Research advances in methods of cyclezation mechanism of sesquiterpenes / 中国中药杂志

Terpenes are the largest group of natural products and contain the widest assortment of structural types. Terpene cyclization is also the most complex reaction found in nature. For a long time, terpenoids with diverse structures have attracted natural product chemists to explore their biosynthesis mechanism. Such a large number of terpene skeletons are catalyzed by enzymes called terpene synthase. Sesquiterpene synthase is a kind of terpene synthase, which can catalyze the cyclization of linear precursor farnesyl pyrophosphate(FPP) to sesquiterpene skeletons. Sesquiterpene synthase cyclize a single precursor FPP into many sesquiterpene skeletons. With the continuous discovery of sesquiterpene synthase, the cyclization mechanism of sesquiterpene synthase has been studied deeply. In recent years, with the development and improvement of isotope labeling of substrate FPP and structural analysis of sesquiterpene synthase, the structure and cyclization mechanism of sesquiterpene synthase have been studied more systematically and accurately. In this review, we reviewed the progress of the research methods on the mechanism of sesquiterpene cyclization by substrate isotope labeling and protein structure, as well as the summary and prospect of sesquiterpene synthase research.

Enhancing thermostability of xylanase from rumen microbiota by molecular cyclization / 生物工程学报

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.

Advances in isopeptide bond-mediated molecular superglue / 生物工程学报

Isopeptide bond-mediated molecular superglue is the irreversible covalent bond spontaneously formed by the side chains of lysine (Lys) and asparagine/aspartic acid (Asn/Asp) residues. The peptide-peptide interaction is specific, stable, and can be achieved quickly without any particular physicochemical factor. In the light of recent progress by domestic and foreign researchers, here we summarize the origin, assembly system and mechanism of isopeptide bond reaction, as well as the molecular cyclization and protein topological structure mediated by it. The prospect for its application in synthetic vaccine, hydrogel and bacterial nanobiological reactor is further discussed.

Filamentous fungal sesterterpenoids and their synthases / 生物工程学报

Although the number of sesterterpenoids is fewer than other terpenoids reported, they have presented a wide range of biological activities and medicinal value. Reported filamentous fungal sesterterpene synthases are special on bifunctional two catalytically independent domains: prenyltransferase and terpene cyclase, but less specific on substrates selection and diverse ways of cyclization. This article reviews the research advances in filamentous fungal sesterterpenoids and their synthases, especially describes filamentous fungal sesterterpenoids and the structure and function characteristics of sesterterpene synthase.

Advances in protein cyclization / 生物工程学报

Proteins, which exist mainly in linear form in vivo, are easily affected by the change of ambient temperature and pH. The application of proteins (enzymes) in the fields of industrial catalyzing, food manufacturing and medicine are restricted due to their properties. The cyclic structure of natural cyclic peptides confers high thermal stability on itself; such mechanism can be referred to in further enhancement of the thermal stability and transformation of the structure of enzymes. This article reviewed the latest progress in the domestic and international studies on protein cyclization and summarized the traditional methods (such as protein trans-splicing, expressed protein ligation and sortase-catalyzed transpeptidation) in protein cyclization. A novel method based on SpyTag/SpyCather-mediated enzyme cyclization was discussed in more detail.

Enzymatic cyclization of peptides using immobilized sortase A / 药学学报

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.

Functional characterization of sll0659 from Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that Sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types and mutants. In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cycilzation. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.

New dammarane type triterpene with cyclization at the side chain from oxidative alkaline-degradation products of PQS / 药学学报

To study the oxidative alkaline-degradation products of PQS (Panax quinquefolium saponin), two compounds were isolated from the crude product of oxidative alkaline-degradation by silica gel column chromatography, Sephadex LH-20 column chromatography and recrystallized methods. On the basis of spectroscopic analysis, their structures were established as (12R, 20S, 24R)-20, 24; 12, 24-diepoxy-24-deisopropyl-dammarane-3beta-ol (1) and (20S, 24R)-20, 24-epoxydammarane-3beta3, 12beta, 25-triol (2). Compounds 1 and 2, dammarane type triterpene with cyclization at the side chain, were obtained for the first time from alkaline-degradation products of total ginsenosides of Panax quinquefolium L., compound 1 is a new compound.

