Phosphonates transition-state analogues

An example of esterase behaviour is provided by a catalytic antibody developed by Tramontano et al. (1988), using a phosphonate transition state analogue [53] as the hapten. The antibody cleaves the carboxylic ester [54, R = Me] with enzyme-like efficiency (kc/ku = 6.25 X 106 = 1.5 mM ... 

Selection with transition-state analogues has also been used to extract catalytic antibodies from libraries of phage-antibodies. McCafFerty et al. [52] have shown that antibodies with nanomolar affinities for a phosphonate transition-state analogue could easily be extracted from a library derived from immunized mice. 

A phosphonate transition-state analogue (218) for phe-gly hydrolysis was successfully employed to generate catalytic antibodies that showed moderate rate accelerations (10 -10 ) however, the uncharged methyl phosphonamidate (219) failed to generate any catalytic activity. ... 

A similar concept was used in the development of artificial chymotrypsin mimics [54]. The esterase-site was modeled by using the phosphonate template 75 as a stable transition state analogue (Scheme 13.19). The catalytic triad of the active site of chymotrypsin - that is, serine, histidine and aspartic acid (carboxy-late anion) - was mimicked by imidazole, phenolic hydroxy and carboxyl groups, respectively. The catalytically active MIP catalyst 76 was prepared using free radical polymerization, in the presence of the phosphonate template 75, methacrylic acid, ethylene glycol dimethacrylate and AIBN. The template removal conditions had a decisive influence on the efficiency of the polymer-mediated catalysis, and best results were obtained with aqueous Na2CC>3. 

Hanson JE, Kaplan AP, Bartlett PA. Phosphonate analogues of 63. earboxypeptidase A substrates are potent transition-state analogue inhibitors. Biochemistry 1989 28(15) 6294-6305. 

It has already been reported that antibodies prepared against the transition state of a reaction show considerable catalytic activity [113]. For example, antibodies prepared against a phosphonic ester (as a transition state analogue for alkaline ester hydrolysis) enhanced the rate of ester hydrolysis by 10 -10" fold. Recently, similar systems based on imprinted polymers which display high catalytic activity have been successfully prepared. Initial attempts were performed by several groups [114-117] with imprinted polymers based on non-stoichiometric, non-covalent interactions, which, however, gave results far below those obtained with antibodies. Rate enhancements up to 6.7-fold were reached in one case. 

A. Sachpatzidis, C. Dealwis, J.B. Lubetsky, P.H. Liang, K.S. Anderson, and E. Lolis. 1999. Crystallographic studies of phosphonate-based alpha-reaction transition-state analogues complexed to tryptophan synth asQ Biochemistry 38 12665-12674. (PubMed)... 

Sachpatzidis, A., Dealwis, C., Lvibetsky, J. R.. Liang, P. H., Anderson, K. S, and Lolis, F. 1999. Crystallographic studies of phosphonate-based a-reaction transition-state analogues complexed to tryptophan syntha.se. Biochemistry 38 12665—12674,... 

The formation of a spatially and functionally specific cavity has an obvious application in enzyme type catalysis [112]. Furthermore, in the light of interest in catalytic antibodies [113], the analogy between antibody and MIP suggests the concept of catalytic plastibodies . Robinson and Mosbach [114] demonstrated the potential for catalytic activity by imprinting a transition state analogue (p-nitrophenylmethyl-phosphonate) of the hydrolysis of p-ni-trophenylacetate (Figure 6.29)... 

Figure 10.18 Catalytic monoclonal antibody (MAb) mediated ester hydrolysis. The MAb was generated through immunoreaction with the illustrated hapten linked to an appropriate carrier protein. The part of the hapten related to the substrate structure is shown in red. The phosphonate link is used as a transition state analogue of the rate determining step transition state leading to the key tetrahedral intermediate of ester hydrolysis. The MAb should optimally bind the rate determining step transition state relative to substrate or products in order to effect maximum catalytic effect.

A second example involves a transesterification reaction catalyzed by an antibody generated against a phosphonate diester transition state analogue [11]. The antibody catalyzes the corresponding acyl transfer reaction of thymidine and an alanyl ester with an effective molarity of 3 x 10 m... 

Much less potent as they are. aldehyde, ketone, and haloketone analogues of substrates are also reversible inhibitors of CPA. For example, (RS)-2-benzyl-4-oxobu-tanoic acid is a competitive inhibitor of CPA with a Ki of 480 nM. These classes of compounds are more or less hydrated in water to form a gem-diol(ate), with a structure that mimics that of a putative intermediate of hydrolysis of substrates (see above). In other words, these compounds seem to serve as a transition-state analogue, just like the phosphonates described above, and hence, they bind more tightly than substrates. It may be noted that the structure of these transition-state analogues more closely resembles that of a putative intermediate of hydrolysis generated by... 

The peptidic a-hydroxyl phosphonates present a promising and structurally unique class of transition-state analogue inhibitors of proteolytic enzymes and make the first disclosure of their application in preparing inhibitors of human renin. 

The phosphonate 55, designed as a transition-state analogue for the inhibition of UDP-glucuronosyltransferase, the enzyme involved in the detoxification of xenobiotics by glucuron-idation, has been made as outlined in Scheme 9, the uronate function being introduced by Jones... 

The preparation of phosphonate derivatives 2 and 3, made as transition state analogues for reactions of 3-gaIactosyl transferase of bovine milk, have been reported using tri-O-acetyl-l-C-formyl-D-galactal as the key intermediate. ... 

Transition states of this tetrahedral nature have now been mimicked effectively by a range of stable analogues, including phosphonic acids, phosphonate esters, a-difluoroketones, and hydroxymethylene functional groups (Jacobs, 1991). Lerner s group elicited antibodies to a tetrahedral anionic phosphonate hapten [3] (Appendix entry 2.9)2 whilst Schultz s group isolated a protein with high affinity for p-nitrophenyl cholyl phosphate [4] (Fig. 4) (Appendix entry 3.2). 

Phosphonates have been widely used as analogues of carboxylic acids. They have been particularly effective as analogues of tetrahedral transition states that occur in the course of enzyme-catalyzed reactions such as hydrolysis of the amide (peptide) bond. As such, they may be used as inhibitors of enzymes (e.g., 82, 83) or as haptens for producing antibodies that are catalytic (e.g., 84). A notable example is H203P— CH2—CH2—CH(—NH2)—COOH, which has effects that are likely to be due to its interference with glutamate as a neurotransmitter (85). 

The most widely used strategy involves the synthesis of the network around a structural analogue of the transition state of the reaction. The imprinted sites then correspond to the conformation of the substrates in the transition state. For ester hydrolysis this state can, for instance, be simulated by a phosphonate derivative as template [156,167]. An imprinted network with an esterase-type catalytic activity can then be obtained. For the MIP represented in Fig. 19(1), the reaction rate is increased 100-fold with respect to the reaction without catalyst and kinetics of the Michaelis-Menten type, as well as inhibition by an analogue of the transition state are observed [156]. 

A kinetic study of the reaction of acyclic, (28), and cyclic, (29) and (30), phosphonates with / -substituted benzaldehydes using ethoxide as the base has been carried out. The results show the reactions to be third order, indicate a build up of negative charge in the transition state of the rate determining step (p = +2), and show an acceleration in the case of five-membered cyclic phosphonates of about 20 X over the acyclic analogues. An analogous study of phosphinates and phosphine oxides gives similar results and shows that phosphinates form alkenes approximately 35 x faster than phosphine oxides in spite of similar p/lTa values for the a-protons in each compound. 

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