EHUD KEINAN

MULTIFARENES

 

Multifarenes: new class of macrocycles, which are constructed of alternating building blocks, are conveniently accessible by three complementary syntheses that provide modularity and scalability. In addition to metal-ion coordination, these cavitands show increased flexibility with increasing ring size, offering opportunities for induced fit to guest molecules (Fig. 1).

SEMITHIO BAMBUSURILS

 

Semithio-bambusurils, which represent a new family of macrocyclic host molecules, have been prepared by a convenient, scalable synthesis. These new cavitands are double functional: they strongly bind a broad variety of anions in their interiors and metal ions at their sulfur-edged portals. The solid-state structure of semithio-bambus[4]uril with HgCl2 demonstrates the ability of these compounds to form linear chains of coordination polymers with thiophillic metal ions. The crystal structure of semithio-bambus[6]uril with tetraphenylphosphonium bromide exhibit the unique anion binding properties of the host cavity and the characteristics of the binding site (Fig.2). 

SYNTHETIC ENZYMES

 

Efforts to generate new enzymatic activities from existing protein scaffolds may not only provide biotechnologically useful catalysts but also lead to a better understanding of the natural process of evolution. Enzymes are usually characterized as catalyzing a specific reaction by a unique chemical mechanism. However, small changes in the amino acid sequence of some enzymes can markedly alter the catalytic properties of the enzymes, affecting the substrate selectivity and subtle aspects of the catalytic mechanism. The catalytic promiscuity displayed in these enzymes may be an important factor in the natural evolution of new catalytic activities and in the development of new catalysts through protein engineering methods.

 

We profoundly changed the catalytic activity and mechanism of 4-oxalocrotonate tautomerase by means of a rationally designed and synthetically accomplished amino acid substitutions table covalent adduct. For example, we have shown that the substrate specificity can be altered in a predictable way by electrostatic manipulation of the enzyme’s active site. A total of 1, 2, or all 3 active-site arginine residues were replaced by citrulline analogs to maintain the steric features of the active site of 4-oxalocrotonate tautomerase while changing its electronic properties. These synthetic changes revealed that the wild-type enzyme binds the natural substrate predominantly through electrostatic interactions. This and other mechanistic insights led to the design of a modified enzyme that was specific for a new substrate that had different electrostatic properties and that bound the enzyme via hydrogen-bonding complementarity rather than by electrostatic interactions. The synthetic analog of 4-oxalocrotonate tautomerase was a poor catalyst of the natural 4-oxalocrotonate substrate but an efficient catalyst for a ketoamide substrate. 

Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes have selenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides and disulfides. The selenol/disufide exchange reaction is common to all of these enzymes and the active site redox potential reflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymes containing selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kinetic role of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond in a protein has not been experimentally determined. In order to fully evaluate the effects of Sec on oxidoreductase redox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved 11CXX14C active site were chemically synthesized. Grx3, Grx3(C11U) and Grx3(C14U) exhibited redox potentials of -194, -260 and -275 mV, respectively. The position of redox equilibrium between Grx3(C11U-C14U) (-309 mV) and thioredoxin (Trx) (-270 mV) suggests a possible role for diselenide bonds in biological systems. Kinetic analysis showed that the lower redox potentials of the Sec variants result primarily from the greater nucleophilicity of the active site selenium rather than its role as either a leaving group or a ‘central atom’ in the exchange reaction. The 102 to 104-fold increase in the rate of Trx reduction by the seleno-Grx3 analogs demonstrates that oxidoreductases containing either selenenylsulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reduction kinetics in comparison with their sulfide counterparts. The research is being done in collaboration with P.E. Dawson, the Skaggs Institute.

CATALYTIC ANTIBODIES

 

A relatively unexplored opportunity in the science of catalytic antibodies is the in vivo modification of an organism phenotype by incorporating the gene that encodes for a catalytic antibody into the genome of that organism. An attractive application of this concept would be the expression of such a catalyst in transgenic plants to award the plant with a beneficial trait. For example, introduction of herbicide-resistance trait in commercial plants is highly desirable because it allows growing the crop plant in the presence of a non-selective herbicide that affects only weeds and other undesired plant species. We have shown that herbicide-resistant plants can be engineered by designing both herbicide and a catalytic antibody that destroys it in planta. We have demonstrated that such a transgenic plant can be achieved via a three-step maneuver: a) development of a new carbamate herbicide, one that can be catalytically destroyed by the aldolase antibody 38C2; b) separate expression of the light chain and half of the heavy chain (Fab) of the catalytic antibody in the endoplasmatic reticulum (ER) of two plant lines of Arabidopsis Thaliana; and c) cross pollination of these two transgenic plants to produce a herbicide-resistant F1 hybrid (Fig. 3). In vivo expression of catalytic antibodies could become a useful, general strategy to achieve desired phenotype modifications not only in plants but also in other organisms. 