Evidence for the Presence of a cis-acting Element in the Coding Region of the Japanese Encephalitis Virus Capsid Protein

Japanese encephalitis virus (JEV) is a member of mosquito-borne flaviviruses. To investigate whether there is a cis-acting genetic element in the coding region of the JEV C protein, which is required for viral replication, we generated four mutants by introducing a various size of deletions in each structural protein-coding region, designated as pJEV/Rep/deltaCC/LUC, pJEV/Rep/deltaC/LUC, pJEV/Rep/deltaprM/LUC, and pJEV/Rep/deltaE/LUC, of these, all replicons except for pJEV/Rep/deltaCC/LUC were competent in replication. Since pJEV/Rep/deltaCC/LUC is the same as pJEV/Rep/ deltaC/LUC except for an additional 5' deletion (nt 132~201) in the coding region of the C protein, this region appeared to be essential for RNA replication. This is consistent with the proposed cyclization sequence motif in the 5' region of the C gene, which has been recently shown to be required for replication in other mosquito-borne flaviviruses such as DV, YFV, KUN, and WNV. Thus, our results suggest that a cis-acting genetic element in the coding region of the JEV C protein may play an important role in RNA replication. This study will facilitate the current understanding of JEV RNA replication.

Development of Macrocyclic Ligands for Stable Radiometal Complexes / 대한핵의학회잡지

Current interest in the regioselective N-functionalization of tetraazacycloalkanes (cyclen and cyclam) stems mainly from their complexes with radioactive metals for applications in diagnostic (64Cu, 111In, 67Ga) and therapeutic (90Y) medicine, and with paramagnetic ions for magnetic resonance imaging (Gd+3). Selective methods for the N-substitution of cyclen and cyclam is a crucial step in most syntheses of cyclen and cyclam-based radiometal complexes and bifunctional chelating agents. In addition, mixing different pendent groups to give hetero-substituted cyclen derivatives would be advantageous in many applications for fine-tuning the compound's physical properties. So far, numerous approaches for the regioselective N-substitution of tetraazacycloalkanes and more specifically cyclen and cyclam are reported. Unfortunately, none of them are general and every strategy has its own strong points and drawbacks. Herein, we categorize numerous regioselective N-alkylation methods into three strategies, such as 1) direct substitution of the macrocycle, 2) introduction of the functional groups prior to cyclization, and 3) protection/functionalization/deprotection. Our discussion is also split into the methods of mono- and tri-functionalization and di-functionalizataion based on number of substituents. At the end, we describe new trials for the new macrocycles which form more stable metal complexes with various radiometals, and briefly mention the commercially available tetraazacycloalkanes which are used for the biconjugation of biomolecules.

Synthesis of new Imidazoline derives by the ethylene diamine cyclization from nitril compounds

The imidazol and imidazoline derives have many advantages in therapy. For the synthesis of new imidazoline derives, the authors implemented the ethylene diamene cyclization from nitril compounds differently substituted. Then, a dehydrogenation of these imidazolines leaded to the imidazol derives. The last reaction consisted of the condensation of these imidazolines and imidazoles. The authors subjected their synthesized products to an ophthalmic protective evaluation (anti-oedema, secondary anti-hyperemia) on the experimental ocular irritancy models

Peptídeos de conformação restrita relacionados ao sítio 2 do fator de crescimento de fibroblastos ácido humano (hFGF-1): estudo sobre estrutura e atividade / Study of structure-activity relationship of synthetic peptides derived from human acidic Fibroblast Growth Factor Site 2 primary sequence

Na busca por agonistas, antagonistas e inibidores de natureza peptídica do fator de crescimento de fibroblastos ácido humano (hFGF-1), iniciamos o presente trabalho fazendo uma análise conformacional teórica do peptídeo Ac-WFVGLKKNGSSKRGPRT-N`H IND. 2ï (107-123[hFGF-1]). Em trabalho anterior, este composto havia se mostrado um agonista da atividade mitogênica da proteína capaz de inibir a ligação de `ANTPOT. 125Iï-hFGF-1 aos seus receptores celulares e de se ligar à resina heparina-Sepharose (Oyama et al. 1996). Os cálculos das propriedades dinâmicas deste peptídeo (I; Tabela 1) demonstraram que ele não adotava nenhuma conformação preferencial, o que poderia justificar a baixa atividade apresentada pelo mesmo (`10 POT. 4ï vêzes menor do que a da proteína nativa)...

Synthesis of some cytidine derivatives

Synthesis of some cytidine derivatives involving 4,5 cyclisation are performed by the individual reactions of both of 5-hydroxy, 5-mercapto and 5-amino cytidines with chloroethyl formate, thionyl chloride and thiophosgene successively. On the other hand, reactions of cytidine with chloroacetone or chloropropionaldehyde give 3,4-cyclized cytidine products

Base catalyzed cycloaddition on N-phenyl-4, 6-diphenyl-3acetyl-2-pyridone

It has been reported that alpha, Beta-unsaturated ketones react with nucleophiles to give under different conditions either the open chain addition products or a new heterocyclic system by normal addition followed by cyclization. In the present investigation the authors introduce N-aryl-2-pyridone heterocyclic ring to investigate to what extent the a 6-unsaturated system can be affected by replacement that pyridone ring and to emphasize the general concept that the carbonyl group in alpha, Beta-unsaturated moiety is still kinetically reactive even if it is present together with the electrical localized carbonyl which inhibits the proton tautomeric effect by N-phenyl replacement