Site-Specific Photochemistry along a Protonated Peptide Scaffold.

We present a detailed study of the time-dependent photophysics and photochemistry of a known conformation of the two protonated pentapeptides Leu-enkephalin (Tyrosine-Glycine-Glycine-Phenylalanine-Leucine, YGGFL) and its chromophore-swapped analogue FGGYL, carried out under cryo-cooled conditions in the gas phase. Using ultraviolet-infrared (UV-IR) double resonance, we record excited state IR spectra as a function of time delay between UV and IR pulses. We identify unique Tyr OH stretch transitions due to the S1 state and the vibrationally excited triplet state(s) formed by intersystem crossing, Tn(v). Photofragment mass spectra are recorded out of the S1 origin and following UV-IR double resonance. Several competing site-specific fragmentation pathways are discovered involving peptide backbone cleavage, Tyr side chain loss, and N-terminal NH3 loss mediated by electron transfer. In YGGFL, IR excitation in the S1 state promotes electron transfer (ET) from the aromatic ring to the N-terminal R-NH3+ group leading to loss of neutral NH3. This product channel is missing in FGGYL due to the larger distance for ET from Y(4) to NH3+. Selective loss of the Tyr side chain occurs out of an excited state process following UV excitation and is further enhanced by IR excitation in S1 and Tn(v) states of both YGGFL and FGGYL. Finally, IR excitation in the S1 or Tn(v) states fragments the peptide backbone exclusively at amide(4), producing the b4 cation. We postulate that this selective fragmentation results from intersystem crossing to produce vibrationally excited triplets with enough energy to launch the proton along a proton conduit present in the known starting structure.

Demystifying DPP III Catalyzed Peptide Hydrolysis-Computational Study of the Complete Catalytic Cycle of Human DPP III Catalyzed Tynorphin Hydrolysis.

Dipeptidyl peptides III (DPP III) is a dual-domain zinc exopeptidase that hydrolyzes peptides of varying sequence and size. Despite attempts to elucidate and narrow down the broad substrate-specificity of DPP III, there is no explanation as to why some of them, such as tynorphin (VVYPW), the truncated form of the endogenous heptapeptide spinorphin, are the slow-reacting substrates of DPP III compared to others, such as Leu-enkephalin. Using quantum molecular mechanics calculations followed by various molecular dynamics techniques, we describe for the first time the entire catalytic cycle of human DPP III, providing theoretical insight into the inhibitory mechanism of tynorphin. The chemical step of peptide bond hydrolysis and the substrate binding to the active site of the enzyme and release of the product were described for DPP III in complex with tynorphin and Leu-enkephalin and their products. We found that tynorphin is cleaved by the same reaction mechanism determined for Leu-enkephalin. More importantly, we showed that the product stabilization and regeneration of the enzyme, but not the nucleophilic attack of the catalytic water molecule and inversion at the nitrogen atom of the cleavable peptide bond, correspond to the rate-determining steps of the overall catalytic cycle of the enzyme.

Identification of a Novel Delta Opioid Receptor Agonist Chemotype with Potential Negative Allosteric Modulator Capabilities.

The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious ß-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit ß-arrestin, as it has been suggested that compounds that efficaciously recruit ß-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a 'NAM-agonist' in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays.

Near UV and Visible Light Induce Iron-Dependent Photodegradation Reactions in Pharmaceutical Buffers: Mechanistic and Product Studies.

Near UV (λ = 320-400 nm) and visible light (λ = 400-800 nm) can lead to the oxidation of pharmaceutical proteins, which can affect efficiency and promote immunogenicity. However, no concise mechanism has been established for the photo-oxidation of pharmaceutical proteins under near UV and visible light. Here, we show that carboxylic acid buffer-Fe3+ complexes can function as photosensitizers, causing peptide degradation via the formation of various radicals and oxidants. Three pharmaceutical relevant carboxylic acid buffers (citrate, acetate, and succinate) were tested under near UV and visible light. Oxidation reactions were monitored for model peptides containing readily oxidizable amino acids, such as methionine- or leucine-enkephalin and proctolin peptide. Oxidation products were evaluated by RP-HPLC coupled to UV or fluorescent detection and RP-HPLC-MS/MS. Specifically for citrate buffer, the light-induced formation of H2O2, •OH, •CO2-, and formaldehyde was demonstrated. The peptides displayed oxidation of Met, hydroxylation of Tyr and Phe, as well as the formation of novel products from Tyr. Experiments with 18O2 resulted in the incorporation of 18O into various reaction products, consistent with a metal-catalyzed activation of O2 into reactive oxygen species. The addition of EDTA and DTPA did not prevent the oxidation of the peptides and, in some cases, enhanced the oxidation. Our results demonstrate that pharmaceutical buffer-Fe3+ complexes, exposed to UV and visible light, can promote various pathways of oxidation reactions in pharmaceutical formulations.

