Walther Schmid

Synthesis of a 13C-methyl-group-labeled methionine precursor as a useful tool for simplifying protein structural analysis by NMR spectroscopy.

In the past decade NMR spectroscopy has emerged as one of the most powerful tools for the determination of protein structure and dynamics. The introduction of uniform N,C labeling, coupled with the development of triple-resonance experiments has permitted structural and dynamic studies of systems that are up to 20 kDa in molecular mass. The characterization of larger macromolecules requires techniques that maximize both spectral resolution and sensitivity due to chemical-shift overlap and increasing linewidth in high molecular-weight systems. Consequently, research has focused on correlation spectroscopy of side-chain methyl groups. New labeling techniques have been established by using a-ketoacids as biosynthetic precursors for the production of isotope-labeled proteins. Attention has therefore been turned towards the synthesis of a-ketobutyric and a-ketovaleric acid, biosynthetic precursors for the aliphatic amino acids isoleucine, as well as leucine and valine, respectively. NMR investigations of the trimeric E. coli enzyme IIA (34 kDa), the monomeric 82 kDa enzyme malate synthase G from E. coli, or studies of the protein p21-KID within a 45 kDa binary complex (p21-KID/Cdk2) are applications of these ACHTUNGTRENNUNGdevelopments. Furthermore, new methods for quantifying the amplitudes of motion of methyl-containing side chains have been developed, which are essential for a better understanding of dynamics and its relationship to function. Here we report a novel synthetic route towards a precursor of the S-C-methyl-bearing amino acid, methionine. The synthesis sequence is outlined in Scheme 1. Starting from easily accessible tert-butyl a-bromomethacrylate (1), homoallylic alcohol 2 was obtained through an indium mediated aqueous Barbier-type reaction between 1 and formaldehyde. The highest yields were obtained by using 4.5 mmol allyl ester (1), 9 mmol indium powder, and 36 mmol depolymerized paraformaldehyde in a mixture of THF and water (5:1) after 16 h of sonication. Primary alcohol 2 was converted to the corresponding tosylate 3 by using standard procedures. Treatment of 3 with potassium thioacetate in acetone gave thioacetyl-compound 4 after 6 h of sonication in excellent yield. Subsequent ozonolysis of the double bond at 78 8C in dichloromethane afforded 2-oxo-derivate 5. The a-keto functionality of 5 was further transformed into its N,N’-dimethylhydrazone 6. Although the thioester of 5 could have been cleaved by N,N’-dimethylhydrazine, the obviously greater reactivity of the a-keto functionality allowed for selective protection in high yield. All attempts to generate a thiol moiety starting from ester 6 by using a ZemplBn saponification protocol with sodium methanolate in dry methanol failed. Compound 7 was finally obtained by treatment of thioester 6 with ethylamine and silver triflate at 20 8C, followed by methylation of the thiolate with CH3I in THF. To prevent disulfide formation, silver triflate was applied in equimolar amounts; this pushed the reaction towards product formation due to silver iodide precipitation. Subsequently, the hydrazone of 7 was cleaved by using an aqueous HCl solution (1m) and THF to obtain a-ketoester 8. Hydrolysis of the tert-butyl ester 8 was performed in a mixture of an aqueous HCl solution (0.025m) and acetonitrile to yield the target precursor compound 9 in 32% overall yield (starting from 1). [a] M. Fischer, Prof. J. H usler, Prof. W. Schmid Institute of Organic Chemistry, University of Vienna W hringer Strasse 38, 1090 Vienna (Austria) Fax: (+43)1-4277-9 521 E-mail : walther.schmid@univie.ac.at

Synthesis of both epimeric 2-deoxy-n-acetylneuraminic acids and their behaviour towards CMP-sialate synthetase-a comparison with 2-β-methylketoside of n-acetylneuraminic acid☆

Methyl-2-β-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminate 2 respectively methyl 1-2,3-didehydro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminate 3 were transformed by catalytic hydrogenation or by reduction with tributyltin hydride to the corresponding 2-deoxy derivatives 4 and 5, which gave after saponification the acids 6 and 7. Both epimers are not inhibitors of CMP-sialate synthetase (EC 2.7.7.43), whereas the 2-β-methylketoside of Neu5Ac behaves as a competitive inhibitor of the activation of Neu5Ac (N-acetylneuraminic acid).

Ultrafast photo-induced charge transfer unveiled by two-dimensional electronic spectroscopy

The interaction of exciton and charge transfer (CT) states plays a central role in photo-induced CT processes in chemistry, biology, and physics. In this work, we use a combination of two-dimensional electronic spectroscopy (2D-ES), pump-probe measurements, and quantum chemistry to investigate the ultrafast CT dynamics in a lutetium bisphthalocyanine dimer in different oxidation states. It is found that in the anionic form, the combination of strong CT-exciton interaction and electronic asymmetry induced by a counter-ion enables CT between the two macrocycles of the complex on a 30 fs timescale. Following optical excitation, a chain of electron and hole transfer steps gives rise to characteristic cross-peak dynamics in the electronic 2D spectra, and we monitor how the excited state charge density ultimately localizes on the macrocycle closest to the counter-ion within 100 fs. A comparison with the dynamics in the radical species further elucidates how CT states modulate the electronic structure and tune fs-reaction dynamics. Our experiments demonstrate the unique capability of 2D-ES in combination with other methods to decipher ultrafast CT dynamics.

