Racemic protein crystallization

An X-ray crystal structure of 28 bound in the thumb-region of the NS5B polymerase showed little interaction of the acetamide moiety with the protein. Alterations at this position were explored in order to improve the physical properties of the compound. Incorporation of basic amines as part of this side-chain, leading to zwitterionic compounds, reduces plasma binding and has a beneficial effect on cell activity and pharmacokinetic profiles. In the cell-based replicon assay, racemic 29 has an EC50 of 152 nM in the presence of 10% fetal calf serum and 376 nM in the presence of 50% normal human serum [71],... 

TThe primary function of D-amino acid oxidase, present at high levels in the kidney, is thought to be the detoxification of ingested D-amino acids derived from bacterial cell walls and from cooked foodstuffs (heat causes some spontaneous racemization of the l-amino acids in proteins). Oxalate, whether obtained in foods or produced enzymatically in the kidneys, has medical significance. Crystals of calcium oxalate account for up to 75% of all kidney stones. ... 

Alanine made in this way must be racemic, because the starting materials are achiral. However, if we isolate alanine from a natural source—by hydrolysing vegetable protein, for example—we find that this is not the case. Natural alanine is solely one enantiomer, the one drawn below. Samples of chiral compounds that contain only one enantiomer are called enan-tiomerically pure. We know that natural alanine contains only this enantiomer from X-ray crystal structures. 

Pentelute BL, Gates ZP, Tereshko V et al (2008) X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers. J Am Chem Soc 130 9695-9701... 

Cross-linked crystals of lipase from Candida rugosa (CRL) were applied in the resolution of racemic ketoprofen chloroethyl ester. In batch-wise operation, the half-life of the catalyst was reached after about 18 cycles or, in terms of enzyme consumption, about 5.6 g of enzyme protein were consumed to prepare 1 kg of (S)-ketoprofen. CRL suffers from a low specific activity towards this poorly water-soluble substrate which may explain the high enzyme input [117]. 

Why Nature uses only one enantiomer of most important biochemicals is an easier question to answer than how this asymmetry came about in the first place, or why L-amino acids and D-sugars were the favoured enantiomers, since, for example, proteins made out of racemic samples of amino acids would be complicated by the possibility of enormous numbers of diastereomers. Some have suggested that life arose on the surface of single chiral quartz crystals, which provided the asymmetric environment needed to make life s molecules enantiomerically pure. Or perhaps the asymmetry present in the spin of electrons released as gamma rays acted as a source of molecular asymmetry. Given that enantiomerically pure living systems should be simpler than racemic ones, maybe it was just chance that the L-amino acids and the D-sugars won out. 

In addition to hydrase properties, crystalline crotonase is believed to possess three different catalytic properties it acts as an isomerase that converts the cis to the trans form it catalyzes the racemization of the d( —) hydroxybutyryl CoA to the l( -h) hydroxy-butyryl CoA (racemase activity) and it catalyzes the transfer of crotonyl-S-pantetheine to CoA to yield crotonyl CoA (transcrotonylase activity). This association of several catalytic properties with a single crystalline protein suggests either that crystallization is no absolute criterion for purity or that pure protein may present several rather different catalytic properties, each of which may be elicited at different active sites. 

As mentioned above, SAMs are organic assemblies formed by the adsorption of molecules from solution or the gas phase onto the surface of solids. If the adsorbed molecules are chiral, the self assembled monolayer is also rendered chiral. The chirality of the molecule can be distributed within the monolayer interior or located at the terminus of the molecule. However, the chirality is only expressed when the chiral constituent is exposed at the monolayer surface. Chiral SAMs are used in chiral systems. The SAMs can be used to specifically interact with chiral species, such as proteins or amino acids. Chiral SAMs have been used in enantioselective crystallization. In this case, a racemic solution of a chiral molecule is crystallized on a chiral SAM. The chiral SAM serves as a nucleating surface for one of the enantiomers, thereby increasing its crystallization on the SAM. Thus, enantioselective crystallization is achieved. 

These processes resulted in racemic mixtures. However, the resolution of this mixture is believed to have occurred by spontaneous crystallization. This process most likely occurred by chance. Minerals such as natural dissymmetric quartz crystals and metal ions may have played a crucial role of optical selection by selective chelation of only one stereoisomer. After all, stereoselective polymerization of olefins by metal surfaces (Ziegler-Natta catalysts) is a well-documented industrial process for the synthesis of isotactic polymers. We also know the importance of metal ion binding in many biochemical transformations. It is essential for the maintenance of the native structure of nucleic acids and numerous proteins and enzymes. Other physical forces through radioactive elements, 7-radiation, or from cosmic rays, may have also been involved in optical selection. For instance, recent experiments with strontium-90 indicate that D-tyrosine is destroyed more rapidly than the naturally occurring L-isomer. It is tempting to incorporate such factors into the origin of dissymmetry in life process (46). 

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