Andrea Vasella

Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin.

Aminoglycosides are potent antibacterials, but therapy is compromised by substantial toxicity causing, in particular, irreversible hearing loss. Aminoglycoside ototoxicity occurs both in a sporadic dose-dependent and in a genetically predisposed fashion. We recently have developed a mechanistic concept that postulates a key role for the mitochondrial ribosome (mitoribosome) in aminoglycoside ototoxicity. We now report on the surprising finding that apramycin, a structurally unique aminoglycoside licensed for veterinary use, shows little activity toward eukaryotic ribosomes, including hybrid ribosomes which were genetically engineered to carry the mitoribosomal aminoglycoside-susceptibility A1555G allele. In ex vivo cultures of cochlear explants and in the in vivo guinea pig model of chronic ototoxicity, apramycin causes only little hair cell damage and hearing loss but it is a potent antibacterial with good activity against a range of clinical pathogens, including multidrug-resistant Mycobacterium tuberculosis. These data provide proof of concept that antibacterial activity can be dissected from aminoglycoside ototoxicity. Together with 3D structures of apramycin-ribosome complexes at 3.5-Å resolution, our results provide a conceptual framework for further development of less toxic aminoglycosides by hypothesis-driven chemical synthesis.

Structural and biochemical evidence for a boat-like transition state in |[beta]|-mannosidases

Enzyme inhibition through mimicry of the transition state is a major area for the design of new therapeutic agents. Emerging evidence suggests that many retaining glycosidases that are active on alpha- or beta-mannosides harness unusual B2,5 (boat) transition states. Here we present the analysis of 25 putative beta-mannosidase inhibitors, whose Ki values range from nanomolar to millimolar, on the Bacteroides thetaiotaomicron beta-mannosidase BtMan2A. B2,5 or closely related conformations were observed for all tightly binding compounds. Subsequent linear free energy relationships that correlate log Ki with log Km/kcat for a series of active center variants highlight aryl-substituted mannoimidazoles as powerful transition state mimics in which the binding energy of the aryl group enhances both binding and the degree of transition state mimicry. Support for a B2,5 transition state during enzymatic beta-mannosidase hydrolysis should also facilitate the design and exploitation of transition state mimics for the inhibition of retaining alpha-mannosidases--an area that is emerging for anticancer therapeutics.

Crystal structure of glucooligosaccharide oxidase from Acremonium strictum: a novel flavinylation of 6-S-cysteinyl, 8alpha-N1-histidyl FAD

Glucooligosaccharide oxidase from Acremonium strictum has been screened for potential applications in oligosaccharide acid production and alternative carbohydrate detection, because it catalyzes the oxidation of glucose, maltose, lactose, cellobiose and cello- and maltooligosaccharides. We report the crystal structures of the enzyme and of its complex with an inhibitor, 5-amino-5-deoxy- cellobiono-1,5-lactam at 1.55- and 1.98-Å resolution, respectively. Unexpectedly, the protein structure demonstrates the first known double attachment flavinylation, 6-S-cysteinyl, 8α-N1-histidyl FAD. The FAD cofactor is cross-linked to the enzyme via the C6 atom and the 8α-methyl group of the isoalloxazine ring with Cys130 and His70, respectively. This sugar oxidase possesses an open carbohydrate-binding groove, allowing the accommodation of higher oligosaccharides. The complex structure suggests that this enzyme may prefer a β-d-glucosyl residue at the reducing end with the conserved Tyr429 acting as a general base to abstract the OH1 proton in concert with the H1 hydride transfer to the flavin N5. Finally, a detailed comparison illustrates the structural conservation as well as the divergence between this protein and its related flavoenzymes.

Enantioselective synthesis of D-erythro-sphingosine

Abstract D- erythro -sphingosine ( 4 ) and the 3-amino-2-hydroxy-L- erythro isomer 15 were synthesized in a highly enantio- and regioselective manner by a modified Sharpless asymmetric epoxidation.

Synthesis of anL-Fucose-Derived Cyclic Nitrone and its Conversion toα-L-Fucosidase Inhibitors

The L-fuco-nitrone 1 has been synthesized from allyl 4,6-O-benzylidene-α-D-glucopyranoside (4) in 11 steps and an overall yield of 18%. The key step is the intramolecular alkylation of an intermediary 1,1-bis(hydroxylamine) derived from the tosyloxy oximes (E/Z)-2. The nitrone 1 has been transformed into the diamine 30, the indolizidines 39 and 40, the indolizidinones 34 and 35, and the imidazole 44, all inhibiting bovine epididymis α-L-fucosidase with IC50 values between 105 nM and 240 μM.

