New Rufomycins from Streptomyces atratus MJM3502 Expand Anti-Mycobacterium tuberculosis Structure-Activity Relationships.

Four new rufomycins, compounds 1-4, named rufomycins 56, 57, 58, and 61, respectively, exhibiting new skeletal features, were obtained from Streptomyces atratus strain MJM3502 and were fully characterized. Compounds 1 and 2 possess a 4-imidazolidinone ring not previously encountered in this family of cyclopeptides, thereby resulting in a [5,17] bicyclic framework. The in vitro anti-Mycobacterium tuberculosis potency of compounds 3 and 4 is remarkable, with minimum inhibitory concentration values of 8.5 and 130 nM, respectively.

The qNMR Summit 5.0: Proceedings and Status of qNMR Technology.

The goal of the qNMR Summit is to take stock of the status quo and the recent developments in qNMR research and applications in a timely and accurate manner. It provides a platform for both advanced and novice qNMR practitioners to receive a well-rounded update and discuss potential qNMR-related applications and collaborations. For over a decade, scientists from academia, industry, nonprofit institutions, and governmental bodies have focused on the standardization of qNMR methodology, as well as its metrological and pharmacopeial utility. This paper reviews key content of qNMR Summits 1.0 to 4.0 and puts into perspective the outcomes and available transcripts of the October 2019 Summit 5.0, with attendees from the United States, Canada, Japan, Korea, and several European countries. Summit presentations focused on qNMR methodology in the pharmaceutical industry, advanced quantitation algorithms, and promising developments.

Prenylated Coumaric Acids from Artemisia scoparia Beneficially Modulate Adipogenesis.

Two new diprenylated coumaric acid isomers (1a and 1b) and two known congeners, capillartemisin A (2) and B (3), were isolated from Artemisia scoparia as bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR methods and LC-MS, with their purity assessed by 1D 1H pure shift qNMR spectroscopic analysis. The bioactivity of compounds was evaluated by enhanced accumulation of lipids, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the plant's 80% EtOH extract, these purified compounds showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This suggests that additional bioactive substances are responsible for the multiple metabolically favorable effects on adipocytes observed with Artemisia scoparia extract.

Reaction of Oxidized Polysialic Acid and a Diaminooxy Linker: Characterization and Process Optimization Using Nuclear Magnetic Resonance Spectroscopy.

Native polysialic acid (natPSA) is a high-molecular-weight glycan composed of repeat units of α-(2 → 8) linked N-acetylneuraminic acid (Neu5Ac). Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used, after attachment of a linker molecule, for terminal attachment of PSA to protein. Previously, we showed that the oxidized PSA (oxoPSA) contained a hemiacetal at the oxidation site and can react with a linker containing an aminooxy group in a conjugation reaction to form a stable oxime linkage. Thus, reagents containing an aminooxy group may be prepared for conjugation of PSA to the carbohydrate moiety of therapeutic proteins, thereby increasing their half-life. These aminooxy-PSA reagents can selectively react with aldehyde groups generated by mild NaIO4 oxidation of glycans on the surface of the target protein. To comprehend the conjugation, unoxidized tetrasialic acid and Neu5Ac were reacted in model reactions with a diaminooxy linker to define the nuclear magnetic resonance (NMR) chemical shifts. Based on these data, we were able to show that, in the case of PSA, the reaction with the linker occurs not only at the expected oxidized end to form an aldoxime but also at the end distal to the oxidation to form a ketoxime. We determined that, in aged solutions, both oxoPSA and PSA aldoxime were hydrolyzed. PSA aldoxime was also shown to disproportionate to form a dimer (PSA-linker-PSA), which then could react further with the released linker at one of its PSA termini. Furthermore, NMR was used to monitor the effects of deliberate process changes so that conditions could be optimized for attachment of linker at the desired end of the PSA chain, which led to a well-defined product.

Screening of complex fucoidans from four brown algae species as procoagulant agents.

Fucoidans are complex sulfated polysaccharides extracted from brown algae. Depending on the concentration, they have been shown to stimulate and inhibit blood coagulation in vitro. Promotion of coagulation is mediated by blocking tissue factor pathway inhibitor (TFPI). We screened fucoidan extracts from four brown algae species in vitro with respect to their potential to improve coagulation in bleeding disorders. The fucoidans' pro- and anticoagulant activities were assessed by global hemostatic and standard clotting assays. Results showed that fucoidans improved coagulation parameters. Some fucoidans also activated the contact pathway of coagulation, an undesired property reported for sulfated glycosaminoglycans. Chemical evaluation of fucoidans' complex and variable structure included molecular weight (Mw), polydispersity (polyD), structural heterogeneity, and organic and inorganic impurities. Herewith, we describe a screening strategy that facilitates the identification of crude fucoidan extracts with desired biological and structural properties for improvement of compromised coagulation like in hemophilia.

