by Academician Alexei KHOKHLOV, Lomonosov Moscow State University,
Valentina VASILEVSKAYA, Dr. Sc. (Phys. & Math.), Nesmeyanov Institute of Organoelemental Compounds, RAS
Fine control of chemical reactions implies the use of various instruments for their initiation, acceleration, or, vice versa, inhibition, and arrest, if necessary. Enzymes are effective natural catalysts, and scientists all over the world work to create their synthetic analogs. Now we shall speak about our recent results in this sphere.
CHARACTERISTICS OF THE MEDIUM AND REACTION VELOCITY
Two methods of regulation of chemical reactions are known in modern science: reduction of their potential barrier by effective catalysts and use of "nanoreactors" (media with heterogeneous particles differing in size by several nanometers, which effectively adsorb the reagents). The mechanism of action in this latter case is rather simple: the reagents placed into the heterogeneous medium are adsorbed in nanocavities, their local concentration increases, and the reaction velocity increases in proportion.
Model presentation of surface nanoreactors: A - emulsion droplets; B - surface of the globule, on which amphiphilic reagents, consisting of hydrophobic and hydrophilic groups, are concentrated.
Structurized polymer lattices, micellar solutions*, and emulsions can serve as nanoreactors. The efficiency of these media is determined not only by their structure, but also by the reagents' affinity to them. For example, a reaction between hydrophobic substances can be stimulated by using an emulsion of two components (nonpolar water-insoluble compound and water**) or aqueous solution of micelles. The reaction between surfactants can be regulated by using media with well-discernible interface between the phases: these reagents, placed into such systems, are concentrating in the layers just several nanometers thick (the surface nanoreactors), in which the process is mainly taking place.
The simplest variant of these nanoreactors is a totality of the outer layers of spherical droplets in the emulsion, the more intricate one is the surface of a soluble globule. This latter variant is realized in enzymatic catalysis***.
The nature created special protein macromolecules for regulation of biological processes: enzymes. Each of them is characterized by the unique structure, determined by the sequence of amino-acid residues in the polypeptide chain, due to which the catalytic function of each protein is "set up" for a certain biochemical reaction. However, these substances have many universal properties, important for their "work". We should like to distinguish the following:
First, the proteins realize their function in a compact (globular) state, and hence, have a well-formed surface between the inner nonpolar nucleus and outer polar phase (the above-mentioned definition of polarity is true of this case too).
The enzyme globules remain in the solution and do not precipitate. This is due to their special organization. As we know, all aminoacids can be divided into two groups: hydrophobic (those attracted to each other and trying to escape contacts with water) and hydrophilic (vice versa, "disliking" contacts with all monomeric components and trying to surround themselves by the maximum number of water molecules). The macromolecular chain in the protein globule is coiled so that the hydrophobic aminoacids are located inside and form the nucleus, while the hydrophilic components are located on its surface and form the "protective membrane", preventing reactions between the protein hydrophobic components and other macromolecules and hence, preventing aggregation and precipitation of these latter ones.
The enzymes' catalytic effect proper (reduction of the potential barrier of the reaction) is attained due to the so-called active center, very often lying on the globule surface and consisting of several monomer components of the protein chain, oriented towards each other in a special way.
* Micellar solutions are solutions of surface-active substances with concentrations sufficient for their self-organization with the formation of aggregations-"micelles", consisting of the nucleus insoluble in this medium and enveloped in a protective membrane. - Auth.
** Polar media are solvents with high dielectric permeability (water), nonpolar ones-with low dielectric permeability (benzene, toluene, hexane). Hydrophilic substances are dissolved in the former, hydrophobic ones in the latter. They are "distributed" in quite the same way in complex mixed systems containing the above media, while surfactants, having both hydrophilic and hydrophobic groups, are located at the interface of these media. - Auth.
*** See: A. Yanenko, "Priorities of Industrial Biotechnology", Science in Russia, No. 5, 2006; A. Sinitsyn, "Versatile Enzymes". Science in Russia, No. 4, 2007. - Ed.
Effect of concentration of amphiphilic substances on the surfaces of emulsion droplets of different radius.
Such are the fundamentals of the natural catalysis organization, simulating which it is possible (we believe) to create their synthetic analogs.
ENZYMES AS SURFACE NANOREACTORS
Thus, we assumed that an enzyme-like catalyst is a sort of a surface nanoreactor, or, in other words, a heterogeneity, due to which the reagents are adsorbed in a thin perisurface layer.
Let us verify this by the simplest reaction, substrate (A) transformation in the presence of catalyst (B). The object of this analysis is the case when the molecules of the reagents are characterized by a complex structure, contain hydrophobic and hydrophilic groups of atoms, and are surface-active. Emulsion (spherical droplets of oil in water) served as surface nanoreactors. Formally we use these droplets by analogy with globular proteins: there is a hydrophobic "nucleus", extensive hydrophilic environment, and clear-cut interface between the phases in both cases.
