| 1. |
- Auman, Dirk, et al.
(författare)
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Peroxy Intermediate Drives Carbon Bond Activation in the Dioxygenase AsqJ
- 2022
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:34, s. 15622-15632
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Tidskriftsartikel (refereegranskat)abstract
- Dioxygenases catalyze stereoselective oxygen atom transfer in metabolic pathways of biological, industrial, and pharmaceutical importance, but their precise chemical principles remain controversial. The α-ketoglutarate (αKG)-dependent dioxygenase AsqJ synthesizes biomedically active quinolone alkaloids via desaturation and subsequent epoxidation of a carbon–carbon bond in the cyclopeptin substrate. Here, we combine high-resolution X-ray crystallography with enzyme engineering, quantum-classical (QM/MM) simulations, and biochemical assays to describe a peroxidic intermediate that bridges the substrate and active site metal ion in AsqJ. Homolytic cleavage of this moiety during substrate epoxidation generates an activated high-valent ferryl (FeIV = O) species that mediates the next catalytic cycle, possibly without the consumption of the metabolically valuable αKG cosubstrate. Our combined findings provide an important understanding of chemical bond activation principles in complex enzymatic reaction networks and molecular mechanisms of dioxygenases.
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| 2. |
- Dorst, Kevin, 1992-, et al.
(författare)
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Conformational Preferences at the Glycosidic Linkage of Saccharides in Solution as Deduced from NMR Experiments and MD Simulations : Comparison to Crystal Structures
- 2024
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Ingår i: Chemistry - A European Journal. - 0947-6539 .- 1521-3765. ; 30:15
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Tidskriftsartikel (refereegranskat)abstract
- Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo- or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles phi and psi have been investigated for O-methyl glycosides of three disaccharides where the substitution takes place at a secondary alcohol, viz., in alpha-l-Fucp-(1 -> 3)-beta-d-Glcp-OMe, alpha-l-Fucp-(1 -> 3)-alpha-d-Galp-OMe and alpha-d-Glcp-(1 -> 4)-alpha-d-Galp-OMe, corresponding to disaccharide structural elements present in bacterial polysaccharides. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one-dimensional 1H,1H-NOESY NMR experiments to obtain transglycosidic proton-proton distances and one- and two-dimensional heteronuclear NMR experiments to obtain 3JCH transglycosidic coupling constants related to torsion angles phi and psi. Computed effective proton-proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the alpha-(1 -> 3)-linked disaccharides and revealed that for the bimodal distribution at the psi torsion angle for the alpha-(1 -> 4)-linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter-residue hydrogen bonding pattern for the alpha-(1 -> 4)-linked disaccharide, including a nonconventional hydrogen bond between H5 ' in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield 1H NMR chemical shift displacement compared to alpha-d-Glcp-OMe. Comparison of population distributions of the glycosidic torsion angles phi and psi in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation. The importance of solvation on the preferred conformation of saccharides in solution and in crystals is unraveled by solution-state NMR and computational MD studies of solvated disaccharides. Crystal structures containing solvated glycan structures have glycosidic linkage conformations similar to those of the carbohydrate molecules in solution.
