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aikido 3d 2.0 crack1. Field of the Invention
This invention relates to nitroxides and in particular to the use of nitroxides as reagents for covalent attachment to biological components.
2. Description of the Prior Art
Reactive derivatives of nitroxides have been proposed as reagents for selectively labelling proteins by covalent attachment to amino acids introduced into a protein using the water-soluble carbodiimide EDC/NHS.sup.+ (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide) (Berridge et al., Nature 302, 716 (1983)). In this approach, an amino acid is placed in a protein of interest and activated by EDC/NHS under mild conditions. A nitroxide is then added and the resulting product is characterised using nuclear magnetic resonance and electrospray mass spectrometry.
There is now considerable evidence that nitroxides are biologically stable and potent inhibitors of metal-catalysed free radical reactions. In addition, nitroxides have been used as model radicals for studies of the mechanisms of radical chain reactions in biological systems.
Unfortunately, while nitroxides are known to be efficient electron spin polarising agents, their usefulness as reagents for selective labelling by covalent attachment to proteins has previously been limited by a number of factors. Most significantly, the nitroxides used in the carbodiimide/EDC/NHS labelling reaction are very bulky and relatively poor spin polarising agents. In addition, the carbodiimide/EDC/NHS reaction conditions cause undesired side reactions, such as hydrolysis of the cysteine thiolates to generate disulphide products. This presents a serious problem for both primary and secondary labelling of proteins, since it is desirable to use the carbodiimide/EDC/NHS reaction to produce structures containing up to ten or more different labelling groups in a protein.Q:
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