Hydrolytic probe

As mentioned in Section 4, the analysis of rate data resulting from unimolecular reactions is considerably easier than the analysis of such data for bimolecular reactions, and the same is true for pseudounimolecular reactions. Kinetic probes currently used to study the micellar pseudophase showing first-order reaction kinetics are almost exclusively compounds undergoing hydrolysis reactions showing in fact pseudofirst-order kinetics. In these cases, water is the second reactant and it is therefore anticipated that these kinetic probes report at least the reduced water concentration (or better water activity in the micellar pseudophase. As for solvatochromic probes, the sensitivity to different aspects of the micellar pseudophase can be different for different hydrolytic probes and as a result, different probes may report different characteristics. Hence, as for solvatochromic probes, the use of a series of hydrolytic probes may provide additional insight. 

Most of the pseudounimolecular hydrolytic probe reactions involve water-catalyzed pH-independent hydrolysis of activated esters, amides, or acid chlorides " " (Scheme 9). The hydrolysis of these probes occurs via a dipolar... 

The effects of the high ionic strength (3) appear to depend rather much on the details of the hydrolytic probe. For example, aqueous rate constants for 4-nitrophenyl 2,2-dichloropropionate 2 decrease as a function of added electrolyte (as typically happens), but the origin of this rate decrease by added electrolytes was interpreted to be different from the rate-retarding effect exerted by micelles based on activation parameter considerations. For another hydrolytic probe, phenyl chloro-formate lb, increasing electrolyte concentrations also decrease the rate of reaction, but this effect is negligible in comparison to the micellar rate effects. " ... 

The hydrolysis of p-nitrophenyl acetate and bis(p-nitrophenyl phosphate) are frequently used to probe hydrolytic activity. A problem with some other dinuclear systems is that the Zn units are held together by bridging ligands which can be cleaved on reaction with the substrate.440 This is not the case in a ditopic ligand such as those designed by Lippard and co-workers based on Kemp s triacid imide with a xylyl spacer.441,442 Both zinc dimers and mixed metal dimers were formed and a structure characterized with a bridging phosphodiester (Figure 6). 

Fig. 6.13. Different designs of FRET sensors. (A) Substrates for hydrolytic enzymes. (B) Sensors for bond formation. (C) Sensors based on conformational or structural change. (D) Environmentally sensitive probes. (E) Quenched activity-based probe to monitor small molecule-enzyme interaction. (F) Small molecule-enzyme interaction using a labeled protein.

A significant number of fluorogenic sensors are based purely on an increase or decrease of fluorescence. As discussed above, these probes, though often very useful, lack the internal reference that ratiometric sensors provide and require therefore difficult calibration. Especially hydrolytic enzymes are easily monitored by a fluor-ophore that is held in a non-fluorescent stage until the enzyme of interest removes the quencher. Certainly, it is beneficial if the... 

Since kinases are not hydrolytic enzymes, a small molecule-based FRET probe does not seem to be a straight forward solution for this enzyme activity. Nevertheless, quite a number of fluorescent probes based on small substrate peptides have been prepared in... 

Another important group of hydrolytic enzymes are phospho- and cyclophosphodiesterases. They catalyze the hydrolysis of phospho-diester bonds and many of the most relevant biological substrates are nucleic acids. Phospholipase C and D are also important examples. Initial attempts to measure phosphodiesterase activity placed a phosphodiester between a fluorophore and a quencher and the probe was tested in vitro [146], This system was slightly modified by Caturla and used for the identification of catalysts with phosphodiesterase activity [147], More recently, Nagano and co-workers used a coumarin donor and fluorescein as a FRET... 

Figure 16 (a) Structures of adenylation domain intermediates and inhibitors aminoacyl-sulfamoyl adenosine (AMS) and cisoid -like macrocyclic inhibitor, (b) Alkyne-functionalized chemical probe for NRPS A and PCP domains, (c) Structure of aminoacyl PCP, SNAC substrate analogue, and hydrolytically stable phosphopantetheinyl analogue, (d) Structure of vinylsulfonamide probe. R represents a peptide component and R an amino acid side chain. 

De Macario, E. C. Macario, A. J. L. Rivard, C. J. Grohmann, K. Use of Antibody Probes to Monitor Hydrolytic Microbes in Municipal Solid Waste Anaerobic Digesters SERI-SP-231-3520 Solar Energy Research Institute Golden, CO, 1989 pp 47-58. 

We have now supplemented LC with °0 labeling as a probe of the relative contributions of hydrolytic attack on the three... 

A method based on inhibition of ABAP-induced oxidation of a fluorogenic probe, 2, 7-dichlorodihydrofluorescein diacetate (H2-DCF-DA), commonly used for detection of production of reactive oxygen species, has been proposed (V2). Oxidation of the probe can be monitored either spectrophotometrically or fluori-metrically. A drawback of the method is the necessity of hydrolysis of the ester because only the deesterified from can be oxidized. The rate of hydrolysis depends on the hydrolytic activity of the samples measured. A modification consisting of chemical hydrolysis of H2-DCF-DA before measurement is impractical because 2,7 -dichlorodihydrofluorescein oxidizes rapidly. 

Several academic partners and Siemens Medical Solutions USA Inc. (Molecular Imaging) in Culver City, USA, made the synthesis of an [18F]fluoride-radiolabeled molecular imaging probe, 2-deoxy-2-[18F]fluoro-D-glucose in an integrated microfluidic device (see Figure 5.1) [21]. Five sequential processes were made, and they are [18F]fluoride concentration, water evaporation, radiofluorination, solvent exchange and hydrolytic deprotection. The half-life of [lsF]fluorine (t1/2 = llOmin) makes rapid synthesis of doses essential. This is one of the first examples of an automated multistep synthesis in microflow fashion. 

In a synthetic context, it is important to note that liberation of the carbonyl from the respective imine photoproducts has so far not yet been probed. A potential drawback of this step could be simultaneous cleavage of the cyclopropane under the necessary hydrolytic conditions, a transformation which is, however, by itself part of the synthetic strategy in many instances. ... 

In the experimental study and interpretation of hydrolytic reaction rates, many kinetic effects provide opportunities for probing the detailed nature of the reaction or have significant practical consequences on drug stability. The topics of temperature dependence, catalysis and solvent effects, were discussed in the first edition of this encyclopedia. 

---