4-Aminoacridine, protonation

The mono- and some di-aminoacridines, 6- and 7-aminoquin-oline, and 3-aminopyridine form monocations in which protonation occurs preferentially at the ring nitrogen atom. However, ultraviolet spectral data show that 1,9-diaminoacridine (237, R = NH2) undergoes proton addition on the amino groups in preference to the cyclic nitrogen atom, whereas l-amino-9-methylacridine (237, R = Me) is monoprotonated principally at the amino group in ethanol... 

Transhydrogenase was also purified somewhat later and independently by a more complicated procedure which, however, also produced the 115000 molecular weight polypeptide [102]. Subsequently, additional improved methods have been published, which involve the use of immobilized antibodies [103] and affinity chromatography on immobilized NAD" [104] or NADP" [105]. With all preparations reconstituted transhydrogenase was shown to be a proton pump by both an indirect assay using 9-aminoacridine as pH probe for the interior space of the vesicles [106], and a direct assay using a pH electrode [107]. 

Proton flux across lipid bilayers can be measured by a variety of techniques. For example, a buffered pH gradient can be established across liposome membranes, and the rate of decay of the gradient can be monitored by any of several methods. Early measurements were carried out by monitoring pH shifts in the external medium with a glass electrode (1) later measurements used pH-sensitive dyes such as pyranine, carboxyfluorescein, and 9-aminoacridine (4-6). Cafiso and Hubbell (7) used spin labels very effectively, and Perkins and Cafiso (8) conducted an extensive series of measurements with this system. 

Various optical methods have also been used to estimate the two components of the proton-motive force. For measurements absorbance changes of carotenoids, chlorophyll and merocyanins have been determined, whereas for evaluation of changes of pH-gradients the fluorescence of compounds like 9-aminoacridine have been measured. These optical methods, however, are not of more than diagnostic value since their quantitative reliability is questionable. 

Using the protocol of titrations of qE against the ionophoric uncoupler nigericin in p>ea and spinach chloroplasts, and using 9-aminoacridine fluorescence as a monitor for the proton gradient, we have investigated some relationships between ApH, "redox state", and zeaxanthin content, in their effects up>on the magnitude of qE. 

This effect may be clarified somewhat by plotting qE against the intrathylakoid proton concentration, which can be calculated from the extent of quenching of 9-aminoacridine fluorescence (Fig.2(a)). The relationship in the absence of ant.A is very similar to data previously... 

Fig. 2 (A) Relationship between qE and internal proton concentration in the presence (closed circles) and absence (open circles) of 0.5 uM antimycin A, (B) Relationship between qE and ApH (in absence of ant.A). Conditions as for Fig.l internal proton concentration and LpH were calculated from extent of quenching of 9-aminoacridine fluorescence.

A more qualitative estimate of endosome pH can be obtained by monitoring the distribution of a permeant, fluorescent amine such as 9-aminoacridine or acridine orange whose fluorescence varies with concentration. Such amines acaimulate in acid compartments because the protonated form of the dye is much less membrane permeant than the free base. Indeed, if the latter equilibrates readily across membranes then the pH within the vesicle i.s given by the relationship (1.5) ... 

On deprotonation, 1-arylindazolium salts form l-arylindazol-3-ylidenes which rearrange spontaneously via ring cleavage, ring closure and proton transfer to 9-aminoacridines (Scheme 45). ... 

Upon the photolysis of 9-azidoacridine hydrochloride, the resulting acridyl nitrene was protonated at the endocyclic nitrogen atom. Evidently, the positive charge of the acridine nucleus prevents the dimerization reaction to the azoacridine dication. Therefore, the consecutive abstraction of two hydrogen atoms from solvent moleeules with formation of protonated 9-aminoacridine (Scheme 3), whieh gives the free amine upon neutralization, is the main reaction route of the aeridyl nitrene eation. 

Aminoacridine (9-A A) has been introduced as a matrix for negative-mode MALDI. " While most traditional MALDI matrices have been acidic in character, 9-AA is a moderately strong base. The mechanism by which 9-AA brings about ionization in the negative mode appears to involve abstraction of a labile proton in an acid-base reaction. 

It is important to note the favorable influence of protonation (iminium formation) on the ease of reduction. The salt form is expected to be present in vivo, similar to other aminoacridines . Although the potentials are fairly negative under our conditions, we believe that they may be appreciably more positive in vivo due to stereochemical effects. 

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