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| 3. |
- Dorst, Kevin M., 1992-
(författare)
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Deciphering Carbohydrate Structure : From NMR Chemical Shifts to Conformational Analysis
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Carbohydrates are ubiquitous in nature and exhibit a multitude of roles. Besides nucleic and amino acids, they can be regarded as the third alphabet of life. They are used as energy source to fuel the cells, as structural building blocks and play a key role in cellular recognition processes. Compared to the other two groups of biomacromolecules, carbohydrates display a higher level of structural complexity by virtue of the number of individual monosaccharide building blocks, as well as the greater number of possibilities of connecting them and additional modifications. This renders a high information content and a good understanding of the structure-function relationship of glycans is important, since the presence or absence of specific structures can make the difference between health and disease.Carbohydrate structures can be characterized and studied by NMR spectroscopy at the atomic level. This process is time-consuming and error-prone, due to the narrow spectral window, in which most carbohydrate resonances are located leading to severe spectral overlap. Computer programs have been developed, aiding this process. This thesis investigates the quality of prediction of NMR chemical shifts of glycopeptides, highly branched oligosaccharide structures and those bearing a non-natural organic aglycone at the reducing end, as well as the automated determination of primary carbohydrate structures from unassigned NMR spectroscopic data thereof. Novel developments of the CASPER program are highlighted.The three-dimensional structure of carbohydrates plays an important role during carbohydrate-protein interactions. This thesis investigates the conformational preferences and dynamics of glycan structures ranging from di- to tetrasaccharides. A particular focus lies on the measurement of transglycosidic 3JCH coupling constants by NMR. Furthermore, the experimental spectroscopic data is compared to results from MD simulations.Synthetic carbohydrate chemistry has a strong focus on stereoselective C−O bond formation for the synthesis of oligo- and polysaccharides. Each glycosylation reaction can produce two stereoisomeric structures. To date, the mechanistic pathway of glycosylation reactions is still not fully understood, since many different parameters influence the stereoselectivity. A combined experimental and computational study exploring the role of the solvent is presented and a linear correlation of the selectivity with a solvatochromic parameter for the polarizability of the solvent was found.
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| 4. |
- Dorst, Kevin, 1992-, et al.
(författare)
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NMR chemical shift prediction and structural elucidation of linker-containing oligo- and polysaccharides using the computer program CASPER
- 2023
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 533
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Tidskriftsartikel (refereegranskat)abstract
- Carbohydrate structures containing alkyl groups as aglycones are useful for investigating enzyme activity and glycan-protein interactions. Moreover, linker-containing oligosaccharides with a spacer group are commonly used to print glycan microarrays or to prepare protein-conjugates as vaccine candidates. The structural accuracy of these synthesized glycans are essential for interpretation of results from biological experiments in which the compounds have been used and NMR spectroscopy can unravel and confirm their structures. An approach for efficient 1H and 13C NMR chemical shift assignments employed a parallel NOAH-10 measurement followed by NMR spin-simulation to refine the 1H NMR chemical shifts, as exemplified for a disaccharide with an azidoethyl group as an aglycone, the NMR chemical shifts of which have been used to enhance the quality of CASPER (http://www.casper.organ.su.se/casper/). The CASPER program has been further developed to aid characterization of linker-containing oligo- and polysaccharides, either by chemical shift prediction for comparison to experimental NMR data or as structural investigation of synthesized glycans based on acquired unassigned NMR data. The ability of CASPER to elucidate structures of linker-containing oligosaccharides is demonstrated and comparisons to assigned or unassigned NMR data show the utility of CASPER in supporting a proposed oligosaccharide structure. Prediction of NMR chemical shifts of an oligosaccharide, corresponding to the repeating unit of an O-antigen polysaccharide, having a linker as an aglycone and a non-natural substituent derivative thereof are presented to exemplify the diversity of structures handled. Furthermore, NMR chemical shift predictions of synthesized polysaccharides, corresponding to bacterial polysaccharides, containing a linker are described showing that in addition to oligosaccharide structures also polysaccharide structures having an aglycone spacer group can be analyzed by CASPER.
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| 5. |
- Dorst, Kevin, 1992-, et al.
(författare)
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On the influence of solvent on the stereoselectivity of glycosylation reactions
- 2024
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 535
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Tidskriftsartikel (refereegranskat)abstract
- Methodology development in carbohydrate chemistry entails the stereoselective formation of C-O bonds as a key step in the synthesis of oligo- and polysaccharides. The anomeric selectivity of a glycosylation reaction is affected by a multitude of parameters, such as the nature of the donor and acceptor, activator/promotor system, temperature and solvent. The influence of different solvents on the stereoselective outcome of glycosylation reactions employing thioglucopyranosides as glycosyl donors with a non-participating protecting group at position 2 has been studied. A large change in selectivity as a function of solvent was observed and a correlation between selectivity and the Kamlet-Taft solvent parameter pi* was found. Furthermore, molecular modeling using density functional theory methodology was conducted to decipher the role of the solvent and possible reaction pathways were investigated.
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