Dihedral Angle Calculations To Elucidate the Folding of Peptides through Its Main Mechanical Forces.

This study reports a general method to calculate dihedral angles (φ and ψ) of a given amino acid sequence, focusing on potential energy and torque moment concepts. By defining these physical measures in relation to the chemical interactions that occur on each single amino acid residue within a peptide, we analyze the folding process as the result of main mechanical forces (MMFs) exerted in the specific amino acid chain of interest. As a proof of concept, Leu-enkephalin was initially used as a model peptide to carry out the theoretical study. Our data show agreement between calculated Leu-enkephalin backbone dihedral angles and the corresponding experimentally determined X-ray values. Hence, we used calcitonin to validate our MMF-based method on a larger peptide, i.e., 32 amino acid residues forming an α-helix. Through a similar approach (although simplified with regard to electrostatic interactions), the calculations for calcitonin also demonstrate a good agreement with experimental values. This study offers new opportunities to analyze peptides' amino acid sequences and to help in the prediction of how they must fold, assisting in the development of new computational techniques in the field.

A Modified Traveling Wave Ion Mobility Mass Spectrometer as a Versatile Platform for Gas-Phase Ion-Molecule Reactions.

Taken individually, chemical labeling and mass spectrometry are two well-established tools for the structural characterization of biomolecular complexes. A way to combine their respective advantages is to perform gas-phase ion-molecule reactions (IMRs) inside the mass spectrometer. This is, however, not so well developed because of the limited range of usable chemicals and the lack of commercially available IMR devices. Here, we modified a traveling wave ion mobility mass spectrometer to enable IMRs in the trapping region of the instrument. Only one minor hardware modification is needed to allow vapors of a variety of liquid reagents to be leaked into the trap traveling wave ion guide of the instrument. A diverse set of IMRs can then readily be performed without any loss in instrument performance. We demonstrate the advantages of implementing IMR capabilities in general, and to this quadrupole-ion mobility-time-of-flight (Q-IM-TOF) mass spectrometer in particular, by exploiting the full functionality of the instrument, including mass selection, ion mobility separation, and post-mobility fragmentation. The potential to carry out gas-phase IMR kinetics experiments is also illustrated. We demonstrate the versatility of the setup using gas-phase IMRs of established utility for biological mass spectrometry, including hydrogen-deuterium exchange, ion-molecule proton transfer reactions, and covalent modification of DNA anions using trimethylsilyl chloride.

Synthesis and biological evaluation of quercetin and resveratrol peptidyl derivatives as potential anticancer and antioxidant agents.

Quercetin and resveratrol are polyphenolic compounds, members of the flavonoid and the stilbene family, respectively, both medicinally important as dietary anticancer and antioxidant agents. They are present in a variety of foods-including fruits, vegetables, tea, wine, as well as other dietary supplements-and are responsible for various health benefits. Different quercetin and resveratrol esters of Leu/Met-enkephalin and tetrapeptide Leu-Ser-Lys-Leu (LSKL) were synthesized as model systems for monitoring the influence of the peptides on biological activity of resveratrol and quercetin. General formula of the main peptidyl-quercetin derivatives is 2-[3-(aa)n-4-hydroxyphenyl]-3,5,7-tri-hydroxy-4H-1-benzopyran-4-on, and the general formula of the main peptidyl-resveratrol derivatives is (E)-5-[4-(aa)n)styryl]benzene-1,3-diol. The antioxidant and anticancer activities of prepared compounds were investigated. Significant anticancer activity was obtained for the LSKL-based both quercetin and resveratrol derivatives. All prepared compounds exhibit antioxidant activity, in particular quercetin derivative containing Met-enkephalin.

Determination of methionine-enkephalin and leucine-enkephalin by LC-MS in human plasma: Study of pre-analytical stability.

The aim of this work was to assess the influence of preanalytical variables on the stability of two endogenous opioid peptides (Methionine-Enkephalin and Leucine-Enkephalin) in human plasma. For this purpose, first a sensitive LC-MS/MS analytical method was developed and validated for the simultaneous quantitative analysis of these two peptides. The methodology consisted of a simple protein precipitation step followed by UPLC separation and MRM quantitative analysis using a stable isotope labelled Methionine-Enkephalin as internal standard. The method with a limit of quantitation of 10 pg/mL showed good reproducibility with excellent accuracy and precision, and was linear up to 2000 pg/mL. An extensive evaluation of the pre-analytical stability of these peptides in human blood was carried out to ensure an adequate sample collection procedure to obtain reliable results in the analysis of clinical samples.

