TSA Approach

Following the first successful examples of catalytic antibodies raised against haptens as transition state analogues (TSAs) reported by Lerner and Schultz, the TSA approach has been applied in a large number of studies in order to generate new biocatalysts for many chemical transformations. According to the transition state theory, the catalytic efficiency ( cat/ uncat) of given enzymatic reaction can be deduced from the thermodynamic cycle (Scheme 1) under ideal conditions. ... 

The identical TSA approach using the S YNAPT G2 (higher mobility at 95 vs. low mobility at 125) instead of the first generation SYNAPT instrument (higher mobility at 89 vs. low mobility at 101) provided a drift resolution enhancement of 18 Bins for the doubly Uthiated palmitic acid over the isobaric ion. Palmitic acid is a saturated fatty acid and one C2 unit shorter as compared to oleic add (Scheme 9.2). 

HCS Hauled-container systems TSA Total systems approach... 

One of the most direct questions to ask in the perspective of enzyme design is whether an already existing protein with a binding pocket might be turned into a new catalyst by introducing catalytic residues directly, rather than by the elaborated TSA mimicry approach used for catalytic antibodies, hoping to create a new biocatalyst that could harness both the activity and the selectivity, in particular stereoselectivity, that is possible with enzymes. 

Results have generally been disappointing. It can be difficult to remove the TSA from the polymer, but a more fundamental problem concerns the efficiency of the catalysis observed. The most efficient systems catalyze the hydrolysis of carboxylate and reactive phosphate esters with Michaelis-Menten kinetics and accelerations (koAJKM)/kunoJ approaching 103,1661 but the prospects for useful catalysis of more complex reactions look unpromising. Apart from the usual difficulties the active sites produced are relatively inflexible, and the balance between substrate binding and product inhibition is particularly acute. 

A second use of this type of analysis has been presented by Stewart and Benkovic (1995). They showed that the observed rate accelerations for some 60 antibody-catalysed processes can be predicted from the ratio of equilibrium binding constants to the catalytic antibodies for the reaction substrate, Km, and for the TSA used to raise the antibody, Kt. In particular, this approach supports a rationalization of product selectivity shown by many antibody catalysts for disfavoured reactions (Section 6) and predictions of the extent of rate accelerations that may be ultimately achieved by abzymes. They also used the analysis to highlight some differences between mechanism of catalysis by enzymes and abzymes (Stewart and Benkovic, 1995). It is interesting to note that the data plotted (Fig. 17) show a high degree of scatter with a correlation coefficient for the linear fit of only 0.6 and with a slope of 0.46, very different from the theoretical slope of unity. Perhaps of greatest significance are the... 

A general approach to the task has been to raise antibodies to TSAs related... 

While indirect selections work quite well for antibodies they have been less successful in the case of catalytic nucleic acids. There are only three examples which prove that it is possible in principle to obtain a ribo- or deoxyribozyme by selecting an aptamer that binds to a TSA A rotamase ribozyme [7], a ribozyme capable of catalyzing the metallation of a porphyrin derivative [92], and one catalytic DNA of the same function [93]. Another study reported the selection of a population of RNA-aptamers which bind to a TSA for a Diels-Alder reaction but the subsequent screen for catalytic activity was negative for all individual RNAs tested [94]. The attempt to isolate a transesterase ribozyme using the indirect approach also failed [95]. 

Another approach employed to increase sensitivity of microarrays is signal amplification at the post-hybridization stage. One such technique is fyramide signal amplification (TSA) which requires 20-100 times less RNA than direct cDNA labeling (40). This method was originally used in immunohistochemistry and has been an important tool for immunofluorescence microscopy (41,42). 

Note that in Eq. 11-3, in contrast to the equation used for describing bulk phase partitioning, CA, is not the total surface area (TSA,) of the organic molecule. Rather it reflects the contact area between the molecule and the surface. But as a first approximation, we will assume that CA, is proportional to TSA,. By doing so, we neglect any special steric aspects that may affect the ability of a sorbate to closely approach the surface moieties with which it interacts. 

