**1. Introduction**

All chemical transformations pass through an unstable structure called the transition state, which is poised between the chemical structures of the substrates and products. The transi‐ tion states for chemical reactions are proposed to have lifetimes near 10-13 sec, the time for a single bond vibration. Thus, the transition state is the critical configuration of a reaction sys‐ tem situated at the highest point of the most favorable reaction path on the potential-energy surface, with its characteristics governing the dynamic behavior of reacting systems deci‐ sively. It is used primarily to understand qualitatively how chemical reactions take place.

Yet transition state structure is crucial to understanding enzymatic catalysis, because enzymes function by lowering activation energy. Linus Pauling coiled an accepted view, that incredible catalytic rate enhancements caused by enzyme is governed by tight binding to the unstable transition state structure in 1948. Because reaction rate is proportional to the fraction of the re‐ actant in the transition state complex, the enzyme was proposed to increase the concentration of these reactive species. This proposal was further formalized by Wolfenden (1972) and cow‐ orkers, who hypothesized that the rate increase imposed by enzymes is proportional to the af‐ finity of the enzyme to the transition state structure relative to the Michaelis complex.

Transition state structures of enzymatic targets for cancer, autoimmunity, malaria and bacteri‐ al antibiotics have been explored by the systematic application of kinetic isotope effects and computational chemistry. Today the combination of experimental and computational access to transition-state information permits the design of transition-state analogs as powerful enzy‐ matic inhibitors and exploration of protein features linked to transition-state structure.

Molecular electrostatic potential maps of transition states serve as blueprints to guide syn‐ thesis of transition state analogue inhibitors of chosen enzymes. Substances, that ideally

© 2013 Gluza and Kafarski; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mimic geometric and electrostatic features of a transition state (or other intermediates of high energy) are considered as excellent enzyme inhibitors (Fig.1). They bind up to 108 times tighter than substrate. Thus, the goal of transition-state analogs design is to create stable chemical structures with van der Waals geometry and molecular electrostatic potential sur‐ faces as close as possible to those of the transition state.

The design of transition-state inhibitors is likely to become more frequent in the future, alongside with the development of theory and technology for understanding enzyme transi‐ tion states. Today the sequence of information required to obtain transition state analog of enzymatic reaction considers: choice of the suitable enzyme (most likely suited to kinetic isotope effect measurement), selection of presumable mechanism(s) of catalyzed reaction, measurement of kinetic isotope effects (KIE), computer-aided calculations matching the in‐ trinsic KIEs, construction of steric and electronic map of transition state and synthesis of sta‐ ble compound(s) matching this map [Schramm, 2007]. This procedure has been developed gradually in parallel with the advances in KIE enzymology, computational chemistry, and

Transition State Analogues of Enzymatic Reaction as Potential Drugs

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At present, the most reliable method to determine three-dimensional architecture of transi‐ tion state is through the use of computational methods in conjunction with experimentally

Isotopic substitution is a useful technique for probing reaction mechanisms. The change of an isotope may affect the reaction rate in a number of ways, providing clues to the pathway of the reaction. The advantage of isotopic substitution is that this is the least disturbing structural change that can be effected in a molecule. Replacement of one isotope of the sub‐ strate by another at vicinity where bonds are being or re-hybridizing typically leads to a change in the rate of the reaction. Thus, kinetic isotope effects measurements compare kcat/KM values between isotope-labeled and natural abundance reactants. This provides in‐ formation about which bonds are broken or formed, and identifies changes in hybridization that occur during the rate limiting step of a reaction. It is reached by conversion of atom-byatom KIE values to a specific static model with fixed bond angles and lengths by computa‐ tional matching to a quantum chemical model of the reaction of interest. Substrate, intermediate and product geometries are located as the global minima. Transition-state structures are located with a single imaginary frequency, characteristic of true potential en‐

Such an analysis was performed recently for human thymidine phosphorylase, an enzyme responsible for thymidine homeostasis, action of which promotes angiogenesis. Thus, inhib‐ itors of this enzyme might be considered as promising anticancer agents. Its transition state was characterized using multiple kinetic isotope effect measurements applying isotopically

H, 14C and 15N) enriched thymidines, which were synthesized enzymatically [Schwartz et al, 2010]. A transition state constrained to match the intrinsic KIEs was found using density functional theory. In the proposed mechanism (Fig.2), departure of the thymine results in a discrete ribocation intermediate. Thymine likely leaves deprotonated at N1 and undergoes enzyme-catalyzed protonation before the next step. In the following step, the intermediate undergoes nucleophilic attack from an activated water molecule to form the products. The latter step is a reaction rate limiting step as determined by energetics of its transition state.

synthetic organic chemistry.

measured kinetic isotope effect (KIE).

ergy saddle points.

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**3. Determination of transition state architecture**

Although some reviews on the subject have been published, this concept has not been re‐ viewed in detail [Wolfenden, 1999; Robertson, 2005; Schramm, 2005; Schramm, 2007; Dyba‐ ła-Defratyka et al. 2008; Schramm, 2011]. In this review the current trends, alongside with appropriate case studies in designing of such inhibitors will be presented.
