Phase equilibria in a system of aqueous arginine with an octane solution of sulfonic acid

The extraction of arginine (Arg) from aqueous salt (0.1 M NaCl) solutions with a sulfo extractant in a wide range of pH values and amino acid concentrations was studied. The 0.1 M solution of dinonylnaphthalenesulfonic acid (HD) in octane was used as an extractant. The degree of extraction was found to be high at pH 0.8–9.0. This can be explained by the effect of additional intermolecular interactions in the extractant phase involving the guanidine group of Arg.     

Synthesis, NMR Characterization, and a Simple Application of Lithium Borotritide

LiBH(4) is a powerful and selective reagent for regiospecific reduction reactions. A simple synthesis of LiB(3)H(4) at near theoretical specific radioactivity is reported. We have treated Li(3)H synthesized from tritium gas ((3)H(2), approximately 98%) with BBr(3) to produce LiB(3)H(4) (specific activity = 4120 GBq/mmol = 110 Ci/mmol. The maximum theoretical specific activity of LiB(3)H(4) is 4252 GBq/mmol = 115.04 Ci/mmol; 1 matom of (3)H = 1063 GBq = 28.76 Ci.) The tritium labeling performance of the reagent was tested by an exemplary reduction of 2-naphthaldehyde to 2-naphthalenemethanol. LiB(3)H(4) and the reduction products were characterized by a combination of (1)H, (3)H, and (11)B NMR techniques, as appropriate.     

The arginine anomaly: arginine radicals are poor hydrogen atom donors in electron transfer induced dissociations

Arginine amide radicals are generated by femtosecond electron transfer to protonated arginine amide cations in the gas phase. A fraction of the arginine radicals formed (2-amino-5-dihydroguanid-1'-yl-pentanamide, 1H) is stable on the 6.7 micros time scale and is detected after collisional reionization. The main dissociation of 1H is loss of a guanidine molecule from the side chain followed by consecutive dissociations of the 2-aminopentanamid-5-yl radical intermediate. Intramolecular hydrogen atom transfer from the guanidinium group onto the amide group is not observed. These results are explained by ab initio and density functional theory calculations of dissociation and transition state energies. Loss of guanidine from 1H is calculated to require a transition state energy of 68 kJ mol(-)(1), which is substantially lower than that for hydrogen atom migration from the guanidine group. The loss of guanidine competes with the reverse migration of the arginine alpha-hydrogen atom onto the guanidyl radical. RRKM calculations of dissociation kinetics predict the loss of guanidine to account for >95% of 1H dissociations. The anomalous behavior of protonated arginine amide upon electron transfer provides an insight into electron capture and transfer dissociations of peptide cations containing arginine residues as charge carriers. The absence of efficient hydrogen atom transfer from charge-reduced arginine onto sterically proximate amide group blocks one of the current mechanisms for electron capture dissociation. Conversely, charge-reduced guanidine groups in arginine residues may function as radical traps and induce side-chain dissociations. In light of the current findings, backbone dissociations in arginine-containing peptides are predicted to involve excited electronic states and proceed by the amide superbase mechanism that involves electron capture in an amide pi* orbital, which is stabilized by through-space coulomb interaction with the remote charge carriers.     

Arginine-mimic labeling with guanidinoethanethiol to increase mass sensitivity of lysine-terminated phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) generally shows better mass sensitivity for arginine-terminated peptides than for lysine-terminated peptides, presumed to arise from the higher proton affinity of the guanidine group in arginine. Here, we report a new method for analyzing phosphopeptides in which phosphopeptides are labeled with a novel chemical tag, guanidinoethanethiol (GET), by a beta-elimination/Michael addition before MS analysis. GET labeling converts phosphoserine into guanidinoethylcysteine (Gec) containing a guanidine moiety, along with an increase in mass of 21.1 Da. GET-labeled peptides are detected by MALDI MS with greatly increased peak intensities compared to those of intact phosphopeptides. In particular, GET labeling of lysine-terminated phosphopeptides dramatically increased peak intensity. GET labeling of lysine-terminated phosphopeptides improved sensitivity up to 22 times compared to that of the corresponding aminoethanethiol (AET) labeling, in which AET was used as a labeling tag containing an amino group instead of the guanidine group. These results show the guanidine group plays a very important role in increasing the observed sensitivity of MALDI MS for labeled peptide, derivatized from serine-phosphorylated peptides.     

