Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Pyridoxamine driven selective turn-off detection of picric acid using glutathione stabilized fluorescent copper nanoclusters and its applications with chemically modified cellulose strips.

The present work reports the interaction of various vitamin B6 cofactors with the red emitting glutathione stabilized copper nanoclusters (GSH-CuNCs). Addition of pyridoxamine (PM) resulted a new turn-on band at 410nm due to the possible adsorption over the surface of GSH-CuNCs. The nano-assembly PM-GSH-CuNCs was applied for the selective detection of nitro-aromatic compounds. Upon addition of picric acid (PA), the fluorescence of PM-GSH-CuNCs was selectively quenched at 410nm and ~ 625nm among the other tested nitro-aromatic compounds. With a linearity range from 9.9µM to 43µM, the concentration of PA can be detected down to 2.74µM. The high selectivity exhibited by the nano-assembly allows to detect PA in real samples like tap water, river water and matchstick. Advantageously, the nano-assembly PM-GSH-CuNCs was chemically adsorbed over the cellulosic strips and applied for the naked-eye detection of PA down to 1µM.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Scavenging 4-Oxo-2-nonenal.

4-Oxo-2-nonenal (ONE), a product of cellular lipid oxidation, reacts nonspecifically with the lysine residues of proteins and is generated in increased amounts during degenerative diseases and cancer. We show that pyridoxamine, salicylamine, and related 2-aminomethylphenols react with ONE, to form pyrrolo[2,1-b][1,3]oxazines with the participation of both the amino and the phenolic groups. 2-Aminomethylphenols react with ONE as well as with the Michael adducts of ONE much more rapidly than lysine, suggesting their use for therapeutically scavenging ONE.

Investigation of the use of Maillard reaction inhibitors for the production of patatin-carbohydrate conjugates.

Selected Maillard reaction inhibitors, including aminoguanidine, cysteine, pyridoxamine, and sodium bisulfite, were evaluated for their effect on the production of carbohydrate conjugated proteins with less cross-linking/browning. Patatin (PTT), a major potato protein, was glycated with galactose, xylose, galactooligosaccharides, xylooligosaccharides, galactan, and xylan under controlled conditions. The effectiveness of the inhibitors to control the glycation reaction was assessed by monitoring the glycation extent, the protein cross-linking, and the formation of dicarbonyl compounds. Sodium bisulfite was the most effective inhibitor for PTT-galactose and PTT-xylan reaction systems (reaction control ratios of 210.0 and 12.8). On the other hand, aminoguanidine and cysteine led to the highest reaction control ratios for the PTT-xylose/xylooligosaccharide (160.0 and 143.0) and PTT-galactooligosaccharides/galactan (663.0 and 71.0) reaction systems, respectively. The use of cysteine and aminoguanidine as inhibitors led to 1.7-99.4% decreases in the particle size distribution of the PTT conjugates and to 0.4-9.3% increases in their relative digestibility, per 5% blocked lysine.

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.

The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for ß-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate ß-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.

Isolation and structural characterization of acrylamide-pyridoxamine adducts.

Pyridoxamine (PM) is an effective inhibitor of the formation of the carcinogen acrylamide (AA) from its precursors in low-moisture model systems. Although AA is widely assumed to act by scavenging carbonyl compounds, no alternative pathways have to date been explored. In this work, we found AA to directly react with PM in a low-moisture acrylamide-pyridoxamine model system heated at 140 °C for up to 40 min. The reaction products gave four major chromatographic peaks that were assigned to acrylamide-pyridoxamine adducts. Two of the adducts (AA-PM-1 and AA-PM-3) were selected for isolation and structural characterization with various spectroscopic (UV, fluorescence, IR, and NMR) and mass spectrometric techniques (MS, MS/MS). As shown by the proposed reaction scheme, PM can directly react with AA via Michael addition. The reaction involves a nucleophilic attack of the PM amine group on AA (an α,ß-unsaturated carbonyl compound) to give adduct AA-PM-3, which was identified as 3-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)amino)propanamide. However, AA-PM-3 further reacts with any additional AA present in the medium to give adduct AA-PM-1 identified as 3,3'-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)azanediyl)dipropanamide. The time courses of these adduct formation reactions were studied in cookies supplemented with PM, where AA-PM-3 was found to be the predominant structure.

Characterization of scavengers of gamma-ketoaldehydes that do not inhibit prostaglandin biosynthesis.

