Structure Elucidation, Total Synthesis, Antibacterial In Vivo Efficacy and Biosynthesis Proposal of Myxobacterial Corramycin.

Herein, we describe the myxobacterial natural product Corramycin isolated from Corallococcus coralloides. The linear peptide structure contains an unprecedented (2R,3S)-γ-N-methyl-ß-hydroxy-histidine moiety. Corramycin exhibits anti-Gram-negative activity against Escherichia coli (E. coli) and is taken up via two transporter systems, SbmA and YejABEF. Furthermore, the Corramycin biosynthetic gene cluster (BGC) was identified and a biosynthesis model was proposed involving a 12-modular non-ribosomal peptide synthetase/polyketide synthase. Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule's absolute configuration. Importantly, intravenous administration of 20 mg kg-1 of Corramycin in an E. coli mouse infection model resulted in 100 % survival of animals without toxic side effects. Corramycin is thus a promising starting point to develop a potent antibacterial drug against hospital-acquired infections.

New Insights in Thrombin Inhibition Structure-Activity Relationships by Characterization of Octadecasaccharides from Low Molecular Weight Heparin.

Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline ß-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.

Analytical comparison of a US generic enoxaparin with the originator product: The focus on comparative assessment of antithrombin-binding components.

Enoxaparin sodium, a low-molecular-weight heparin (LMWH) prepared from porcine intestinal heparin, is widely used for the prevention and treatment of venous thromboembolism. The antithrombotic activity of heparin is mediated mainly through its activation of antithrombin (AT) and subsequent inhibition of coagulation factors. Heparin is a complex heteropolymer and the sulfation pattern of its alternating uronic acid and glucosamine sugar units is a major factor influencing its biological activity. The manufacturing process itself is associated with the introduction of exogenous microheterogeneities that may further affect its biological efficacy. This is important since enoxaparin is prepared by depolymerizing the heparin with the aim of optimizing its biological activity and safety. Changes during its manufacture could thus affect its biological activity and safety. The current study was performed to assess potential differences between the originator enoxaparin and a new generic enoxaparin commercialized by Teva. Heparinase digestion, AT affinity chromatography, gel permeation chromatography, anion exchange chromatography, and nuclear magnetic resonance methodologies were used. The results indicated differences in oligosaccharides related to the cleavage selectivity around the heparin AT-binding sequences of the Teva Enoxaparin Sodium Injection, USP and the originator Sanofi enoxaparin. These differences influence the strength of the AT-binding affinity of the individual oligosaccharides, their ability to activate AT and, therefore, the inhibitory potency on the proteases of the coagulation cascade. This study, together with other published analytical reports, describes specific compositional differences between generics and originator LWMHs. However, it is yet to be established whether such variations might have any clinical relevance.

Synthesis of new conformationally constrained pentasaccharides as molecular probes to investigate the biological activity of heparin.

We have synthesized three new antithrombin activating pentasaccharides displaying various sulfation patterns on the reducing end unit (H). We found that when L-iduronic acid stands in the 2S(0) conformation, the sulfate groups at positions 3 and 6 of the reducing end unit are practically devoid of influence on the activation of antithrombin. This suggests that the positive role of these sulfates is more related to their ability to shift the conformational equilibrium of L-iduronic acid towards 3S(0) than to directly interact with the protein.

A simple scheme for the design of solvent-suppression pulses.

Excitation sculpting was first introduced as a way to efficiently suppress solvent signals. It requires a pulse sequence that acts as a null pulse at the solvent-resonance frequency and as an inversion pulse everywhere else. In this article, it is shown that such a goal can be achieved starting with "top-hat" inversion shaped pulses such as I-BURP-2 or gaussian cascade G3. The result is a Globally Antisymmetric Selective Pulse, or GASP. Numerical optimization was used to extend the performance of such pulses. Multifrequency signal suppression was shown to be possible through application of successive excitation sculpting modules.