Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phase

In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host-guest interactions might contribute to this enantiomer separation.     

Chiral separation using molecularly imprinted heteroaromatic polymers

Novel molecularly imprinted polymer systems utilizing 4-vinylpyridine and 1-vinylimidazole as functional monomers have been developed for enantioselective recognition of carboxylic and N-protected amino acids. Non-covalent interactions between the functional monomers and the template molecules were the source of the subsequent recognition sites in the resultant polymers. The capacity of the polymers for molecular recognition was investigated by using them as stationary phases in the HPLC mode. Polymers prepared with 4-vinylpyridine were found to be more efficient in racemic resolution than those prepared with 1-vinylimidazole. When applying a racemic mixture of the template molecule, the polymers showed highest affinity for the enantiomer used as template. Imprints of a racemic template molecule, as expected, did not exhibit enantioselectivity. The optimal molar ratio of 4-vinylpyridine to the template Cbz-L -Asp-OH in the polymerization mixture was determined to be 12:1. In addition to enantioselectivity, the investigated polymers demonstrated ‘ligand selectivity’ e.g., a Cbz-L -Asp-OH-imprinted polymer was able to separate Cbz-D ,L -Asp-OH, but was unable to separate Cbz-D ,L -Glu-OH.     

Surface imprinting polymers for the recognition of nucleotides

A highly selective polymer has been prepared for the selective separation of nucleotides by the surface imprinting polymerization. A dialkyl quaternary ammonium chloride was effective as the functional molecule for recognizing the difference in the structure of nucleotides. Adsorptive behavior of the ionic species of the structural analogues, inosine-5'-monophosphoric acid (IMP) and guanosine-5'-monophosphoric acid (GMP), could be controlled by changing the pH condition. Surface imprinting polymers were prepared under different pH conditions; pH 9.0 and pH 8.5. The IMP-imprinted polymers exhibited higher template effect for IMP than for a structural analogue, GMP. A reference polymer prepared without the imprint molecule neither exhibit any selectivity to IMP nor to GMP. The adsorption behavior was quantitatively evaluated by the binding constants for the IMP-imprinted polymer. The imprinting polymer was found to recognize a small structural difference in nucleotides.     

Dendritic star polymers for efficient DNA binding and stimulus-dependent DNA release

Water-soluble core-shell star polymers consisting of a dendritic polyphenylene core and an outer shell containing a defined number of amino groups have been synthesized via atom transfer radical polymerization (ATRP). All macromolecules efficiently interacted with a diverse set of DNA fragments, and stable complexes were formed and visualized by atomic force microscopy. The observed tight binding of DNA, which was found in the sub-nanomolar range, was mainly attributed to strong electrostatic interactions. Complex stoichiometries between the polyelectrolytes were controlled via the number of amino groups of the star polymers, and well-defined nanoscopic architectures were formed. DNA was released from the complexes after treatment with high concentrations of sodium chloride in aqueous solution. Such star polymers, which allow the binding and release of DNA, represent attractive candidates for the development of novel anion-exchange resins for DNA purification or as nonviral vector systems for gene delivery.     

Structure of thermal polymers of amino acids

When glutamic acid is a predominant amino acid in a thermally polymerized mixture of amino acids, pyro Glu is exclusively found at the N-terminal end of the poly-amino acid polymer. It probably initiates the polymerization process. Lysine-containing polymers will probably contain epsilon N-(glutamyl)L-lysine cross links which may account for the higher molecular weights observed in these polymers (100-200 000). Incorporation of some amino acids facilitates the incorporation of others. When utilizing mixtures of three to eight amino acids with glutamic acid as one of the amino acids, some fractions are obtained which include all the amino acids in the polymerization mixture. The biosynthesis of glutathione, gramicidin, tyrocidine and cell-wall polypeptides has demonstrated that non-random amino acid sequence peptides can be biologically synthesized without the direct participation of nucleic acids. That is, the enzymes appear to provide adequate chemical specificity to form non-random amino acid sequence peptides. The properties and replication of the scrapie agent may provide us with more profound insight as to the evolution of purely physical-chemical systems into biological systems.     

