Hydrazide functionalized magnetic nanoparticles for specific extraction of N-terminal serine and threonine peptides.

In this study, we present hydrazide functionalized magnetic nanoparticles as a sorbent prepared by a new and facile method. Scanning electron microscope and Fourier transform infrared were used for characterizing the synthesized nanoparticles. The ability of the sorbent to extract N-terminal serine and threonine peptides was evaluated. The peptides were modified by oxidation of the hydroxyl group in the 1,2-amino alcohol structure before extraction. These aldehyde-forms of peptides were specifically bonded to the hydrazide groups of the sorbent. The formed hydrazone bonds were cleaved in the presence of hydroxylamine reagent. Finally, the oximated peptides were released and quantified with a high-performance liquid chromatography-diode array spectroscopy. The effects of experimental parameters including extraction time, elution time and elution volume on extraction efficiency were also investigated. The required time for the extraction process to reach equilibrium and elution time was only 8 h. The adsorption efficiency of the sorbent was 79 and 77% for peptides with N-terminal serine and threonine, respectively. The sorbent showed good specificity for extracting the peptides. In addition, the extraction efficiency of the sorbent remained constant in the presence of a non-N-terminal serine and threonine peptide as interference.

Depsidomycins B and C: New Cyclic Peptides from a Ginseng Farm Soil-Derived Actinomycete.

LC/MS-based chemical profiling of a ginseng farm soil-derived actinomycete strain, Streptomyces sp. BYK1371, enabled the discovery of two new cyclic heptapeptides, depsidomycins B and C (1 and 2), each containing two piperazic acid units and a formyl group at their N-terminus. The structures of 1 and 2 were elucidated by a combination of spectroscopic and chemical analyses. These new compounds were determined to possess d-leucine, d-threonine, d-valine, and S-piperazic acid based on the advanced Marfey's method and a GITC (2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization of their hydrolysates, followed by LC/MS analysis. Depsidomycins B and C displayed significant antimetastatic activities against metastatic breast cancer cells (MDA-MB-231).

Deciphering the streamlined genome of Streptomyces xiamenensis 318 as the producer of the anti-fibrotic drug candidate xiamenmycin.

Streptomyces xiamenensis 318, a moderate halophile isolated from a mangrove sediment, produces the anti-fibrotic compound xiamenmycin. The whole genome sequence of strain 318 was obtained through long-read single-molecule real-time (SMRT) sequencing, high-throughput Illumina HiSeq and 454 pyrosequencing technologies. The assembled genome comprises a linear chromosome as a single contig of 5,961,401-bp, which is considerably smaller than other reported complete genomes of the genus Streptomyces. Based on the antiSMASH pipeline, a total of 21 gene clusters were predicted to be involved in secondary metabolism. The gene cluster responsible for the biosynthesis of xiamenmycin resides in a strain-specific 61,387-bp genomic island belonging to the left-arm region. A core metabolic network consisting of 104 reactions that supports xiamenmycin biosynthesis was constructed to illustrate the necessary precursors derived from the central metabolic pathway. In accordance with the finding of a putative ikarugamycin gene cluster in the genome, the targeted chemical profiling of polycyclic tetramate macrolactams (PTMs) resulted in the identification of ikarugamycin. A successful genome mining for bioactive molecules with different skeletons suggests that the naturally minimized genome of S. xiamenensis 318 could be used as a blueprint for constructing a chassis cell with versatile biosynthetic capabilities for the production of secondary metabolites.

Facile preparation of protein stationary phase based on polydopamine/graphene oxide platform for chip-based open tubular capillary electrochromatography enantioseparation.

