Peptides Research Articles

Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.

Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer-target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics.

Late-Stage Diversification of Peptides via Pd-Catalyzed Site-Selective δ-C(sp2)-H Fluorination and Amination.

Site-selective C-H fluorination is an attractive strategy for directly transforming inert C-H bonds into C-F bonds, yet it remains a significant challenge. Herein, we have developed an efficient and versatile strategy for site-selective fluorination and amination of phenylalanine-containing peptides via late-stage Pd-catalyzed δ-C(sp2)-H activation, providing a valuable tool for the in situ synthesis of fluorinated indoline scaffolds within peptides.

Site-Selective Modification of Peptides via Late-Stage Pd-Catalyzed Tandem Reaction of Phenylalanine with Benzoquinone.

An efficient and straightforward approach for site-selective functionalization of phenylalanine and phenylalanine-containing peptide via a Pd-catalyzed tandem reaction has been developed. The robust method underwent dual C-H activation, including C-C coupling with benzoquinone and intramolecular C-N cyclization, providing a feasible and rapid synthetic route to incorporate 4-benzoquinone-indoline fragments into peptides.

Palladium-Catalyzed Acylation of Phenylalanine-Containing Peptides

Key words palladium catalysis - C–H functionalization - acylation - regioselectivity

FLUORIMETRY APPLICATION FOR DIFFERENTIAL DIAGNOSIS OF COLORECTAL CANCER AND PRECANCEROUS LESIONS ON HISTOLOGICAL SECTIONS

In the investigation we compared autofluorescence spectra of colon cancer (CC) and precancerous lesions by fluorimetry in different parts of colon. 78 biopsies from different pathological processesin colon, reflecting the basic steps of CC, were investigated. It is shown, that autofluorescence spectra of all types of adenomas as well as adenocarcinomas have two maxima at 260÷270 nm and at 330÷340 nm. The first maximum is primarily defined by tryptophan- and phenylalanine-containing peptides. The second maximum of the spectra is mainly defined by collagen and NADH presence. The results demonstrate not only peculiarities in spectral characteristics of benign and malignant tumors depending on their localization, but also explain different risk of adenomas to malignant transformation, depending on theirtype The obtained results can be used for further improving of differential diagnosis of colonic neoplasia.

Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase.

We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. Graphical Abstract ᅟ.

Simultaneous Time-Dependent Surface-Enhanced Raman Spectroscopy, Metabolomics, and Proteomics Reveal Cancer Cell Death Mechanisms Associated with Gold Nanorod Photothermal Therapy.

In cancer plasmonic photothermal therapy (PPTT), plasmonic nanoparticles are used to convert light into localized heat, leading to cancer cell death. Among plasmonic nanoparticles, gold nanorods (AuNRs) with specific dimensions enabling them to absorb near-infrared laser light have been widely used. The detailed mechanism of PPTT therapy, however, still remains poorly understood. Typically, surface-enhanced Raman spectroscopy (SERS) has been used to detect time-dependent changes in the intensity of the vibration frequencies of molecules that appear or disappear during different cellular processes. A complete proven assignment of the molecular identity of these vibrations and their biological importance has not yet been accomplished. Mass spectrometry (MS) is a powerful technique that is able to accurately identify molecules in chemical mixtures by observing their m/z values and fragmentation patterns. Here, we complemented the study of changes in SERS spectra with MS-based metabolomics and proteomics to identify the chemical species responsible for the observed changes in SERS band intensities during PPTT. We observed an increase in intensity of the bands at around 1000, 1207, and 1580 cm-1, which were assigned in the literature to phenylalanine, albeit with dispute. Our metabolomics results showed increased levels of phenylalanine, its derivatives, and phenylalanine-containing peptides, providing evidence for more confidence in the SERS peak assignments. To better understand the mechanism of phenylalanine increase upon PPTT, we combined metabolomics and proteomics results through network analysis, which proved that phenylalanine metabolism was perturbed. Furthermore, several apoptosis pathways were activated via key proteins (e.g., HADHA and ACAT1), consistent with the proposed role of altered phenylalanine metabolism in inducing apoptosis. Our study shows that the integration of the SERS with MS-based metabolomics and proteomics can assist the assignment of signals in SERS spectra and further characterize the related molecular mechanisms of the cellular processes involved in PPTT.

Fragmentation mechanism of UV-excited peptides in the gas phase.

