Immunochemically Significant Fluoresceins: Structure Determination by Nuclear Magnetic Resonance Spectroscopy

A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 1: Preparation and properties of a triplet probe

“…Comparison of the proton NMR spectrum of the product with that of fluorescein is'othiocyanate (see Corey and Churchill, 1966) demonstrated that the chemical shifts, splittings, and intensities of the resonances are consistent with the replacement of four protons on the xanthene moiety by bromine.…”

Synthesis and characterisation of pure isomers of iodoacetamidotetramethylrhodamine

“…The two isomers were separated by fractional crystallisation which, although laborious and rather inefficient (see Experimental section), was able to produce sufficient quantities of pure materials to carry forward to give useful quantities of pure IATR isomers. With the individual isomers 5 and 6 in hand it was necessary to assign the structures unambiguously. Simple calculation using incremental chemical shifts l o suggests that the two compounds should have almost identical 'H NMR spectra, but in fact dramatic differences were evident.…”

“…A sample sublimed in oacuo (50 "C, 2 mmHg) gave clear needles, m.p. 122-123 "C (lit.," J3, 5 (21.25 g, 155 mmol) in dry toluene (500 cm3) was heated under reflux for 6 h and cooled. The toluene was evaporated off under reduced pressure and the residue was dissolved in CHCl, (500 cm3), washed successively with dil.…”

Fluorescent Protein Conjugates

“…Borek (45) presented evidence from IR spectra that isomer I is 5' -substituted, i.e., para to the carboxylic acid group, whereas isomer II would be 4'-substituted, i.e., meta to the acid group. Corey and ChurchiII, (46) however, came to exactly the opposite conclusion by examining NMR spectra, and it appears certain now that isomer I is the meta isomer. Attempts to introduce a fluorescein label into proteins by means of a reactive group other than isocyanate, isothiocyanate or dichlorotriazine, such as a diazonium salt, (47) an acid chloride, (34) or a sulfonyl chloride, (48) did not produce useful conjugates, mostly because the fluorescence intensity was sharply reduced after any of these modifications.…”

“…The decay of I with time, t, follows a simple exponential relationship: [46] in which 10 is the value of I at the moment of excitation, and T is the decay time of the fluorescence. From the definition of r [Eq.…”