Mass spectrometric evidence for mechanisms of fragmentation of charge-derivatized peptides

Mass spectrometry of charged derivatives of peptides has been a growing area of interest in the past decade. Fragmentation of charged derivatives of peptides is believed to be different from than that of protonated peptides when analyzed by collisionally activated dissociation-tandem mass spectrometry (CAD-MS/MS). The charged derivatives fragment by charge-remote fragmentation mechanisms, which are usually classified as high-energy (HE)-CAD processes. Our objective in the present study is to investigate the mechanism of fragmentation of charged derivatives of peptides when analyzed by matrix-assisted laser desorption/ionization-postsource decay-mass spectrometry (MALDI-PSD-MS) and electrospray ionization (ESI)-CAD-MS/MS (ion trap), which involve low-energy processes. Three major types of hydrogens (alpha, beta, and amide) are available for migration during the formation of the *a(n) ions (the predominant ion series produced from these charged derivatives). To pinpoint which of the three hydrogens is involved in the formation of the *a(n) ions, deuterium-labeled peptide derivatives with labels at specific sites were synthesized and analyzed by MALDI-PSD-MS and ESI-CAD-MS/MS. Our results suggest that the amide hydrogen of the residue at which the cleavage occurs shifts during the formation of *a(n); this observation serves as evidence for the mechanism proposed earlier by Liao et al. for fragmentation of such charged derivatives. The results also help elucidate the structure of the *a(n) ions, *b(n) ions, and others formed during cleavage at the proline residue, as well as the ions formed during loss of the C-terminal residue from these charged derivatives.     

Characterization of the 310-helix in model peptides by HRMAS NMR spectroscopy

A tetra- and a hepta-homopeptide from the C(alpha)-tetrasubstituted Aib (alpha-aminoisobutyric acid) residue were covalently linked to the POEPOP resin by the fragment-condensation approach. The conformational preferences of the two model peptides were determined for the first time on a solid support by means of high-resolution magic angle spinning NMR spectroscopy. The results obtained indicate that the Aib homopeptides adopt a regular 3(10)-helical structure even when they are covalently bound to a polymeric matrix, and thus confirm the remarkable conformational stability of the peptides rich in this amino acid. An ATR-FTIR spectroscopic investigation, performed in parallel, also confirmed that these polymer-bound peptides do indeed adopt a helical conformation. The results of this study open the possibility to exploit the peptide-resin conjugates based on C(alpha)-tetrasubstituted alpha-amino acids as helpful, structurally organized templates in molecular recognition studies or as catalysts in asymmetric synthesis.     

C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus

Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.     

Alpha-gamma hybrid peptides that contain the conformationally constrained gabapentin residue: characterization of mimetics of chain reversals

The crystal structures of four dipeptides that contain the stereochemically constrained gamma-amino acid residue gabapentin (1-(aminomethyl)cyclohexaneacetic acid Gpn) are described. The molecular conformation of Piv-Pro-Gpn-OH (1), reveals a beta-turn mimetic conformation, stabilized by a ten atom C[bond]H...O hydrogen bond between the Piv CO group and the pro S hydrogen of the Gpn CH(2)[bond]CO group. The peptides Boc-Gly-Gpn-OH (2), Boc-Aib-Gpn-OH (3), and Boc-Aib-Gpn-OMe (4) form compact, folded structures, in which a distinct reversal of polypeptide chain direction is observed. In all cases, the Gpn residue adopts a gauche,gauche (g,g) conformation about the C(gamma)[bond]C(beta) (theta(1)) and C(beta)[bond]C(alpha) (theta(2)) bonds. Two distinct Gpn conformational families are observed. In peptides 1 and 3, the average backbone torsion angle values for the Gpn residue are phi=98 degrees, theta(1)=-62 degrees, theta(2)=-73 degrees, and psi=79 degrees, while in peptide 2 and 4 the average values are phi=-103 degrees, theta(1)=-46 degrees, theta(2)=-49 degrees, and psi=-92 degrees. In the case of 1 and 3, an intramolecular nine-membered O[bond]H...O hydrogen bond is formed between the C[double bond]O of the preceding residue and the terminal carboxylic acid OH group. All four alpha-gamma dipeptide sequences yield compact folded backbone conformations; this suggests that the Gpn residue may be employed successfully in the design of novel folded structures.     

Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.     

Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/&mgr;ES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.     

A tandem MS precursor-ion scan approach to identify variable covalent modification of albumin Cys34: a new tool for studying vascular carbonylation

We developed a liquid chromatography electrospray ionisation multi-stage mass spectrometry (LC-ESI-MS/MS) approach based on precursor-ion scanning and evaluated it to characterize the covalent modifications of Cys34 human serum albumin (HSA) caused by oxidative stress and reactive carbonyl species (RCS) adduction. HSA was isolated and digested enzymatically to generate a suitable-length peptide (LQQCPF) containing the modified tag residue. The resulting LQQCPF peptides were identified by LC-ESI-MS/MS in precursor-ion scan mode and further characterized in product-ion scan mode. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of a series of LQQCPF derivatives containing Cys34 modified with different alpha,beta-unsaturated aldehydes and di and ketoaldehydes. We used a Boolean logic to enhance the specificity of the method: this reconstitutes a virtual current trace (vCT) showing the peaks in the three precursor-ion scans, marked by the same parent ion. The method was first evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4-hydroxy-hexenal, 4-hydroxy-trans-2-nonenal (HNE) and acrolein (ACR). Then we studied the Cys34 modification of human plasma incubated with mildly oxidized low-density lipoproteins (LDL), and the method easily identified the LQQCPF adducts with HNE and ACR. In other experiments, plasma was oxidized by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or by Fe2+/H2O2. In both conditions, the sulfinic derivative of LQQCPF was identified and characterized, indicating that the method is suitable not only for studying RCS-modified albumin, but also to check the oxidative state of Cys34 as a marker of oxidative damage.     

Toward direct determination of conformations of protein building units from multidimensional NMR experiments VI: chemical shift analysis of his to gain 3D structure and protonation state information

NMR--chemical shift structure correlations were investigated by using GIAO-RB3LYP/6-311++G(2d,2p) formalism. Geometries and chemical shifts (CSI values) of 103 different conformers of N'-formyl-L-histidinamide were determined including both neutral and charged protonation forms. Correlations between amino acid torsional angle values and chemical shifts were investigated for the first time for an aromatic and polar amino acid residue whose side chain may carry different charges. Linear correlation coefficients of a significant level were determined between chemical shifts and dihedral angles for CSI[(1)H(alpha)]/phi, CSI[(13)C(alpha)]/phi, and CSI[(13)C(alpha)]/psi. Protonation of the imidazole ring induces the upfield shift of CSI[(13)C(alpha)] for positively charged histidines and an opposite effect for the negative residue. We investigated the correspondence of theoretical and experimental (13)C(alpha), (13)C(beta), and (1)H(alpha) chemical shifts and the nine basic conformational building units characteristic for proteins. These three chemical shift values allow the identification of conformational building units at 80% accuracy. These results enable the prediction of additional regular secondary structural elements (e.g., polyProlineII, inverse gamma-turns) and loops beyond the assignment of chemical shifts to alpha-helices and beta-pleated sheets. Moreover, the location of the His residue can be further specified in a beta-sheet. It is possible to determine whether the appropriate residue is located at the middle or in a first/last beta-strand within a beta-sheet based on calculated CSI values. Thus, the attractive idea of establishing local residue specific backbone folding parameters in peptides and proteins by employing chemical shift information (e.g., (1)H(alpha) and (13)C(alpha)) obtained from selected heteronuclear correlation NMR experiments (e.g., 2D-HSQC) is reinforced.     

