Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C-18 column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2 x 10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N = 3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain. (C) 2008 Elsevier B.V. All rights reserved.

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

D-Amino acid peptide residualizing agents bearing N-hydroxysuccinimido- and maleimido-functional groups and their application for trastuzumab radioiodination.

INTRODUCTION: Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N(ϵ)-(3-[(*)I]iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-D-GEEEK (Mal-D-GEEEK-[(*)I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new D-amino acid based agent that might avoid these potential problems. METHODS: N(α)-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-D-EEEG (NHS-IB-D-EEEG), which contains 3 D-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-D-EEEG and Mal-D-GEEEK-IB was compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated D-peptide trastuzumab conjugates. RESULTS: NHS-[(131)I]IB-D-EEEG was produced in 53.8%±13.4% and conjugated to trastuzumab in 39.5%±7.6% yield. Paired-label internalization assays with trastuzumab-NHS-[(131)I]IB-D-EEEG and trastuzumab-Mal-D-GEEEK-[(125)I]IB demonstrated similar intracellular trapping for both conjugates at 1h ((131)I, 84.4%±6.1%; (125)I, 88.6%±5.2%) through 24h ((131)I, 60.7%±6.8%; (125)I, 64.9%±6.9%). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[(131)I]IB-D-EEEG, 29.8%±3.6%ID/g; trastuzumab-Mal-D-GEEEK-[(125)I]IB, 45.3%±5.3%ID/g) and was significantly higher for (125)I at all time points. In general, normal tissue levels were lower for trastuzumab-NHS-[(131)I]IB-D-EEEG, with the differences being greatest in kidneys ((131)I, 2.2%±0.4%ID/g; (125)I, 16.9%±2.8%ID/g at 144 h). CONCLUSION: NHS-[(131)I]IB-D-EEEG warrants further evaluation as a residualizing radioiodination agent for labeling internalizing antibodies/fragments, particularly for applications where excessive renal accumulation could be problematic.

Vibrational spectroscopy and DFT calculations of the di-amino acid peptide L-aspartyl-L-glutamic acid in the zwitterionic state

Solid state IR and Raman as well as aqueous solution state Raman spectra are reported for the linear di-amino acid peptide L-aspartyl-L-glutamic acid (L-Asp-L-Glu); the solution state Raman spectrum has also been obtained for the N,O-deuterated derivative. SCF-DFT calculations at the B3-LYP/cc-pVDZ level established that the structure and vibrational spectra of L-Asp-L-Glu can be interpreted using a model of the peptide with ten hydrogen-bonded water molecules, in conjunction with the conductor-like polarizable continuum solvation method. The DFT calculations resulted in the computation of a stable zwitterionic structure, which displays trans-amide conformation. The vibrational spectra were computed at the optimised molecular geometry, enabling normal coordinate analysis, which yielded satisfactory agreement with the experimental IR and Raman data. Computed potential energy distributions of the normal modes provided detailed vibrational assignments.

Vibrational spectroscopy and DFT calculations of di-amino acid cyclic peptides. Part I: cyclo(Gly-Gly), cyclo(L-Ala-L-Ala) and cyclo(L-Ala-Gly) in the solid state and in aqueous solution

Investigations of the vibrational spectra of cyclo(Gly-Gly), cyclo(L-Ala-L-Ala) and cyclo(t-Ala-Gly) are reported. Raman scattering and Fourier transform infrared (FTIR) spectra of solid-state and aqueous protonated samples, as well as their corresponding N-deuterated isotopomers, have been examined. In addition, density functional theory (DFT) (B3-LYP/cc-pVDZ) calculations of molecular structures and their associated vibrational modes were carried out. In each case, the calculated structures of lowest energy for the isolated gas-phase molecules have boat conformations. Assignments have been made for the observed Raman and FTIR vibrational bands of the cyclic di-amino acid peptides (CDAPs) examined. Raman polarization studies of aqueous phase samples are consistent with C-2 and C-1 symmetries for the six-membered rings of cyclo(L-Ala-L-Ala) and cydo(L-Ala-Gly), respectively. There is a good correlation between experimental and calculated vibrational bands for the three CDAPs. These data are in keeping with boat conformations for cydo(L-Ala-L-Ala) and cyclo(L-Ala-Gly) molecules, predicted by the ab initio calculations, in both the solid and aqueous solution states. However, Raman spectroscopic results might infer that cyclo(L-AlaGly) deviates only slightly from planarity in the solid state. The potential energy distributions of the amide I and II modes of a cis-peptide linkage are shown to be significantly different from those of the trans-peptides. For example, deuterium shifts have shown that the cis-amide I vibrations found in cyclo(Gly-Gly), cyclo(L-Ala-L-Ala), and cyclo(L-Ala-Gly) have larger N-H contributions compared to their trans-amide counterparts. Compared to trans-amide II vibrations, cis-amide II vibrations show a considerable decrease in N-H character.