Indium-mediated allylation of aldehydes: A convenient route to 2-deoxy and 2,6-dideoxy carbohydrates

Abstract The allylation of protected polyhydroxy aldehydes 1 and 14 has been achieved by indium metal with ultrasound promotion generating the diastereomeric pair of homoallylic polyols 3 , 4 and 16 , 17 respectively with moderate to good stereoselectivity. Pursuing this allylation strategy with the corresponding deprotected polyhydroxy aldehydes led to the same pair of homoallylic polyols but with a quite different ratio of the diastereomers generated. The polyols were further transformed to 2-deoxy ( 5 and 6 ) and 2,6-dideoxy ( 18 and 19 ) carbohydrates by ozonolysis.

Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection?

One of the first events taking place when a crystal of a metalloprotein is exposed to X-ray radiation is photoreduction of the metal centres. The oxidation state of a metal cannot always be determined from routine X-ray diffraction experiments alone, but it may have a crucial impact on the metals environment and on the analysis of the structural data when considering the functional mechanism of a metalloenzyme. Here, UV-Vis microspectrophotometry is used to test the efficacy of selected scavengers in reducing the undesirable photoreduction of the iron and copper centres in myoglobin and azurin, respectively, and X-ray crystallography to assess their capacity of mitigating global and specific radiation damage effects. UV-Vis absorption spectra of native crystals, as well as those soaked in 18 different radioprotectants, show dramatic metal reduction occurring in the first 60 s of irradiation with an X-ray beam from a third-generation synchrotron source. Among the tested radioprotectants only potassium hexacyanoferrate(III) seems to be capable of partially mitigating the rate of metal photoreduction at the concentrations used, but not to a sufficient extent that would allow a complete data set to be recorded from a fully oxidized crystal. On the other hand, analysis of the X-ray crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than metal centre reduction, and suggests further testing of HEPES and 2,3-dichloro-1,4-naphtoquinone as potential scavengers.

Selective Isotope Labelling of Leucine Residues by Using α-Ketoacid Precursor Compounds

You can have one without the other: A new metabolic precursor compound can selectively introduce (13)C and (2)H patterns at leucine residues in proteins in cell-based expression systems without interfering with valine metabolism. The protocol provides selectively labelled macromolecules well suited for probing structure and dynamics in high-molecular-weight proteins by NMR spectroscopy.

Galactosylation by use of β-galactosidase: Enzymatic syntheses of disaccharide nucleosides

Abstract The synthesis of various galactose containing disaccharide nucleosides has been achieved by utilizing the transgalactosylation potential of s-galactosidase from Aspergillus oryzae. Thus, using p-nitrophenyl-s-D-galactoside 1 as galactosyl donor, 2-deoxyuridine 2a, uridine 2b, thymidine 2c and adenosine 2d have proven to be useful acceptors for the enzyme catalyzed disaccharide nucleoside formation. The regiochemistry of the products 4a – 4d formed after acetylation has been assigned unambiguously by using modern NMR-techniques.

α-Ketoacids as precursors for phenylalanine and tyrosine labelling in cell-based protein overexpression

Abstract13C-α-ketoacid metabolic precursors of phenylalanine and tyrosine effectively enter the metabolism of a protein overexpressing E. coli strain to label Phe- and Tyr-residues devoid of any cross-labelling. The methodology gives access to highly selective labelling patterns as valuable tools in protein NMR spectroscopy without the need of 15N-chiral amino acid synthesis using organic chemistry.

Galactosylation by use of β-galactosidase : chemo-enzymatic syntheses of di- and trisaccharides

Abstract The synthesis of various galactose containing disaccharides has been achieved by utilizing the transgalactosylation potential of β-galactosidase from Aspergillus oryzae. Thus, using p- nitrophenyl-β-D-galactoside 1 as galactosyl donor, thio-glucosides 2a-c, thio-galactosides 5a, 5b and pent-4-enyl-glucoside 2d have proved to be useful acceptors for the enzyme catalyzed disaccharide formation. We further demonstrated the synthetic potential of the disaccharides obtained by enzymatic synthesis in preparing trisaccharide 10 starting with 4a and 4d and applying chemical glycosylation procedures developed for thio- and pent-4-enyl glycosides.

Synthesis of fluorinated maltose derivatives for monitoring protein interaction by 19F NMR

Summary A novel reporter system, which is applicable to the 19F NMR investigation of protein interactions, is presented. This approach uses 2-F-labeled maltose as a spy ligand to indirectly probe protein–ligand or protein–protein interactions of proteins fused or tagged to the maltose-binding protein (MBP). The key feature is the simultaneous NMR observation of both 19F NMR signals of gluco/manno-type-2-F-maltose-isomers; one isomer (α-gluco-type) binds to MBP and senses the protein interaction, and the nonbinding isomers (β-gluco- and/or α/β-manno-type) are utilized as internal references. Moreover, this reporter system was used for relative affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation between 19F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein–ligand interaction interfaces.