Synthesis and antiviral activity of novel glycyrrhizic acid conjugates with D-amino acid esters

Glycyrrhizic acid (GA) conjugates with methyl and ethyl esters of D-amino acids (D-Trp, D-Phe, D-Tyr, D-Val, D-Leu) have been synthesized by the activated esters method using mixtures of N-hydroxybenzotriazole or N-hydroxysuccinimide with N,N′-dicyclohexylcarbodiimide. GA conjugate with D-Trp ethyl ester exhibited antiviral activity against influenza viruses A/H3N2, A/H1N1/pdm09, A/H5N1, B (SI > 10–29), and HRSV (SI > 25). GA conjugate with D-Trp methyl ester inhibited influenza virus A/H1N1/pdm09 (SI > 30).     

New aspects of biosynthesis and metabolism of <Emphasis Type="Italic">N</Emphasis>-acyldopamines in rat tissues

Possible biosynthetic pathways of N-acyldopamines in rat tissues were compared. It was shown that an insignificant amount of the conjugation products was formed during the incubation of arachidonic acid and dopamine, whereas the substitution of tyrosine for dopamine resulted in the productive biosynthesis of N-arachidonoyldopamine. The biosynthesis presumably involves several closely conjugated enzymatic stages, and free fatty acids rather than their CoA esters served as the starting substrates. The decarboxylation stage probably precedes the stage of catechol system formation, because N-acetyltyramine (a probable intermediate) was easily oxidized by monophenol monooxygenase to N-acyldopamine, whereas N-acyltyrosine is hydrolyzed under these conditions. Biosynthesis of N-acyldopamines in a cell-free medium was accompanied by their methylation. The possibility of oxidative metabolism of N-acyldopamines, which could serve as co-substrates or inhibitors of different oxidoreductases, was shown for the first time.     

Modification of fatty acid selectivity of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase A by error-prone PCR

Objective

To generate Candida antarctica lipase A (CAL-A) mutants with modified fatty acid selectivities and improved lipolytic activities using error-prone PCR (epPCR).

Results

A Candida antarctica lipase A mutant was obtained in three rounds of epPCR. This mutant showed a 14 times higher ability to hydrolyze triacylglycerols containing conjugated linoleic acids, and was 12 and 14 times more selective towards cis-9, trans-11 and trans-10, cis-12 isomers respectively, compared to native lipase. Lipolytic activities towards fatty acid esters were markedly improved, in particular towards butyric, lauric, stearic and palmitic esters.

Conclusion

Directed molecular evolution is an efficient method to generate lipases with desirable selectivity towards CLA isomers and improved lipolytic activities towards esters of fatty acids.

     

“Thioureidopeptide”: Novel Synthon for the Synthesis of <Emphasis Type="Italic">N,N′, N″</Emphasis>-Trisubstituted Guanidinopeptide Mimics

The synthesis of N ^α-protected N,N′,N″-trisubstituted guanidinopeptide mimic molecules suitably decorated in peptide backbone has been delineated in one pot employing HgCl₂ as a desulphurizing agent. Chiral N ^α -protected thioureidopeptide esters were employed as synthons for the synthesis of title molecules. The protocol is simple and the reaction conditions employed were mild, amenable to the amino acid chemistry.     

Synthesis and SAR Studies of Bisthiourea Derivatives of Dipeptides <Emphasis Type="Italic">Lys/lys</Emphasis>-<Emphasis Type="Italic">Asp,Lys/lys</Emphasis>-<Emphasis Type="Italic">Trp</Emphasis> Conjugated Benzo[d]isoxazole as Promising Antioxidants

A novel series of bisthiourea derivatives of four dipeptides consisting of Lys-Asp, lys-Asp, Lys-Trp and lys-Trp conjugated to 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole were synthesized and characterized by physical method and spectroscopic data. The molecules 1–24 were evaluated for their in vitro antioxidant activity and compared with commercial antioxidants ascorbic acid (AA) and gallic acid (GA), employing 1,1-diphenyl-2-picryl-hydrazyl (DPPH), N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sufonic acid) (ABTS) assays. The results revealed that IC₅₀ of 8, 11, 20 and 23 with electron donating OCH₃ group were lower than the IC₅₀ of commercial standards AA and GA in all the three performed antioxidant assays indicating the good activities of these compounds. The analogues with Trp (13–24) showed better activity than the corresponding analogues with Asp (1–12). Further, the dipeptide derivatives with d-configuration (lys) were found to be more potent than the dipeptide derivatives with l-configuration (Lys).     

