Cascade Dissociations of Peptide Cation-Radicals. Part 2. Infrared Multiphoton Dissociation and Mechanistic Studies of z-Ions from Pentapeptides

Dissociations of z(4) ions from pentapeptides AAXAR where X=H, Y, F, W, and V produce dominant z(2) ions that account for >50 % of the fragment ion intensity. The dissociation has been studied in detail by experiment and theory and found to involve several isomerization and bond-breaking steps. Isomerizations in z(4) ions proceed by amide trans→cis rotations followed by radical-induced transfer of a β-hydrogen atom from the side chain, forming stable C(β) radical intermediates. These undergo rate-determining cleavage of the C(α)-CO bond at the X residue followed by loss of the neutral AX fragment, forming x(2) intermediates. The latter were detected by energy-resolved resonant excitation collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) experiments. The x(2) intermediates undergo facile loss of HNCO to form z(2) fragment ions, as also confirmed by energy-resolved CID and IRMPD MS(4) experiments. The loss of HNCO from the x(2) ion from AAHWR is kinetically hampered by the Trp residue that traps the OCNH radical group in a cyclic intermediate.     

Near-UV Photodissociation of Tryptic Peptide Cation Radicals. Scope and Effects of Amino Acid Residues and Radical Sites

Peptide cation-radical fragment ions of the z-type, [^●AXAR⁺], [^●AXAK⁺], and [^●XAR⁺], where X = A, C, D, E, F, G, H, K, L, M, N, P, Y, and W, were generated by electron transfer dissociation of peptide dications and investigated by MS³-near-ultraviolet photodissociation (UVPD) at 355 nm. Laser-pulse dependence measurements indicated that the ion populations were homogeneous for most X residues except phenylalanine. UVPD resulted in dissociations of backbone CO─NH bonds that were accompanied by hydrogen atom transfer, producing fragment ions of the [y_n]⁺ type. Compared with collision-induced dissociation, UVPD yielded less side-chain dissociations even for residues that are sensitive to radical-induced side-chain bond cleavages. The backbone dissociations are triggered by transitions to second (B) excited electronic states in the peptide ion R-CH^●-CONH- chromophores that are resonant with the 355-nm photon energy. Electron promotion increases the polarity of the B excited states, R-CH⁺-C^●(O^–)NH-, and steers the reaction to proceed by transfer of protons from proximate acidic C_α and amide nitrogen positions.

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A Database of Transition-Metal-Coordinated Peptide Cross-Sections: Selective Interaction with Specific Amino Acid Residues

Ion mobility mass spectrometry (IMS-MS) techniques were used to generate a database of 2288 collision cross sections of transition-metal-coordinated tryptic peptide ions. This database consists of cross sections for 1253 [Pep + X]²⁺ and 1035 [Pep + X + H]³⁺, where X²⁺ corresponds to Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺. This number of measurements enables the extraction of structural trends for transition-metal-coordinated peptide ions. The range of structures and changes in collision cross sections for X²⁺-coordinated species (compared with protonated species of the same charge state) is similar to Mg²⁺-coordinated species. This suggests that the structures are largely determined by similarities in cation size with differences among the cross section distributions presumably caused by X²⁺ interactions with specific functional groups offered by the residue R-groups or the peptide backbone. Cross section contributions for individual residues upon X²⁺ solvation are assessed with the derivation of intrinsic size parameters (ISPs). The comparison of the [Pep + X]²⁺ ISPs with those previously reported for [Pep + Mg]²⁺ ions displays a lower contribution to the cross section for His, carboxyamidomethylated Cys, and Met, and is consistent with specific metal-residue interactions identified within protein X-ray crystallography databases.

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Dissociations of Complexes Between Monovalent Metal Ions and Aromatic Amino Acid or Histidine

The fragmentations of [AA + M]⁺ complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag^?).

