Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623.

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.

Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug.

OBJECTIVES: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. RESULTS: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug. CONCLUSIONS: The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.

Aziridine Formation by a FeII /α-Ketoglutarate Dependent Oxygenase and 2-Aminoisobutyrate Biosynthesis in Fungi.

Aziridine is a characteristically reactive molecule with increased bioactivity due to its strained ring structure. Here, we investigated the biosynthesis of 2-aminoisobutyric acid (AIB) in Penicillium, and successfully reconstituted the three-step biosynthesis from L-Val to AIB in vitro. This previously unknown aziridine formation pathway proceeded with the non-heme iron and α-ketoglutarate-dependent (FeII /αKG) oxygenase TqaL, followed by aziridine ring opening by the haloalkanoic acid dehalogenase (HAD)-type hydrolase TqaF, and subsequent oxidative decarboxylation by the NovR/CloR-like non-heme iron oxygenase TqaM. Furthermore, the X-ray crystal structure of the C-N bond forming FeII /αKG oxygenase TqaL was solved at 2.0 Šresolution. This work presents the first molecular basis for aziridine biogenesis, thereby expanding the catalytic repertoire of the FeII /αKG oxygenases. We also report the unique aziridine ring opening by a HAD-type hydrolase and the remarkable oxidative decarboxylation by a non-heme iron oxygenase to produce AIB.

Hypoxia-Induced Pro-Protein Therapy Assisted by a Self-Catalyzed Nanozymogen.

The success of intracellular protein therapy demands efficient delivery and selective protein activity in diseased cells. Therefore, a cascaded nanozymogen consisting of a hypoxia-activatable pro-protein, a hypoxia-inducing protein, and a hypoxia-strengthened intracellular protein delivery nanovehicle was developed. RPAB, an enzymatically inactive pro-protein of RNase, reversibly caged with hypoxia-cleavable azobenzene, was delivered with glucose oxidase (GOx) using hypoxia-responsive nanocomplexes (NCs) consisting of azobenzene-cross-linked oligoethylenimine (AOEI) and hyaluronic acid (HA). Upon NC-mediated delivery into cancer cells, GOx catalyzed glucose decomposition and aggravated tumoral hypoxia, which drove the recovery of RPAB back to the hydrolytically active RNase and expedited the degradation of AOEI to release more protein cargoes. Thus, the catalytic reaction of the nanozymogen was self-accelerated and self-cycled, ultimately leading to a cooperative anti-cancer effect between GOx-mediated starvation therapy and RNase-mediated pro-apoptotic therapy.

Distinct activation mechanisms trigger the trypanocidal activity of DNA damaging prodrugs.

Quinone-based compounds have been exploited to treat infectious diseases and cancer, with such chemicals often functioning as inhibitors of key metabolic pathways or as prodrugs. Here, we screened an aziridinyl 1,4-benzoquinone (ABQ) library against the causative agents of trypanosomiasis, and cutaneous leishmaniasis, identifying several potent structures that exhibited EC50 values of <100 nM. However, these compounds also displayed significant toxicity towards mammalian cells indicating that they are not suitable therapies for systemic infections. Using anti-T. brucei ABQs as chemical probes, we demonstrated that these exhibit different trypanocidal modes of action. Many functioned as type I nitroreductase (TbNTR) or cytochrome P450 reductase (TbCPR) dependent prodrugs that, following activation, generate metabolites which promote DNA damage, specifically interstrand crosslinks (ICLs). Trypanosomes lacking TbSNM1, a nuclease that specifically repairs ICLs, are hypersensitive to most ABQ prodrugs, a phenotype exacerbated in cells also engineered to express elevated levels of TbNTR or TbCPR. In contrast, ABQs that contain substituent groups on the biologically active aziridine do not function as TbNTR or TbCPR-activated prodrugs and do not promote DNA damage. By unravelling how ABQs mediate their activities, features that facilitate the desired anti-parasitic growth inhibitory effects could be incorporated into new, safer compounds targeting these neglected tropical diseases.

Study of Bioreductive Anticancer Agent RH-1-Induced Signals Leading the Wild-Type p53-Bearing Lung Cancer A549 Cells to Apoptosis.

Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-cyclohexa-2,5-diene-1,4-dione) is a potential anticancer agent. RH-1 action is associated with NAD(P)H: quinone oxidoreductase (NQO1) which reduces this diaziridinylbenzoquinone into DNA-alkylating hydroquinone and is overexpressed in many tumors. Another suggested mechanism of RH-1 toxicity is the formation of reactive oxygen species (ROS) arising from its redox cycling. In order to improve anticancer action of this and similar antitumor quinones, we investigated the involvement of different signaling molecules in cytotoxicity induced by RH-1 by using wild-type tumor suppressor p53 bearing nonsmall cell lung carcinoma A549 cells as a model. Gradual and prolonged increase of mitogen-activated protein kinases (MAPK) ERK, P38, and JNK phosphorylation was observed during 24-h RH-1 treatment. In parallel, activation of DNA damage-sensing ATM kinase, upregulation, and phosphorylation of TP53 (human p53) took place. Inhibition studies revealed that RH-1-induced A549 apoptosis involved the NQO1-ATM-p53 signaling pathway and ROS generation. TP53 participated in ROS- and DNA damage-induced cell death differently. Moreover, MAP kinase JNK was another TP53 activator and death inducer in A549 cells. At the same time, rapid and prolonged activation of AKT kinase during RH-1 treatment was found, and it proved to be antiapoptotic kinase in our model system. Therefore, we identified that different and opposite cell death regulating signaling pathways, which may counteract one another, are induced in cancer cells during chemotherapeutic RH-1 treatment.

Broad-range glycosidase activity profiling.

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.

A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining ß-glucosidases.

Retaining ß-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining ß-glucosidases. Whereas ß-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining ß-glucosidases, ß-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining ß-glucosidases by the combined use of cyclophellitol ß-epoxide- and ß-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent ß-aziridine but not ß-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the ß-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining ß-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining ß-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.

Synthesis of 2-aryl-3-(2-cyanoethyl)aziridines and their chemical and enzymatic hydrolysis towards γ-lactams and γ-lactones.

Trans- and cis-2-aryl-3-(2-cyanoethyl)aziridines, prepared via alkylation of the corresponding 2-aryl-3-(tosyloxymethyl)aziridines with the sodium salt of trimethylsilylacetonitrile, were transformed into variable mixtures of 4-[aryl(alkylamino)methyl]butyrolactones and 5-[aryl(hydroxy)methyl]pyrrolidin-2-ones via KOH-mediated hydrolysis of the cyano group, followed by ring expansion. In addition, next to this chemical approach, enzymatic hydrolysis of the former aziridinyl nitriles by means of a nitrilase was performed as well, interestingly providing a selective route towards the above-mentioned functionalized γ-lactams.

Modulation of polyplex release from biodegradable microparticles through poly(ethylenimine) modification and varying loading concentration.

PURPOSE: This work investigates the effects of hyaluronic acid (HA) conjugated onto branched poly(ethylenimine) (bPEI) and varying loading concentrations of these polymers complexed with DNA on their release from poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and the transfection of target cells. METHODS: To examine the effect of alteration of the gene delivery polymer on the system, we observed the morphology, size, loading efficiency, polymer and DNA release, and the transfection efficiency for the microparticles formed with three internal phase loading concentrations during microparticle formation. RESULTS: Addition of HA to this vector allowed for increased loading concentration within these systems and significantly altered release kinetics without changing the morphology of the particles. The incorporation of HA onto the bPEI backbone significantly increased the transfection efficiency of the complexes released from the corresponding microparticle formulation. CONCLUSIONS: The results show that the modification of bPEI with HA and the concentration of loaded polymer/DNA complexes can significantly alter the entrapment and release profiles from PLGA microparticles. This is significant in that it offers insight into the effects of modification of gene delivery vectors on a controlled release system designed to achieve a sustained therapeutic response.

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the ß3 subunit of the GABA(A) receptor.

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5ß)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-ß3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-ß3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 µM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-ß3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the ß3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3ß,5ß)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-ß3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole ß3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