Intrinsic structure of pentapeptide Leu-enkephalin: geometry optimization and validation by comparison of VSCF-PT2 calculations with cold ion spectroscopy.

The intrinsic structure of an opioid peptide [Ala2, Leu5]-leucine enkephalin (ALE) has been investigated using first-principles based vibrational self-consistent field (VSCF) theory and cold ion spectroscopy. IR-UV double resonance spectroscopy revealed the presence of only one highly abundant conformer of the singly protonated ALE, isolated and cryogenically cooled in the gas phase. High-level quantum mechanical calculations of electronic structures in conjunction with a systematic conformational search allowed for finding a few low-energy candidate structures. In order to identify the observed structure, we computed vibrational spectra of the candidate structures and employed the theory at the semi-empirically scaled harmonic level and at the first-principles based anharmonic VSCF levels. The best match between the calculated "anharmonic" and the measured spectra appeared, indeed, for the most stable candidate. An average of two spectra calculated with different quantum mechanical potentials is proposed for the best match with experiment. The match thus validates the calculated intrinsic structure of ALE and demonstrates the predictive power of first-principles theory for solving structures of such large molecules.

Photodissociation spectroscopy of protonated leucine enkephalin.

Protonated leucine enkephalin (YGGFL) was studied by ultraviolet photodissociation (UVPD) from 225 to 300 nm utilizing an optical parametric oscillator tunable wavelength laser system (OPO). Fragments were identified by absolute mass measurement in a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Bond cleavage was preferred in the vicinity of the two aromatic residues, resulting in high ion abundances for a4, a1, b3, y2 and y1 fragments. a, b and y ions dominated the mass spectrum, and full sequence coverage was achieved for those types. Photodissociation was most effective at the short wavelength end of the studied range, which is assigned to the onset of the La π-π* transition of the tyrosine chromophore, but worked well also at the Lb π-π* chromophore absorption maxima in the 35 000-39 000 cm-1 region. Several side-chain and internal fragments were observed. H atom loss is observed only above 41 000 cm-1, consistent with the requirement of a curve crossing to a repulsive 1πσ* state. It is suggested that the photochemically generated mobile H atom plays a role in further backbone cleavages, similar to the mechanism for electron capture dissociation. The b4 fragment is most intense at the Lb chromophore absorptions, undergoing additional fragmentation at higher photon energies. The high resolution of the FT-ICR MS revealed that out of all x and z-type fragments only x3 and x4 were formed, with low intensity. Other previously reported x- and z-fragments were re-assigned to internal fragments, based on exact mass measurement.

Gas-Phase Folding of a Prototypical Protonated Pentapeptide: Spectroscopic Evidence for Formation of a Charge-Stabilized ß-Hairpin.

Ultraviolet and infrared-ultraviolet (IR-UV) double-resonance photofragment spectroscopy has been carried out in a tandem mass spectrometer to determine the three-dimensional structure of cryogenically cooled protonated C-terminally methyl esterified leucine enkephalin [YGGFL-OMe+H](+). By comparing the experimental IR spectrum of the dominant conformer with the predictions of DFT M05-2X/6-31+G(d) calculations, a backbone structure was assigned that is analogous to that previously assigned by our group for the unmodified peptide [ Burke, N.L.; et al. Int. J. Mass Spectrom. 2015 , 378 , 196 ], despite the loss of a C-terminal OH binding site that was thought to play an important role in its stabilization. Both structures are characterized by a type II' ß-turn around Gly(3)-Phe(4) and a γ-turn around Gly(2), providing spectroscopic evidence for the formation of a ß-hairpin hydrogen bonding pattern. Rather than disrupting the peptide backbone structure, the protonated N-terminus serves to stabilize the ß-hairpin by positioning itself in a pocket above the turn where it can form H-bonds to the Gly(3) and C-terminus C═O groups. This ß-hairpin type structure has been previously proposed as the biologically active conformation of leucine enkephalin and its methyl ester in the nonpolar cell membrane environment [ Naito, A.; Nishimura, K. Curr. Top. Med. Chem. 2004 , 4 , 135 - 143 ].

Synthesis, photophysical properties of triazolyl-donor/acceptor chromophores decorated unnatural amino acids: Incorporation of a pair into Leu-enkephalin peptide and application of triazolylperylene amino acid in sensing BSA.