A novel chip-based enantioselective open-tubular capillary electrochromatography (OT-CEC) was developed employing bovine serum albumin (BSA) conjugated polydopamine-graphene oxide (PDA/GO) nanocomposites (PDA/GO/BSA) as stationary phase. After the poly(dimethylsiloxane) (PDMS) microfluidic chip was filled with a freshly prepared solution containing dopamine and graphene oxide, PDA/GO nanocomposites were formed and deposited on the inner wall of microchannel as permanent coating via the oxidation of dopamine by the oxygen dissolved in the solution. The PDA/GO-coated PDMS microchips not only have the adhesion of PDA that make them easily immobilized in the microchannel, but also have the larger surface and excellent biocompatibility of graphene which can incorporate much more biomolecules and well maintain their biological activity. In addition, incorporation of GO in PDA film can make surface morphology more rough, which is beneficial for enhancing the loading capacity of proteins in the microchannels and increasing sample capacity of OT-CEC columns. BSA was stably immobilized in the PDMS microchannel to fabricate a protein-stationary phase. Compared with the native PDMS microchannels, the modified surfaces exhibited much better wettability, more stable electroosmotic mobility, and less nonspecific adsorption. The efficient separation of chiral amino acids (tryptophan and threonine) and chiral dipeptide demonstrate that the constructed OT-CEC columns own ideal enantioselectivity. The presented strategy using PDA/GO coating as a versatile platform for facile conjugation of proteins may offer new processing strategies to prepare a functional surface designed on microfluidic chips.

Mechanism of N6-threonylcarbamoyladenonsine (t(6)A) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-AMP.

Genetic and biochemical studies have recently implicated four proteins required in bacteria for the biosynthesis of the universal tRNA modified base N6-threonylcarbamoyl adenosine (t(6)A). In this work, t(6)A biosynthesis in Bacillus subtilis has been reconstituted in vitro and found to indeed require the four proteins YwlC (TsaC), YdiB (TsaE), YdiC (TsaB) and YdiE (TsaD). YwlC was found to catalyze the conversion of L-threonine, bicarbonate/CO(2) and ATP to give the intermediate L-threonylcarbamoyl-AMP (TC-AMP) and pyrophosphate as products. TC-AMP was isolated by HPLC and characterized by mass spectrometry and (1)H NMR. NMR analysis showed that TC-AMP decomposes to give AMP and a nearly equimolar mixture of L-threonine and 5-methyl-2-oxazolidinone-4-carboxylate as final products. Under physiological conditions (pH 7.5, 37 °C, 2 mM MgCl(2)), the half-life of TC-AMP was measured to be 3.5 min. Both YwlC (in the presence of pyrophosphatase) and its Escherichia coli homologue YrdC catalyze the formation of TC-AMP while producing only a small molar fraction of AMP. This suggests that CO(2) and not an activated form of bicarbonate is the true substrate for these enzymes. In the presence of pyrophosphate, both enzymes catalyze clean conversion of TC-AMP back to ATP. Purified TC-AMP is efficiently processed to t(6)A by the YdiBCE proteins in the presence of tRNA substrates. This reaction is ATP independent in vitro, despite the known ATPase activity of YdiB. The estimated rate of conversion of TC-AMP by YdiBCE to t(6)A is somewhat lower than the initial rate from L-threonine, bicarbonate and ATP, which together with the stability data, is consistent with previous studies that suggest channeling of this intermediate.

Preparation of optically active allothreonine by separating from a diastereoisomeric mixture with threonine.

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH(3)) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH(3) by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.

Heart-cutting 2D-CE with on-line preconcentration for the chiral analysis of native amino acids.

The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on-line preconcentration of native amino acids in heart-cutting 2D-CE with multiple detection points using contactless conductivity detection. The tMCRB focusing was obtained by using ammonium formate (pH 8.56) as sample matrix and acetic acid (pH 2.3) as a BGE in the first dimension of the heart-cutting 2D-CE. Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection. A stacked fraction from the first dimension was selected, isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid). This on-line tMCRB preconcentration coupled with heart-cutting 2D-CE was applied with success to the chiral separation of D,L-phenylalanine, and D,L-threonine in a mixture of 22 native amino acids. The sample mixture was diluted in 0.8 M of ammonium formate, and injected at a concentration of 2.5 muM for each enantiomer with a volume corresponding to 10% of the total capillary volume. An LOD (S/N=3) of 2 muM was determined for L-threonine.

Preparation of a new crown ether-based chiral stationary phase containing thioester linkage for the liquid chromatographic separation of enantiomers.

