Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

Summary The synthesis of peptides employing 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtained in good yield (70–95%). Furthermore, both the¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

9-Fluorenylmethyl chloroformate (Fmoc-Cl) as a useful reagent for the synthesis of pentafluorophenyl, 2,4,5-trichlorophenyl,pentachlorophenyl, p-nitrophenyl,o-nitrophenyl and succinimidyl esters of Nα-urethane protected amino acids

Summary 9-Fluorenylmethyl chloroformate has been demonstrated to be useful reagent for the synthesis of several commonly used active esters of Fmoc-/Boc-/Z-amino acids. These include pentafluorophenyl, 2,4,5-trichlorophenyl, pentachlorophenyl,p-nitrophenyl,o-nitrophenyl, and succinimidyl esters. The method is simple, rapid and efficient. All the compounds made have been isolated as crystaline solids in good yield and optical purity. They were fully characterized by IR, and¹H NMR.     

1-(t-Butyldimethylsilyloxy) benzotriazole (TBDMS-OBt): A new and novel reagent for the synthesis of peptides

Synthesis and use of 1-(t-butyldimethylsilyloxy)benzotriazole (TBDMS-OBt) in the coupling of Fmoc-amino acid chlorides to amino free amino acid esters in homogeneous solution phase is described. The coupling required no addition of base and was fast and racemization free. Work up and isolation of products were easy. Yield, purity and ¹H NMR analysis of peptides, synthesised by this method, were satisfactory.     

Synthesis of mimetic peptides containing glucosamine

A convenient synthesis of 2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-d-glucopyranoside was described from the readily available starting material 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine). Herein, the coupling of different lipophilic amino acids with 2-amino-3,4,6-tri-O-benzyl-2-deoxy-β-d-glucose was reported via an amide linkage as useful building blocks for the synthesis of glycopeptides. Of particular interest, bioactive peptide Arg-Gly-Asp (RGD) was incorporated into the building block containing valine was also reported. The 15 examples of corresponding di-, tri- and tetra-peptides were obtained as single αanomers.     

Antibacterial activities of peptides designed as hybrids of antimicrobial peptides

Hybrid peptides (HP-MA, HP-ME), each of 20 residues and incorporating 2–9 residues of Helicobacter pylori ribosomal protein L1 (HP) and 1–12 residues of magainin 2 and melittin, were designed. The antibiotic activities of these peptides were evaluated using bacterial, tumor and human erythrocyte cells. HP-MA had a stronger antibacterial activity against Gram-positive bacteria and Gram-negative bacteria than HP (2-20) and magainin 2, and HP-ME was similar to melittin. None of the hybrids had anti-tumor or hemolytic activity. These peptides were further investigated using an artificial liposomal vesicle and 1,6-diphenyl-1,3,5-hexatriene as a membrane probe, and confirmed to have similar antibacterial activities. The antibacterial effect of these hybrids is probably caused by their ability to damage the bacterial plasma membrane. Additional circular dichroism spectra suggested that the -helical structure of these peptides plays an important role in their antibiotic effect but that -helical property is less connected with the enhanced antibiotic activity.     

Determination of peptides and amino acids from wool and beer with sensitive fluorescent reagent 2-(9-carbazole)-ethyl chloroformate by reverse phase high-performance liquid chromotography and liquid chromotography mass spectrometry

