Photolysis of (trimethylsilyl)-, bis(trimethylsilyl)-, and pentamethyldisilanylketene in hydrocarbon solution in the presence of aliphatic alcohols affords alkoxysilanes from trapping of silene reactive intermediates. The silenes (1,1,2-trimethylsilene, 1,1,2-trimethyl-2-(trimethylsilyl)silene, and 1,1-dimethyl-2-trimethylsilene) can be viewed as the products of [1,2]-methyl or -trimethylsilyl migration in the silyl carbene formed by photodecarbonylation of the ketene, although a competing direct excited-state pathway cannot be ruled out. Far-UV (193 nm) laser flash photolysis of the compounds in hydrocarbon solution affords transients which are formed during the ∼20 ns laser pulse and are assignable to the silenes on the basis of their UV absorption spectra and reactivity toward alcohols. 1,1,2-Trimethylsilene has also been generated by laser flash photolysis of (trimethylsilyl)diazomethane and -diazirine in hexane and acetonitrile solution, and its spectrum and rate constants for reaction with MeOH, MeOD, and t-BuOH have been determined. Those in hexane agree with those obtained using the corresponding ketene as the precursor. The rate constants for addition of ROH to these and three other 2-substituted 1,1-dimethylsilenes correlate with the resonance substituent parameter σR°, affording ρR values of +8.0 ± 2.2 and +6.5 ± 2.6 for MeOH and t-BuOH, respectively. This allows the conclusion that the reactivity of simple silenes toward nucleophiles is enhanced by resonance electron-acceptor substituents at carbon. To probe for the possible intermediacy of (trimethylsilyl)carbene in the formation of 1,1,2-trimethylsilene from these compounds, flash photolysis experiments with (trimethylsilyl)ketene, -diazomethane, and -diazirine in the presence of pyridine have been carried out. Absorptions assignable to the carbene−pyridine ylide were observed from the ketene and the diazirine, allowing an estimate of between 0.1 and 0.4 ns for the lifetime of singlet (trimethylsilyl)carbene in hydrocarbon solvents at room temperature.

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The most frequent determinant of aminoglycoside antibiotic resistance in Gram-positive bacterial pathogens is a bifunctional enzyme, aminoglycoside acetyltransferase-6 -aminoglycoside phosphotransferase-2؆ (AAC(6 )-aminoglycoside phosphotransferase-2؆, capable of modifying a wide selection of clinically relevant antibiotics through its acetyltransferase and kinase activities. The aminoglycoside acetyltransferase domain of the enzyme, AAC(6 )-Ie, is the only member of the large AAC(6 ) subclass known to modify fortimicin A and catalyze Oacetylation. We have demonstrated through solvent isotope, pH, and site-directed mutagenesis effects that Asp-99 is responsible for the distinct abilities of AAC(6 )-Ie. Moreover, we have demonstrated that small planar molecules such as 1-(bromomethyl)phenanthrene can inactivate the enzyme through covalent modification of this residue. Thus, Asp-99 acts as an active site base in the molecular mechanism of AAC(6 )-Ie. The prominent role of this residue in aminoglycoside modification can be exploited as an anchoring site for the development of compounds capable of reversing antibiotic resistance in vivo.Clinical usage of aminoglycoside-aminocylitol antibiotics is blocked by the presence of aminoglycoside modifying enzymes (AMEs) 1 in resistant organisms (for review see Refs. 1 and 2). Bacteria become protected from aminoglycosides, because the modified antibiotics can no longer bind with high affinity to their target, the A-site of the small ribosomal subunit, because of unfavorable steric and/or electrostatic constraints (3). The AMEs are a diverse set of proteins composed of three families: aminoglycoside nucleotidyltransferases, aminoglycoside acetyltransferases (AACs), and aminoglycoside phosphotransferases (APHs).The most clinically important AME in Gram-positive bacterial pathogens such as Staphylococcus aureus is AAC(6Ј)-APH(2Љ) (4). This enzyme is unique in that it is bifunctional, comprising activities from two classes of AMEs: an N-terminal AAC (AAC(6Ј)-Ie) and a C-terminal APH (APH(2Љ)-Ia) ( Fig. 1) (5, 6). AAC(6Ј)-APH(2Љ) has an extraordinary ability to detoxify a wide selection of aminoglycosides (7, 8) not only as a result of its bifunctional nature but also because of the special characteristics of each activity. For example, APH(2Љ)-Ia has a tremendous ability to accommodate a vast range of antibiotics and binding conformations as evidenced by the remarkably broad regiospecificity of phosphoryl transfer that enables modification to occur on hydroxyl groups from four different aminoglycoside ring systems (see Fig. 1) (8). Conversely, AAC(6Ј)-Ie, has a very stringent regiospecificity of acetyl transfer, because it is restricted to acetylation of the 6Ј-position of aminoglycosides only (see Fig. 1) (8). AAC(6Ј)-Ie is the sole member of the very large AAC(6Ј) subclass known to acetylate the antibiotic fortimicin A (9), and moreover, in addition to N-acetylation activity, this enzyme has been shown to have O-acetylation capabilities (8). The unusual activities of AAC(6Ј)-...

[structure: see text] The 1,6-dipivaloyl derivative of 3,4,7,8-tetramethyl-2,5-dithioglycoluril (6) was prepared and the crystal structure determined by X-ray diffraction; 6 is a twisted amide in which severe ring strain and nonbonded interactions compel both pivaloyl groups to twist dramatically out of the ring plane. The amide oxygen atoms point in opposite directions with respect to the mean plane through the glycoluril core, and the bridgehead methyl groups are forced out of the symmetric syn geometry (eta = 30.5 degrees ). The structure of 7, a rearrangement product generated during synthesis of 6, was also determined by single-crystal X-ray diffraction.