Substances A and B, placed into the emulsion, adsorbed preferably on its droplets, their concentration in the perisurface layer increasing and the reaction velocity increasing in proportion. It was found that the reaction in such systems, in comparison with homogeneous (aqueous or oily) solvent, can be several hundred and even thousand times more rapid. Estimations show that this jump-wise increment is determined by the surface activity of the reagents (their affinity to the surface) and depends on the size of emulsion droplets. If the droplets are small and, hence, numerous, the reagents are distributed on a larger surface and their local concentration is not much higher than in a homogeneous solvent. If the droplets are large and/or they are not numerous, the negligible total area of the emulsion is completely covered by the small percentage of the reagents, while the major part is diffused in the solution (in water and inside oily droplets). The reaction velocities in such system and in a homogeneous solvent differ slightly. The maximum effect can be expected with emulsion droplets of intermediate size, when all molecules of reagents are placed on their surface, without leaving any sites free.
For experimental verification of our estimate, we studied model reactions of hydrolytic cleavage of n-nitrophenyl esters of different acids (all of them are surfactants) under the effect of various catalysts (by the way, these ionic exchange reactions of dissolved substances with water can be spontaneous as well). Any substances containing imidazole groups* (let us remember this term, as the choice of reagents for experiments described in this paper is determined by the presence of these groups) and some basal ions can initiate these transformations. In general, these catalysts are soluble in water and, in order to render them the surfactant characteristics, imidazole groups were sutured to a special matrix.
Hydrolysis of n-nitrophenyl esters was carried out in water emulsions and emulsions of nonpolar carbohydrates with normal structure (tetradecane and dodecane**). A significant (several-fold) increase in the velocity of this reaction could be attained only by using surface-active catalysts, the desired result in this case being exclusively due to the reagents' concentration on the surfaces of dispersed droplets of the emulsion.
One of the distinguishing features of enzymatic catalysis is compliance with the Michaelis-Menten law***,
* Imidazole is a group of atoms in many important natural compounds and in molecules of synthetic drugs. It is a catalyst for organic substances hydrolysis. - Auth.
** Tetradecane and dodecane are threshold (saturated) CnH2n+2 carbohydrate series. - Ed.
*** Michaelis-Menten's equation (introduced by the German biochemists L. Michaelis and M. Menten in 1913) describes the relationship between the enzymatic reaction velocity and the substrate concentration on condition that the total number of the enzyme molecules is permanent and is significantly less than the number of the substrate molecules. - Auth.
Dependence of reaction velocity (M) on the size of emulsion droplets (R) at different values of adsorption energy (ε); Mo - reaction velosity in the homogeneous solution; d - typical size of molecules of reagents.
indicating that an increase in the substrate concentration in the presence of permanent concentration of the catalyst at first leads to increase in the reaction velocity, and then, at the maximum value "reaches the plateau", i.e., changes no longer. Estimations showed that in our system of surface nanoreactors, the kinetics of the process in many cases was in agreement with this regularity.
The main difference between the enzyme proteins and emulsion droplets, discussed here, is that in the former the catalytic groups are connected to the globule by strong chemical bonds, while in the latter the relationship of catalyst B molecules and the surface is not "strong" and it can be partially or completely forced out from the surface by the substrate A molecules if its concentrations are high.
The Michaelis-Menten law in the studied systems is valid only for cases when the surface activity of catalyst B is many times higher than that of substrate A; moreover, it is so great that we can say that B is "strongly fixed" to the surface. The surface nanoreactor, which can be considered as an enzyme-like catalyst, "is born" on this condition.
Though formally speaking, surface reactors emerging on the base of emulsions and globule solutions are similar, the latter ones have an indisputable advantage: the catalytic groups can be "strongly" sutured only to a macromolecule, while in emulsions the substance essential for the reaction velocity is at a high risk of being forced out from the surface. In addition, the size of a surface nanoreactor can be more precisely set up in macromolecular systems ("the droplet radius" for the model described above), they are easier to modify by varying the "quality" of the solvent (temperature, composition, pH values, etc.).
In a "bad" solvent the monomeric components, strongly attracted to each other, accumulate at a minimally possible distance and the macromolecule forms the globule or collapses. In a "good" solvent they push each other away, and the macromolecule "grows", it is in a state of a "tangle". Its size in the course of the tangle transformation into a globule increases several-fold. If we compare a macromolecule to a low-molecular substance, we can say, that a "tangle" is like gas, while the globule is like liquid.
The advantages of using man-made globular systems as surface nanoreactors are obvious. But we had to overcome a fundamental difficulty: make them water-soluble. The specialists know: as soon as the quality of the solvent is so "poor" that a synthetic macromolecule forms a globule, it aggregates with other similar structures and precipitates, as this becomes energetically advantageous. In order to prevent this process, a hydrophilic coating should be created on the globular surface, similar to the membrane of natural protein formations.