Regional Heterogeneity of L-Glutamate and L-Aspartate High-Affinity Uptake Systems in the Rat CNS

The high-affinity uptake of L-[3H]glutamate and L-[3H]aspartate into synaptosomes prepared from rat cerebral cortical, hippocampal, and cerebellar tissue was reduced by a number of structural analogues of L-glutamate and L-aspartate. threo-3-Hydroxy-L-aspartic acid was a more potent inhibitor of L-glutamate uptake than of L-aspartate uptake in the cerebral cortex, but not in the hippocampus or cerebellum. A similar pattern of selectivity was observed for cis-1-aminocyclobutane-1,3-dicarboxylic acid. Dihydrokainate was also more potent against L-glutamate than against L-aspartate in the cerebral cortex, but in the hippocampus, it was more potent against L-aspartate than against L-glutamate. By contrast, L-alpha-aminoadipate was significantly more potent in the cerebellum than in the cerebral cortex and hippocampus as an antagonist of both L-glutamate and L-aspartate. These results support other evidence that there is regional heterogeneity in acidic amino acid uptake sites and that the amino acids L-glutamate and L-aspartate may be taken up by a number of transport systems with overlapping substrate specificity but different inhibitor profiles.     

Folic acid conjugated amino acid-based star polymers for active targeting of cancer cells

Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 μg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.     

Membrane fusion between liposomes composed of acidic phospholipids and neutral phospholipids induced by melittin: a differential scanning calorimetric study

Melittin-induced membrane fusion between neutral and acidic phospholipids was examined in liposome systems with a high-sensitivity differential scanning calorimeter. Membrane fusion could be detected by calorimetric measurement by observing thermograms of mixed liposomal lipids. The roles of hydrophobic and electrostatic interactions were investigated in membrane fusion induced by melittin. Melittin, a bee venom peptide, is composed of a hydrophobic region including hydrophobic amino acids and a positively charged region including basic amino acids. When phosphatidylcholine liposomes were prepared in the presence of melittin, reductions in the phase transition enthalpies were observed in the following order; dimyristoylphosphatidylcholine (DMPC) > dipalmitoylphosphatidylcholine (DPPC) > distearoylphosphatidylcholine (DSPC) > dielaidoylphosphatidylcholine (DEPC). The plase transition enthalpy of an acidic phospholipid, dipalmitoylphosphatidylserine (DPPS), was raised by melittin at low concentrations, then reduced at higher concentrations. DPPC liposomes prepared in melittin solution were fused with DPPS liposomes when the liposomal dispersions were mixed and incubated. Similar fusion was observed between dipalmitoylphosphatidylcholine and dimyristoylphosphatidic acid (DMPA) liposomes. These results indicate that a peptide including hydrophobic and basic regions can mediate membrane fusion between neutral and acidic liposomes by hydrophobic and electrostatic interactions.     

Elucidating the substrate specificity and condensation domain activity of FkbP, the FK520 pipecolate-incorporating enzyme

Rapamycin, FK506, and FK520 are potent immunosuppressant natural product macrocycles generated by hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) systems in streptomycetes. An important functional element within these molecules is an l-pipecolate moiety that is incorporated into the completed polyketide chain by the action of RapP/FkbP, a four-domain NRPS that also putatively serves to cyclize the chain after amino acid insertion. Here we report the expression and purification of recombinant FkbP from the FK520 biosynthetic pathway. Using a combination of radioassays and Fourier transform mass spectrometry, we demonstrate that once FkbP has been phosphopantetheinylated in vitro, its peptidyl carrier protein domain can be successfully loaded with l-pipecolic acid and, to a lesser extent, l-proline. The first condensation domain of FkbP is shown to be active through the successful acetylation of aminoacyl-S-FkbP using the appropriately loaded terminal acyl carrier protein from the PKS array, FkbA, as the chain donor. Site-directed mutagenesis confirmed that the N-terminal condensation domain catalyzes the transfer reaction. Acetylation of prolyl-S-FkbP was more rapid and occurred to a greater extent than that of pipecolyl-S-FkbP, a trend which was also observed with alternative acyl chain donors. These observations suggest that the adenylation domain of FkbP serves as the primary selectivity filter for pipecolate incorporation.     

