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.

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.

Probing Structural Information of Gas-Phase Peptides by Near-Edge X-ray Absorption Mass Spectrometry.

Near-edge X-ray absorption mass spectrometry (NEXAMS) is an action-spectroscopy technique of growing interest for investigations into the spatial and electronic structure of biomolecules. It has been used successfully to give insights into different aspects of the photodissociation of peptides and to probe the conformation of proteins. It is a current question whether the fragmentation pathways are sensitive toward effects of conformational isomerism, tautomerism, and intramolecular interactions in gas-phase peptides. To address this issue, we studied the cationic fragments of cryogenically cooled gas-phase leucine enkephalin ([LeuEnk+H]+) and methionine enkephalin ([MetEnk+H]+) produced upon soft X-ray photon absorption at the carbon, nitrogen, and oxygen K-edges. The interpretation of the experimental ion yield spectra was supported by density-functional theory and restricted-open-shell configuration interaction with singles (DFT/ROCIS) calculations. The analysis revealed several effects that could not be rationalized based on the peptide's amino acid sequences alone. Clear differences between the partial ion yields measured for both peptides upon C 1s → π*(C═C) excitations in the aromatic amino acid side chains give evidence for a sulfur-aromatic interaction between the methionine and phenylalanine side chain of [MetEnk+H]+. Furthermore, a peak associated with N 1s → π*(C═N) transitions, linked to a tautomeric keto-to-enol conversion of peptide bonds, was only present in the photon energy resolved ion yield spectra of [MetEnk+H]+.

Collision energies on QTof and Orbitrap instruments: How to make proteomics measurements comparable?

Quadrupole time-of-flight (QTof) collision-induced dissociation (CID) and Orbitrap higher-energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry-based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q-Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap-specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).

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.

A Tilted Surface and Ion Carpet Array for SID.

The development of native mass spectrometry (MS) has provided structural biologists an additional tool to probe the structures of large macromolecular systems. Surface-induced dissociation (SID) is one activation method used within tandem MS experiments that has proven useful in interrogating the connectivity and topology of biologically-relevant protein complexes. We present here the use of a tilted surface and ion carpet array within a new SID device design, enabling decreased dimensions along the ion path and fewer lenses to tune. This device works well in fragmenting ions of both low (peptides) and high (protein complexes) m/z. Results show that the ion carpet array, while enabling simplification of the back-end of the device, has deficiencies in product collection and subsequently signal at higher SID energies when fragmenting protein complexes. However, the use of the tilted surface is advantageous as an effective way to shorten the device and reduce the number of independent voltages.

NCI-ELMO: A New Method To Quickly and Accurately Detect Noncovalent Interactions in Biosystems.

Accurate determinations of noncovalent interactions in biological systems are fundamental to rationalize the structure and to get insights into the functions and the dynamics of macromolecules. Here we propose a new tool for the efficient identification of noncovalent interactions in proteins. The noncovalent interaction (NCI) method, a well-established strategy to detect noncovalent interactions in chemical systems, is coupled with the libraries of extremely localized molecular orbitals (ELMOs), which allow instantaneous reconstruction of quantum mechanically rigorous electron distributions of polypeptides and proteins. Test calculations performed on different interactions show that the new NCI-ELMO strategy always outperforms the original NCI method based on the promolecular approximation, while it is in close agreement with original NCI analyses based on fully quantum mechanical calculations. The new technique allows for unraveling the network of interactions in biological systems and to rapidly monitor their evolutions with time, with possible consequences on the design of new drugs.

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.

Hydrophilic interaction liquid chromatography for dalargin separation from its structural analogues and side products.