A new method for the sensitive determination of amino acids and peptides using the tagging reagent 2-(9-carbazole)-ethyl chloroformate (CEOC) with fluorescence (FL) detection has been developed. Identification of derivatives was carried out by liquid chromotography mass spectrometry. The chromophore in the 2-(9-fluorenyl)-ethyl chloroformate (FMOC) reagent was replaced by carbazole, which resulted in a sensitive fluorescence lerivatizing agent CEOC. CEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. Studies on derivatization demonstrate excellent derivative yields over the pH range 8.8-10.0. Maximal yields close to 100% are observed with three- to fourfold molar reagent excess. Derivatives exhibit strong fluorescence and allow direct injection of the reaction mixture with no significant disturbance from the major fluorescent reagent degradation by-products, such as 2-(9-carbazole)-ethanol and bis-(2-(9-carbazole)-ethyl) carbonate. In addition, the detection responses for CEOC derivatives are compared to those obtained with FMOC. The ratios AC(CEOC)/AC(FMOC) = 1.00-1.82 for fluorescence (FL) response and AC'(CEOC)/AC'(FMOC) = 1.00-1.21 for ultraviolet (UV) response are observed (here, AC and AC' are, respectively, FL and UV response). Separation of the derivatized peptides and amino acids has been optimized on a Hypersil BDS C18 column. Excellent linear responses are observed. This method was used successfully to analyze protein hydrolysates from wool and from direct-derivatized beer.     

Synthesis of peptides employing Fmoc-/Boc-/Z-amino acid fluorides and activated commercial zinc dust

Summary The coupling of urethane protected amino acid fluorides is accomplished in the presence of activated, commercial zinc dust to synthesize several di- and tripeptides. The coupling was fast and racemization free. The yield as well as purity of the peptides was satisfactory. The method was extended for the incorporation of sterically hindered α,α-dialkylamino acids and N-methylamino acids as well.     

Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: Use of AUA as a restart codon

Summary An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the -galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active -galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the -galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.     

Three newAspergillus species isolated from clinical sources as a causal agent of human aspergillosis

Three new species ofAspergillus isolated from clinical sources in China are described and illustrated:A. beijingensis, A. qizutongii andA. wangduanlii. The first species is characterized by spreading colonies, yellow to grayish green conidial heads, smooth-walled conidiophores with a clavate vesicle, uniseriate aspergilla and nearly globose, micro-verrucose conidia. The second is characterized by spreading colonies, olive-yellow conidial heads, conspicuously roughened conidiophores with a flask-shaped vesicle, uniseriate but often secondarily proliferating aspergilla and globose, smooth conidia. The third is characterized by rapidly growing colonies, dull green conidial heads, smooth to irregularly roughened conidiophores which are often surrounded by coiled hyphae in the basal part and are terminally swollen into a globose or irregular shaped vesicle, uniseriate aspergilla and globose, micro-verrucose conidia.     

Parasitism as a biological control agent of dinoflagellate blooms in the California Current System

Amoebophrya is a marine parasite recently found to infect and kill bloom-forming dinoflagellates in the California Current System (CCS). However, it is unknown whether parasitism by Amoebophrya can control dinoflagellate blooms in major eastern boundary upwelling systems, such as the CCS. We quantified the abundance of a common bloom-forming species Akashiwo sanguinea and prevalence of its parasite (i.e., % infected cells) in surface water samples collected weekly from August 2005 to December 2008 at the Santa Cruz Wharf (SCW), Monterey Bay, CA. Additionally, we measured physical and chemical properties at the SCW and examined regional patterns of wind forcing and sea surface temperature. Relative abundance of the net phytoplankton species was also analyzed to discern whether or not parasitism influences net phytoplankton community composition. Epidemic infection outbreaks (>20% parasite prevalence in the host species) may have contributed to the end or prevented the occurrence of A. sanguinea blooms, whereas low parasite prevalence was associated with short-term (≤2 weeks) A. sanguinea blooms. The complete absence of parasitism in 2007 was associated with an extreme A. sanguinea bloom. Anomalously strong upwelling conditions were detected in 2007, suggesting that A. sanguinea was able to outgrow Amoebophrya and ‘escape’ parasitism. We conclude that parasitism can strongly influence dinoflagellate bloom dynamics in upwelling systems. Moreover, Amoebophrya may indirectly influence net phytoplankton species composition, as species that dominated the net phytoplankton and developed algal blooms never appeared to be infected.     