Noninvasive optical imaging of nitroreductase gene-directed enzyme prodrug therapy system in living animals.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising and emerging strategy that attempts to limit the systemic toxicity inherent to cancer chemotherapy by means of tumor-targeted delivery and expression of an exogenous gene whose product converts nontoxic prodrug(s) into activated cytotoxic agent(s). The bacterial nitroreductase (NTR) enzyme, coupled with its substrate prodrug 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), is a promising GDEPT strategy that has reached clinical trials. However, no strategy exists to visually monitor and quantitatively evaluate the therapeutic efficacy of NTR/CB1954 prodrug therapy in cells and imaging in living animals. As the success of any GDEPT is dependent upon the efficiency of transgene expression in vivo, we developed a safe, sensitive and reproducible noninvasive imaging method to monitor NTR transgene expression that would allow quantitative assessment of both therapeutic efficacy and diagnostic outcome of NTR/CB1954 prodrug therapy in the future. Here, we investigate the use of a novel fluorescent imaging dye CytoCy5S (a Cy5-labeled quenched substrate of NTR enzyme) on various cancer cell lines in vitro and in NTR-transfected tumor-bearing animals in vivo. CytoCy5S-labeled cells become fluorescent at 'red-shifted' wavelengths (638 nm) when reduced by cellular NTR enzyme and remains trapped within the cells for extended periods of time. The conversion and entrapment was dynamically recorded using a time-lapsed microscopy. Systemic and intratumoral delivery of CytoCy5S to NTR-expressing tumors in animals indicated steady and reproducible signals even 16 h after delivery (P<0.001). This is the first study to address visual monitoring and quantitative evaluation of NTR activity in small animals using CytoCy5S, and establishes the capability of NTR to function as an imageable reporter gene.

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis.

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of ß-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-ß-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z = 1287.77), a ß-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and ß-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.

An unusually cold active nitroreductase for prodrug activations.

A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.

Counteracting vasopressin-mediated water reabsorption by ATP, dopamine, and phorbol esters: mechanisms of action.

Water homeostasis is regulated by a wide variety of hormones. When in need for water conservation, vasopressin, released from the brain, binds renal principal cells and initiates a signaling cascade resulting in the insertion of aquaporin-2 (AQP2) water channels in the apical membrane and water reabsorption. Conversely, hormones, including extracellular purines and dopamine, antagonize AVP-induced water permeability, but their mechanism of action is largely unknown, which was investigated here. Addition of these hormones to mpkCCD cells decreased total and plasma membrane abundance of AVP-induced AQP2, partly by increasing its internalization to vesicles and lysosomal degradation. This internalization was ubiquitin dependent, because the hormones increased AQP2 ubiquitination, and the plasma membrane localization of AQP2-K270R, which cannot be monoubiquitinated, was unaffected by these hormones. Both hormones also increased AQP2 phosphorylation at S261, which followed ubiquitination, but was not essential for hormone-induced AQP2 degradation. A similar process occurs in vivo, as incubation of dDAVP-treated kidney slices with both hormones also resulted in the internalization and S261 phosphorylation of AQP2. Both hormones also reduced cAMP and AQP2 mRNA levels, suggesting an additional effect on AQP2 gene transcription. Interestingly, phorbol esters only reduced AQP2 through the first pathway. Together, our results indicate that ATP and dopamine counteract AVP-induced water permeability by increasing AQP2 degradation in lysosomes, preceded by ubiquitin-dependent internalization, and by decreasing AQP2 gene transcription by reducing the AVP-induced cAMP levels.

Site-specific introduction of an acetyl-lysine mimic into peptides and proteins by cysteine alkylation.

Protein acetylation on Lys residues is recognized as a significant post-translational modification in cells, but it is often difficult to discern the direct structural and functional effects of individual acetylation events. Here we describe a new tool, methylthiocarbonyl-aziridine, to install acetyl-Lys mimics site-specifically into peptides and proteins by alkylation of Cys residues. We demonstrate that the resultant thiocarbamate modification can be recognized by the Brdt bromodomain and site-specific antiacetyl-Lys antibodies, is resistant to histone deacetylase cleavage, and can confer activation of the histone acetyltransferase Rtt109 by simulating autoacetylation. We also use this approach to obtain functional evidence that acetylation of CK2 protein kinase on Lys102 can stimulate its catalytic activity.

Efficient gene transfection using novel cationic polymers poly(hydroxyalkylene imines).

A series of novel cationic polymers poly(hydroxyalkylene imines) were synthesized and tested for their ability to transfect cells in vitro and in vivo. Poly(hydroxyalkylene imines), in particular, poly(2-hydroxypropylene imine) (pHP), poly(2-hydroxypropylene imine ethylene imine) (pHPE), and poly(hydroxypropylene imine propylene imine) (pHPP) were synthesized by polycondensation reaction from 1,3-diamino-2-propanol and the appropriate dibromide. Electron microscopic examination demonstrated that the resulting polymers condensed DNA into toroid shape complexes of 100-150 nm in size. Transfection studies showed that all three polymers were able to deliver genetic material into the cell, with pHP being superior to pHPP and pHPE. pHP acted as an efficient gene delivery agent in a variety of different cell lines and outcompeted most of the widely used polymer or lipid based transfection reagents. Intravenous administration of pHP-DNA polyplexes in mice followed by the reporter gene analysis showed that the reagent was suitable for in vivo applications. In summary, the results indicate that pHP is a new efficient reagent for gene delivery in vitro and in vivo.