Retention behaviour of Dalargin and five peptide analogues of Leu-enkephalin, has been extensively studied by hydrophilic interaction liquid chromatography (HILIC) on a bare silica stationary phase (Atlantis® HILIC silica). The influence of buffer pH, ionic strength, and organic modifier content on peptide retentions was examined. Variation of organic modifier content (70-90% ACN) shows that, as expected, the most polar peptide, Dalargin, is the most retained. Moreover, at acidic pH, the retention mechanism for all the peptides studied seems to rely, mainly, on adsorption phenomenon. By varying the pswH buffer (between 4.4-7.5), we observed that the retention of all the peptides was mainly governed by their total number of charges, whatever the variation (increase or decrease) of their retention factor. At pswH 7.5, an increase of the cationic counter-ion concentration (NH4+) lead to a decrease of the retention factor of Dalargin, suggesting a weak cation exchange for this peptide. For the other peptides, the variation of the retention factors was negligible between 5-15mM. Above 15mM, the retention factors of all the peptides increased, probably due to an increase of the water layer thickness at the surface of the stationary phase. In the second part of the study, qualitative analysis of non-purified dalargin, resulting from solid-phase synthesis, was realized. Optimisation of the separation of the target peptide from its side products has been first performed with UV detection. Then, by coupling the HILIC column with ESI-MS, using the optimal separation conditions, it was possible to identify Dalargin and to propose the amino-acids sequence of its side-products.

Synthesis of Gly-ψ[(Z)CF═CH]-Phe, a Fluoroalkene Dipeptide Isostere, and Its Incorporation into a Leu-enkephalin Peptidomimetic.

A new Leu-enkephalin peptidomimetic designed to explore the hydrogen bond acceptor ability of the third peptide bond has been prepared and studied. This new analog is produced by replacing the third amide of Leu-enkephalin with a fluoroalkene. An efficient and innovative synthesis of the corresponding dipeptide surrogate Fmoc-Gly-ψ[(Z)CF═CH]-Phe-OH is described. The key step involves the alkylation of a tin dienolate from the less hindered face of its chiral sulfonamide auxiliary derived from camphor. Once its synthesis was complete, its incorporation into the peptidomimetic sequence was achieved on a solid support with chlorotrityl resin following the Fmoc strategy. The peptidomimetic was characterized using competition binding with [125I]-deltorphin I on membrane extracts of HEK293 cells expressing the mouse delta opioid receptor (DOPr) and based on its abilities to inhibit the electrically induced contractions of the mouse vas deferens and to activate the ERK1/2 signaling pathway in DRGF11/DOPr-GFP cells. Together with our previous observations, our findings strongly suggest that the third amide bond of Leu-enkephalin primarily acts as a hydrogen bond acceptor in DOPr. Consequently, this amide bond can be successfully replaced by an ester, a thioamide, or a fluoroalkene without greatly impacting the binding or biological activity of the corresponding analogs. The lipophilicity (LogD7.4) of the active analog was also measured. It appears that fluoroalkenes are almost as efficient at increasing the lipophilicity as normal alkenes.

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 ].

Determination of internal energy distributions of laser electrospray mass spectrometry using thermometer ions and other biomolecules.

The internal energy distributions for dried and liquid samples that were vaporized with femtosecond duration laser pulses centered at 800 nm and postionized by electrospray ionization-mass spectrometry (LEMS) were measured and compared with conventional electrospray ionization mass spectrometry (ESI-MS). The internal energies of the mass spectral techniques were determined by plotting the ratio of the intact parent molecular features to all integrated ion intensities of the fragments as a function of collisional energy using benzylpyridinium salts and peptides. Measurements of dried p-substituted benzylpyridinium salts using LEMS resulted in a greater extent of fragmentation in addition to the benzyl cation. The mean relative internal energies, were determined to be 1.62 ± 0.06, 2.0 ± 0.5, and 1.6 ± 0.3 eV for ESI-MS, dried LEMS, and liquid LEMS studies, respectively. Two-photon resonances with the laser pulses likely caused lower survival yields in LEMS analyses of dried samples but not liquid samples. In studies with larger biomolecules, LEMS analyses of dried samples from glass showed a decrease in survival yield compared with conventional ESI-MS for leucine enkephalin and bradykinin of ~15% and 11%, respectively. The survival yields for liquid LEMS analyses were comparable to or better than ESI-MS for benzylpyridinium salts and large biomolecules.

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.

Effects of pH and charge state on peptide assembly: the YVIFL model system.

Peptide oligomerization is necessary but not sufficient for amyloid fibril formation. Here, we use a combination of experiments and simulations to understand how pH influences the aggregation properties of a small hydrophobic peptide, YVIFL, which is a mutant form of [Leu-5]-Enkephalin. Transmission electron microscopy and atomic force microscopy measurements reveal that this peptide forms small aggregates under acidic conditions (pH = 2), but that extensive fibrillization only occurs under basic conditions (pH = 9 and 11). Ion-mobility mass spectrometry identifies key oligomers in the oligomerization process, which are further characterized at an atomistic level by molecular dynamics simulations. These simulations suggest that terminal charges play a critical role in determining aggregation propensity and aggregate morphology. They also reveal the presence of steric zipper oligomers under basic conditions, a possible precursor to fibril formation. Our experiments suggest that multiple aggregation pathways can lead to YVIFL fibrils, and that cooperative and multibody interactions are key mechanistic elements in the early stages of aggregation.