Chemical Modification of Amino Acid Residues in Human Plasminogen

The chemical modification of human plasminogen (HPg) was studied with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-acetylimidazole (NAI), 1,2-cyclohexanedione (CHD), chloramine T(Ch-T)and N-bromosuccinimide (NBS) as modifying reagents at its carboxyl group, tyrosine, arginine, methionine and tryptophan residues, respectively. The results indicate that tyrosine and arginine residues are not essential for HPg activity, while carboxyl groups, methionine and tryptophan residues are important for the activi-ty of HPg. The Keech and Farrant‘s kinetic analysis reveals that one tryptophan residue, one methionine residue and two carboxyl groups are essential for HPg activity.     

气相色谱-质谱联用法分析手印残留物中氨基酸的研究

氨基酸是汗液中的重要成分,也是显现手印的重要物质成分.针对手印物质中氨基酸含量少,检测要求灵敏度高的特点,本文利用微波衍生化气相色谱质潜联用技术分析手印中的氨基酸成分,该方法高效、快速、灵敏,为刑事科学技术手印显现部分的发展提供帮助.     

含丝氨酸和组氨酸残基手性肽核酸单体的合成

设计合成了含组氨酸残基碱基为胸腺嘧啶的手性肽核酸单体, 咪唑氨基的最终保护基为2,4-二硝基苯基(Dnp). 对文献合成方法进行了适当改进, 制备了两种含丝氨酸和组氨酸残基碱基为腺嘌呤的手性肽核酸单体, 以上化合物均可作为制备手性肽核酸的基本构建单元.     

Rapid Characterization by Thin-Layer Chromatography of Amino Acid and Peptide Derivatives Enzymically Prepared During Protease-Mediated Peptide Synthesis

Abstract

Thin-layer chromatographic position matching combined with color matching appeared to be a convenient method for characterization of the products of individual synthetic reactions during the protease-catalyzed synthesis of Leu- and Met-enkephalin.     

Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins

Peptide nucleic acid (PNA) is a nucleic acid analog which consists of purines, pyrimidines bases, and a neutrally charged peptide backbone. The PNA has the potential as a very useful biological probe for protein analysis since it has more in vivo biological stability as compared to DNA- or RNA-based aptamers. Usually, the addition of amino acids or peptide to the PNA backbone is used to improve its water-solubility and cell-permeability, but these modifications may affect the interaction between PNA and proteins. To date, the investigation of the interaction between PNA and proteins is rare, and there is no reported study about the effects of modifications. In this work, we designed two types of amino acid modified PNAs, (Lys)₂-PNA and (Glu)₂-PNA, which kept the same base sequence with 15-mer thrombin aptamer and had two basic lysine and two acidic glutamic acid residues on N-terminal of the peptide backbone, respectively. To rapidly assess the binding affinity and specificity of modified PNA and proteins, the online CE reaction method was developed to analyze the interactions of (Lys)₂-PNA/(Glu)₂-PNA and three proteins: thrombin (THB), single-strand DNA-binding protein (SSB) and human serum albumin (HSA). Meanwhile, the interactions of (Lys)₂-PNA/(Glu)₂-PNA and thrombin were compared with that of the corresponding complementary base sequence (Lys)₂-cPNA/(Glu)₂-cPNA and thrombin. The online CE reaction results showed that the interaction of (Lys)₂-PNA and (Glu)₂-PNA with three proteins was in the order of THB > SSB > HSA. However, (Lys)₂-PNA and (Lys)₂-cPNA showed similar binding affinity with thrombin; while the binding affinity of (Glu)₂-PNA with thrombin was stronger than that of (Glu)₂-cPNA with thrombin. Moreover, the binding constant K_b of (Glu)₂-PNA and three proteins was determined by affinity capillary electrophoresis (ACE). The online CE reaction eliminates the requirement of incubation, and thus it is fast in detection, and easy to operate with minimum cost. The method is particularly suitable for the interaction studies of expensive modified PNAs and proteins, and can assist the design of PNA probe that binds to proteins.     