Novel Synthesis of <Emphasis Type="Italic">N</Emphasis>-Glycosyl Amino Acids Using T3P<Superscript>®</Superscript>: Propylphosphonic Acid Cyclic Anhydride as Coupling Reagent

In this paper is presented a novel and simple synthetic pathway for obtaining new protected and unprotected N-glucosyl amino acids from 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl amine and Fmoc-l-amino acids. Three methodologies were evaluated, using the coupling reagents: N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate, diisopropylcarbodiimide and propylphosphonic acid cyclic anhydride. The obtained products using propylphosphonic acid cyclic anhydride showed less undesired species, easy purification and higher yields than the other two methodologies. Deprotection strategies widely used in solid phase peptide synthesis were applied to develop the synthetic pathway reported and achieve the final products. The protected and unprotected N-glucosyl amino acids were purified using solid phase extraction chromatography and characterized by high performance liquid Chromatography and nuclear magnetic resonance spectroscopy. Different amino acids (Fmoc-l-Asp(OtBu)OH, Fmoc-l-Phe(OH) and Fmoc-l-Lys(Boc)-OH) have been employed to demonstrate the simple and reproducible coupling methodology using propylphosphonic acid cyclic anhydride. The results showed that new protected and unprotected N-glucosyl amino acids can be obtained with high purity and the methodology could be used with any Fmoc-amino acid. The methodology developed could be considered as a synthetic tool for obtaining building blocks for glycopeptide synthesis and potential drugs candidates based on glycoconjugates.     

Coating polypropylene surfaces with protease weakens the adhesion and increases the dispersion of <Emphasis Type="Italic">Candida albicans</Emphasis> cells

Objectives

To investigate the ability of the proteases, subtilisin and α-chymotrypsin (aCT), to inhibit the adhesion of Candida albicans biofilm to a polypropylene surface.

Results

The proteases were immobilized on plasma-treated polypropylene by covalently linking them with either glutaraldehyde (GA) or N′-diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS). The immobilization did not negatively affect the enzyme activity and in the case of subtilisin, the activity was up to 640% higher than that of the free enzyme when using N-acetyl phenylalanine ethyl ester as the substrate. The efficacies against biofilm dispersal for the GA-linked SubC and aCT coatings were 41 and 55% higher than the control (polypropylene coated with only GA), respectively, whereas no effect was observed with enzymes immobilized with DIC and NHS. The higher dispersion efficacy observed for the proteases immobilized with GA could be both steric (proper orientation of the active site) and dynamic (higher protein mobility/flexibility).

Conclusions

Proteases immobilized on a polypropylene surface reduced the adhesion of C. albicans biofilms and therefore may be useful in developing anti-biofilm surfaces based on non-toxic molecules and sustainable strategies.

     

Chemical Composition of Lipids from Peat-Forming Leafy Mosses of Eutrophic Swamps of Western Siberia and Altai

The molecular composition of lipids in three samples of leafy mosses (Aulacomnium palustre, Warnstorfia fluitans, and Calliergon giganteum) has been determined. The revealed acyclic compounds included normal and isoprenoid alkanes, isoprenoid alkenes, normal and isoprenoid ketones, carboxylic acids and their esters, alcohols, and aldehydes. Among cyclic compounds, bi-, tri- and tetracyclic polycycloaromatic hydrocarbons (PAHs), bicyclic and pentacyclic terpenoids, steroids and tocopherols have been observed. The identified organic compounds consisted mainly of carbocyclic acids and n-alkanes with the prevalence of C₂₇ homologues. A. palustre is characterized by a reduced content of isoprenoid compounds, alcohols, and ketones, while the content of unsaturated acids, pentacyclic terpenoids, and aldehydes is rather heightened. A. palustre differs from W. fluitans and C. giganteum in the steroid composition and contains eremophylene, a sesquiterpenoid, which is absent in the mosses of the family Amblystegiaceae. Compared to C. giganteum, W. fluitans has a higher content of lycopadiene, carboxylic acids, n-alkanes, phyt-2-ene, aldehydes, esters, squalene, diploptene, α-tocopherol, and triphenyl phosphates.     

Regulation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu fermentation

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.     