Hydrogen‐deuterium exchange of α‐carbon protons and fragmentation pathways in N‐methylated glycine and alanine‐containing peptides derivatized by quaternary ammonium salts

Recently, we developed a selective and efficient method of hydrogen‐deuterium exchange (HDX) at the α‐carbon (α‐C) of sarcosine residue (N‐methylglycine) in model peptides [B?chor et al. J. Mass Spectrom. 2014, 49, 43]. Here, we report the influence of quaternary ammonium (QA) group on HDX at the α‐C of sarcosine and N‐methylalanine in peptides. The obtained results suggest a significant acceleration of the HDX in sarcosine residue caused by the presence of QA. The effect depends on the distance between the sarcosine residue and QA moiety. The deuterons, introduced at α‐C, are resistant to the back‐exchange in acidic aqueous solution. The collision induced dissociation of the deuterium‐labeled analogs of QA‐tagged oligosarcosine peptides without mobile hydrogen revealed the mobilization of the hydrogens localized at α‐C of sarcosine residue. Copyright © 2014 John Wiley & Sons, Ltd.     

Differentiation of Boc- alpha,beta- and beta,alpha-peptides and a pair of diastereomeric beta,alpha-dipeptides by positive and negative ion electrospray tandem mass spectrometry (ESI-MS/MS)

Positive and negative ion electrospray ionization (ESI) tandem mass spectral study of a new series of hybrid peptides, viz, BocN-alpha,beta-peptides and BocN-beta,alpha-peptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and L-Ala has been carried out. The alpha,beta-peptides have been differentiated from beta,alpha-peptides by the collision-induced dissociation (CID) of [M + H]+ and [M - H]- ions in positive and negative ion ESI-MS respectively. The fragment ion [M + H - C(CH3)3 + H]+ formed from [M + H]+ ions by the loss of 2-methyl-prop-2-ene in alpha,beta-peptides with L-Ala at the N-terminus is insignificant or totally absent for beta,alpha-peptides which have the Caa (S) at N-terminus. The fragment ion [M - H-C(CH3)3OH - HNCO]- formed from [M - H]- of beta,alpha-peptide acids is totally absent for alpha,beta-peptide acids. This has been attributed to the absence of the beta-methylene group in alpha,beta-peptides, and the participation of the beta-methylene group in the loss of HNCO in beta,alpha-peptide acids is confirmed by the deuteration experiments. The CID of [M + H-Boc + H]+ ions of these peptides also produce characteristic fragmentation. In the CID spectra of alpha,beta-peptides, the b(n)+ ions and the resulting y(n)+ ions occur at a mass difference of 243 and 71 Da corresponding to the successive losses of Caa and L-Ala, whereas a mass difference of 71 and 243 Da is observed for beta,alpha-peptides. In contrast to the CID of protonated peptides, the CID of [M - H]- ions of the alpha,beta- and beta,alpha-peptide acids do not give b(n)- ions and show abundant z(n) (-) ions. Further, a pair of diastereomeric dipeptide esters and acids have been distinguished by the CID of [M + H]+ ions. The loss of 2-methyl-prop-2-ene is more pronounced for Boc-NH-Caa(R)-D-Ala-OCH3 (21) and Boc-NH-Caa(R)-D-Ala-OH (23) with Caa (R) at the N-terminus, whereas it is totally absent for Boc-NH-Caa (S)-D-Ala-OCH3 (22) and Boc-NH-Caa(S)-D-Ala-OH (24) peptides, which have Caa (S) at the N-terminus. Thus, on the basis of our previous and present studies, we propose that the CID of [M + H]+ ions provides a simple and useful method for distinguishing the configuration of Caa (S or R) at the N-terminus of BocN-carbo beta,alpha- and beta,beta-dipeptides.     