Improving fragmentation of poorly fragmenting peptides and phosphopeptides during collision-induced dissociation by malondialdehyde modification of arginine residues

Despite significant technological and methodological advancements in peptide sequencing by mass spectrometry, analyzing peptides that exhibit only poor fragmentation upon collision-induced dissociation (CID) remains a challenge. A major cause for unfavorable fragmentation is insufficient proton 'mobility' due to charge localization at strongly basic sites, in particular, the guanidine group of arginine. We have recently demonstrated that the conversion of the guanidine group of the arginine side chain by malondialdehyde (MDA) is a convenient tool to reduce the basicity of arginine residues and can have beneficial effects for peptide fragmentation. In the present work, we have focused on peptides that typically yield incomplete sequence information in CID-MS/MS experiments. Energy-resolved tandem MS experiments were carried out on angiotensins and arginine-containing phosphopeptides to study in detail the influence of the modification step on the fragmentation process. MDA modification dramatically improved the fragmentation behavior of peptides that exhibited only one or two dominant cleavages in their unmodified form. Neutral loss of phosphoric acid from phosphopeptides carrying phosphoserine and threonine residues was significantly reduced in favor of a higher abundance of fragment ions. Complementary experiments were carried out on three different instrumental platforms (triple-quadrupole, 3D ion trap, quadrupole-linear ion trap hybrid) to ascertain that the observation is a general effect.     

Solid-phase synthesis of arginine-containing peptides and fluorogenic substrates using a side-chain anchoring approach

Attachment of an amino acid to a solid support by its side chain is sometimes necessary to take advantage of an alpha-carboxylic group available for diverse modifications, including the incorporation of a fluorophore for the preparation of fluorogenic substrates. In contrast to most other amino acids, anchoring the guanidinium group of an arginine to a resin requires the use of a supplementary linker. To avoid the usually multistep synthesis of such a linker as well as its difficult attachment to the guanidine group, we developed a simple method where the guanidine group is built on a Rink amide resin. Our strategy followed the steps of guanidine formation: (i) addition of an isothiocyanate derivative of ornithine to the amino group of a solid support, yielding Nomega-linked thiocitrulline; (ii) S-methylation of thiourea; (iii) guanidinylation using ammonium acetate. Cleavage of the resin generated the arginine-containing compound, the amine group of the resin becoming part of the guanidine. We have demonstrated the usefulness of this method by the synthesis of a series of fluorogenic substrates for trypsin-like serine proteases, which were obtained in high yield and purity. Then, our strategy also allowed generation from the same precursor differentially substituted arginine derivatives, including Nomega-methyl- and Nomega-ethylarginines. The ability to prepare such analogues together with the intermediates thiocitrulline and S-methylisothiocitrulline from a unique precursor while the alpha-amine and carboxylic groups remain available for modification also makes this method a powerful tool for combinatorial solid-phase synthesis of NO synthase inhibitors.     

Deuterium labeling of glyprolines by isotope exchange

Deuterium-labeled 5-oxo-Pro-Arg-Pro, 5-oxo-Pro-His-Pro-Gly-Pro-NH₂, and Phe-Pro-LeuPro-Ala were synthesized. The effect of the method of peptide deposition on the catalyst, the catalyst nature, temperature, and structure of peptides on the inclusion of deuterium with the formation of the appropriate number of isotopomers was demonstrated. The 5-oxo-Pro-Arg-Pro, 5-oxo-Pro-His-Pro-Gly-Pro-NH₂, and Phe-Pro-Leu-Pro-Ala peptides containing, on average, 4.6, 7.7, and 5.4 deuterium atoms per peptide molecule, respectively, were obtained.     

Tritiation of Peptides to High Specific Radioactivity. Part 1. Synthesis and Biological Properties of [13-(3H4)Norvaline]-α-MSH and of [2,23-Bis((3H2)tyrosine)]ACTH(1–24)

α-MSH and ACTH(1–24) were tritiated to high specific radioactivity (> 100 Ci/mmol) using a new tritiation apparatus with which the tritiation reaction can be performed at slightly elevated pressure. This allows short reaction times with the least possible damage to the molecule. The starting compounds for the tritiation were [13-propargylglycine]α-MSH and [2,23-Bis(3′,5′-diiodotyrosine)]ACTH(1–24). Both tritiations were quantitative and yielded products of high purity, full biological activity, and with a specific radioactivity of 115 Ci/mmol and 100 Ci/mmol, respectively.     