Vibrational spectroscopic studies of the structure of di-amino acid peptides. Part II: cyclo(L-Asp-L-Asp) in the solid state and in aqueous solution

Solid-state protonated and N,O-deuterated Fourier transform infrared (IR) and Raman scattering spectra together with the protonated and deuterated Raman spectra in aqueous solution of the cyclic di-amino acid peptide cyclo(L-Asp-L-Asp) are reported. Vibrational band assignments have been made on the basis of comparisons with previously cited literature values for diketopiperazine (DKP) derivatives and normal coordinate analyses for both the protonated and deuterated species based upon DFT calculations at the B3-LYP/cc-pVDZ level of the isolated molecule in the gas phase. The calculated minimum energy structure for cyclo(L-Asp-L-Asp), assuming C-2 symmetry, predicts a boat conformation for the DKP ring with both the two L-aspartyl side chains being folded slightly above the ring. The C=O stretching vibrations have been assigned for the side-chain carboxylic acid group (e.g. at 1693 and 1670 cm(-1) in the Raman spectrum) and the cis amide I bands (e.g. at 1660 cm(-1) in the Raman spectrum). The presence of two bands for the carboxylic acid C=O stretching modes in the solid-state Raman spectrum can be accounted for by factor group splitting of the two nonequivalent molecules in a crystallographic unit cell. The cis amide II band is observed at 1489 cm(-1) in the solid-state Raman spectrum, which is in agreement with results for cyclic di-amino acid peptide molecules examined previously in the solid state, where the DKP ring adopts a boat conformation. Additionally, it also appears that as the molecular mass of the substituent on the C-alpha atom is increased, the amide II band wavenumber decreases to below 1500 cm(-1); this may be a consequence of increased strain on the DKP ring. The cis amide II Raman band is characterized by its relatively small deuterium shift (29 cm(-1)), which indicates that this band has a smaller N-H bending contribution than the trans amide II vibrational band observed for linear peptides.

IR/Raman spectroscopy and DFT calculations of cyclic di-amino acid peptides. Part III: comparison of solid state and solution structures of cyclo(L-Ser-L-Ser)

B3-LYP/cc-pVDZ calculations of the gas-phase structure and vibrational spectra of the isolated molecule cyclo(L-Ser-L-Ser), a cyclic di-amino acid peptide (CDAP), were carried out by assuming C-2 symmetry. It is predicted that the minimum-energy structure is a boat conformation for the diketopiperazine (DKP) ring with both L-Beryl side chains being folded slightly above the ring. An additional structure of higher energy (15.16 kJ mol(-1)) has been calculated for a DKP ring with a planar geometry, although in this case two fundamental vibrations have been calculated with imaginary wavenumbers. The reported X-ray crystallographic structure of cyclo(L-Ser-L-Ser), shows that the DKP ring displays a near-planar conformation, with both the two L-Beryl side chains being folded above the ring. It is hypothesized that the crystal packing forces constrain the DKP ring in a planar conformation and it is probable that the lower energy boat conformation may prevail in the aqueous environment. Raman scattering and Fourier-transform infrared (FT-IR) spectra of solid state and aqueous solution samples of cyclo(L-Ser-L-Ser) are reported and discussed. Vibrational band assignments have been made on the basis of comparisons with the calculated vibrational spectra and band wavenumber shifts upon deuteration of labile protons. The experimental Raman and IR results for solid-state samples show characteristic amide I vibrations which are split (Raman:1661 and 1687 cm(-1), IR:1666 and 1680 cm(-1)), possibly due to interactions between molecules in a crystallographic unit cell. The cis amide I band is differentiated by its deuterium shift of ~ 30 cm(-1), which is larger than that previously reported for trans amide I deuterium shifts. A cis amide II mode has been assigned to a Raman band located at 1520 cm(-1). The occurrence of this cis amide II mode at a wavenumber above 1500 cm(-1) concurs with results of previously examined CDAP molecules with low molecular weight substituents on the C-alpha atoms, and is also indicative of a relatively unstrained DKP ring.

Vibrational spectra and crystal structure of the di-amino acid peptide cyclo(L-Met-L-Met): Comparison of experimental data and DFT calculations

Experimental Raman and FT-IR spectra of solid-state non-deuterated and N-deuterated samples of cyclo(L-Met-L-Met) are reported and discussed. The Raman and FT-IR results show characteristic amide I vibrations (Raman: 1649 cm-1, infrared: 1675 cm-1) for molecules exhibiting a cis amide conformation. A Raman band, assigned to the cis amide II vibrational mode, is observed at sim1493 cm-1 but no IR band is observed in this region. Cyclo(L-Met-L-Met) crystallises in the triclinic space group P1 with one molecule per unit cell. The overall shape of the diketopiperazine (DKP) ring displays a (slightly distorted) boat conformation. The crystal packing employs two strong hydrogen bonds, which traverse the entire crystal via translational repeats. B3-LYP/cc-pVDZ calculations of the structure of the molecule predict a boat conformation for the DKP ring, in agreement with the experimentally determined X-ray structure. Copyright © 2009 John Wiley & Sons, Ltd.

An Electron-Microscope X-Ray-Diffraction And Amino-Acid-Analysis Study Of The Opercular Filament Cuticle Calcareous Opercular Plate And Habitation Tube Of Pomatoceros Lamarckii Quatrefages (Polychaeta Serpulidae)

The structure, X-ray diffraction and amino acid compositions of the opercular filament cuticle, calcareous opercular plate and habitation tube of the polychaete serpulid, Pomatoceros lamarckii quatrefages, are reported. The opercular filament cuticle is made up of protein and chitin. The chitin is probably in the crystallographic α form. The structure and amino acid composition of the organic components of the opercular filament cuticle and calcareous opercular plate have similarities but are distinctly different from those of the calcareous habitation tube. The opercular plate and habitation tube are composed of different polymorphs of calcium carbonate, aragonite and calcite respectively. Comparisons are made with other chitin-protein systems, structural and calcified proteins.