Sintesis de fotosondas derivadas de ampicilina

Abstract

The Gram-positive bacteria Staphylococcus aureus is the main cause of hospital- and community-associated infections. S. aureus is the most frequent cause of surgical, lower respiratory tract, and cardiovascular infections. In addition, it is the second most common cause of health-care associated pneumonia and of bloodstream infections.1,2,5 Methicillin-resistant S. aureus (MRSA) is a high priority concern in the world and in particular in Latin America, both in hospitals and in the community. MRSA has become the first cause of hospital-associated infections in Latin America, and there has been an increasing number of reports of community acquired MRSA infections. The acquisition of resistance to b-lactam antibiotics, generally concurrent with the acquisition of resistance to other antibacterial agents, represents a huge challenge for the prevention and treatment of S. aureus associated infections. Very few new antibacterial are in advanced stages of clinical evaluation for the treatment of bacterial infections, including MRSA. S. aureus presents two main mechanisms of resistance to b-lactam antibiotics: the expression of the PC1 b-lactamase, capable of hydrolyzing and inactivating the b-lactam antibiotics, and the acquisition of a PBP (PBP2a) with low affinity for b-lactam antibiotics, and which is hence not inhibited by them. S. aureus presents another important system that coordinates the response to antibiotics that inhibit the biosynthesis of the peptidoglycan: the VraSRT system. The three-component system VraSRT controls the peptidoglycan crosslinking process, and is activated by β-lactam and glycopeptide antibiotics. VraSRT has a critical role in glycopeptide-resistance. The VraSRT system is composed of three components: VraS and VraT are membrane proteins that could quickly detect stress in the cell wall and transmit the signal to the cell cytoplasm. The understanding of the signal transduction mechanism employed by the sensor proteins of the VraSRT system is of great interest because they are possible targets for the design of inhibitors that can be used in conjunction with antibiotics for the treatment of S. aureus infections. Photoaffinity labeling is a useful technique employed to study noncovalent interactions between protein-ligands and protein-protein complexes. A photoaffinity labeling reagent is a molecule that contains a photoreactive group which produces highly reactive intermediates upon photolysis. The most widely used classes of photoactive functionality include benzophenones, trifluoromethylphenyldiazirines, and arylazides, which give rise to diradicals, carbenes and nitrenes by UV irradiation. These generated intermediates can produce hydrogen abstraction reactions, initiate highly efficiency double bond addition reactions, insertion reactions to C-H and N-H bonds (carbenes, nitrenes) with neighboring biomolecules giving stable covalent adducts. Each of these families of compounds have advantages and disadvantages. For example, benzophenones are chemically more stable than azides and diazirines, can be manipulated in ambient light and like diazirines are activated at 350 nm avoiding wavelengths that cause protein damage. However, its bulky size and hydrophobicity can complicate the binding with the macromolecule. On the other hand, arylazides give reactions with high efficiency, present excellent stability for storage and their synthesis is easy but they are activated at wavelengths that could affect the biological system under study. Finally, trifluoromethylphenyldiazirines meet most of the characteristics of an ideal photoreactive, although their synthesis needs more steps. Herein, we report the synthesis of modified variants of β-lactam antibiotics that can be used as affinity photoprobes in order to elucidate the molecular events that lead to the induction of resistance systems in bacteria. Using these photoprobes we demonstrate that the membrane protein VraS interacts directly with β-lactam antibiotics, which results in its autophosphorylation and phosphotransfer to VraR.