Synthesis of Tritium and Deuterium Labelled Agmatine

Agmatine is a promising anti-opioid dependence and relapse drug1~2 discovered and developed by Beijing Institute of Pharmacology and Toxicology, but the physiological role of agmatine in inhibition of tolerance to and dependence on opioid are not well est…     

Preparation of substituted cinnamic acids labelled with deuterium and tritium at the ring positions

A series of substituted cinnamic acids labelled at the ring positions with deuterium or tritium has been easily obtained via the Doebner modification of Knoevenagel condensation reaction between labelled benzaldehyde derivatives and malonic acid in pyridine. The substituted benzaldehyde precursors were synthesized by isotope exchange method in deuterated or tritiated water at 80 °C in acidic medium. Ring-tritiated substituted cinnamic acids with specific activity ranging from 207 GBq /5.6 Ci/ mol^–1 to 4366 GBq /118 Ci/ mol^–1 was obtained using ca. 37 GBq /1 Ci/ of HTO.     

Hydrogen-deuterium exchange and selective labeling of deprotonated amino acids and peptides in the gas phase

Hydrogen-deuterium exchange reactions of deprotonated amino acids and small peptides were studied. Selective labeling can be carried out at the alpha-amino group of lysine (2 of 4 labile hydrogens undergo exchange with CF3CH2OD) and the guanidine side chain of arginine (3 of 6 labile hydrogens undergo exchange with CH3CH2OD and C6H5CH2OD). Differential labeling of peptides also was accomplished, and the extent of H/D exchange is dependent on the amino acids which are present as well as their order in the chain.     

A density functional theory study of a concerted mechanism for proton exchange between amino acid side chains and water

 A concerted mechanism for proton exchange between water and the amino acid side chains of cysteine, serine, arginine and glutamic acid has been investigated with hybrid density functional theory. The models used include, besides the amino acid side chain, a number of water molecules ranging from one to five in some cases. The modeling of the amino acids without their backbones is shown to be an excellent approximation. Long-range polarization effects were incorporated through a dielectric cavity method allowing a better comparison to existing measurements for free amino acids in water. The barriers converge rather fast with the number of water molecules for all the present amino acids and the converged values are in reasonable agreement with experiments with discrepancies in the range 2–6 kcal/mol. The dielectric effects were found to be small for all systems except cysteine, where there is a lowering of the barrier by 3–5 kcal/mol. The transition states for these concerted pathways form rings in which the separated charges can be stabilized. Received: 25 October 1999 / Accepted: 5 April 2000 / Published online: 21 June 2000     

Synthesis of Deuterium- or Tritium-Labeled Norepinephrine and Evaluation of Its Biological Activity

A labeled norepinephrine analogue was prepared by bromination of norepinephrine with subsequent debromination with tritium or deuterium gas. The yield of tritiated norepinephrine was 10–20% with a molar radioactivity of 27 Ci/mmol. The biological activity of tritiated norepinephrine was evaluated by analyzing its specific capture by noradrenergic neutrons in thick sections of the brainstem and midbrain of Wistar rats, and in a suspension of cells of pheochromocytoma PC12 expressing the membrane transporter of norepinephrine. It is shown that tritium-labeled norepinephrine synthesized by the proposed method does not differ in biological activity from the natural norepinephrine.     

Experimental evidence for an inverse hydrogen migration in arginine radicals

Radicals formed by electron transfer to protonated arginine have been predicted by theory to undergo an inverse migration of the hydrogen atom from the C(alpha) position to the guanidine carbon atom. Experiments are reported here that confirm that a fraction of arginine and arginine amide radicals undergo such an inverse hydrogen migration. The rearranged arginine and arginine amide C(alpha) radicals are detected as stable anions after charge inversion by collisions with Cs atoms of precursor cations at 3 and 50 keV kinetic energies. RRKM calculations on the B3-PMP2/aug-cc-pVTZ potential energy surface indicate that arginine radicals undergo rapid rotations of the side chain to reach conformations suitable for C(alpha)-H transfer, which is calculated to be fast (k > 10(9) s(-1)) in radicals formed by electron transfer. By contrast, H-atom transfer from the guanidine group onto the carboxyl or amide C=O groups is >50 times slower than the C(alpha)-H atom migration. The guanidine group in arginine radicals is predicted to be a poor hydrogen-atom donor but a good H-atom acceptor and thus can be viewed as a radical trap. This property can explain the frequent observation of nondissociating cation radicals in electron capture and electron transfer mass spectra of arginine-containing peptides.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

Viscosities and apparent molar volumes of some amino acids in water and in 6M guanidine hydrochloride at 25°C

Apparent molar volumes and viscosity B- and D- coefficients of amino acids glycine, valine, proline, serine and arginine have been determined in water and in aqueous 6M guanidine hydrochloride (GuHCl) solution at 25°C. Transfer volumes and transfer viscosity B-coefficients were evaluated for the amino acids studied in going from water to 6M GuHCl. These transfer properties which were all positive were interpreted in terms of strong interactions of GuHCl molecules with the charged centers of amino acid molecules. A comparison of results obtained in this work for GuHCl and those obtained from literature for urea has shown that GuHCl has stronger interactions than urea with amino acids. This finding explained the previous experimental observations on GuHCl being a stronger denaturing agent than urea for proteins.     

