The Biosynthetic Gene Cluster of Mushroom-Derived Antrocin Encodes Two Dual-Functional Haloacid Dehalogenase-like Terpene Cyclases

(−)-Antrocin ( 1 ), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase-like terpene cyclase AncC that biosynthesizes the drimane-type sesquiterpene (+)-albicanol ( 2 ) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2 . Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)-trans-γ-monocyclofarnesol instead of 2 . Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane-type scaffold.     

Discovery and Biosynthesis of Cihunamides,Macrocyclic Antibacterial RiPPs with a Unique C−N Linkage Formed by CYP450 Catalysis

Cihunamides A–D ( 1 – 4 ), novel antibacterial RiPPs, were isolated from volcanic-island-derived Streptomyces sp. The structures of 1 – 4 were elucidated by ¹H, ¹³C, and ¹⁵N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C−N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co-expression of the core genes demonstrated the biosynthesis of cihunamides through P450-mediated oxidative Trp-Trp cross-linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including that of tryptorubins, which possess a distinct Trp-Trp linkage. Cihunamides do not display the non-canonical atropisomerism shown in tryptorubins, which are the founding members of the “atropitide” family. Therefore, we propose to use a new RiPP family name, “bitryptides”, for cihunamides, tryptorubins, and their congeners, wherein the Trp-Trp linkages define the structural class rather than non-canonical atropisomerism.     

Non-canonical Biosynthesis of the Brexane-Type Bishomosesquiterpene Chlororaphen through Two Consecutive Methylation Steps in Pseudomonas chlororaphis O6 and Variovorax boronicumulans PHE5-4

A non-canonical biosynthetic pathway furnishing the first natural brexane-type bishomosesquiterpene (chlororaphen, C₁₇H₂₈) was elucidated in the γ-proteobacterium Pseudomonas chlororaphis O6. A combination of genome mining, pathway cloning, in vitro enzyme assays, and NMR spectroscopy revealed a three-step pathway initiated by C10 methylation of farnesyl pyrophosphate (FPP, C₁₅) along with cyclization and ring contraction to furnish monocyclic γ-presodorifen pyrophosphate (γ-PSPP, C₁₆). Subsequent C-methylation of γ-PSPP by a second C-methyltransferase furnishes the monocyclic α-prechlororaphen pyrophosphate (α-PCPP, C₁₇), serving as the substrate for the terpene synthase. The same biosynthetic pathway was characterized in the β-proteobacterium Variovorax boronicumulans PHE5-4, demonstrating that non-canonical homosesquiterpene biosynthesis is more widespread in the bacterial domain than previously anticipated.     

Synthesis of Neocaesalpin A,AA, and Nominal Neocaesalpin K**

The first total synthesis of heavily oxidized cassane-type diterpenoid neocaesalpin A ( 1 ) is disclosed. At the heart of the synthesis lies an intermolecular Diels–Alder reaction that rapidly assembles the target framework from commercial materials. A carefully orchestrated sequence of oxidations secured the desired oxygenation pattern. Late-stage release of the characteristic butenolide occurred through a novel mercury(II)-mediated furan oxidation. Successful extension of the route allowed preparation of neocaesalpin AA ( 2 ) as well as nominal neocaesalpin K ( 3 ) and suggested structural revision of neocaesalpin K, leading to the hypothesis that the two are likely the same natural product with correct assignment as 2 .     

Fractional derivatives of products of Airy functions

Fractional derivatives of the products of Airy functions are investigated, and Dα{Ai(x)×Bi(x)}, where Ai(x) and Bi(x) are the Airy functions of the first and second type, respectively. They turn out to be linear combinations of Dα{Ai(x)} and Dα{Gi(x)}, where Gi(x) is the Scorer function. It is also proved that the Wronskian W(x) of the system of half integrals {D−1/2Ai(x),D−1/2Gi(x)} and its Hilbert transform can be considered special functions in their own right since they are expressed in terms of and Ai(x)Bi(x), respectively. Various integral relations are established. Integral representations for Dα{Ai(x−a)Ai(x+a)} and its Hilbert transform −HDα{Ai(x−a)Ai(x+a)} are derived.     

Analysis of Low Hamming Weight Products

Hoffstein and Silverman suggested a use of Low Hamming Weight Product (LHWP) to compute a random power in a group or a multiple of an element in a ring. It reduces the computation of powers in a group with fast endomorphisms such as the Galois field F_2ⁿ and Koblitz elliptic curves. In this paper, we introduce a reduced representation of LHWP and apply them to attack the relevant cryptosystems.     

The assessment of π-π selective stationary phases for two-dimensional HPLC analysis of foods: Application to the analysis of coffee

Differences between alkyl, dipole-dipole, hydrogen bonding, and π-π selective surfaces represented by non-resonance and resonance π-stationary phases have been assessed for the separation of ‘Ristretto’ café espresso by employing 2DHPLC techniques with C18 phase selectivity detection. Geometric approach to factor analysis (GAFA) was used to measure the detected peaks (N), spreading angle (β), correlation, practical peak capacity (n_p) and percentage usage of the separations space, as an assessment of selectivity differences between regional quadrants of the two-dimensional separation plane. Although all tested systems were correlated to some degree to the C18 dimension, regional measurement of separation divergence revealed that performance of specific systems was better for certain sample components. The results illustrate that because of the complexity of the ‘real’ sample obtaining a truly orthogonal two-dimensional system for complex samples of natural origin may be practically impossible.     

Spectrophotometric Determination of Amoxycillin in Dosage Forms

Abstract

Two spectrophotometric methods are proposed for the determination of amorycillin and its doeage fonne. Both procedures depend on the phenolic nature of the compound. The first method is based on condeneation with 4-aminoautipyrine in the presence of an alkaline oxidising agent. A red coloured antipyrine dye peaking at 500 nm. is produced. The second method involves the use of folin's reagent, a blue colour with λ max at 670 nm is obtained. The proposed methods could be applied successfully for the determination of dosage forms containing amoxycillin, and the results obtained were compared favourably with those obtained with the official methods.     

New Immobilized Enzyme Membranes for Tailor-Made Biosensors

Abstract

Commercially available polyamide preactivated membranes for immunodiagnostic use were found suitable for the preparation of immobilized enzyme membranes adaptable to biosensors. Membranes with immobilized glucose oxidese as model enzyme, tested with a microprocessor-based device involving an enzymatic electrode, gave excellent results. The extremely simple and fast procedure allows anyone to prepare such bioactive membranes easily, possibly from his own enzyme preparation and within a few minutes set up a specific probe from commercially available sensors.