Insight into enzyme-catalyzed aziridine formation mechanism in ficellomycin biosynthesis.

Ficellomycin is an aziridine-containing antibiotic, produced by Streptomyces ficellus. Based on the newly identified ficellomycin gene cluster and the assigned functions of its genes, a possible pathway for aziridine ring formation in ficellomycin was proposed, which is a complex process involving at least 3 enzymatic steps. To obtain support for the proposed mechanism, the targeted genes encoding sulfate adenylyltransferase, adenylsulfate kinase, and a putative sulfotransferase were respectively disrupted and the subsequent analysis of their fermentation products revealed that all the three genes were involved in aziridine formation. To further confirm the mechanism, the key gene encoding a putative sulfotransferase was over expressed in Escherichia coli Rosseta (DE3). Enzyme assays indicated that the expressed sulfotransferase could specifically transfer a sulfo group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto the hydroxyl group of (R)-(-)-2-pyrrolidinemethanol. This introduces a good leaving group in the form of the sulfated hydroxyl moiety, which is then converted into an aziridine ring through an intramolecular nucleophilic attack by the adjacent secondary amine. The sulfation/intramolecular cyclization reaction sequence maybe a general strategy for aziridine biosynthesis in microorganisms. Discovery of this mechanism revealed an enzyme-catalyzed route for the synthesis of aziridine-containing reagents and provided an important insight into the functional diversity of sulfotransferases.

Steady-state and stopped-flow kinetic studies of three Escherichia coli NfsB mutants with enhanced activity for the prodrug CB1954.

The enzyme nitroreductase, NfsB, from Escherichia coli has entered clinical trials for cancer gene therapy with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, CB1954 is a poor substrate for the enzyme. Previously we made several NfsB mutants that show better activity with CB1954 in a cell-killing assay in E. coli. Here we compare the kinetic parameters of wild-type NfsB with CB1954 to those of the most active single, double, and triple mutants isolated to date. For wild-type NfsB the global kinetic parameters for both k(cat) and K(m) for CB1954 are about 20-fold higher than previously estimated; however, the measured specificity constant, k(cat)/K(m) is the same. All of the mutants are more active with CB1954 than the wild-type enzyme, the most active mutant showing about 100-fold improved specificity constant with CB1954 over the wild-type protein with little effect on k(cat). This enhancement in specificity constants for the mutants is not seen with the antibiotic nitrofurazone as substrate, leading to reversed nitroaromatic substrate selectivity for the double and triple mutants. However, similar enhancements in specificity constants are found with the quinone menadione. Stopped-flow kinetic studies suggest that the rate-determining step of the reaction is likely to be the release of products. The most active mutant is also selective for the 4-nitro group of CB1954, rather than the 2-nitro group, giving the more cytotoxic reduction product. The double and triple mutants should be much more effective enzymes for use with CB1954 in prodrug-activation gene therapy.

Molecular basis for the stereoselective ammoniolysis of N-alkyl aziridine-2-carboxylates catalyzed by Candida antarctica lipase B.

Candida antarctica lipase B catalyzed the stereoselective ammoniolysis of N-alkyl aziridine-2-carboxylates in tBuOH saturated with ammonia and yielded the (2S)-aziridine-2-carboxamide and unreacted (2R)-aziridine-2-carboxylate. Varying the N-1 substituent on the aziridine ring changed the rate and stereoselectivity of the reaction. Substrates with a benzyl substituent or a (1'R)-1-phenylethyl substituent reacted approximately ten times faster than substrates with a (1'S)-1-phenylethyl substituent. Substrates with a benzyl substituent showed little stereoselectivity (E=5-7) while substrates with either a (1'R)- or (1'S)-1-phenylethyl substituent showed high stereoselectivity (D>50). Molecular modeling by using the current paradigm for enantioselectivity-binding of the slow enantiomer by an exchange-of-substituents orientation-could not account for the experimental results. However, modeling an umbrella-like-inversion orientation for the slow enantiomer could account for the experimental results. Steric hindrance between the methyl in the (1'S)-1-phenylethyl substituent and Thr138 and Ile189 in the acyl-binding site likely accounts for the slow reaction. Enantioselectivity likely stems from an unfavorable interaction of the methine hydrogen with Thr40 for the slow enantiomer and from subtle differences in the orientations of the other three substituents. This success in rationalizing the enantioselectivity supports the notion that an umbrella-like-inversion orientation can contribute to enantioselectivity in lipases.