Mechanism of 2-hydropropyl-beta-cyclodextrin in the stabilization of frozen formulations.

Freezing of commonly used parenteral products to increase pharmaceutical stability for cost-saving purposes is a common practice in patient care. However, frozen meropenem, a model drug, in saline has a shelf life of less than a month due to the low glass transition temperature (Tg'): below -40°C. When meropenem is formulated with the 2-hydroxypropyl-ß-cyclodextrin (HPBC) the shelf life (⩾90% potency) is extrapolated to be greater than one year at -25°C based on data for storage at 6months. The mechanisms that may explain meropenem-HPBC formulation frozen stability include vitrification and/or formation of an inclusion complex. Although NMR data indicated complexation of meropenem by HPBC in a ratio of 0.6:1, inclusion was unlikely to be the mechanism as stability was not extended to the thawed solutions. Therefore, vitrification is concluded to be the stabilization mechanism. The Tg' for meropenem-HPBC (13.3%) formulation at pH 7.9 was -17.75°C which was similar to that of a meropenem solution formulated with a known vitrifying agent, Dextran 40. This higher Tg' for HPBC was unexpected based on trends predicted by the Fox-Flory equation. Trial formulations containing either Dextran 1, Dextran 40, hydroxyethyl starch, or sulfobutyl-beta-cyclodextrin heptasodium (Captisol®) were also unable to stabilize meropenem as the Tg' values were below the frozen storage temperature. Upon 6-month storage, potency losses were -3.0% and -7.7% for meropenem frozen premix formulated in 13.3% HPBC (pH 7.9) at -25 and -20°C storage, respectively; versus -31.2% and -60.8% for controls. Frozen premixes with high ionic strength (containing NaCl or Captisol®) and/or at pH 7.3 were also found to be unstable.

Complete structural elucidation of an oxidized polysialic acid drug intermediate by nuclear magnetic resonance spectroscopy.

Polysialic acid (PSA) is a high molecular weight glycan composed of repeat units of α(2→8) linked 5-N-acetyl-neuraminic acid. Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used for terminal attachment of PSA to therapeutic proteins. The work presented here permitted complete NMR peak assignments of not only the repeat units, but also the two terminal units at each end of oxidized PSA, an intermediate, which can be used to improve drug performance. The assignments were made using a variety of NMR techniques on oligomers of sialic acid as well as oxidized PSA with molecular masses of 4 and 20 kDa. This enabled structure elucidation that showed the actual moiety formed was not the expected aldehyde or its hydrate, but is a hemiacetal between the oxidation site on the terminal sialic acid ring and the penultimate ring. The existence of a hemiacetal structure has major implications on stability, reactivity, and conjugation chemistry of oxidized PSA. The assignment process also revealed deuterium exchange of the axial hydrogen at the 3- (methylene) position of the ring, which was in agreement with the literature.

A comparison of quantitative nuclear magnetic resonance methods: internal, external, and electronic referencing.

The performance of three quantitative NMR methods was compared in terms of short-term and long-term precision and accuracy, robustness, linear range, and general applicability. The Internal Reference method employs a reference material co-dissolved with sample; the External Reference method employs a reference material contained in a separate solution; and the third method, known as Electronic REference To access In vivo Concentrations (ERETIC), employs an externally calibrated digital reference peak. The Internal Reference method results were the most precise and remained stable within 0.1% for at least 4 weeks. The results from the External Reference and ERETIC methods were practically equivalent to each other during this time. These methods exhibited small differences relative to the standard set by the Internal Reference method and slightly lower precision, establishing them as practical alternatives to the Internal Reference method. In contrast to the Internal Reference method, the External Reference and ERETIC methods possess several advantages that address peak overlap, flexibility of calibration, and duration of applicability. The study was designed such that each spectrum contained the information needed to compare the three methods while all other variables were kept constant. Applicability of pulse width compensation is addressed. ERETIC software compensation and minor adjustments to 90° pulse width were concluded to be unnecessary for this system. Although each of the methods was applied here to specifically calculate and compare chemical purity values, this evaluation applies generally to absolute quantitation by NMR.

NMR of heparin API: investigation of unidentified signals in the USP-specified range of 2.12-3.00 ppm.

This article addresses the identification and quantification of the chemical species resulting in resonances at 2.17 and 2.25 ppm in the (1)H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade heparin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through (13)C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: (1)H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12-3.00 ppm for heparin API.