Configurationally selective transition state analogue inhibitors of glycosidases. A study with nojiritetrazoles, a new class of glycosidase inhibitors

Abstract “Mannonojiretetrazole” ( 7 ), a novel mannosidase inhibitor, has been synthesized in six steps from 2,3,4,6-tetra- O -benzyl- d -mannose oxime. The structure of 7 has been established by X-ray analysis. The solid state conformation of 7 is 6 H 7 (= 4 H 3 , numbering based on carbohydrate nomenclature), and the conformation in CD 3 OD is close to S 7 (sofa; = S 3 , numbering based upon carbohydrate nomenclature), while the conformation of the previously synthesized analogue with the gluco configuration ( 6 ) is 6 H 7 , both in the solid state and in solution in D 2 O or CD 3 OD. Both 6 and 7 have been tested as inhibitors of each of a series of five α- and β-glucosidases and -mannosidases as well as of a β-galactosidase, and inhibition constants have been determined. A good correlation (ϱ = 0.9) was found between log K i for each inhibitor—enzyme pair and log ( V m / K m ) for the corresponding substrate—enzyme pair, thereby providing the first such proof for any glycosidase inhibitor being a transition state analogue. This clearly demonstrates a case where true transition state analogue inhibitors of glycosidases are configurationally selective.

The kinetic anomeric effect. Additions of nucleophiles and of dipolarophiles to N-glycosylnitrones and to N-pseudoglycosylnitrones

Abstract To prove the hypothesis of the role of a kinetic anomeric effect in the highly diastereoselective additions of nucleophiles and of dipolarophiles to N-glycosylnitrones we compared these additions to those to analogous N-pseudoglycosylnitrones having a methylene group in place of the furan ring oxygen. An almost complete loss of the diastereoselectivity was found for the addition of lithium dimethyl phosphite tris(trimethylsilyl)phosphite and methyl methacrylate to the N-pseudoglycosylnitrones which moreover reacted more slowly as predicted by the hypothesis of the kinetic anomeric effect. Pseudo first order kinetics for the ZnCl2 promoted addition of P(OSiMe3)3 to nitrones were measured; activation energies diastereoselectivities and the influence of Lewis acids are discussed.

1H‐NMR Analysis of Intra‐ and Intermolecular H‐Bonds of Alcohols in DMSO: Chemical Shift of Hydroxy Groups and Aspects of Conformational Analysis of Selected Monosaccharides, Inositols, and Ginkgolides