Expression of cyclooxygenase-2 (COX-2) is associated with the development of many pathologic conditions. The product of COX-2, prostaglandin H(2) (PGH(2)), can spontaneously rearrange to form reactive gamma-ketoaldehydes called levuglandins (LGs). This gamma-ketoaldehyde structure confers a high degree of reactivity on the LGs, which rapidly form covalent adducts with primary amines of protein residues. Formation of LG adducts of proteins has been demonstrated in pathologic conditions (e.g., increased levels in the hippocampus in Alzheimer's disease) and during physiologic function (platelet activation). On the basis of knowledge that lipid modification of proteins is known to cause their translocation and to alter their function, we hypothesize that modification of proteins by LG could have functional consequences. Testing this hypothesis requires an experimental approach that discriminates between the effects of protein modification by LG and the effects of cyclooxygenase-derived prostanoids acting through their G-protein coupled receptors. To achieve this goal, we have synthesized and evaluated a series of scavengers that react with LG with a potency more than 2 orders of magnitude greater than that with the epsilon-amine of lysine. A subset of these scavengers are shown to block the formation of LG adducts of proteins in cells without inhibiting the catalytic activity of the cyclooxygenases. Ten of these selective scavengers did not produce cytotoxicity. These results demonstrate that small molecules can scavenge LGs in cells without interfering with the formation of prostaglandins. They also provide a working hypothesis for the development of pharmacologic agents that could be used in experimental animals in vivo to assess the pathophysiological contribution of levuglandins in diseases associated with cyclooxygenase up-regulation.

Enantioselective reductive amination of alpha-keto acids by papain-based semisynthetic enzyme.

Alkylation of a cysteine residue in papain with a pyridoxamine (PX) cofactor was carried out. The resulting semisynthetic enzyme (papain-PX) has no detectable protease activity but has the ability to catalyze enantioselective reductive amination of alpha-keto acids. The conjugate was characterized by ion-exchange chromatography, and the optimal reaction conditions were found. We report that papain-PX reductively aminates the alkyl side chain of functionalized alpha-keto acids to give the respective alpha-amino acids with high enantioselectivities, greater than 70%. Based on these studies, we propose a new model for the catalytic activity of the semisynthetic enzyme with Interchem software. The results of the study demonstrate the effectiveness of the modified enzyme and its potential for engineering new catalytic specificity.

A comparative study of the chemical reactivity of pyridoxamine, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds.

Pyridoxamine (PM) has long been known to inhibit protein glycation via various mechanisms of action. One such mechanism involves the scavenging of carbonyl compounds with glycating ability. Despite the abundant literature on this topic, few quantitative kinetic studies on the processes involved have been reported. In this work, we conducted a comparative kinetic study under physiological pH and temperature conditions of the reactions of PM, Ac-Phe-Lys and Ac-Cys with various glycating carbonyl compounds (viz. aldehydes, alpha-oxoaldehydes and ketones). The microscopic formation rate constants for Schiff bases of PM and various carbonyl compounds, k(1), are of the same order of magnitude as those for the Schiff bases of Ac-Phe-Lys. However, because PM exhibits a higher proportion of reactive form at physiological pH, its observed second-order rate constant is ca. five times greater than that for Ac-Phe-Lys. That could explain PM ability to compete with amino residues in protein glycation. On the other hand, the observed formation rate constant for thiohemiacetals is four orders of magnitude greater than the formation constants for the Schiff bases of PM, which excludes PM as a competitive inhibitor of Cys residues in protein glycation.

Pyridoxamine analogues scavenge lipid-derived gamma-ketoaldehydes and protect against H2O2-mediated cytotoxicity.

Isoketals and levuglandins are highly reactive gamma-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge alpha-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive gamma-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic gamma-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogues of pyridoxamine, salicylamine and 5'-O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogues, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by alpha-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogues appear to preferentially scavenge gamma-ketoaldehydes. Both pyridoxamine and its lipophilic analogues inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogues provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogues to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.

Chiral polyamines from reduction of polypeptides: asymmetric pyridoxamine-mediated transaminations.

BH3.THF can reduce polypeptides to polyamines with retention of chirality. The resulting polyamines are intriguing general platforms for asymmetric catalysis, given the diverse structures available and their relative ease of synthesis. We have constructed a number of chiral pyridoxamine catalysts based on reduced peptides. These compounds transaminate alpha-ketoacids with moderate to good enantioselectivity, while their peptidyl counterparts show almost no chiral induction.

Conformational changes in the reaction of pyridoxal kinase.