The research in the field of design and synthesis of unnatural amino acids is growing at a fast space for the increasing demand of proteins of potential therapeutics and many other diversified novel functional applications. Thus, we report herein the design and synthesis of microenvironment sensitive fluorescent triazolyl unnatural amino acids (UNAA) decorated with donor and/or acceptor aromatic chromophores via click chemistry. The synthesized fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical properties and DFT/TDDFT calculation. HOMO-LUMO distribution shows that the emissive states of some of the amino acids are characterized with more significant electron redistribution between the triazolyl moiety and the aromatic chromophores linked to it leading to modulated emission property. A pair of donor-acceptor amino acid shows interesting photophysical interaction property indicating a FRET quenching event. Furthermore, one of the amino acid, triazolyl-perylene amino acid, has been exploited for studying interaction with BSA and found that it is able to sense BSA with an enhancement of fluorescence intensity. Finally, we incorporated a pair of donor/acceptor amino acids into a Leu-enkephalin analogue pentapeptide which was found to adopt predominantly type II ß-turn conformation. We envisage that our investigation is of importance for the development of new fluorescent donor-acceptor unnatural amino acids a pair of which can be exploited for generating fluorescent peptidomimetic probe of interesting photophysical property for applications in studying peptide-protein interaction.

Enhancement of Ion Activation and Collision-Induced Dissociation by Simultaneous Dipolar Excitation of Ions in x- and y-Directions in a Linear Ion Trap.

Collision-induced dissociation (CID) in linear ion traps is usually performed by applying a dipolar alternating current (AC) signal to one pair of electrodes, which results in ion excitation mainly in one direction. In this paper, we report simulation and experimental studies of the ion excitation in two coordinate directions by applying identical dipolar AC signals to two pairs of electrodes simultaneously. Theoretical analysis and simulation results demonstrate that the ion kinetic energy is higher than that using the conventional CID method. Experimental results show that more activation energy (as determined by the intensity ratio of the a4/b4 fragments from the CID of protonated leucine enkephalin) can be deposited into parent ions in this method. The dissociation rate constant in this method was about 3.8 times higher than that in the conventional method under the same experimental condition, at the Mathieu parameter qu (where u = x, y) value of 0.25. The ion fragmentation efficiency is also significantly improved. Compared with the conventional method, the smaller qu value can be used in this method to obtain the same internal energy deposited into ions. Consequently, the "low mass cut-off" is redeemed and more fragment ions can be detected. This excitation method can be implemented easily without changing any experimental parameters.

IR spectroscopy of protonated leu-enkephalin and its 18-crown-6 complex embedded in helium droplets.

Ultracold IR spectra of the protonated five amino acid peptide leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) embedded in superfluid helium droplets have been recorded using a free-electron laser as radiation source. The results show resolved spectra, which are in good agreement with theoretical calculations, as well as with the available gas-phase data indicating that the helium environment does not induce a significant matrix-shift. In addition, the effect of the interaction between the charge and the peptide backbone has been further investigated by complexing protonated leu-enkephalin with one 18-crown-6 molecule. Good agreement between the experimental and theoretical results allow for an assignment of a preferred molecular structure.

Synthesis and analgesic activity of alkylated, reduced and constrained oligoheterocyclic peptidomimetic analogs of Leu-enkephalin.

We report the design and the parallel solid phase synthesis of linear and oligoheterocyclic peptidomimetic analogs of Leu-enkephalin. The described peptidomimetics represent different unique scaffolds that distribute in the space the peptidyl side chains of amino acids essential for biological activity and mimic the bioactive conformation of the Leu-enkephalin peptide. All the compounds were screened in competitive radioligand binding assays to determine their affinities for µ-(MOR), and κ-(KOR) opioid receptors. A reduced analog of Leu-enkephalin TPI1879-26 with activity Ki=60 nM for the mu receptor was identified.

Ion mobility spectrometry, infrared dissociation spectroscopy, and ab initio computations toward structural characterization of the deprotonated leucine-enkephalin peptide anion in the gas phase.

Although the sequencing of protonated proteins and peptides with tandem mass spectrometry has blossomed into a powerful means of characterizing the proteome, much less effort has been directed at their deprotonated analogues, which can offer complementary sequence information. We present a unified approach to characterize the structure and intermolecular interactions present in the gas-phase pentapeptide leucine-enkephalin anion by several vibrational spectroscopy schemes as well as by ion-mobility spectrometry, all of which are analyzed with the help of quantum-chemical computations. The picture emerging from this study is that deprotonation takes place at the C terminus. In this configuration, the excess charge is stabilized by strong intramolecular hydrogen bonds to two backbone amide groups and thus provides a detailed picture of a potentially common charge accommodation motif in peptide anions.