Essential Amino Acid Residues Controlling the Unique Regioselectivity of Heme Oxygenase in Pseudomonas aeruginosa

Heme oxygenase (HO), an amphipathic microsomal protein, catalyzes the oxygen-dependent degradation of heme (iron-protoporphyrinIX) to alpha-biliverdin, CO, and free iron ion. Interestingly, all of HO regiospecifically oxidize the alpha-meso position of the heme to form alpha-biliverdin isomer while nonenzymatic heme degradation forms all four possible alpha-, beta-, gamma-, delta-biliverdin isomers at nearly identical yield. Recently, an interesting example has been found in HO (PigA) of the Gram-negative bacterium Pseudomonas aeruginosa, which does not produce alpha-biliverdin at all, but forms the mixture of beta- and gamma-biliverdins at a ratio of 3:7. While studying the mechanism of the unique regioselectivty of PigA, we found essential amino acid residues, Lys34, Lys132, and Phe189, controlling the unique regioselectivity of PigA. In this communication, we show that Lys34 and Lys132 are essential amino acid residues to hold the rotated heme in the active site of PigA via hydrogen-bonding interaction with the heme propionate and that Phe189 controls the product ratio of beta- and delta-biliverdins via steric interaction with heme substituents. These interactions place the beta- or delta-meso position of the heme at the oxidation site of PigA, leading to the unique regioselectivity.     

鸟嘌呤与氨基酸残基体系的极化力场

发展了应用于鸟嘌呤G和氨基酸残基体系的浮动电荷力场, 该力场明确定义了孤对电子和键的电荷和位置, 通过电荷随着环境的浮动来体现极化效应; 通过氢键拟合函数k_HB描绘了氢键键能. 应用量子化学方法, 对G与氨基酸残基体系从氢键、 几何结构及电荷分布3个方面展开计算及分析, 并以其为基准, 确定参数发展了适用于G与氨基酸残基氢键体系的ABEEMσπ PFF. 采用3种不同力场模拟目标分子的结构和性质. 模拟结果表明, 发展的ABEEMσπ PFF与量子化学方法具有最好的一致性, 可用于模拟生物大分子体系.     

The Early Life of a Peptide Cation-Radical. Ground and Excited-State Trajectories of Electron-Based Peptide Dissociations During the First 330 Femtoseconds

We report a new approach to investigating the mechanisms of fast peptide cation-radical dissociations based on an analysis of time-resolved reaction progress by Ehrenfest dynamics, as applied to an Ala-Arg cation-radical model system. Calculations of stationary points on the ground electronic state that were carried out with effective CCSD(T)/6-311++G(3df,2p) could not explain the experimental branching ratios for loss of a hydrogen atom, ammonia, and N–C_α bond dissociation in (AR + 2H)^+●. The Ehrenfest dynamics results indicate that the ground and low-lying excited electronic states of (AR + 2H)^+● follow different reaction courses in the first 330 femtoseconds after electron attachment. The ground (X) state undergoes competing loss of N-terminal ammonia and isomerization to an aminoketyl radical intermediate that depend on the vibrational energy of the charge-reduced ion. The A and B excited states involve electron capture in the Arg guanidine and carboxyl groups and are non-reactive on the short time scale. The C state is dissociative and progresses to a fast loss of an H atom from the Arg guanidine group. Analogous results were obtained by using the B3LYP and CAM-B3LYP density functionals for the excited state dynamics and including the universal M06-2X functional for ground electronic state calculations. The results of this Ehrenfest dynamics study indicate that reaction pathway branching into the various dissociation channels occurs in the early stages of electron attachment and is primarily determined by the electronic states being accessed. This represents a new paradigm for the discussion of peptide dissociations in electron based methods of mass spectrometry.     