To understand the processes involved in the catalytic mechanism of pyridoxal kinase (PLK),1 we determined the crystal structures of PLK.AMP-PCP-pyridoxamine, PLK.ADP.PLP, and PLK.ADP complexes. Comparisons of these structures have revealed that PLK exhibits different conformations during its catalytic process. After the binding of AMP-PCP (an analogue that replaced ATP) and pyridoxamine to PLK, this enzyme retains a conformation similar to that of the PLK.ATP complex. The distance between the reacting groups of the two substrates is 5.8 A apart, indicating that the position of ATP is not favorable to spontaneous transfer of its phosphate group. However, the structure of PLK.ADP.PLP complex exhibited significant changes in both the conformation of the enzyme and the location of the ligands at the active site. Therefore, it appears that after binding of both substrates, the enzyme-substrate complex requires changes in the protein structure to enable the transfer of the phosphate group from ATP to vitamin B(6). Furthermore, a conformation of the enzyme-substrate complex before the transition state of the enzymatic reaction was also hypothesized.

Crystal structures of human mitochondrial branched chain aminotransferase reaction intermediates: ketimine and pyridoxamine phosphate forms.

The three-dimensional structures of the isoleucine ketimine and the pyridoxamine phosphate forms of human mitochondrial branched chain aminotransferase (hBCATm) have been determined crystallographically at 1.9 A resolution. The hBCATm-catalyzed transamination can be described in molecular terms together with the earlier solved pyridoxal phosphate forms of the enzyme. The active site lysine, Lys202, undergoes large conformational changes, and the pyridine ring of the cofactor tilts by about 18 degrees during catalysis. A major determinant of the enzyme's substrate and stereospecificity for L-branched chain amino acids is a group of hydrophobic residues that form three hydrophobic surfaces and lock the side chain in place. Short-chain aliphatic amino acid side chains are unable to interact through van der Waals contacts with any of the surfaces whereas bulky aromatic side chains would result in significant steric hindrance. As shown by modeling, and in agreement with previous biochemical data, glutamate but not aspartate can form hydrogen bond interactions. The carboxylate group of the bound isoleucine is on the same side as the phosphate group of the cofactor. These active site interactions are largely retained in a model of the human cytosolic branched chain aminotransferase (hBCATc), suggesting that residues in the second tier of interactions are likely to determine the specificity of hBCATc for the drug gabapentin. Finally, the structures reveal a unique role for cysteine residues in the mammalian BCAT. Cys315 and Cys318, which immediately follow a beta-turn (residues 311-314) and are located just outside the active site, form an unusual thiol-thiolate hydrogen bond. This beta-turn positions Thr313 for its interaction with the pyridoxal phosphate oxygens and substrate alpha-carboxylate group.

Specific host-guest interactions in a protein-based artificial transaminase.

Artificial enzymes can be created by covalent attachment of a catalytic active group to a protein scaffold. Recently, we assembled an artificial transaminase by conjugation of intestinal fatty acid binding protein (IFABP) with a pyridoxamine derivative via a disulfide bond; the resulting construct catalyzed a transamination reaction 200-fold faster than free pyridoxamine. To identify the origin of this increased catalytic efficiency computer modeling was first used to identify two putative residues, Y14 and R126, that were in close proximity to the gamma-carboxylate group of the substrate, alpha-ketoglutartate. These positions were mutated to phenylalanine and methionine, respectively, and used to prepare semisynthetic transaminases by conjugation to pyridoxamine (Px) or an N-methylated derivative (MPx). Kinetic analysis of the resulting constructs showed that the R126M mutation reduced substrate affinity 3- to 6-fold while the additional Y14F mutation had a negligible effect. These results are consistent with a model for substrate recognition that involves an electrostatic interaction between the cationic guanidinium group of R126 and the anionic carboxylate from the substrate. Interestingly, one of the conjugates that contains an N-methylated pyridoxamine catalyzes a transamination reaction with a k(cat)' value of 1.1h(-1) which is the fastest value for k(cat) we have thus far obtained and is 34-fold greater than that for the free cofactor in the absence of the protein.

Enzymes by design: chemogenetic assembly of transamination active sites containing lysine residues for covalent catalysis.

Artificial enzymes can be created by covalent conjugation of a catalytic active group to a protein scaffold. Here, two transamination catalysts were designed via computer modeling and assembled by chemically conjugating a pyridoxamine moiety within the large cavity of intestinal fatty acid binding protein. Each catalyst included a lysine residue, introduced via site-directed mutagenesis, that promotes catalysis by covalent interactions with the pyridoxamine group. Evidence for such interactions include the formation of a Schiff base with the pyridoxal form of the catalyst and a rate versus pH dependence that is bell shaped; both of these features are manifested in natural transaminases. The resulting constructs operate with high enantioselectivity (83-94% ee) and increase the rate of reaction as much as 4200-fold over the rate in the absence of the protein; this is a modest (12-fold) increase in catalytic efficiency (kcat/KM) compared to the conjugate lacking the lysine residue. Most importantly, these artificial aminotransferases are the first examples of designed bioconjugates capable of covalent catalysis, highlighting the potential of this chemogenetic approach.