Novel thioester reagents afford efficient and specific S-acylation of unprotected peptides under mild conditions in aqueous solution.

S-acylated peptides have many potential uses for elucidating the biophysical, structural and other properties of the numerous S-acylated proteins of mammalian cells. However, with the currently available reagents, preparation of specifically S-acylated derivatives of peptides is generally laborious or simply unfeasible. We here show that novel, easily preparable aryl and alkyl thioester derivatives of palmitic acid can mediate S-acylation of peptides corresponding to physiologically S-acylated sequences from the proteins p56(lck) and H-ras and the Po glycoprotein of peripheral myelin, with high selectivity for cysteine over other amino acid functional groups (including hydroxyl and both alpha- and epsilon-amino residues), and with much greater efficiency than is obtained using acyl-coenzyme A derivatives. Efficient and selective S-acylation can be accomplished under very mild conditions in aqueous systems containing lipid vesicles or detergent micelles, or in homogenous aqueous/acetonitrile mixtures. Using these novel thioesterifying reagents, we confirm previous suggestions that the N-terminal cysteine residue of Hedgehog proteins can exhibit rapid, uncatalyzed S-to-N acyl transfer following S-acylation to produce the N-palmitoylated amino terminus found in the mature protein. By contrast, we demonstrate that spontaneous S-to-N acyl transfer from the cysteine to the terminal glycine residue in the amino-terminal peptide of G(alphas) is far less rapid and is likely too slow to explain the physiological N-palmitoylation of the amino terminus of this protein.     

Influence of initiator and different polymerisation conditions on performance of molecularly imprinted polymers

A set of polymers was imprinted with (-)-ephedrine using two different initiators. A chemometrics approach was used to optimise experiments aimed at analysis of the interplay of parameters such as polymerisation time, temperature and percentage of initiator. The results presented demonstrate the importance of keeping the right balance between these various parameters of polymerisation conditions. It is shown that enhancing one single parameter such as polymer rigidity does not necessarily improve polymer performance. In general it could be concluded that MIPs should be synthesised over a long period of time using low concentration of initiator and low temperature. The best selectivity was achieved for polymers prepared by photo-initiation with 2,2-dimethoxy-2-phenylacetophenone as initiator.     

Facile synthesis of multiamino vinyl poly(amino acid)s for promising bioapplications

We presented a general and facile strategy to prepare biocompatible multiamino polymers. Series of new monomers were synthesized by esterification of 2-hydroxyethyl methacrylate (HEMA) and Boc-amino acids, such as Boc-l-phenylalanine, Boc-glycine, Boc-l-alanine, Boc-l-valine, and Boc-l-lysine. Subsequent vinyl polymerization of monomers gave rise to vinyl poly(amino acid)s with a side primary amino group at each unit if deprotected. Both atom transfer radical polymerization (ATRP) and conventional free radical polymerization (FRP) were employed to prepare the multiamino polymers. A well controlled effect upon molecular weight with the standard first-order kinetics was achieved in cases of ATRP, and high molecular weight polymers were obtained via FRP. MTT assay showed that cell survival rates for the multiamino polymers were almost maintained above 90% and that their cytotoxicities were much lower than that of linear PEI (PEI 25000). Zeta potential measurements demonstrated that the vinyl poly(amino acid)s are electropositive, and AFM measurements showed that the vinyl poly(amino acid)s could tightly condense DNA into granular structures at a suitable concentration. The combination of facile availability, controlled productivity, low cytotoxicity and strong binding ability with DNA promises the great potential of the novel multiamino polymers in bioapplications.     