Peptide backbone reconstruction using dead-end elimination and a knowledge-based forcefield

A novel, yet simple and automated, protocol for reconstruction of complete peptide backbones from C(alpha) coordinates only is described, validated, and benchmarked. The described method collates a set of possible backbone conformations for each set of residue triads from a structural library derived from the PDB. The optimal permutation of these three residue segments of backbone conformations is determined using the dead-end elimination (DEE) algorithm. Putative conformations are evaluated using a pairwise-additive knowledge-based forcefield term and a fragment overlap term. The protocol described in this report is able to restore the full backbone coordinates to within 0.2-0.6 A of the actual crystal structure from C(alpha) coordinates only. In addition, it is insensitive to errors in the input C(alpha) coordinates with RMSDs of 3.0 A, and this is illustrated through application to deliberately distorted C(alpha) traces. The entire process, as described, is rapid, requiring of the order of a few minutes for a typical protein on a typical desktop PC. Approximations enable this to be reduced to a few seconds, although this is at the expense of prediction accuracy. This compares very favorably to previously published methods, being sufficiently fast for general use and being one of the most accurate methods. Because the method is not restricted to the reconstruction from only C(alpha) coordinates, reconstruction based on C(beta) coordinates is also demonstrated.     

Fragmentation characteristics of peptide-metal ion adducts under matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometric conditions

Fragmentation reactions of sodium-cationized enkephalin peptides generated by matrix-assisted laser desorption/ionization were studied using post-source decay (PSD) with a reflectron time-of-flight mass spectrometer. Several matrices and analyte-matrix sample preparation methods were evaluated for high-intensity ion currents that could last for the entire PSD analysis. A triple dried-droplet sample preparation procedure with 2,5-dihydroxybenzoic acid as the matrix was found to yield abundant longer-lasting ion signals of the peptide-Na(+) ion adducts. The principal decay product of these adduct ions is the [b(n-1) + Na + OH](+) ion, which provides an unambiguous identification of the C-terminal residue of a peptide. In some peptides, the loss of a second residue from the C-terminus is also observed. No other sequence-specific ions were observed.     

Effect of the basic residue on the energetics, dynamics, and mechanisms of gas-phase fragmentation of protonated peptides

The effect of the basic residue on the energetics, dynamics, and mechanisms of backbone fragmentation of protonated peptides was investigated. Time-resolved and collision energy-resolved surface-induced dissociation (SID) of singly protonated peptides with the N-terminal arginine residue and their analogues, in which arginine is replaced with less basic lysine and histidine residues, was examined using a specially configured Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). SID experiments demonstrated different kinetics of formation of several primary product ions of peptides with and without arginine residue. The energetics and dynamics of these pathways were determined from Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data. Comparison between the kinetics and energetics of fragmentation of arginine-containing peptides and the corresponding methyl ester derivatives provides important information on the effect of dissociation pathways involving salt bridge (SB) intermediates on the observed fragmentation behavior. Because pathways involving SB intermediates are characterized by low threshold energies, they efficiently compete with classical oxazolone and imine/enol pathways of arginine-containing peptides on a long time scale of the FTICR instrument. In contrast, fragmentation of histidine- and lysine-containing peptides is largely determined by canonical pathways. Because SB pathways are characterized by negative activation entropies, fragmentation of arginine-containing peptides is kinetically hindered and observed at higher collision energies as compared to their lysine- and histidine-containing analogues.     

Matrix-assisted laser desorption/ionisation mass spectrometric response factors of peptides generated using different proteolytic enzymes

Matrix-assisted laser desorption/ionisation (MALDI) mechanisms and the factors that influence the intensity of the ion signal in the mass spectrum remain imperfectly understood. In proteomics, it is often necessary to maximise the peptide response in the mass spectrum, especially for low abundant proteins or for proteolytic peptides of particular significance. We set out to determine which of the common proteolytic enzymes give rise to peptides with the best response factors under MALDI conditions. Standard proteins were enzymatically digested using four common proteases. We assessed relative response factors by coanalyzing the resulting digests. Thus, when tryptic peptides were added in equimolar quantities to their corresponding Asp-N, chymotrypsin and Glu-C digests, tryptic peptide signals were always predominant in the resulting MALDI mass spectra. Observable peaks attributable to non-tryptic peptides generally contained a terminal basic residue. It was proposed that a terminal basic residue has a disproportionate influence upon gas-phase basicity, and this hypothesis was supported by experiments with model isotopically labelled peptides. Experiments applying Cook's kinetic method showed that the peptide with a C-terminal arginine residue was more basic than the equivalent peptide with an N-terminal arginine, which was more basic than the peptide in which the arginine was mid-chain. Thus, the observation of the higher MALDI mass spectrometry response factors of tryptic peptides in comparison with peptides derived using other proteolytic enzymes corresponds with higher gas-phase basicities and may, along with other factors such as the complexity of the digest, influence the choice of enzyme in "bottom-up" proteomic experiments.     

