Assays Based on IRThermography

Photovoltaic IR cameras equipped with focal plane array detectors provide a two-dimensional thermal image as a spatial map of the temperature and emis-

sivity distribution of all objects in the picture (33). It is customary to use different colors to visualize different photon intensities of the detected infrared radiation, i.e., red areas indicate "hot spots" and blue areas denote "cold spots".

Recently, emissivity corrected IR-thermography of large libraries of heterogeneous catalysts was developed, a technique that requires only very small amounts of catalysts (<200 pg) (34). The main object of this study was to visualize temperature differences solely owing to the catalytic activity of the catalysts, which was achieved by applying a linear correction to the detector response and subtracting the IR image of the library just before the start of the reaction, i.e., as background (offset) from the images during catalytic experiments. This means that local emissivity differences are no longer visible, and the heat evolution from the catalytic reactions on a microtiter plate can then be reliably detected.

Thereafter, application in the area of enantioselective homogeneous transition metal catalysis and enzyme catalysis was reported (35). A commercially available Eppendorf-Thermomixer was modified such that the top was replaced by an aluminum plate into which holes were drilled for cyclidrical glass reaction vessels about 8 mm in diameter and 35 mm in height. The whole microtiter plate can be shaken so as to ensure agitation of the reaction contents in each well. The method was illustrated by experiments involving kinetic resolution of chiral substrates, i.e., the (R)- and (S)-compounds were reacted separately pairwise.

Time-resolved detection of an enantioselective enzyme-catalyzed kinetic resolution was demonstrated (35). The enzyme (lipase from Candida antarctica) was added to the wells of the microtiter plate in immobilized form, i.e., the reaction was catalyzed by a heterogeneous catalyst. Using (S)- and (R)-1-phenylethanol 21 as the substrates separately and vinyl acetate as the acylating agent, it was demonstrated that the reaction is highly (R)-selective, i.e., hot spots appeared above the wells of the microtiter plate containing (R)-21 (see Fig. 9). The result is in perfect agreement with the literature data according to which the ee-value of the acylated form at 50% conversion is >99% in favor of (R)-22.

These and further developments (36) show that IR-thermography is a useful tool in the high-throughput identification of highly active and enantioselective enzymes (or other catalysts) in exothermic processes. The method allows one to distinguish such "hits" from other members of a library of catalysts much less active or less enantioselective. However, quantitative correlation with enantioselectivity is not possible so far (36). The disadvantage of the method hinges on the fact that small differences in enantioselectivity, as usually observed in sequential rounds of enzyme mutagenesis, cannot be picked up by IR-thermo-graphic assays. The fact that meaningful comparisons on microtiter can only be made if the same amount of enzyme is present in each well also needs to be kept in mind when attempting to apply this technology.

rac (S) (R)

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Fig. 9. Time-resolved IR thermographic imaging of the lipase-catalyzed enantioselective acylation of 21 after (A) 0.5, (B) 0.5 and (C) 3.5 min (35). The control experiment without enzyme is given in the bottom row in each case. The bar on the far right is the temperature/color key of the temperature window used [°C],

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