Multiplexed Discrimination of Single Amino Acid Residues in Polypeptides in a Single SERS Hot Spot

The SERS-based detection of protein sequences with single-residue sensitivity suffers from signal dominance of aromatic amino acid residues and backbones, impeding detection of non-aromatic amino acid residues. Herein, we trap a gold nanoparticle in a plasmonic nanohole to generate a single SERS hot spot for single-molecule detection of 2 similar polypeptides (vasopressin and oxytocin) and 10 distinct amino acids that constitute the 2 polypeptides. Significantly, both aromatic and non-aromatic amino acids are detected and discriminated at the single-molecule level either at individual amino acid molecules or within the polypeptide chains. Correlated with molecular dynamics simulations, our results suggest that the signal dominance due to large spatial occupancy of aromatic rings of the polypeptide sidechains on gold surfaces can be overcome by the high localization of the single hot spot. The superior spectral and spatial discriminative power of our approach can be applied to single-protein analysis, fingerprinting, and sequencing.     

Residues of nortestosterone esters at injection sites. Part 1. Oral bioavailability.

Injectable esters of 17 beta-19-nortestosterone (beta-NT) have been used illegally for growth promotion in European Union (EU) cattle production. There are no data on oral bioavailability of free beta-NT from beta-NT esters. Adult non-pregnant female Landrace pigs (n = 4) were fitted with jugular vein cannulae and were treated as follows with an appropriate 'flushing out' period between each treatment: an intravenous bolus of beta-NT at 0.1 mg kg-1 body weight (NTi.v.), 300 mg nortestosterone phenylpropionate (NTPP) in oil of arachis administered per os (NTPPoil) and 300 mg NTPP in aqueous suspension administered per os (NTPPaq). Blood samples were taken for up to 24 h and analysed for free beta-NT by enzyme immunoassay. Calculation of the area under the plasma time concentration curve (AUC), allowed absolute bioavailability estimations for both oral treatments. Mean bioavailability of beta-NT following NTPPaq was 0.35% (range 0.25-0.41%) compared to 2.25% (range 0.86-2.85%) for NTPPoil. Mean +/- standard error of mean time of maximum plasma concentration (Tmas) of free beta-NT occurred earlier (2.3 +/- 0.6 h) with NTPPoil compared to NTPPaq (10.3 +/- 1.03 h) and the maximum plasma concentration (Cmax) was also greater for NTPPoil compared to NTPPaq (36.1 +/- 6.49 vs. 3.2 +/- 0.31 micrograms l-1). It is concluded that the presence of arachis oil enhances the absorption of NTPP. Notwithstanding the possible effects that cooking and other food processing may have on such injection site residues, extrapolation of these results to man suggests that consumption of NTPP-containing injection sites may result in peak plasma concentrations of free beta-NT which are significantly greater than those observed following injection of NT esters.     

Re-examination of the Reactivity of N-Carboxy Amino Acid Anhydrides 1. Polymerisation of Amino Acid NCAs in Acetonitrile and in the Solid State in Hexane

Ring-opening polymerisation of N-carboxy anhydrides of γ-benzyl-L- glutamate, L-alanine and L-leucine by a primary amine initiator in acetonitrile and in hexane was examined, with care taken to avoid contamination by moisture. The polymerisation of amino acid NCAs initiated by butylamine in hexane proceeded in the crystalline state (solid state) because the NCA crystals did not dissolve in hexane. Although amino acid NCAs were believed to polymerise completely in acetonitrile, polymerisation of the amino acid NCAs in acetonitrile was found to stop at around 20% conversion. As resulting polypeptides did not dissolve in acetonitrile, the polymer terminals were considered to be occluded in the polymer precipitate. On the other hand, each amino acid NCA was much more reactive in the solid state in hexane than in acetonitrile. Especially, L-leucine NCA showed remarkable reactivity in the solid state. The reactivity in the solid state was explained with reference to the crystal structure.     