The interpretation of 1H-NMR chemical shifts, coupling constants, and coefficients of temperature dependence (δ(OH), J(H,OH), and Δδ(OH)/ΔT values) evidences that, in (D6)DMSO solution, the signal of an OH group involved as donor in an intramolecular H-bond to a hydroxy or alkoxy group is shifted upfield, whereas the signal of an OH group acting as acceptor of an intramolecular H-bond and as donor in an intermolecular H-bond to (D6)DMSO is shifted downfield. The relative strength of the intramolecular H-bond depends on co-operativity and on the acidity of OH groups. The acidity of OH groups is enhanced when they are in an antiparallel orientation to a C−O bond. A comparison of the 1H-NMR spectra of alcohols in CDCl3 and (D6)DMSO allows discrimination between weak and strong intramolecular H-bonds. Consideration of IR spectra (CHCl3 or CH2Cl2) shows that the rule according to which the downfield shift of δ(OH) for H-bonded alcohols in CDCl3 parallels the strength of the H-bond is valid only for alcohols forming strong intramolecular H-bonds. The combined analysis of J(H,OH) and δ(OH) values is illustrated by the interpretation of the spectra of the epoxyalcohols 14 and 15 (Fig. 3). H-Bonding of hexopyranoses, hexulopyranoses, alkyl hexopyranosides, alkyl 4,6-O-benzylidenehexopyranosides, levoglucosans, and inositols in (D6)DMSO was investigated. Fully solvated non-anomeric equatorial OH groups lacking a vicinal axial OR group (R=H or alkyl, or (alkoxy)alkyl) show characteristic J(H,OH) values of 4.5 – 5.5 Hz and fully solvated non-anomeric axial OH groups lacking an axial OR group in β-position are characterized by J(H,OH) values of 4.2 – 4.4 Hz (Figs. 4 – 6). Non-anomeric equatorial OH groups vicinal to an axial OR group are involved in a partial intramolecular H-bond (J(H,OH)=5.4 – 7.4 Hz), whereas non-anomeric equatorial OH groups vicinal to two axial OR form partial bifurcated H-bonds (J(H,OH)=5.8 – 9.5 Hz). Non-anomeric axial OH groups form partial intramolecular H-bonds to a cis-1.3-diaxial alkoxy group (as in 29 and 41: J(H,OH)=4.8 – 5.0 Hz). The persistence of such a H-bond is enhanced when there is an additional H-bond acceptor, such as the ring O-atom (43 – 47: J(H,OH)=5.6 – 7.6 Hz; 32 and 33: 10.5 – 11.3 Hz). The (partial) intramolecular H-bonds lead to an upfield shift (relative to the signal of a fully solvated OH in a similar surrounding) for the signal of the H-donor. The shift may also be related to the signal of the fully solvated, equatorial HO−C(2), HO−C(3), and HO−C(4) of β-D-glucopyranose (16: 4.81 ppm) by using the following increments: −0.3 ppm for an axial OH group, 0.2 – 0.25 ppm for replacing a vicinal OH by an OR group, ca. 0.1 ppm for replacing another OH by an OR group, 0.2 ppm for an antiperiplanar C−O bond, −0.3 ppm if a vicinal OH group is (partially) H-bonded to another OR group, and −0.4 to −0.6 for both OH groups of a vicinal diol moiety involved in (partial) divergent H-bonds. Flip-flop H-bonds are observed between the diaxial HO−C(2) and HO−C(4) of the inositol 40 (J(H,OH)=6.4 Hz, δ(OH)=5.45 ppm) and levoglucosan (42; J(H,OH)=6.7 – 7.1 Hz, δ(OH)=4.76 – 4.83 ppm; bifurcated H-bond); the former is completely persistent and the latter to ca. 40%. A persistent, unidirectional H-bond C(1)−OH⋅⋅⋅O−C(10) is present in ginkgolide B and C, as evidenced by strongly different δ(OH) and Δδ(OH)/ΔT values for HO−C(1) and HO−C(10) (Fig. 9). In the absence of this H-bond, HO−C(1) of 52 resonates 1.1 – 1.2 ppm downfield, while HO−C(10) of ginkgolide A and of 48 – 50 resonates 0.5 – 0.9 ppm upfield.

Inhibition of O-Glcnacase by a Gluco-Configured Nagstatin and a Pugnac-Imidazole Hybrid Inhibitor

Synthesis of a PUGNAc-imidazole hybrid and its characterization as an inhibitor of human O-GlcNAcase through enzyme kinetics and X-ray structural analysis.

Very Strong Inhibition of Glucosidases by C(2)-Substituted Tetrahydroimidazopyridines

The C(2)-substituted imidazoles 11, 15 – 17, 19, 21, 23/24, 28 – 31, 37, and 38 have been prepared from the known 2,3-unsubstituted imidazole 7via the iodoimidazole 10, and tested as inhibitors of β- and α-glucosidases. Introduction of hydrophobic and flexible substituents, such as in 28 and 29, led to a very strong inhibition of β-glucosidases, with Ki values for 29 of 1.2 and 0.11 nM against β-glucosidases from almonds and Caldocellum saccharolyticum, respectively. A slow onset of the inhibition was observed for the strongly inhibiting 16, 28 – 31, 37, and 38. While the introduction of a hydroxymethyl or a phenethyl substituent as in 17 and 30 led to stronger inhibition, the 1′-hydroxyphenethyl derivatives 37 and 38 were weaker inhibitors than 16 and 29. This result is interpreted in the light of a conformational change of the substrate on the way to the transition state. The substituent at C(2) has only a moderate influence on the selectivity of the inhibition of two β- and one α-glucosidases, increasing it by a maximal factor of ca. 10 (16), or decreasing it by a maximal factor of ca. 15 (37).