The role of gibberellins and auxin on the tomato cell layers in pericarp via the expression of ARFs regulated by miRNAs in fruit set

Phytohormones, such as auxin (IAA) and gibberellin (GA), are known to be essential for fruit development. We utilized GA-deficient (gib-3) and diageotropica (dgt) tomato mutants to elucidate the effects of single hormones in the pericarp. The application of IAA or GA, respectively, to gib-3 or dgt single mutants induced a significant morphological difference in the fruit set. We found that IAA application induced cell division in the gib-3 pericarp and that GA application did not increase the cell layers in the dgt pericarp. In molecular studies, the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were downregulated by IAA application, whereas the expression of their regulators miRNA160 and miRNA167 was upregulated by IAA application in gib-3 plants. Furthermore, the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were upregulated by GA application, whereas the expression levels of miRNAs were reduced in the dgt mutant. These results imply that the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were negatively correlated with the number of cell layers in the pericarp during fruit set. To further support this hypothesis, 35s:mSlARF10 transgenic plants resistant to SlmiR160 cleavage of SlARF10 mRNA were used to investigate the cell layers in fruit. These results revealed that mSlARF10 overexpression indeed resulted in fewer cell layers than in wild type fruit. Together, our data suggest that GA- and IAA-mediated miRNAs and their target ARFs influence the formation of pericarp cell layers during fruit set development.     

Heterologous expression of a novel <Emphasis Type="Italic">Poa pratensis</Emphasis> gibberellin 2-oxidase gene, <Emphasis Type="Italic">PpGA2ox</Emphasis>, caused dwarfism,late flowering,and increased chlorophyll accumulation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin 2-oxidases (GA2oxs) irreversibly convert bioactive gibberellins (GAs) and their immediate precursors into inactive GAs via 2-β hydroxylation and so regulate gibberellin content in plants. However, to the best of our knowledge, little has been known about the GA2oxs and its function in cool season turfgrass Poa pratensis. In this study, rapid amplification of cDNA end (RACE) was employed to isolate PpGA2ox from P. pratensis. The open reading frame of PpGA2ox was 1 047 bp in length, corresponding to 348 amino acids. PpGA2ox was localized in both nucleus and cytoplasm. The expression of PpGA2ox could be up-regulated by 10 μM gibberellic acid, 5 μM methyl jasmonate, or 10 μM indole-3-acetic acid. In addition, its native promoter could drive GUS expression in both leaf apex and shoot apical region. Moreover, overexpression of PpGA2ox in Arabidopsis led to GA-deficiency leading to dwarf phenotype, delayed flowering time, and increased chlorophyll content. Our study suggests that PpGA2ox could be a candidate gene for breeding new cultivars of P. pratensis.     

Neuro-and psychotropic activity of <Emphasis Type="Italic">N</Emphasis>-uronoylamino acids and <Emphasis Type="Italic">N</Emphasis>-uronoylpeptides

Peculiarities of the rat behavior were studied in a series of experimental stress models after a systemic administration of new N-uronoyl derivatives of amino acids. The psychotropic effect was shown to be determined by the nature of the amino acid fragment. N-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyraneuronoyl)-glycylglycine exhibited an anxiolytic effect more pronounced than that of pyracetam, whereas N-(1,2:3,4-di-O-isopropilidene-α-D-galactopyranuronoyl)-glycylglutamic acid has antidepressant action stronger than that of amitriptyline. Mechanisms for the psychotropic effects of the examined derivatives are discussed.     

Synthesis and Protective Activity of β-Glycosides of <Emphasis Type="Italic">N</Emphasis>-Acetylmuramyl-<Emphasis Type="Italic">L</Emphasis>-Alanyl-<Emphasis Type="Italic">D</Emphasis>-Isoglutamine with Alkylalicyclic and Arylaliphatic Aglycons

The following glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) were synthesized: β-4-tert-butylcyclohexyl MDP, β-2-(adamant-1-yl)ethyl MDP, β-2,2-diphenylethyl MDP, and β-2-(p-biphenyl) ethyl MDP. The starting peracetylated β-N-acetylglucosaminides were prepared by the oxazoline method. They were converted into 4,6-O-isopropylidene-N-acetyl-D-muramic acids, which were coupled with L-Ala-D-Glu(NH₂)OBn. The target glycopeptides were obtained after their deprotection. The stimulation of the anti-infection resistance of mice against Staphylococcus aureus by the MDP glycosides was studied.     

New Charge-Deficient Agmatine Analogues

N,N′-Di-Boc-N″-triflylguanidine was demonstrated to be an efficient guanidinylation reagent for O-substituted hydroxylamines. N-(3-Aminooxypropyl)- and N-(3-aminopropoxy)guanidines, previously unknown isosteric and charge-deficient agmatine analogues, have been synthesized. The possibilities of using these compounds in studying polyamine metabolism are discussed.     