Cross-oxidation of angiotensin II by glycerophosphatidylcholine oxidation products

Peptide and protein lipoxidation is a deleterious process which has been related to several degenerative conditions. In the present study, the interaction of lipid secondary oxidation products with peptides was investigated by evaluating the modifications occurring to angiotensin II (Ang-II) in the presence of an oxidizing polyunsaturated glycerophospholipid (1-palmitoyl-2-arachidonoyl-glycerophosphatidylcholine, PAPC). PAPC oxidation was promoted by Fenton chemistry and the oxidation products were incubated with Ang-II. The reaction products were finally analysed by off-line nanospray high-performance liquid chromatography/matrix-assisted laser desorption/ionization tandem mass spectrometry (nano-HPLC/MALDI-TOF-MS/MS). Ang-II was found to form adducts with 26 different aldehydes, leading to 37 distinct reaction products. Modification of Ang-II occurred through reaction with reactive carbonyl species (RCS) originating from fatty acyl chain cleavage, while interactions with the oxidized phospholipid could not be detected. Adduction was observed to occur both by Michael and Schiff base mechanisms, most prevalently taking place at the peptide N-terminus or the arginine residue. Histidine modification could only be demonstrated to occur via Michael addition with two aldehydes: 4-hydroxy-2-nonenal (HNE) and 2-octenal. The highly reactive 4-oxo-2-nonenal (ONE) was shown to react preferentially with the arginine side chain, while malondialdehyde addition could only be confirmed at the N-terminus. Aspartic acid oxidative decarboxylation, amino acid side chain oxidation, multiple adduction or peptide cross-links could not be perceived. The inability to detect these reaction products is indicative of their low abundance or non-existence in competitive reaction conditions. The multiplicity of peptide modifications described emphasizes the complexity of lipoxidation, the effects of which are not possible to fully understand by the evaluation of independent reaction products.     

Probing of arginine residues in peptides and proteins using selective tagging and electrospray ionization mass spectrometry

A general labelling method is presented which allows the determination of the number of guanidine groups (related to arginine and homoarginine in peptides and proteins) by means of mass spectrometry. It implies a guanidine-selective derivatization step with 2,3-butanedione and an arylboronic acid under aqueous, alkaline conditions (pH 8-10). The reaction mixture is then directly analysed by electrospray ionization mass spectrometry without further sample pretreatment. Other amino acids are not affected by this reaction although it is demonstrated that lysine side-chains may be unambiguously identified when they are converted to homoarginine prior to derivatization. Guanidine functionalities, as e.g. in the amino acid arginine, are easily identified by the characteristic mass shift between underivatized and derivatized analyte. The tagging procedure is straightforward and selective for guanidine groups. The influence of several experimental parameters, especially the pH of the solution and the choice of reagents, is examined and the method is applied to various arginine-containing peptides and to lysozyme as a representative protein. Possible applications of this technique and its limitations are discussed.     

Labelling indole-3-acetic acid with tritium

A simple method of obtaining indole-3-acetic acid/ growth stimulating plant hormone/ labelled with tritium has been described. The preparate has specific radioactivity of 455 MBq/mmol, and 50% yield as compared to the initial amount of used substance.     

Use of the arginine-specific butanedione/phenylboronic acid tag for analysis of peptides and protein digests using matrix-assisted laser desorption/ionization mass spectrometry

We have applied an arginine-specific labeling technique to the study of peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The reaction converts the guanidine group of the arginine side chain by reacting it with 2,3-butanedione and an arylboronic acid. Despite the general chemical lability of the tag under acidic conditions, it was possible to employ acidic matrices like alpha-cyano-4-hydroxycinnamic acid without adverse effects, using the thin-layer technique for preparation. After optimizing the method using arginine-containing model peptides--for which sensitivity down to the low fmol range was demonstrated--the procedure was applied to enzymatic digests of several model proteins in solution and to protein spots in gels obtained by two-dimensional electrophoretic separation of cell lysate samples. Information on the presence of arginine in peptides can be easily obtained from the mass spectra by the characteristic mass shift and the isotope pattern resulting from the incorporation of boron. This information might serve as a valuable additional search constraint for achieving a higher degree of confidence for protein identification by peptide mass fingerprinting.