An example of this imprinting approach is illustrated in Scheme 1, where a polymer with hydrolytic properties towards the diphenyl-carbonate (1) was imprinted with the corresponding phosphate template (4), which represents a TSA for the hydrolytic reaction. This structure, in fact, mimics the tetrahedral intermediate formed during the hydrolysis of the carbonate, and therefore it allows imprinting a cavity with functional groups placed in the right spatial position. 

In 1994 Shea et al. reported the preparation of gel-like imprinted polymers with enantioselective esterolytic activity toward the Boc-D-phenyl-alanine p-nitrophenol ester (28) [19]. The polymers were prepared using a covalent approach, rather than metal complexes or non-covalent interactions, by attaching the catalytic phenol-imidazole unit to the TSA phosphonate via ester linkage (29). The imprinted polymer, containing the catalytic unit (30), showed little selectivity toward the D-enantiomer used for the imprinting. 

The result of this approach was a 100-fold increase in the hydrolytic activity of the imprinted polymer compared with the background at pH = 7.6. As a control, another polymer was made using a complex between amidine and benzoate, showing a surprisingly 20-fold increase in the hydrolysis of the substrate. The authors also reported a kinetic investigation of the TSA-imprinted and the benzoate-imprinted polymers, in addition to the free catalyst in solution. Although the ratio substrate/catalyst is not specified, and therefore the steady-state conditions could not be verified, the authors claimed for the two polymers a Michaelis-Menten kinetic behaviour, with a higher profile for the TSA-imprinted polymer. On the other hand, the free catalyst in solution showed, as expected, a linear dependence of the rate from the substrate concentration. The TSA also showed a moderate selectivity towards its own substrate. 

Scheme 8 Imprinting approach used to catalyse the reaction between tetrachlorothiophene dioxide (43) and maleic anhydride (44) to give (45). The chlorendic anhydride (46), representing the TSA, is used as the template for imprinting.

The use of polymerisable imidazole as functional monomer has been largely used for the preparation of hydrolytic imprinted polymers from other groups as well. Ohkubo et al. [38], for instance, described the preparation of imprinted polymers with esterolytic activity towards /V-dodecanoyl leucine-p-nitrophenyl ester (57) using a system similar to the Shea one but based on a stoichiometric non-covalent approach. The imprinting was carried out with a phosphonate TSA and the activity of the resulting imprinted polymer was twice the non-imprinted. 

A different approach was used by Emgenbroich and Wulff [42] to develop imprinted polymers with enantioselective esterase activity. The system was based on the use of an amidinium functional monomer (33), already developed earlier by the same group, but in this case a chiral phosphonate (62) was used as imprinting TSA in order to catalyse the hydrolysis of the corresponding chiral ester (63). The polymer imprinted with the L-enantiomer was able to enhance the esterolytic activity 325-fold when compared with the background and 80 times compared to the non-imprinted polymer. The ratio between the two rates constant, Lai-i/L ii-i) = 1 -4, can be taken as a measure of the enantioselective efficiency of the reaction. 

Markowitz et al. developed a different approach, again in an attempt to overcome some of the inherent difficulties that arise when imprinted bulk materials are used as catalysts [82], Here, the authors used a template-directed method to imprint an a-chymotrypsin TSA at the surface of silica nanoparticles, prepared with a number of organically modified silanes as functional monomers. Silica particle formation was performed in a microemulsion, where a mixture of a non-ionic surfactant and... 

The second aim is selection of molecules with new catalytic function, called ribozymes. Two different approaches are used to find catalytic nucleic acids. One is to synthesize a transition-state analog (TSA) of the corresponding reaction [2], The TSA is then used as target molecule in the affinity selection scheme described above. The selected aptamers are screened to find molecules that catalyze the respective reaction that proceeds via this transition state. This concept has been successfully used for catalytic anti-... 

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