Overexpression of aThellungiella halophila CBl9 homolog,ThCBL9, confers salt and osmotic tolerances in transgenicarabidopsis thaliana

The calcineurin B-like (CBL) proteins, comprising a large subfamily of calcium sensors in plant cells, play an important role in many stress responses. We cloned a gene from the halophyteThellungiella halophila that is homologous toAtCBL9 inArabidopsis thaliana. The 1008-bpThCBL9 contains an ORF of 639 bp and encodes 213 amino acids, with a 5“-untranslated region of 193 bp and a 3”-untranslated region of 176 bp. Its amino acid sequence shares high homology with AtCBLs.ThCBL9 is up-regulated by ABA, NaCI, and PEG inThellungiella leaves. Using molecular biological methods, we over-expressedThCBL9 inA. thaliana and found that this enhanced tolerances to both high salt and osmotic stress in transgenicArabidopsis. These authors contributed equally to this work.     

Modified chemotactic peptides: Synthesis and activity of an azaTic-containing fMLP-OMe analogue

Summary The synthesis and the biological activity of a pseudopeptide analogue of the chemotacticN-formyltripeptide fMLP-OMe, containing the aza Tic (3,4-dihydro-2(1H)-phthalazinecarboxylic acid) residue replacing the native phenylalanine, is described. Whereas pseudopeptides containing lineara-azaammo acids are currently studied, data on the new group of analogues containing cyclic-aza residues capable of limiting the rotameric distribution of the side chains (topological control) are just emerging in the literature. At our best knowledge, the here described [azaTic³]fMLP-OMe represents the first example of the introduction of this new type of-aza residue into a natural bioactive peptide.     

Two-hybrid interaction of a human UBC9 homolog with centromere proteins ofSaccharomyces cerevisiae

Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of theSaccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity toS. cerevisiae Ubc9p and 64% identity toSchizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses aS. cerevisiae ubc9 temperature-sensitive mutation, indicating that theUBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However,S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction betweenS. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G₂/M phase ofS. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.     

The Synthesis and Characterization of a Helical Miniature Protein Mimicking the OGT Active Domain

The dynamic modification of many nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) on serine or threonine is catalyzed by O-GlcNAc transferase (OGT). The conserved GPGTF (glycogen phosphorylase/glycosyl transferase) motif, one of the α-helices of the second domain in OGT, was identified as a putative UDP-GlcNAc binding site. A miniature protein was designed which contains all of the conserved residues of GPGTF motif in the O-GlcNAc transferase, and was shown to adopt an alpha helix in 10% trifluoroethanol. It was anticipated that the miniature protein could shed light on the mechanism of dynamic O-GlcNAc modification and provide a potential drug for the diabetes and neurodegenerative diseases.     

Nuclear-accumulation kinetics of p9 CksHs1 and p9 CksHs2 in live plant cells correlate with immunochemical characteristics

Summary The two human homologues of the fission yeast cell cycle protein p13 ^suc1 displayed structural characteristics consistent with their existing in solution as differently folded monomers despite 81% identity with respect to their primary structures and both being capable of fulfilling the functions of their homologues in fission and budding yeasts. Carboxyfluorescein-labelled p9 ^CksHs1 and p9 ^CksHs2 retained their native structures. When microinjected into live stamen hair cells ofTradescantia virginiana, the labelled proteins accumulated in the nuclei of the cells. Markedly different nuclearaccumulation kinetics indicated that the human proteins interact differently with other cellular constituents, which supports the proposition that they may have different roles in cellular regulation.Abbreviations Cdk cyclin-dependent kinase - tris tris(hydroxymethyl)aminomethane - Hepes N-(2-hydroxyethyl)piperazine-N-(3-ethanesulphonic acid) - CF 5(6)-carboxyfluorescein-N-hydroxysuccinamide ester - SDS-PAGE sodium dodecyl sulphatepolyacrylamide gel electrophoresis - IEF isoelectric focusing - DEAE Sephacel diethylaminoethyl Sephacel - ELISA enzyme-linked immunosorbent assay - IgG immunoglobulin     

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

Summary The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.     

