HPLC and mass spectrometry analysis of the free radical degradation of chondroitin sulfate and its implications in osteoarthritis

Chondroitin sulfate (CS) is vital component of bone and cartilage; it is widely used as a daily supplement in the management of arthritis. Reactive oxygen species (ROS) are involved in vast array of biological processes ranging from regulatory functions to damaging effects in disease pathogenesis. Mass spectrometry is a major tool in deciphering unknown chemical structures, particularly tandem MS. In this study mass spectrometry coupled with High performance liquid chromatography (HPLC) gives some insight into the potential mechanisms involved when ROS are made to attack CS. HPLC and coupled mass spectrometry showed that the free radical depolymerisation of CS yielded an N-acetylgalactosamine (GalNAc) and a uronic acid with varied sulfation. This led us to investigate the possibility of other means of free radical generation with the potential to degrade CS. Alkaptonuria an ultra-rare (1:100000-1:250000) in born error of metabolism resulting in the accumulation of homogentisic acid (HGA) due to a deficiency in homogentisate1,2-dioxygenase. Polymerised HGA is excreted in the urine and deposited as an ochronotic pigment in cartilage. The mechanism of HGAs polymerisation is yet to be concluded due to the complexity of its reactions. This study aims to breakdown individual steps in HGAs biochemistry using mass spectrometry. This will provide further insight into the potential reactions HGA can undergo in the body and propose some mechanisms on how it may polymerise. Using mass spectrometry it became apparent that HGA polymerised when in the presence of copper and whilst HGA in water also yielded a dimer, the structure of which was proposed computationally. Little is known on the mechanisms that underpin the displayed symptoms of AKU, it is known however that the polymerisation of HGA does play a role and is responsible for the ochronotic pigmentation in AKU patients. HPLC and ESI-MS was used to investigate the potential interactions between HGA and CS and propose that HGA is able to degrade CS disaccharides when in the presence of a copper catalyst. And that this interaction will play a role in the early onset of arthritis in AKU patients

Enzymatic synthesis of N-acetyllactosamine from lactose enabled by recombinant β1,4-galactosyltransferases

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial β1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-β-LacNAc from lactose using β1,4-galactosyltransferase NmLgtB-B as the only biocatalyst

Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades

From Wiley via Jisc Publications RouterHistory: received 2021-03-17, rev-recd 2021-04-12, pub-electronic 2021-05-19Article version: VoRPublication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/ L013762/1, BB/M027791/1, BB/M02903411, BB/ M028836/1Funder: H2020 European Research Council; Grant(s): 788231-ProgrES-ERC-2017-ADGAbstract: A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof‐of‐principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed‐bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross‐reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O‐methylnorbelladine

Biocatalytic cascades towards iminosugar scaffolds reveals promiscuous activity of shikimate dehydrogenases

Protecting group-free chemoenzymatic and biocatalytic cascade synthesis of iminosugars from sugar aminopolyols has been achieved using Galactose oxidase F2 and chemical or enzymatic imine reduction. Putative iminosugar oxidoreductases were identified through genome mining, with several shikimate dehydrogenases (SDH) found to effect iminosugar formation. The SDH catalysed transformation is the first example of such activity on an iminosugar scaffold, and provides support for the reduction of iminosugar intermediates by endogenous enzymes in iminosugar biosynthesis

Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios**

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees of drug‐to‐antibody ratio (DAR), in particular on native antibodies. Here, a glycoengineering methodology was developed to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel–Michael addition chemistry. This four‐step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions

Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees antibody-to-drug (DAR) ratios, in particular on native antibodies. Here we report a glycoengineering methodology to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This three step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions

Production of High Value Amine Intermediates via Biocatalytic Cascades in Continuous Flow

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out compartmentalized multistep cascades in a controlled and selective way. As biocatalytic cascades get longer and more complex, reactions become unattainable under typical batch conditions. Here a continuous flow multipoint injection reactor was combined with switching valves to overcome batch incompatibility, thus allowing for successful biocatalytic reaction cascades. As proof-of-principle, several reactive carbonyl intermediates were generated in situ using galactose oxidase and engineered choline oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase-transaminase-imine reductase sequence, introducing different amine reagents at each step without cross reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product alkaloid precursor 4O-methylnorbelladine. The flow biocatalysis platform shown here significantly increases the scope of novel biocatalytic cascades, removing previous limitations due to reaction and reagent batch incompatibility.</p