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Marine Biopolymers
Published in Se-Kwon Kim, Marine Biochemistry, 2023
Beside the seaweed, alginate produced from bacteria via biosynthesis. The two genera of bacteria that can secrete alginate are Pseudomonas and Azotobacter in which the two species gaining more interest are the opportunistic human pathogen Pseudomonas aeruginosa (Linker & Jones, 1964) and the soil-dwelling Azotobacter vinelandii (Gorin & Spencer, 1966). The mechanism of alginate biosynthesis is similar in the two species but they produce alginate for different purposes. Pseudomonas aeruginosa secretes alginate to form the biofilm, whereas Azotobacter vinelandii produces alginate to build the resistant cysts. The alginate biosynthesis in bacteria is very complicated and includes many steps to produce mannuronic acid to build the polymannuronic chain. After that the acetylation in C2 and C3 of the pyranose rings, then the C5 epimerization changes L-mannuronic residues into D-glurunonic residues under the catalyst of C5-mannuronan epimerase. The alginate from Azotobacter vinelandii has the M/G ratio similar to alginate from seaweed, but the alginate from Pseudomonas aeruginosa shows a high content of guluronate (up to 40%) (Hay et al., 2013). However, when using the C5 epimerase to catalyze the epimerization of polymannuronate in vitro, the equilibrium content of guluronate can reach the value of 75% (Schürks et al., 2002).
High-Performance Liquid Chromatography
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Joel J. Kirschbaum, Adorjan Aszalos
Amoxicillin in formulations was analyzed using an octylsilane column and a mobile phase of methanol-0.05 P/I phosphate buffer (pH 5); (6:94) flowing at 1 ml/min into a 254 nm detector. Responses were linear from 0.5 to 2.5 ng/ml. Phenoxyacetic acid was used as internal standard. Penicilloic acid (0.3–0.6%) and 6-aminopenicillanic acid were also resolved. HPLC content results were verified using a colorimetric assay [166]. Epimerization of the penicilloic acid was also studied by HPLC [167].
Ene-Reductases in Pharmaceutical Chemistry
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Ethyl-(S)-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP) (entry 5) is a precursor for the synthesis of antidiabetic drugs like PPAR-α,γ agonists (e.g., Tesaglitazar). Production of EEHP was improved by introducing an OYE3 catalyzed bioreduction step into the synthetic route (Bechtold et al., 2012). Enzymes (OYE3 and glucose dehydrogenase for cofactor regeneration) were overexpressed in E. coli and used after purification. The substrate was bound to a resin simplifying product recovery and minimizing epimerization. NaClO2 oxidation completed the chemoenzymatic synthesis. The overall yield over both steps was 94% with 98% ee.
The piperazine scaffold for novel drug discovery efforts: the evidence to date
Published in Expert Opinion on Drug Discovery, 2022
Maria Novella Romanelli, Dina Manetti, Laura Braconi, Silvia Dei, Alessio Gabellini, Elisabetta Teodori
An important motivation for inserting those bicyclic structures can be related to the discovery of epibatidine (40), an alkaloid found in the skin extracts from the Ecuadorian poison frog Epipedobates tricolor [65], exhibiting high potency on several nAChR receptor subtypes [66]. The remarkable analgesic activity of this compound stimulated the synthesis of a high number of analogues deriving from replacement of the 7-azabicyclo[2.2.1]heptane ring, with the aim of obtaining potent subtype selective compounds. Epibatidine has an exo configuration; epimerization of the stereogenic center in position 2 gave the endo isomer endowed with much lower activity. The critical stereogenic center could be eliminated by substituting the bicyclic moiety with diazabicycloalkanes and using the second N atom as attachment point for the aromatic heterocycle. Toward this aim, the bicyclic moiety of epibatidine has been replaced with 2,5-DBO, 2,5-DBH, 3,6-DBH, and 3,8-DBO (compounds 41–44, Figure 6). 2,5-DBH is a rigid moiety carrying the piperazine ring locked into a twisted boat geometry; theoretical calculations predicted that in 3,6-DBH and 3,8-DBO, the piperazine ring could assume a boat or chair conformation, with the latter being energetically favored [64,67].
Advances in the discovery and development of selective heme-displacing IDO1 inhibitors
Published in Expert Opinion on Drug Discovery, 2020
PF-06840003 [5] is an indole derivative and the third class I IDO1 inhibitor that entered Phase I clinical trial but there lacked evidence to support its further development as a cancer immunotherapy. Similar to epacadostat and navoximod, PF-06840003 occupies the catalytic site of IDO1 enzyme in the presence of the heme group but does not form any coordinative interaction with the heme iron atom [70]. Instead, the succinimide NH of PF-06840003 forms H-bond interaction with the propionate side chain of the heme. PF-06840003 contains a chiral carbon center that is subjected to rapid epimerization. Therefore, the racemic mixture (IC50: 0.41 µM) was advanced toward development even though only the R-enantiomer is active against hIDO1 (IC50: 0.20 µM). PF-06840003 preferentially bound to the ferric (Fe3+) form of hIDO1 with a higher apparent binding affinity (KD: 0.32 µM) than the ferrous form (KD: 14 µM). In cellular assays measuring the concentration of Kyn, the IC50 of PF-06840003 was reported as 1.8 µM and 4.7 µM in Hela cells and human whole blood, respectively. Preclinical studies indicated that PF-06840003 was orally available albeit required twice daily (BID) dosing due to short half-life. It was shown to cross the blood-brain barrier (unbound AUC ratio of brain to plasma: 0.20 for the active isomer). Similar to epacadostat and navoximod, only moderate anticancer activity was observed for PF-06840003 in preclinical syngeneic tumor models [71].
Improving protein glycan coupling technology (PGCT) for glycoconjugate vaccine production
Published in Expert Review of Vaccines, 2020
Jennifer Mhairi Dow, Marta Mauri, Timothy Alexander Scott, Brendan William Wren
GET is now greatly expedited by the availability of competitively priced gene synthesis services. Recent advances in molecular cloning techniques, such as Gibson Assembly [73], Golden Gate Assembly [74], and ligase cycling reaction [75] have also helped to ease the cloning of large gene clusters. Streamlined and faithful expression of the polysaccharide to be coupled can be improved via manipulation of the host bacterial strain by genome engineering, involving removal of interfering glycosylation pathways or genes. For example, the introduction of the epimerase-encoding gene gne into E. coli, which promotes the epimerization of GlcNAc to GalNAc (the reducing end sugar of S. pneumoniae CPS4), improved glycoconjugate yield [76]. Similarly, the co-expression of the targeted glycan with the transcriptional activator RmpA improved Klebsiella capsules expression [62]. Further considerations, such as the introduction or removal of chain length determinants (cld), may be also needed to achieve native-like glycan production [72]. Recently it has been shown that, using a heterologous cld, higher O-antigen polymerization was achieved promoting a stronger immune response of a glycoconjugate vaccine against Paratyphi A [59].