The analysis showed that this problem can be solved if hydrophilic components are included in the macromolecular chain, and in order to form a solid membrane,
Amphiphilic monomeric components, consisting of groups with different affinity to water, are presented as two connected beads: A - amphiphilic monomeric component; B - polymeric chain of amphiphilic components; C - protein-like globule of amphiphilic polymer.
really protecting the hydrophobic nucleus, these components should be in fact amphiphilic (include hydrophobic groups as well). Moreover, no components along the chain should be distributed at random, but in accordance with special so-called protein-like statistics.
We found that protein-like macromolecules (forming soluble globules) can be prepared either by modifying a polymer or by synthesis via co-polymerization or polycondensation of monomeric components with significantly different hydrophobic/hydrophilic characteristics.
Having chosen the second approach, we obtained protein-like polymer chains on the base of copolymers of thermosensitive N-vinylcaprolactam* and N-vinylimidazole, accelerating (initiating) hydrolysis reaction (due to the presence of imidazole groups in it). The chain was organized so that when it was coiled into the globule, the nucleus was formed from N-vinylcaprolactam components, while the surface was formed from N-vinylimidazole groups alone. The resultant structures were tested as catalysts of p-nitrophenylpropionate (surfactant, propionic acid ester) hydrolysis reaction. It was found that they were little effective in this role, if shaped as a tangle, "gas-like", loose, without fixed structure, so important for the processes of surface interface concentration.
At the same time it was established that the velocity of hydrolysis reaction of the above-mentioned substance in the presence of protein-like globules increased 12-fold in comparison with the solution containing the same concentration of imidazole catalytic groups, not "sutured" to a polymeric globule.
If the distribution statistics of groups with different affinity to water is incidental, the "hydrophilic membrane" on the globule surface is not solid, as in similar protein-like formations. Several globules can unite in soluble aggregations of an optimal size, in which hydro-phobic nuclei of several chains are compactly enveloped in a membrane from hydrophilic components. For example, vinylcaprolactam and imidazole copolymer with a stray distribution of components collapses at high temperatures, uniting with macromolecules similar to it so that the inner part of the aggregation is formed from vinylcaprolactam components and the outer part from imidazole hydrophilic groups. Due to availability of imidazole groups exposed in the solvent these complexes can be rather effective as catalysts of hydrolysis reaction.
In order to verify our assumption we measured Arrhenius* dependencies for the model reaction of
* N-vinylcaprolactam is a thermosensitive polymer with conformation easily modified by temperature variations. - Auth.
* Svante August Arrhenius (1859 - 1927), Swedish physicist and chemist, Member of Swedish Academy of Sciences, Honored Member of academies and societies of many countries, including the USSR Academy of Sciences. In his works on chemical kinetics he suggested an equation, taking into account the dependence of chemical reaction velocity constant on temperature (1889). - Ed.
Dependencies of p - nitrophenylacetate hydrolysis reaction velocity (V) on temperature (T) in the presence of poly (N - vinylcaprolactam) and poly (N - vinylimidazole) copolymers of four different compositions and 1 - methylimidazole and poly (N - vinylimidazole).
p-nitrophenylacetate hydrolysis in the presence of four differing copolymer catalysts, and the catalysts of different structure, but with the same concentrations of imidazole groups-low molecular (methyl-imidazole) and homopolymer (poly(N-vinylimidazole)) catalysts. These dependencies are linear in the two latter cases, which is in agreement with the Arrhenius law. By contrast, a sharply pronounced acceleration at temperatures from 35 to 45°C is observed in the first four cases, caused (in this case) by transition of copolymer macromolecules into a more compact state and formation of aggregations with clear-cut interface, containing catalytic groups in the perisurface layer.
Let us note that as thermosensitive "polymeric" catalysts are easily regulated (using them, it is possible to "trigger" and "arrest" the reaction with an accuracy of several degrees), they can be used in special cases (for example, for reactions associated with strongly pronounced endothermal effect, carried out in aggressive media).
At present the final required condition for the creation of enzyme-like catalysts is creation of an analog of the natural enzyme protein active center. In order to solve this problem, we have to correctly select and place on the globular surface the groups, whose desirable effects (initiation, acceleration, etc.) on one or another reaction is possible only on condition of their certain mutual location and/or orientation.
We proceed with studies in this direction, accurately inserting hydroxyl, carboxyl, and imidazole components in the thermosensitive polymer chain with the aim of creating synthetic analogs of the so-called serine hydrolases (enzymes responsible for hydrolysis of complex ester bonds). Their active center contains the above-mentioned "catalytic triad"-hydroxyl, carboxyl, and imidazole atomic groups, oriented in a peculiar way towards each other.
Our article deals with the basic approaches, models, and results of studies aimed at creation of synthetic analogs of natural protein enzymes. Our first publications on the topic appeared not long ago, in 2004. All studies described in this paper are carried out at Nesmeyanov Institute of Organoelemental Compounds, RAS, by a team of scientists including Yuri Belokon, Valery Grinberg, Vladimir Lozinsky, Drs. Sc. (Chem.), the authors of this paper, and a number of young researchers.
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