We present evidence that following near-UV excitation, protonated tyrosine- or phenylalanine-containing peptides undergo intersystem crossing to produce a triplet species. This pathway competes with direct dissociation from the excited electronic state and with dissociation from the electronic ground state subsequent to internal conversion. We employ UV-IR double-resonance photofragment spectroscopy to record conformer-specific vibrational spectra of cold peptides pre-excited to their S1 electronic state. The absorption of tunable IR light by these electronically excited peptides leads to a drastic increase in fragmentation, selectively enhancing the loss of neutral phenylalanine or tyrosine side-chain, which are not the lowest dissociation channels in the ground electronic state. The recorded IR spectra evolve upon increasing the time delay between the UV and IR pulses, reflecting the dynamics of the intersystem crossing on a timescale of ~80 ns and <10 ns for phenylalanine- and tyrosine-containing peptides, respectively. Once in the triplet state, phenylalanine-containing peptides may live for more than 100 ms, unless they absorb IR photons and undergo dissociation by the loss of an aromatic side-chain. We discuss the mechanism of this fragmentation channel and its possible implications for photofragment spectroscopy and peptide photostability.

The effect of increasing hydrophobicity on the self-assembly of amphipathic β-sheet peptides

Peptide self-assembly processes are central to the etiology of amyloid diseases. Much effort has been devoted to characterizing amyloid structure and the mechanisms of peptide self-assembly leading to amyloid. It has been proposed that aromatic side-chain interactions play a central role in early self-assembly recognition events, but this contention remains somewhat controversial. Recent studies have indicated that in some amyloid peptides, aromatic residues can be exchanged for other hydrophobic residues and these nonaromatic variant peptides still retain competency to form amyloid, although with attenuated kinetics. In an effort to understand the relative contributions of aromatic versus generic hydrophobic interactions, studies to quantify the self-assembly properties of amyloid peptides as a function of increasing hydrophobicity and altered aromatic character have been undertaken. In the present study, the amphipathic (FKFE)(2) peptide has been chosen as a model system. The aromatic phenylalanine residues have been globally replaced with nonaromatic natural residues with lower hydrophobicity (alanine, valine, and leucine) and a nonnatural residue with greater hydrophobicity (cyclohexylalanine). The self-assembly properties of these peptides have been characterized by secondary structure analysis and microscopic analysis of the resulting aggregate structures. These studies confirm that aromatic interactions are not strictly required for amyloid formation and that the nonaromatic, but highly hydrophobic, cyclohexylalanine appears to have unique self-assembly characteristics and enhanced hydrogelation properties. The aromatic phenylalanine-containing peptide displays intriguing solvent- and concentration-dependent polymorphism, suggesting that aromatic interactions, while not essential for self-assembly, may give rise to unique structural features.

Enantioselective synthesis of N(alpha)-Fmoc protected (2S,3R)-3-phenylpipecolic acid. A constrained phenylalanine analogue suitably protected for solid-phase peptide synthesis.

Reported herein is the first enantioselective preparation of (2S,3R)-3-phenylpipecolic acid as a conformationally constrained phenylalanine analogue bearing N(alpha)-protection suitable for solid-phase peptide synthesis. Stereochemistries at both the 2- and 3-positions are derived inductively from a single chiral center provided by the commercially available Evans chiral auxiliary, (4S)-4-benzyl-1,3-oxazolidin-2-one. By constraining phi and chi(1) torsion angles, this novel amino acid analogue can serve as a useful tool for the induction of defined geometry in phenylalanine-containing peptides.

Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases

Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases

Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases

Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPbeta, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and selective inhibitor of PTP1B. This peptide inhibits PTP1B with an IC50 of 40 nM, which is at least 100-fold lower than with other PTPs. A second tripeptide, Pro-Phe(CF2P)-Phe(CF2P), is most potent against PTPbeta, with an IC50 of 200 nM, and inhibits PTP1B with an IC50 of 300 nM. These data suggest that it is possible to develop selective, active-site-directed, reversible, potent inhibitors of PTPs.

Expression of the tpr protease gene of Porphyromonas gingivalis is regulated by peptide nutrients.