PLP and PMP radicals: a new paradigm in coenzyme B6 chemistry.

Enzymes frequently rely on a broad repertoire of cofactors to perform chemically challenging transformations. The B6 coenzymes, composed of pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP), are used by many transaminases, racemases, decarboxylases, and enzymes catalyzing alpha,beta and beta,gamma-eliminations. Despite the variety of reactions catalyzed by B6-dependent enzymes, the mechanism of almost all such enzymes is based on their ability to stabilize high-energy anionic intermediates in their reaction pathways by the pyridinium moiety of PLP/PMP. However, there are two notable exceptions to this model, which are discussed in this article. The first enzyme, lysine 2,3-aminomutase, is a PLP-dependent enzyme that catalyzes the interconversion of L-lysine to L-beta-lysine using a one-electron-based mechanism utilizing a [4Fe-4S] cluster and S-adenosylmethionine. The second enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase, is a PMP-dependent enzyme involved in the formation of 3,6-dideoxysugars in bacteria. This enzyme also contains an iron-sulfur cluster and uses a one-electron based mechanism to catalyze removal of a C-3 hydroxy group from a 4-hexulose. In both cases, the participation of free radicals in the reaction pathway has been established, placing these two B6-dependent enzymes in an exclusive class by themselves.

Synthesis of a cationic pyridoxamine conjugation reagent and application to the mechanistic analysis of an artificial transaminase.

An N-methylated, cationic pyridoxamine conjugation reagent was synthesized and tethered via a disulfide bond to a cysteine residue inside the cavity of intestinal fatty acid binding protein. The conjugate was characterized and the kinetic parameters compared to its nonmethylated pyridoxamine analogue. Kinetic isotope effects were used for further mechanistic analysis. Taken together, these experiments suggest that a step distinct from deprotonation of the ketimine in the pyridoxamine to pyridoxal reaction is what limits the rate of the artificial transaminase IFABP-Px. However, the internal energetics of reactions catalyzed by the conjugate containing the N-methylated cofactor appear to be different suggesting that the MPx reagent will be useful in future experiments designed to alter the catalytic properties of semisynthetic transaminases.

Modulating pyridoxamine-mediated transamination through a betabeta alpha motif peptide scaffold.

A pyridoxamine coenzyme amino acid chimera (Pam) was incorporated into a designed betabeta alpha motif peptide to explore the ability of a small synthetic peptide scaffold to influence coenzyme mediated transamination. Structural characterization of this peptide by CD and NMR spectroscopy suggested that the pyridoxamine containing residue was accommodated into the sheet region of the motif without gross structural perturbations. To investigate the ability of the peptide architecture to influence the amount and distribution of transamination product in the conversion of pyruvic acid to alanine, a family of 18 related peptides, CBP01-CBP18, was rapidly synthesized and purified in parallel. These peptides were designed to generate different peptide environments for the pyridoxamine functionality within the context of the structured betabeta alpha peptide motif. Studies of peptide-mediated transamination revealed clear trends in stereospecific production of L-alanine as a function of substitutions at positions five and seven of the motif. Furthermore, new trends favoring the other enantiomeric product resulted from the addition of copper(II) ion, a known chelator of the transamination reaction intermediates. In the presence of copper(II) ion the amount of alanine product generated was increased by up to 32-fold relative to a pyridoxamine model compound in the presence of copper(II) ion. These functional results, accompanied by further CD and NMR spectroscopic analysis of CBP14, one of the CBP family of peptides, suggest that small synthetic betabeta alpha motif peptides can be used to influence the functional properties of coenzymes.

Structural characterization of two synthetic catalysts based on adipocyte lipid-binding protein.

Adipocyte lipid-binding protein (ALBP) is a small (14.5 kDa) 10-stranded beta-barrel protein found in mammalian fat cells. The crystal structures of various holo-forms of ALBP have been solved and show the fatty acid ligand bound in a large (approximately 400 A3) cavity isolated from bulk solvent. Examination of the cavity suggests that it would be a good site for the creation of an artificial catalyst, as numerous well defined crystal structures of ALBP are available and past studies have shown the conformation to be reasonably tolerant to modification and mutagenesis. Previous work has shown ALBP to be a good protein scaffold for exploring enantio- and stereoselective reactions; two constructs, ALBP attached to either a pyridoxamine or a phenanthroline group at C117, have been chemically characterized. Both modified proteins have been crystallized and their structures solved and refined. The X-ray models have been used to examine the origin of the chiral selectivity seen in the products. It is apparent that these covalent adducts reduce the internal cavity volume, sterically limiting substrate interactions with the reactive groups, as well as solvent access to potential intermediates in the reaction pathway.