BIOMOLECULAR COMPUTING DEVICES

 

In fully autonomous molecular computing devices, all components, including input, output, software, and hardware, are specific molecules that interact with each other through a cascade of programmable chemical events, progressing from the input molecule to the molecular output signal. DNA molecules and DNA enzymes have been used as convenient, readily available components of such computing devices because the DNA materials have highly predictable recognition patterns, reactivity, and information-encoding features. Furthermore, DNA-based computers can become part of a biological system, generating outputs in the form of biomolecular structures and functions.

 

Our previously reported 2-symbol–2-state finite automata computed autonomously, and all of their components were soluble biomolecules mixed in solution. The hardware consisted of 2 enzymes, an endonuclease and a ligase, and the software and the input were double-stranded DNA oligomers. More recently, we designed and created 3-symbol–3-state automata that can carry out more complex computations. In addition, we found that immobilization of the input molecules on chips allowed parallel computation, a system that can be used for encryption of information. 

 

The main advantage of autonomous biomolecular computing devices over the electronic computers is their ability to interact directly with biological systems. No interface is required since all components of molecular computers, including hardware, software, input and output are molecules that interact in solution along a cascade of programmable chemical events. We have demonstrated for the first time that the output of a molecular finite automaton can be a visible bacterial phenotype. Our 2-symbol-2-state finite automaton utilizes linear double-stranded DNA inputs prepared by inserting a string of 6-basepair symbols into the lacZ gene on plasmid pUC18. The computation resulted in a circular plasmid that differed from the original pUC18 by either a 9-baspair (accepting state) or 11-basepair insert (unaccepting state) within the lacZ gene. Upon transformation and expression of the resultant plasmids in E. coli, either blue or white colonies were formed on X-gal medium, respectively (Fig. 4). 

SYNTHETIC CAPSIDS

 

Stable structures of icosahedral symmetry can serve numerous functional roles, including chemical micro-encapsulation and delivery of drugs and bio-molecules, observation of encapsulated reactive intermediates, epitope presentation to allow for an efficient immunization process, synthesis of nanoparticles of uniform size and formation of structural elements for molecular supramolecular constructs and molecular computing. By examining physical models of spherical virus assembly we have arrived at a general synthetic strategy for producing chemical capsids at size scales between fullerenes and spherical viruses. Such capsids can be formed by self-assembly from a class of novel symmetric molecules developed from a pentagonal core. By designing chemical complementarity into the five interface edges of the molecule, we can produce self-assembling stable structures of icosahedral symmetry. We considered three different binding mechanisms, including hydrogen bonding, metal binding and formation of disulfide bonds. These structures can be designed to assemble and disassemble under controlled environmental conditions. We have conducted molecular dynamics simulation on a class of corannulene-based molecules to demonstrate the characteristics of self-assembly and to aid in the design of the molecular subunits. This research was done in collaboration with A. Olson of The Scripps Research Institute.

PEROXIDE EXPLOSIVES

 

We are using both x-ray crystallography and electronic structure calculations to study the explosives triacetone triperoxide (TATP) and diacetone diperoxide. The structure, vibrational spectrum, and thermal decomposition of TATP were calculated by using functional density theory. The calculated thermal decomposition pathway of the TATP molecule was a complicated multistep process with several highly reactive intermediates, including singlet molecular oxygen and various biradicals. Of note, the calculations predict formation of acetone and ozone as the main decomposition products and not the intuitively expected oxidation products.

 

The key conclusion from this study is that the explosion of TATP is not a thermochemically highly favored event. Rather, the explosion involves entropy burst, which is the result of formation of 4 gas-phase molecules from every molecule of TATP in the solid state. Quite unexpectedly, the 3 isopropylidene units of the TATP molecule do not play the role of fuel that can be oxidized and release energy during the explosion. Instead, these units function as molecular scaffolds that hold the 3 peroxide units close together spatially in the appropriate orientation for the decomposition chain reaction. This research was done in collaboration with R. Kosloff and Y. Zeiri. Hebrew University of Jerusalem.

FIGURE 1

Solid-state molecular structures at capped sticks presentation. (A) Multifarene[2,2], 1, shown also at spacefill presentation (right). (B) Multifarene[3,3], 2, with and without diethyl ether guest. (C) Multifarene[4,4], 3, with and without ethyl acetate guest. Color codes: red – oxygen, blue – nitrogen, yellow – sulfur, grey – carbon.

FIGURE 2

X-ray structure of 9*TPPB showing the guest bromide and the TPP counter cation (colour code: oxygen, red; sulphur, yellow; nitrogen, blue; carbon, grey, bromine, brown, phosphorous, orange). All hydrogen atoms and the chloroform molecule were omitted for clarity.

FIGURE 3

Influence of a new herbicide, 1, on the rooting and development of A. thaliana plant lines. The control plants are shown in a, c, and e, while the hybrid plant line (F1) expressing both light and heavy chains are shown in b, d, and f. Plantlets grown on MS medium without 1 are shown in a and b, and those grown with 0.05 mM 1 (c-f).

FIGURE 4

A: Computation with aaba input in the presence of the transition molecules resulting in white bacteria (S1). 

B: control experiment using aaba without transition molecules. 

C: Computation with abba input in the presence of the transition molecules resulting in blue bacteria (S0). 

D: control experiment using abba without transition molecules.