Reductant-Induced Free Radical Fluoroalkylation of Nitrogen Heterocycles and Innate Aromatic Amino Acid Residues in Peptides and Proteins

A series of fluoroalkylated cyclic λ³-iodanes and their hydrochloride salts was prepared and used in a combination with sodium ascorbate in buffer or aqueous methanol mixtures for radical fluoroalkylation of a range of substituted indoles, pyrroles, tryptophan or its derivatives, and Trp residues in peptides. As demonstrated on several peptides, the aromatic amino acid residues of Trp, Tyr, Phe, and His are targeted with high selectivity to Trp. The functionalization method is biocompatible, mild, rapid, and transition-metal-free. The proteins myoglobin, ubiquitin, and human carbonic anhydrase I were also successfully functionalized.     

Multiplexed Discrimination of Single Amino Acid Residues in Polypeptides in a Single SERS Hot Spot

The SERS‐based detection of protein sequences with single‐residue sensitivity suffers from signal dominance of aromatic amino acid residues and backbones, impeding detection of non‐aromatic amino acid residues. Herein, we trap a gold nanoparticle in a plasmonic nanohole to generate a single SERS hot spot for single‐molecule detection of 2 similar polypeptides (vasopressin and oxytocin) and 10 distinct amino acids that constitute the 2 polypeptides. Significantly, both aromatic and non‐aromatic amino acids are detected and discriminated at the single‐molecule level either at individual amino acid molecules or within the polypeptide chains. Correlated with molecular dynamics simulations, our results suggest that the signal dominance due to large spatial occupancy of aromatic rings of the polypeptide sidechains on gold surfaces can be overcome by the high localization of the single hot spot. The superior spectral and spatial discriminative power of our approach can be applied to single‐protein analysis, fingerprinting, and sequencing.     

Peptide Enolates. C-Alkylation of Glycine Residues in linear tri-, tetra-, and pentapeptides via dilithium azadienediolates

The Boc-protected tripeptides Boc-Val-Gly-Leu-OH ( 1 ), Boc-Leu-Sar-Leu-OH ( 2 ), Boc-Leu-Gly-MeLeu-OH ( 3 ), and Boc-Val-BzlGly-Leu-OMe ( 64 ), tetrapeptide Boc-Leu-Gly-Pro-Leu-OH ( 9 ), and pentapeptides Boc-Val-Leu-Gly-Abu-Ile-OH ( 4 ), Boc-Val-Leu-Sar-MeAbu-Ile-OH ( 5 ), Boc-Val-Leu-Gly-MeAbu-Ile-OH ( 6 ), Boc-Val-Leu-BzlGly-BzlAbu-Ile-OH ( 7 ), and Boc-Val-Leu-Gly-BzlAbu-Ile-OH ( 8 ) are prepared by conventional methods (Schemes 4–7) or by direct benzylation of the corresponding precursors (Scheme 8). Polylithiations in THF give up to Li₆ derivatives containing glycine, sarcosine or N-benzylglycine Li enolate moieties ( A–H ). The polylithiated systems with a dilithium azadienediolate unit ( C, F–H ) are best generated by treatment with t-BuLi. The yields of alkylation of the glycine or sarcosine residues are up to 90%, with diastereoselectivities from nil to 9:1. Normally, the newly formed stereogenic center has (R)-configuration (i.e. a D -amino-acid residue is incorporated in the peptide chain). Electrophiles which can be employed with the highly reactive azadienediolate moiety are: MeI, EtI, i-PrI, allyl and benzyl bromide, ethyl bromoacetate, CO₂, and Me₂S₂ (Schemes 11–13). No epimerizations of the starting materials (racemization of the amino-acid residues) are observed under the strongly basic conditions. Selected conformations of the peptide precursors, generated by shock-freezing or by very slow cooling from room temperature to ?75° before lithiation, give rise to different stereoselectivities (Scheme 11). The latter and the yields can also be influenced by tempering the lithiated species before (Scheme 9) or after addition of the electrophiles (Scheme 12). Besides the desired products, starting peptides are recovered in the chromatographic purification and isolation procedures (material balance 80–95%). The results described are yet another demonstration that peptides may be backbone-modified through Li enolates, and that whole series of analogous peptide derivatives with various side chains may thus be produced from a given precursor.