Absorbing Chromophore from Absorption Maximum

Chromophores free in solution and bound to macromolecules do not display identical s values and absorption peaks. For example, free hemin absorbs at 390 nm. However, in the cytochrome b2 core extracted from the yeast Hansenula anomala, the absorption maximum of heme is located at 412 nm with a molar extinction coefficient equal to 120 mM-1 cm-1 (Albani 1985). In the same way, protoporphyrin IX dissolved in 0.1 N NaOH absorbs at 510 nm, whereas when it is bound to apohemoglobin, it absorbs in the Soret band at around 400 nm. 

For a more detailed analysis of the absorption properties, the UV spectrum of the model compound (Scheme 7), which was also synthesized, was calculated using semiempirical methods (MOPAC/ZINDO). The experimental UV spectrum of the model compound is nearly identical to the spectrum of the polymer. From the calculation it was derived that four UV transitions contributed to the absorption maximum at 330 nm. In detail, these are the HOMO LUMO, the HOMO->LUMO+l, the HOMO LUMO+2, and the HOMO—LUMO+3 transitions. The first two orbital excitations showed a large involvement of the triazene group, whereas the other two are mainly localized at the phenyl moieties. Similar results were previously reported for aryl dialkyl triazenes [119, 184] which have the same structural unit. Starting from simple chemical considerations, it could be thought that the number of chromophores responsible for the absorbance at around 300 nm is a low value, for example 2 or 4 per unit. On the other hand, the semiempirical calculations indicated the involvement of the phenyl moieties in the absorption properties therefore, the chromophore number in the calculation was not restricted to low values. As a starting point for the calculation, numbers close to the expected value were chosen. 

Most of the naturally occurring bacteriochlorins (e.g., 132) have absorptions between 760 and 780 nm and are extremely sensitive to oxidation, resulting in a rapid transformation into the chlorin state 133 which generally has an absorption maximum at or below 660 nm (Scheme 37). Furthermore, if a laser is used to excite the bacteriochlorin in vivo, oxidation may result in the formation of a new chromophore absorbing outside the laser window, reducing its photodynamic efficacy. Due to the desirable photophysical properties of bacteriochlorins, there has been increasing interest in the synthesis of stable bacteriochlorins from bacteriochlorophyll a or other similar tetrapyrrolic systems. 

DNA photolyases, which use the energy of blue light to split pyrimidine dimers formed by UV irradiation of DNA, provide other examples of large and variable shifts in the absorption spectmm of a bound chromophore. These enzymes contain a bound pterin (methylenetetrahydrofolate, MTHF) or deazaflavin, which serves to absorb light and transfer energy to a flavin radical in the active site [81]. The absorption maximum of MTHF occurs at 360 nm in solution, but ranges from 377 to 415 nm in the enzymes from different organisms [82]. 

The next logical step toward chromophore design was to conduct a spectral survey of commercially available organic compounds in order to learn some general structure-property relationships for minimization of the residual absorbance. As an easily measured figure of merit, the ratio between the minimum and maximum molar absorptivities has been used. In many cases, this ratio (expressed in percent, or more conveniently, as the minimum molar absorptivity per 100,000 L/mol-cm of maximum absorbance) is 5-10% (5000-10,000 per 100,000). (The lower the number the better the dye.) An improved figure of merit would take into account the area under the absorption curve as well as the location of the transparent window relative to the peak in the absorption. This is tantamount to calculating the dispersion from the absorption spectrum, which was too complex for this type of survey. 

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