Experimental investigation on the origin of the genetic code

Summary One of the essential relationships between nucleic acids and amino acids in present biological systems, and perhaps in precursors to these systems is expressed in binding interactions. Such interactions depend on the size, composition and conformation of the interacting species. A simplified model of such complex systems was tested in an attempt to assess first the compositional effect, i.e., the binding behavior of monomeric nucleic acid and protein components. Nine representative amino acids were immobilized individually on a prepared chromatographic support by the formation of an amide linkage. Selective binding of ribonucleoside 5-phosphates was exhibited by these amino acids under standardized conditions and the binding was characterized by a site-binding model. It was found that binding behavior was dependent of the nature of the base and the nature of the amino acid. Basic information is thus provided which should be useful in the interpretation of more complex nucleic acid-protein systems and the study of their role in the evolution of the cell.     

Polymers from Amino acids: Development of Dual Ester-Urethane Melt Condensation Approach and Mechanistic Aspects

A new dual ester-urethane melt condensation methodology for biological monomers-amino acids was developed to synthesize new classes of thermoplastic polymers under eco-friendly and solvent-free polymerization approach. Naturally abundant l-amino acids were converted into dual functional ester-urethane monomers by tailor-made synthetic approach. Direct polycondensation of these amino acid monomers with commercial diols under melt condition produced high molecular weight poly(ester-urethane)s. The occurrence of the dual ester-urethane process and the structure of the new poly(ester-urethane)s were confirmed by (1)H and (13)C NMR. The new dual ester-urethane condensation approach was demonstrated for variety of amino acids: glycine, β-alanine, l-alanine, l-leucine, l-valine, and l-phenylalanine. MALDI-TOF-MS end group analysis confirmed that the amino acid monomers were thermally stable under the melt polymerization condition. The mechanism of melt process and the kinetics of the polycondensation were studied by model reactions and it was found that the amino acid monomer was very special in the sense that their ester and urethane functionality could be selectively reacted by polymerization temperature or catalyst. The new polymers were self-organized as β-sheet in aqueous or organic solvents and their thermal properties such as glass transition temperature and crystallinity could be readily varied using different l-amino acid monomers or diols in the feed. Thus, the current investigation opens up new platform of research activates for making thermally stable and renewable engineering thermoplastics from natural resource amino acids.     

The evolution of the protein synthesis system

An evolutionary picture of the early protein synthesis was presented in relation to the problem of the origin and the evolution of life. A model of an autocatalytic system was studied in this connection. The system in this model included a template nucleotide and two activated amino acid polymerases with or without a nucleotide polymerase. Variables defining the system were: (1) Catalytic activity of the polymerases, (2) Number of amino acid residues at the activity site of the polymerases, (3) Number of amino acid residues at the selectivity site of the polymerases, (4) Number of the polymerases, (5) Accuracy of polymerization and activity of the polymerases, (6) Number of evolutionary improvements, and (7) The probability of an occurrence of beneficial mutations. The population changes of the systems were obtained by computer calculations. The simulation results indicated that even a very small enzymic activity and specificity of the polymerases could eventually lead the system to the most accurate protein synthesis, as far as transitions to systems with higher accuracy were allowed. The model study would encourage further quantiative investigations on catalytic activities of synthetic peptides, and on interactions between nucleotides and amino acids, and constructions of autocatalytic systems from a chemical evolutionary point of view.This is a part of a dissertation of HM to be presented to the Graduate School of the University of Maryland in partial fulfilment of the requirements for the Ph.D.     

Influence of polymerization temperature on the molecular recognition of imprinted polymers

