Resolution function library

Esterases have a catalytic function and mechanism similar to those of lipases, but some structural aspects and the nature of substrates differ [4]. One can expect that the lessons learned from the directed evolution of lipases also apply to esterases. However, few efforts have been made in the directed evolution of enantioselective esterases, although previous work by Arnold had shown that the activity of esterases as catalysts in the hydrolysis of achiral esters can be enhanced [49]. An example regarding enantioselectivity involves the hydrolytic kinetic resolution of racemic esters catalyzed by Pseudomonasfluorescens esterase (PFE) [50]. Using a mutator strain and by screening very small libraries, low improvement in enantioselectivity was... 

Internal DCR of Nitroaldol Libraries (Scheme 6.10) [5,6] iDCR was demonstrated by using a conceptual nitroaldol library including five benzaldehyde derivatives (24,36, and 47-49) and one nitroalkane (50, DCL-F, Scheme 6.11). The benzaldehydes, all with a unique substitution pattern, were selected in order to make analysis clear and simple. However, one of the henzaldehydes contained a cyano functionality in the 2-position (49), deliberately making it a candidate for subsequent tandem cyclization following nitroalcohol formation. 5-exo-dig type cyclizations of hydrox-ynitriles to the corresponding iminolactones are expected [40,41], albeit unexplored [42 5], intramolecular transformations, which in this case could lead to possible kinetic resolution of the library. 

Miller et al. achieved selective functionalization of the enantiotopic hydroxyl groups of meso-inositols. In particular, they were able to convert myo-inositol 49 to either mono-phosphorylated D-myo-inositol-l-phosphate 50 or D-myo-inositol-3-phosphate mt-50 in high yield and with excellent ee (98%) (Scheme 13.25) [40, 41], This remarkable result was achieved by using the pentapeptides 51 or 52 as catalyst. These catalysts were identified from peptide libraries by a combinatorial approach. The peptides 51 and 52 are highly selective and complementary low-molecular-weight kinase mimics. It is also interesting to note that the opposite enantioselectivity of catalysts 51 and 52 could hardly have been predicted on the basis of the type and sequence of the amino acids involved. (Application of the Miller peptide catalysts to the kinetic resolution of racemic alcohols is discussed in Section 12.1.)... 

Quantitative assays can be performed on large populations, with single cell resolution. Flow cytometric analysis provides the opportunity to examine the distribution of protein functions within a library and to determine the fraction of clones having an activity of interest. 

A second-generation library of 486 PBAEs was generated to examine the structure-function relationships with greater resolution. High-throughput screening of luciferase DNA transfection in COS-7 cells followed by biophysical... 

After infrared spectra at low resolution (usually 4 cm ) have been recorded as a function of time and temperature, the further procedure is similar to GC/FT-IR (see Section 17.5.1.7) and, in fact, GC/FT-IR. software, such as Gram-Schmidt thermogram reconstruction and library search with a vapor-phase spectra database is commonly used. Helpful for the identification in the vapor phase is the virtual absence of interaction between molecules, and therefore the nondestructive IR detection enables in many cases an easy and fast interpretation of single and overlapping absorption bands. 

In thermolysis FTIR the sample (typically 200 /ug) is loaded onto a quartz boat, which is inserted straight into a platinum coil filament. With the beam focused several mm above the filament surface, the IR-active gas products from the fast heated sample can be detected in near real-time. Fast thermolysis/FTIR spectroscopy combines rapid-scan FTIR (20 scans/s) with pyrolysis of a material and realtime measurement of the gas spectra [376]. Temperature, mass changes and spectral data of IR active gases are thus measured simultaneously as a function of time during the rapid heating phase. High-resolution vapour phase libraries are used for identification. 

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