A new chiral stationary phase (CSP) containing thioester linkages was prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to mercaptopropylsilica gel. The chiral recognition ability of the new CSP was found to be greater than that of the previously reported CSP containing amide linkages in the resolution of the various alpha-amino acids that were tested, except for that of Met, Ser and Thr. In the resolution of racemic amines and amino alcohols, the new CSP was always better than the one containing amide linkages in terms of the separation factors (alpha) and the resolutions (RS). Given the identical elution orders on the two CSPs, it was concluded that the chiral recognition mechanism is not affected by the change of the linkage type. In addition, the new CSP was found to be quite stable under the acidic mobile phase conditions that were utilized, indicating that the thioester linkage is useful as a tethering group.

Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata.

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitata (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral P. capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine.

Immobilization of phospholipid-avidin on fused-silica capillaries for chiral separation in open-tubular capillary electrochromatography.

Phospholipid-coated fused-silica capillaries with immobilized avidin were applied in the chiral separation of D,L-tryptophan, D,L-PTH-serine, and D,L-PTH-threonine at pH 7.4 by open-tubular CEC. Liposomes prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap biotinyl), or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Biotinyl) with different amounts of phosphatidylserine were assessed as phospholipid coating materials. The stability of the coating and the success of the coating procedure were evaluated in terms of the repeatability of the enantiomer migration times and the resolution of enantiomers. The coating procedure itself significantly affected the migration times and resolution of the enantiomers. Reliable chiral separations with high separation efficiencies were achieved through careful choice of the coating method.

Resolution of enantiomers of DL-amino acids on silica gel plates impregnated with optically pure (-)-quinine.

TLC resolution of enantiomers from racemic amino acids was achieved on silica gel plates impregnated with optically pure (-)-quinine. The successful solvent systems were butanol-chloroform-acetic acid (3:7:5, v/v) for DL-methionine; 6:8:4, v/v for alanine; 10:1:4; v/v for threonine; and ethyl acetate-carbon tetrachloride-propionic acid (10.5:6.5:3.5, v/v) for valine. Minimum detection limits were found to be different for each of the amino acid, ranging between 0.9 and 3.7 microg. The effects of concentration of impregnating reagent, temperature and pH on resolution of enantiomers have been studied in details.

Trace analysis in chiral separation of selected amino enantiomers.

A Crownpack CR(+) column was used for the highly sensitive quantitation of amino acid enantiomers. The use of a mobile phase of exclusively aqueous perchloric acid enabled the use of low-wavelength UV detection (200 nm) before on-line derivatization with o-phthaldialdehyde for fluorimetric detection. The use of simultaneous UV and fluorimetric detection permitted the simultaneous quantification of the impurity and its parent compound. The method was used for the evaluation of some commercially available amino acid standards. Enantiomeric impurities as low as 0.001% (10 ppm) can be determined in some cases. High precision for the determination of trace levels of D-amino acids in the presence of large amounts of corresponding L-enantiomer is demonstrated.

Biosynthesis of fluorinated secondary metabolites by Streptomyces cattleya.

The biosynthesis of organofluorine compounds by Streptomyces cattleya NRRL 8057 was examined using 19F NMR spectroscopy. The organism produced 1.2 mM fluoroacetate and 0.5 mM 4-fluorothreonine as secondary metabolites when cultured for 28 d on a chemically defined medium containing 2 mM fluoride. Cell suspensions from batch cultures harvested at the growth maximum of 4 d were not capable of fluoride uptake or fluorometabolite biosynthesis, but by 6 d had developed an efficient fluoride-uptake system and biosynthesized the two fluorometabolites in almost equal proportions. As the harvest age increased, the proportion of fluoroacetate to 4-fluorothreonine formed by cell suspensions rose progressively so that 16-d-old cells showed a ratio of 76:26 for the two compounds. Fluoride uptake and fluorometabolite production by cell suspensions were highly dependent on pH, with both processes showing a maximum rate at pH 6.0 but declining rapidly at higher pH values. This decrease was particularly marked in the case of fluoroacetate biosynthesis which was barely detectable at pH 7.5. Fluoroacetate and 4-fluorothreonine showed only low levels of interconversion by cell suspensions, suggesting that the carbon skeleton of neither was derived by metabolism of the other. The limited interconversion observed is explicable in terms of a small degree of biological defluorination occurring with each compound, followed by reincorporation of the resulting fluoride ion into the organic form by the active fluorinating system, a phenomenon also noted on incubation of cell suspensions with a number of other fluorinated biochemical intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine, the characteristic structural element of cyclosporins, from a blocked mutant of Tolypocladium inflatum.