The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing chromogenic substrates for trypsin and bacterial collagenases. A previous study by us indicated that Tpr expression was increased under conditions of nutrient limitation. In the present study, we further characterized expression of the tpr gene using a tpr::lacZ reporter gene construct under a range of nutrient conditions. In P. gingivalis, transcription of tpr was initiated 215 bp upstream of the coding region and regulation of tpr expression was at the level of transcription. Deletion mutations in the tpr upstream region identified the promoter region immediately upstream of the transcription start site, determined by primer extension analysis. Three identical 17-bp direct repeats identified within the 5' end of tpr mRNA were involved in tpr regulation. In an Escherichia coli background, tpr transcription was initiated after an AT-rich region upstream of tpr but not at the P. gingivalis start site. Tpr expression in P. gingivalis was suppressed by the addition of peptide and protein nutrients to a peptide-limited growth medium but was only slightly affected by addition of free amino acids. Low-molecular-weight fractions of brain heart infusion rich in phenylalanine, proline, and alanine had the greatest inhibitory effects on expression of the tpr::lacZ construct. Addition of the dipeptide phenylalanyl-phenylalanine to the growth medium resulted in a 10-fold decrease in tpr expression. This suggests that specific phenylalanine-containing peptides are a major factor controlling Tpr expression. Neither hemin starvation, heat shock, nor pH change had significant effects on Tpr expression.

Peptide models XX. Aromatic side-chain–backbone interaction in phenylalanine-containing diamide model system. A systematic search for the identification of all the ab initio conformers of N-formyl-L-phenylalanine-amide

Phenylalanine is the simplest among the four natural amino acid residues that have aromatic side chains. The ab initio conformational analysis performed at the RHF/3-21G level on a phenylalanine-containing diamide model system (N-Formyl-L-Phe-NH2) revealed 19 different structures. Single-point energy calculations were performed using RHF/6-31+G* and DFT(B3LYP)/6-311++G** levels for all conformers. The inverse (γL) and the normal (γD) gamma turn, the extended (βL), the left-handed helical [Formula: see text], and the inverse polyproline II [Formula: see text] backbone conformers each have three (g+, a, and g−) side-chain (χ1,) rotamers. The [Formula: see text] and the [Formula: see text] type main-chain conformers have only two side-chain orientations, respectively. No minima have been found for the conformational building unit of the right-handed helical [Formula: see text] and for the polyproline II [Formula: see text] structures. The present ab initio conformational analysis for For-L-Phe-NH2 is a unique example in which a systematic and complete conformational set was established for a diamide system with an aromatic side chain. Analytic vibrational frequency calculations were established for all stationary points found as minima on the potential energy surface. These data may be used in the future as reference conformers in more detailed vibrational and (or) chemical shielding calculations or during the structural analysis of peptides and proteins by X-ray or NMR techniques. Keywords: ab initio peptide conformers, all RHF/3-21G structures of For-L-Phe-NH2, backbone – side-chain interaction in phenylalanine-containing peptide models.

Further insights on the inhibition of HIV type 1 infection in vitro by CD4-modified synthetic peptides containing phenylalanine.

Phenylalanine-containing peptides from CD4 were synthesized on the basis of chemical similarity with active CD4(81-92)-benzylated peptides. Systematic replacement of amino acids of these peptides bearing the benzyl group by phenylalanine, afforded several peptides that were able to block the binding of gp120 to CD4 and to inhibit HIV-induced syncytium formation. These experiments showed that substitution of residues 81 and 85 by phenylalanine was the most important for activity. Following optimization of the length of phenylalanine-substituted peptides it was found that FYICFVED and FYICFVEDE were the most active. Their IC50 for the inhibition of syncytium formation was around 1.2-1.6 microM. This activity is at least 30 times higher than that of the parent peptide FYIFFVEDQKEEDD previously reported (Lasarte et al., J Acquir Immune Defic Syndr 1994;7:129-134). Binding competition experiments with two different anti-peptide antisera recognizing the V3 region of gp120 and FYICFVEDE, show that the active peptides bind to V3 or to a sterically near region of V3. None of the active peptides was toxic to cells in vitro. The enhanced activity and simplicity of these new phenylalanine-substituted CD4 peptides might be a good starting point for the development of mimotopes of potential use for the treatment of AIDS.