This paper aimed at investigating the influence of polymerization temperature on the molecular recognition of molecularly imprinted polymers (MIPs) based on multiple non-covalent interactions. 3-l-Phenylalanylaminopyridine (3-l-PheNHPy) imprinted polymers were prepared using azobisnitriles as either thermal initiators or photoinitiators at various temperatures of 10, 40 and 60 degrees C, respectively. These polymers were subsequently evaluated in the high-performance liquid chromatographic (HPLC) mode for enantioselectivity. An unexpected result shows that polymer prepared at 40 degrees C has the highest enantioselectivity, but not the polymer prepared at lower temperature of 10 degrees C. Further, the effect of elution temperature and sample load on the selectivity of polymers was investigated in detail. In order to get a better understanding of the "exception", the influence of polymerization temperature on the polymerization extent and polymer morphology was studied by FT-IR spectrum test, cross-polarization magic angle spinning (CP-MAS) (13)NMR spectra experiment and pore analysis. Based on these results we attribute this "exception" to that there is a tradeoff between the extent of polymerization and stabilization of the template-functional monomer complexes. And an optimal polymerization temperature can be found for each combination of template and monomer.     

Fundamental studies of biomolecule partitioning in aqueous two-phase systems

Phase diagram data at 4 degrees C was determined for the aqueous two-phase systems composed of polyethylene glycol, dextran, and water. The Flory-Huggins theory of polymer thermodynamics was used to correlate partitioning of biomolecules in these aqueous two-phase systems resulting in a simple linear relationship between the natural logarithm of the partition coefficient and the concentration of polymers in the two phases. This relationship was verified by partitioning a series of dipeptides which differ from one another by the addition of a CH(2) group on the c-terminal amino acid residue and by utilizing a set of low-molecular-weight proteins. The slope of the line could be expressed in terms of the interactions of the biomolecule with the phase forming polymers and water. The main result for the dipeptides was that knowledge of the partition coefficient in any of the PEG/dextran/water systems, regardless of polymer molecular weight, enabled prediction of the coefficient in all of the systems. The dipeptides were also used for determination of the Gibbs free energy of transfer of a CH(2) group between the phases. This quantity was correlated with polymer concentration, thus establishing a hydrophobicity profile for the PEG/ dextran/water systems. The methodology for predicting dipeptide partition coefficients was extended to proteins, where it was found that low-molecular-weight proteins gave a linear relationship with the tie line compositions of a phase diagram.     

Amino acid requirements and peptidase activities of Streptococcus salivarius subsp. thermophilus

The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp. thermophilus strains. A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids. The strains showed different amino acid requirements. Some amino acids proved to be essential, some were required, while others did not affect growth. In the synthetic medium, only leucine and glutamic acid were essential for growth. In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth. Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed. Strains with higher amino acid requirements were also found to express a wider range of peptidases.     

Combinations of turns in proteins.

We observed that beta- and gamma-turns in protein structure may be associated as peptides representing combinations of turns that span between nine and 26 amino acid residues along the polypeptide backbone chain and often correspond to loops in the protein structure. Around 475 peptides resulted from the analysis of a non-redundant data set corresponding to 248 protein crystal structures selected from the Protein Data Bank. Nearly 40% protein chains are associated with two or more peptides and the peptides with nine and 10 amino acid residues are more frequent. A maximum of four distinct peptides varying in number of amino acid residues were observed in at least 10 proteins along the same protein chain. Nearly 80% peptides comprise type IV beta-turns that are associated with irregular dihedral angle values suggesting this may be important for the conformational diversity associated with the loops in proteins. In general, predominant interactions that possibly stabilize these peptides involve main-chain and side-chain interactions with solvent, in addition to hydrogen bond, salt-bridge and non-bonded interactions. Majority of the peptides were observed in hydrolase, oxidoreductase, transferase, serine proteinase/inhibitor complex, electron transport/electron transfer and lyase proteins.     

Xterra RP18柱高效液相色谱法快速分离测定氨基酸

建立了一种用XterraRP1 8色谱柱快速分离测定水解氨基酸的方法。所采用的色谱条件是 :WatersAlliance系统 ,柱温 5 6℃ ,流速 1 .8ml/min ,检测波长 2 4 8nm ,梯度分离 ,运行周期 2 5min,柱反压低于 2 0 0 0Psi。在 1 7.5min内分离了包括AMQ、NH3 和牛磺酸在内的 2 1种氨基化合物 ,适应于复合氨基酸注射液、含牛磺酸的氨基酸口服液及水解氨基酸样品的分析测定