By mutagenic treatment of a strain of Tolypocladium inflatum, a cyclosporin non-producing mutant was obtained which accumulated the characteristic building unit of cyclosporins, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation Bmt; systematic name: (2S,3R,4R,6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid) in free form. The isolation from a culture filtrate was performed by extraction, chromatographic separation and final crystallization from methanol - water. The structure and stereochemistry of this amino acid was determined by chemical transformation and correlation to dihydro-MeBmt, with known chirality [(2S,3R,4R)-3-hydroxy-4-methyl-2-methylamino-octanoic acid], obtained by hydrolysis of dihydrocyclosporin A.

Enzymatic formation of glidobactamine: a peptide nucleus of glidobactins A, B and C, new lipopeptide antitumor antibiotics.

Glidobactin deacylating activity was found in a bacterial strain of Pseudomonas sp. Glidobactamine, a key intermediate for acyl analogues of glidobactin, was isolated from the enzymatic degradation products of glidobactins after treatment using a column of fibrous active gel on which the cells of the Pseudomonas strain were immobilized. The chemical structure of glidobactamine was confirmed as the intact peptide moiety of glidobactins by chemical reformation of glidobactin A from glidobactamine and 2,4-dodecadienoic acid which is the constitutive fatty acid of glidobactin A.

Cloning and sequencing of a sheep metallothionein cDNA.

A partially purified metallothionein mRNA fraction from copper-injected sheep liver was used to synthesize double-stranded cDNA, which was dC-tailed, annealed to dG-tailed pBR322 and used to transform Escherichia coli MC1061. Of the 1500 recombinant clones only one gave a positive signal when screened with a mouse metallothionein 1 probe. This clone (pSMT-1) contained an insert which included the entire coding region of a sheep metallothionein, the whole 3'-untranslated region, part of the poly(A)-tail and 25 bases of the 5'-untranslated region. DNA sequence analysis showed that this sheep metallothionein was very similar to other mammalian metallothioneins except for a threonine to proline change at amino acid 27. The clone also contained a different polyadenylation signal d(A-G-T-A-A-A) from that usually found d(A-A-T-A-A-A). Comparison of the DNA sequence of the sheep metallothionein with those of other species revealed an interesting region of homology close to the poly(A) addition signal in the 3'-untranslated region of the mRNA.

Gingival matrix collagen in chronic periodontitis.

The isolation and characterization of cyanogen bromide peptides derived from the human gingival collagen of patients with chronic periodontitis revealed the presence of both Type I and Type III collagens in this tissue. The amount of TYPE III collagen, however, was found to be lower than that in normal gingival tissue. In addition, a non-collagenous protein fraction, accounting for approximately 20% of the insoluble matrix, was relatively rich in acidic, hydrophobic, and hydroxy-containing amino acids. Amino acid analysis, likewise, revealed qualitative and quantitative differences between the normal and diseased tissues.

Ligand-exchange chromatography of amino acids on copper-, cobalt- and zinc-chelex 100.

Procedures for the ligand-exchange chromatography of amino acids on copper-, cobalt-and zinc-Chelex 100 have been examined. Ligand exchange on the copper complex affords a simple and rapid method for the removal of amino acids (except for aspartic and glutamic acids) from dilute solutions. The influence of the pH on the binding of amino acids to the metal complex was also studied. The bound amino acids could be eluted with ammonium hydroxide which also causes a slight metal leakage. Chromatography on cobalt- and zinc-Chelex 100 showed that only the basic amino acids were quantitatively attached to these complexes at pH 8.3-9.5, whereas the others were predominantly EXCLUDED. This procedure can be used for the selective concentration and removal of basic amino acids in the presence of other amino acids.