Probing the role of the C-H...O hydrogen bond stabilized polypeptide chain reversal at the C-terminus of designed peptide helices. Structural characterization of three decapeptides

The structural characterization in crystals of three designed decapeptides containing a double d-segment at the C-terminus is described. The crystal structures of the peptides Boc-Leu-Aib-Val-Xxx-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe, (Xxx = Gly 2, (D)Ala 3, Aib 4) have been determined and compared with those reported earlier for peptide 1 (Xxx = Ala) and the all l analogue Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-OMe, which yielded a perfect right-handed alpha-helical structure. Peptides 1 and 2 reveal a right-handed helical segment spanning residues 1 to 7, ending in a Schellman motif with (D)Ala(8) functioning as the terminating residue. Polypeptide chain reversal occurs at residue 9, a novel feature that appears to be the consequence of a C-H.O hydrogen bond between residue 4 C(alpha)H and residue 9 CO groups. The structures of peptides 3 and 4, which lack the pro R hydrogen at the C(alpha) atom of residue 4, are dramatically different. Peptide 3 adopts a right-handed helical conformation over the 1 to 7 segment. Residues 8 and 9 adopt alpha(L) conformations forming a C-terminus type I' beta-turn, corresponding to an incipient left-handed twist of the polypeptide chain. In peptide 4, helix termination occurs at Aib(6), with residues 6 to 9 forming a left-handed helix, resulting in a structure that accommodates direct fusion of two helical segments of opposite twist. Peptides 3 and 4 provide examples of chiral residues occurring in the less favored sense of helical twist; (D)Ala(4) in peptide 3 adopts an alpha(R) conformation, while (L)Val(7) in 4 adopts an alpha(L) conformation. The structural comparison of the decapeptides reported here provides evidence for the role of specific C-H.O hydrogen bonds in stabilizing chain reversals at helix termini, which may be relevant in aligning contiguous helical and strand segments in polypeptide structures.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric observation of a peptide triplet induced by thermal cleavage of cystine.

Heat-induced (90 degrees C, 30 min) beta-elimination of a cystine residue leads to cleavage of a disulfide bond and produces a set of three peptides with a cysteine residue, a thiocysteine residue (+32Da), and a dehydroalanine residue (-34Da). This characteristic feature was observed from somatostatin and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Mass spectrometric observation of this triplet is useful in identifying the presence of a cystine residue in a peptide, and could assist mass spectrometric identification of the peptide from a database.     

Diethylhydrogensilyl-cyclic diethylsilylene derivatives in gas chromatography-mass spectrometry of prostanoids. III. F alpha-prostaglandins and thromboxane B2 derivatives