Synthesis and evaluation of azaproline peptides as potential inhibitors of dipeptidyl peptidase IV and prolyl oligopeptidase

Summary A series of azaproline dipeptides with various N-substituents were synthesized as possible active-site-directed inhibitors of two proline-specific serine proteases, dipeptidyl peptidase IV and prolyl oligopeptidase. Compounds with semicarbazide, carbazate, acylhydrazine and sulphonylhydrazine structures were tested. Some compounds show moderate activity, i.e., in the millimolar range.     

The entomogenous nematode Neoaplectana bibionis as a biological control agent of Agrotis segetum in lettuce

Biological control of the soil-inhabitating larvae (cutworms) of Agrotis segetum Schiff. using the entomogenous nematode Neoaplectana bibionis Bovien was investigated under field conditions. Cutworms were introduced to plots planted with lettuce, Lactuca sativa L. After planting and infection with cutworms the plots were treated with either N. bibionis, the insecticide endosulfan or water. The trials were sited on locations with loamy and sandy soil. Adequate control of cutworms was obtained using 2.5×10⁵ nematodes/m² on sandy soil and 1×10⁶ nematodes/m² on loamy soil. Effects of these treatments with nematodes were equal to the effect of endosulfan.

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Neoaplectana bibionis ein entomophager nematode zur biologischen bekämpfung von Agrotis segetum an salat

Zusammenfassung Unter Freilandbedingungen wurde die biologische Bekämpfung von Erdraupen (Agrotis segetum Schiff., Lepidoptera, Noctuidae) mit dem entomophagen Nematoden Neoaplectana bibionis Bovien geprüft. Mit Salat bepflanzte Parzellen wurden mit Erdraupen besetzt. Anschliessend wurde auf die Parzellen N. bibionis, das Insektizid Endosulfan oder Wasser ausgebracht. Die Versuche wurden in Gebieten mit lehmigen und sandigen Böden durchgeführt. Für eine ausreichende Bekämpfung der Erdraupen waren auf dem sandigen Boden 2.5×10⁵ Nematoden/m² und auf dem lehmigen Boden 1×10⁶ Nematoden/m² erforderlich. Die Wirkung dieser Behandlungen entsprach der mit Endosulfan.

     

Identification of 9-O-acetylated sialoglycans on peripheral blood mononuclear cells in Indian Visceral Leishmaniasis

Although the existence of O-acetylated sialic acids is well known, it is only in recent years that steady refinement of analytical techniques have enabled detailed mapping of their structural diversity [1]. Fluorimetric analysis of peripheral blood mononuclear cells (PBMC) of patients with Visceral Leishmaniasis (VL) showed six fold increase in the percentage of surface 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) as compared to normal human donors. Using Achatinin-H, a 9-O-acetyl sialic acid- binding lectin, an enhanced presence of 9-O-AcSGs in an 2 6 linkage was demonstrated by flow cytometry; abolition of its binding by pretreatment with a recombinant 9-O-acetylesterase corroborated the presence of this glycotope. Western blotting of PBMC from VL patients indicated the presence of five O-acetylated sialoglycans corresponding to 144, 65, 56, 36 and 19 kDa as compared to 144 and 36 kDa in normal individuals. Taken together our data indicates that during active disease, there is an overexpression of 9-O-AcSGs on the surface of PBMC of VL patients, thus opening up new research avenues wherein the expression of this biomarker could be exploited to monitor the clinical status of VL patients. Published in 2004..     

Synthesis of peptides employing Fmoc-/Boc-/Z-amino acid fluorides and activated commercial zinc dust

The coupling of urethane protected amino acidfluorides is accomplished in the presence ofactivated, commercial zinc dust to synthesize severaldi- and tripeptides.The coupling was fast and racemization free. The yieldas well as purity of the peptides was satisfactory.The method was extended for the incorporation ofsterically hindered ,-dialkylaminoacids and N-methylamino acids as well.