Induction of drug specific antibody and the controlled release of drug by 6- O-carboxymethyl-chitin

Biopolymeric properties of a water-soluble and biodegradable chitin derivative, 6- O-carboxymethyl-chitin (CM-chitin), have been investigated to demonstrate the immunological function serving to induce a hapten-specific antibody and the chemotherapeutic function as a drug carrier of controlled release. When CM-chitin was linked by methamphetamine (MA) through a nonbiodegradable spacer, 1-aminobutane (MABA-CM-chitin), MA-specific antibody was produced by the subcutaneous injection, in rabbits, of MABA-CM-chitin in combination with Freund's complete adjuvant, and when injected without intense immunoadjuvant, MABA-CM-chitin oligomer was secreted into blood for more than 120 h. Two-step hydrolysis of pendant-type of polymeric drug was also investigated in order to design a more sophisticated drug delivery system. Phenylalanine-containing peptide spacers were designed to be hydrolysed by enzymes other than lysozyme as a model drug. In the model system, the chromophore-bound phenylalanine peptide spacer was stabilized against enzymatic hydrolysis until CM-chitin was hydrolysed by lysozyme.

The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV.

Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine-containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N-terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411-PGYRHV-416 is the tyrosine-containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410-PPGY-413 forms a well-ordered beta turn structure in solution. Two-dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine-containing peptide and by approximately 50% in the alanine-containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor.

Tetrapeptide inhibitors of protein farnesyltransferase: amino-terminal substitution in phenylalanine-containing tetrapeptides restores farnesylation.

Protein farnesyltransferase from rat brain transfers farnesyl residues to cysteine residues in tetrapeptides that conform to the sequence CA1A2X, where C is cysteine, A1 and A2 are aliphatic amino acids, and X is methionine or serine. When the A2 residue is aromatic [e.g., phenylalanine as in Cys-Val-Phe-Met (CVFM)], the tetrapeptide continues to bind to the enzyme, but it can no longer accept a farnesyl group, and it becomes a pure inhibitor. The current studies show that this resistance to farnesylation also requires a positive charge on the cysteine amino group. Derivatization of this group with acetyl, octanoyl, or cholic acid residues or extension of the peptide with an additional amino acid restores the ability of phenylalanine-containing peptides to accept a farnesyl residue. The same result was obtained when the amino group of cysteine was deleted (mercaptopropionyl-VFM). These data suggest that the positive change on the cysteine amino group acts in concert with an aromatic residue in the A2 position to render peptides resistant to farnesylation by the rat brain enzyme.

Identification of peptides containing tryptophan, tyrosine, and phenylalanine using photodiode-array spectrophotometry

The characteristic absorption spectra of aromatic amino acids between 240 and 310 nm were used to identify tryptophan, tryosine, and phenylalanine-containing peptides. In acidic solution, the absorption spectra of these amino acids exhibit minima or maxima at 255, 270, and 286 nm. Based on these characteristics, the content of the aromatic amino acid in peptide can be estimated. For this study, 2 nmol of tryptic peptides from human apolipoprotein A-1 was separated by high-performance liquid chromatography using a reverse-phase column. The peptide fragments were monitored by a photodiode-array spectrophotometer. This new approach offers a rapid, simple, sensitive, and direct identification of peptides containing aromatic amino acids. Those containing Trp, which may be of interest for DNA sequencing and important in sequence analysis of proteins, can be selectively purified using this technique.

Role and location of "protease I" from Escherichia coli.

Pacaud and Uriel described an enzyme from Escherichia coli ("protease I") that hydrolyzes acetyl phenylalanine naphthyl ester (APNE). We examined the possible involvement of this enzyme in intracellular protein degradation, its subcellular distribution, and its proteolytic activity. Although the APNE-hydrolyzing activity is localized primarily in the periplasm, proteolytic activity against casein was found in the periplasm, membrane, and cytoplasm with similar specific activities. The APNE-hydrolyzing enzyme did not appear to contribute to the proteolytic activity of the periplasm. A mutant deficient in APNE-hydrolyzing activity lacked all activity in the periplasm but showed a slight percentage of residual activity in the cytoplasm. Extracts of such cells were normal in their ability to hydrolyze casein. The mutant was indistinguishable from wild-type cells in its rate of protein degradation during growth or glucose starvation and in the ability to rapidly degrade puromycin-containing polypeptides. Nitrogen starvation, which increased protein breakdown severalfold, affected neither the total amount nor the distribution of APNE-hydrolyzing activity. The mutant showed no defect in its ability to cleave small phenylalanine-containing peptides released during protein degradation. The mutant and wild-type cells are equally able to hydrolyze exogenously supplied phenylalanyl peptides. These experiments suggest that the APNE-hydrolyzing enzyme is not required for protein degradation and that "protease I" is probably not a protease.