Amadori and Heyns rearrangement products of bioactive peptides as potential new ligands of galectin-3.

Non-enzymatic cascade reactions between amines and reducing sugars are known as Maillard reaction. The late phase of these reactions consists of advanced glycation end products (AGEs), which have been implicated in the pathogenesis of numerous human diseases. Recent evidence suggests that galectin-3 acts as a receptor for AGEs and some early products of the Maillard reaction. The early phase of the Maillard reaction, which consists of 1-amino-1-deoxyketoses (Amadori compounds) and 2-amino-2-deoxyaldoses (Heyns compounds), was the subject of our study. The binding interactions between galectin-3 and the Amadori and Heyns compounds of leucine-enkephalin (YGGFL), leucine-enkephalin methyl ester (YGGFL-OMe), truncated enkephalin (YGG and Y) and tetrapeptide (LSKL) were measured using the AlphaScreen competitive binding assay. The affinity of galectin-3 for Amadori and Heyns compounds depends on both the sugar moiety and the amino acid sequence of the model compounds. The best results were obtained with Leu-enkephalin derivatives of Amadori (IC50 = 6.06 µm) and Heyns (IC50 = 8.6 µm) compound, respectively.

Benzotriazole-mediated synthesis of aza-peptides: en route to an aza-leuenkephalin analogue.

Novel N-(N-Pg-azadipeptidoyl)benzotriazoles 20a-e couple efficiently with α-amino acids 21a-e, dipeptides 22a-c, aminoxyacetic acid 23a, depsidipeptide 23b, and α-hydroxy-ß-phenylpropionic acid 27 yielding, respectively, azatripeptides 24a-g, azatetrapeptides 25a,b, a hybrid azatripeptide with an oxyamide bond 26a, a hybrid azatetrapeptide with an ester bond 26b, and a hybrid azatripeptide with an ester bond 28. A new protocol for the synthesis of N-Pg-azatripeptides 33a,b and 35a,b, each containing a natural amino acid at the N-terminus, avoids the low coupling rates of the aza-amino acid residue and enables the solution-phase synthesis of an azaphenylalanine analogue of Leu-enkephalin 40.

Systematic replacement of amides by 1,4-disubstituted[1,2,3]triazoles in Leu-enkephalin and the impact on the delta opioid receptor activity.

Using Cu(I)-catalyzed azide-alkyne cycloaddition in a mixed classical organic phase and solid phase peptide synthesis approach, we synthesized four analogs of Leu-enkephalin to systematically replace amides by 1,4-disubstituted[1,2,3]triazoles. The peptidomimetics obtained were characterized by competitive binding, contractility assays and ERK1/2 phosphorylation. The present study reveals that the analog bearing a triazole between Phe and Leu retains some potency, more than all the others, suggesting that the hydrogen bond acceptor capacity of the last amide of Leu-enkephalin is essential for the biological activity of the peptide.

Amyloid-ß-neuropeptide interactions assessed by ion mobility-mass spectrometry.

Recently, small peptides have been shown to modulate aggregation and toxicity of the amyloid-ß protein (Aß). As such, these new scaffolds may help discover a new class of biotherapeutics useful in the treatment of Alzheimer's disease. Many of these inhibitory peptide sequences have been derived from natural sources or from Aß itself (e.g., C-terminal Aß fragments). In addition, much earlier work indicates that tachykinins, a broad class of neuropeptides, display neurotrophic properties, presumably through direct interactions with either Aß or its receptors. Based on this work, we undertook a limited screen of neuropeptides using ion mobility-mass spectrometry to search for similar such peptides with direct Aß binding properties. Our results reveal that the neuropeptides leucine enkephalin (LE) and galanin interact with both the monomeric and small oligomeric forms of Aß(1-40) to create a range of complexes having diverse stoichiometries, while some tachyknins (i.e., substance P) do not. LE interacts with Aß more strongly than galanin, and we utilized ion mobility-mass spectrometry, molecular dynamics simulations, gel electrophoresis/Western blot, and transmission electron microscopy to study the influence of this peptide on the structure of Aß monomer, small Aß oligomers, as well as the eventual formation of Aß fibrils. We find that LE binds selectively within a region of Aß between its N-terminal tail and hydrophobic core. Furthermore, our data indicate that LE modulates fibril generation, producing shorter fibrillar aggregates when added in stoichiometric excess relative to Aß.