The gas chromatographic (GC) and GC-mass spectrometric properties of the diethylhydrogensilyl-cyclic diethylsilylene (DEHS-DES) derivatives of prostaglandin (PG) F1 alpha methyl ester, PGF2 alpha methyl ester, 6-keto-PGF1 alpha methyl ester-alkyloxime and thromboxane (TX) B2 methyl ester-alkyloxime and the DES derivative of 13,14-dihydro-15-keto-PGF2 alpha methyl ester-alkyloxime were studied. When the ketonic PGs and TXB2 were converted into their methyloxime derivatives, the methylene unit values of these five prostanoid derivatives were slightly greater than those of the corresponding dimethylethylsilyl ether derivatives. When the ketonic PGs were converted into their corresponding ethyloxime derivatives, baseline separation was achieved in 20 min by use of a methylsilicone cross-linked fused-silica capillary column. The mass spectra of these derivatives were characterized by the ion at m/z 157 for F alpha prostaglandins and m/z 269 for TXB2. The major fragmentations were directed by the DES group, and other fragmentations common to the prostanoid derivatives were losses of an ethyl radical at the silicon atom, C5H11 hydrocarbon fragment, diethylhydrogensilanol and C15-C20 hydrocarbon fragment. The mass fragmentations of these prostanoid derivatives are briefly discussed. GC with high-resolution selected-ion monitoring was carried out for the TXB2 derivative at a resolution of 8000 by monitoring the ion at m/z 269.1573. A 25-pg amount of this derivative showed a well shaped doublet with a signal-to-noise ratio of more than 300:1.     

Hydrazone Photoswitches for Structural Modulation of Short Peptides

Molecules that undergo light-driven structural transformations constitute the core components in photoswitchable molecular systems and materials. Among various families of photoswitches, photochromic hydrazones have recently emerged as a novel class of photoswitches with superb properties, such as high photochemical conversion, spectral tunability, thermal stability, and fatigue resistance. Hydrazone photoswitches have been adopted in various adaptive materials at different length scales, however, their utilization for modulating biomolecules still has not been explored. Herein, we present new hydrazone switches that can photomodulate the structures of short peptides. Systematic investigation on a set of hydrazone derivatives revealed that installation of the amide group does not significantly alter the photoswitching behaviors. Importantly, a hydrazone switch comprising an upper phenyl ring and a lower quinolinyl ring was effective for structural control of peptides. We anticipate that this work, as a new milestone in the research of hydrazone switches, will open a new avenue for structural and functional control of biomolecules.     

Ultrafast excited state dynamics in protonated GWG and GYG tripeptides

The excited state dynamics of two protonated tripeptides GWG and GYG has been investigated by pump/probe femtosecond measurements on photofragments, to explore the behavior of peptides where the terminal protonated amino group is not directly linked to the aromatic residue. The dynamics observed are short and surprisingly similar to the dynamics observed on the free protonated tryptophan and tyrosine aromatic amino acids. Specific photofragments observed for protonated GWG are related to the formation of a radical species WG degrees (+) after cleavage of the C(alpha)-N bond near the tryptophan residue.     

Peptide backbone folding induced by the C(alpha)-tetrasubstituted cyclic alpha-amino acids 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) and 1-aminocyclopentane-1-carboxylic acid (Ac5c). A joint computational and experimental study

The conformational study of a new group of synthetic peptides containing 4-amino-1,2-dithiolane-4-carboxylic acid (Adt), a cysteine-related achiral residue, has been carried out through a joint application of computational and experimental methodologies. Molecular Dynamics simulations clearly suggest the tendency of this molecule to adopt a gamma-turn conformation in vacuum and help in analyzing the complex and crucial conformational behaviour of the dithiolane ring which appears to preferentially adopt a C(S)-like structure. Electronic structure calculations carried out in solution using the Density Functional Theory also indicate the preservation of the gamma-like folding in apolar solvents and the helix-like one in more polar solvents. A comparison with the achiral 1-aminocycloalkane-1-carboxylic acid (Ac5c) has been carried out using the same computational tools. NMR and IR data on dipeptide derivatives containing the Adt or Ac5c residue show that in chloroform solution all the models prefer a gamma-turn structure, centered at the cyclic residue, stabilized by an intramolecular H-bond, whereas in a more polar solvent, i.e. dimethyl sulfoxide, this folding is not maintained. The experimental conformational studies, extended to N-Boc protected tripeptides, clearly indicate the remarkable tendency of both the five-membered C(alpha)-tetrasubstituted cyclic amino acids Adt and Ac5c to induce the gamma-turn structure also in models able to adopt the beta-bend conformation.