Fine-scale differences in genetic and census population size ratios between two stream fishes

Comparing the ratio of effective number of breeders (N _b ) to adult population size (N) among closely related coexisting species can provide insights into the role of life history on N _b /N ratios and inform conservation programs towards limiting the loss of evolutionary potential in natural populations. We estimated N _b and N in two coexisting salmonid fishes (Brook trout and Atlantic salmon) for 3–4 consecutive years in two small, adjacent streams in Newfoundland, Canada, using mark-recapture (N), linkage disequilibrium (N _b(LD)), and sibship frequency approaches (N _b(Sib) ). We found that N _b /N ratios were about 20-fold greater in Atlantic salmon than in brook trout (mean 0.20, range 0.06–0.56 vs. mean 0.02, range 0.01–0.05, respectively). This difference was consistent across N _b estimators. In addition, we found that removing migrants reduced N _b : the strength of the effect was weak for N _b(LD) and much stronger for N _b(Sib). Our results highlight the importance of subtle ecological differences and gene flow in shaping N _b /N. They also provide some evidence that the linkage between demographic and evolutionary processes varies between closely related taxa and suggest that a more complete understanding of the N _b /N range across various species is an important component of conservation genetics and management.     

Enzymatic synthesis of lactosylated and sialylated derivatives of epothilone A

Epothilone A is a derivative of 16-membered polyketide natural product, which has comparable chemotherapeutic effect like taxol. Introduction of sialic acids to these chemotherapeutic agents could generate interesting therapeutic glycoconjugates with significant effects in clinical studies. Since, most of the organisms biosynthesize sialic acids in their cell surface, they are key mediators in cellular events (cell-cell recognition, cell-matrix interactions). Interaction between such therapeutic sugar parts and cellular polysaccharides could generate interesting result in drugs like epothilone A. Based on this hypothesis, epothilone A glucoside (epothilone A 6-O-β-D-glucoside) was further decorated by conjugating enzymatically galactose followed by sialic acids to generate epothilone A 7-O-β-D-glucopyranosyl, 4′-O-α-D-galactoside i.e., lactosyl epothilone A (lac epoA) and two sialosides of epothilone A namely epothilone A 7-O-β-D-glucopyranosyl, 4′-O-α-D-galactopyranosyl 3″-O-α-N-acetyl neuraminic acid and epothilone A 7-O-β-D-glucopyranosyl, 4′-O-α-D-galactopyranosyl 6″-O-α-N-acetylneuraminic acid i.e., 3′sialyllactosyl epothilone A: 3′SL-epoA, and 6′sialyllactosyl epothilone A: 6′SL-epoA, respectively. These synthesized analogs were spectroscopically analyzed and elucidated, and biologically validated using HUVEC and HCT116 cancer cell lines.     

<Emphasis Type="Italic">N</Emphasis>-Glycosylation of an IgG antibody secreted by <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.     

Characterization of <Emphasis Type="Italic">GA20ox</Emphasis> genes in tall and dwarf types coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.)

Coconuts (Cocos nucifera L.) are divided by the height into tall and dwarf types. In many plants the short phenotype was emerged by mutation of the GA20ox gene encoding the enzyme involved in gibberellin (GA) biosynthesis. Two CnGA20ox genes, CnGA20ox1 and CnGA20ox2, were cloned from tall and dwarf types coconut. The sequences, gene structures and expressions were compared. The structure of each gene comprised three exons and two introns. The CnGA20ox1 and CnGA20ox2 genes consisted of the coding region of 1110 and 1131 bp, encoding proteins of 369 and 376 amino acids, respectively. Their amino acid sequences are highly homologous to GA20ox1 and GA20ox2 genes of Elaeis guineensis, but only 57% homologous to each other. However, the characteristic amino acids two histidines and one aspartic acid which are the two iron (Fe²⁺) binding residues, and arginine and serine which are the substrate binding residues of the dioxygenase enzyme in the 20G-FeII_Oxy domain involved in GA biosynthesis, were found in the active site of both enzymes. The evolutionary relationship of their proteins revealed three clusters in vascular plants, with two subgroups in dicots and three subgroups in monocots. This result confirmed that CnGA20ox was present as multi-copy genes, and at least two groups CnGA20ox1 and CnGA20ox2 were found in coconut. The nucleotide sequences of CnGA20ox1 gene in both coconut types were identical but its expression was about three folds higher in the leaves of tall coconut than in those of dwarf type which was in good agreement with their height. In contrast, the nucleotide sequences of CnGA20ox2 gene in the two coconut types were different, but the expression of CnGA20ox2 gene could not be detected in either coconut type. The promoter region of CnGA20ox1 gene was cloned, and the core promoter sequences and various cis-elements were found. The CnGA20ox1 gene should be responsible for the height in coconut, which is different from other plants because no mutation was present in CnGA20ox1 gene of dwarf type coconut.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.