Estimation of activation energy from the survival yields: fragmentation study of leucine enkephalin and polyethers by tandem mass spectrometry.

A simple collision model for multiple collisions occurring in quadrupole type mass spectrometers was derived and tested with leucine enkaphalin a common mass spectrometric standard with well-characterized properties. Implementation of the collision model and Rice-Ramsperger-Kassel-Marcus (RRKM) algorithm into a spreadsheet software allowed a good fitting of the calculated data to the experimental survival yield (SY) versus collision energy curve. In addition, fitting also ensured to estimate the efficiencies of the kinetic to internal energy conversion for Leucine enkephalin in quadrupole-time-of-flight and triple quadrupole instruments. It was observed that the experimental SY versus collision energy curves for the leucine enkephalin can be described by the Rice-Ramsperger-Kassel (RRK) formalism by reducing the total degrees of freedom (DOF) to about one-fifth. Furthermore, this collision model with the RRK formalism was used to estimate the critical energy (E(o)) of lithiated polyethers, including polyethylene glycol (PEG), polypropylene glycol (PPG), and polytetrahydrofurane (PTHF) with degrees of freedom similar to that of leucine enkephalin. Applying polyethers with similar DOF provided the elimination of the effect of DOF on the unimolecular reaction rate constant. The estimated value of E(o) for PEG showed a relatively good agreement with the value calculated by high-level quantum chemical calculations reported in the literature. Interestingly, it was also found that the E(o) values for the studied polyethers were similar.

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.

Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides.

A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.

Near-edge X-ray absorption mass spectrometry of a gas-phase peptide.

We have studied the dissociation of the gas-phase protonated peptide leucine enkephalin [YGGFL+H](+) upon X-ray absorption in the region of the C K-edge. The yield of photodissociation products was recorded as a function of photon energy. The total photoabsorption yield is qualitatively similar to near-edge X-ray absorption fine structure (NEXAFS) spectra recorded from condensed phase peptides and proteins. Fragment specificity reveals distinct quantitative differences between spectra obtained for different masses. Fragmentation channels can be assigned to specific electronic transitions some of which are site specific. For instance, C 1s → π(★) excitations in the leucine enkephalin aromatic side chains lead to relatively little fragmentation, whereas such excitations along the peptide backbone induce strong fragmentation.

Superoxide reaction with tyrosyl radicals generates para-hydroperoxy and para-hydroxy derivatives of tyrosine.

Tyrosine-derived hydroperoxides are formed in peptides and proteins exposed to enzymatic or cellular sources of superoxide and oxidizing species as a result of the nearly diffusion-limited reaction between tyrosyl radical and superoxide. However, the structure of these products, which informs their reactivity in biology, has not been unequivocally established. We report here the complete characterization of the products formed in the addition of superoxide, generated from xanthine oxidase, to several peptide-derived tyrosyl radicals, formed from horseradish peroxidase. RP-HPLC, LC-MS, and NMR experiments indicate that the primary stable products of superoxide addition to tyrosyl radical are para-hydroperoxide derivatives (para relative to the position of the OH in tyrosine) that can be reduced to the corresponding para-alcohol. In the case of glycyl-tyrosine, a stable 3-(1-hydroperoxy-4-oxocyclohexa-2,5-dien-1-yl)-L-alanine was formed. In tyrosyl-glycine and Leu-enkephalin, which have N-terminal tyrosines, bicyclic indolic para-hydroperoxide derivatives were formed ((2S,3aR,7aR)-3a-hydroperoxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indole-2-carboxylic acid) by the conjugate addition of the free amine to the cyclohexadienone. It was also found that significant amounts of the para-OH derivative were generated from the hydroxyl radical, formed on exposure of tyrosine-containing peptides to Fenton conditions. The para-OOH and para-OH derivatives are much more reactive than other tyrosine oxidation products and may play important roles in physiology and disease.