P-nitrobenzyl Ester Research Articles

Radiosynthesis of [18F]brequinar for in vivo PET imaging of hDHODH for potential studies of acute myeloid leukemia and cancers.

Dihydroorotate dehydrogenase (DHODH), an enzyme that plays a critical role in the de novo pyrimidine biosynthesis, has been recognized as a promising target for the treatment of diseases that involve cellular proliferation, such as autoimmune diseases and cancers. Pharmacological inhibition of human DHODH (hDHODH) that offers a potential therapeutic strategy for the treatment in adult subjects with acute myeloid leukemia (AML) has recently been supported by phase I/II clinical trials for the treatment of patients with relapsed/refractory AML. To facilitate the development of optimized hDHODH inhibitors, the presence of an in vivo imaging probe that is able to demonstrate in vivo target engagement is critical and desirable. Brequinar is one of the most potent hDHODH inhibitors so far discovered. In this work, we use a copper-mediated radiofluorination (CMRF) strategy and compare the chemical design and radiosynthesis starting from either pinacole boronate p-nitrobenzyl ester (4) or tributylstannate (tin) p-nitrobenzyl ester (5), chosen for their suitability as a precursor to [18F]brequinar. We report here the design, synthesis, radiolabeling and characterization of [18F]brequinar, and a preliminary PET imaging study of DHODH in vivo. This study provides the strategies to create [18F]brequinar, the first hDHODH inhibitor PET radiotracer, which will facilitate its use as a tool (theranostics) for hDHODH drug development and for diagnosis and monitoring therapeutic efficacy in AML and cancers.

Synthesis and In Vitro Antibacterial Activity of New C-3-Modified Carbapenems

New C-3 modified carbapenems have been synthesized by the AdNE-substitution of the enol phosphate group of 4-nitrobenzyl (4R)-3-[(diphenylphosphoryl)oxy]-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-en-2-carboxylate by the corresponding thiols in acetonitrile in the presence of diisopropylethylamine (DIPEA). Mercaptoacetic acid methyl ester, furan-1-methylthiol, methyl N-(mercaptoacetyl)methioninate, and 2-(4-methylpiperazin-1-yl)-2-oxoethanethiol as thiols have been used. As a result, we have obtained the expected 4-nitrobenzyl esters of 3-[(2-methoxy-2-oxoethyl)thio]-, 3-[(2-furylmethyl)thio], 3-{[2-(4-methylpiperazin-1-yl)-2-oxoethyl]thio}, and 3-[((2-(S)-1-methoxy-4-methylthio-1-oxopropan-2-yl)amino)-2-oxoethyl]thio derivatives of (4R,5S,6S)-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid in yields of 67–87% after purification by column chromatography. The p-nitrobenzyl (pNb) esters of carbapenems have been converted to the corresponding acids by hydrogenolysis over 10% Pd/C in methanol. The antibacterial activity of the resulting carbapenems and their preceding pNb-esters has been studied against microorganisms of Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Streptococcus oralis. The compounds that have been found exceed in their activity the known drugs Meropenem and Cilapenem.

Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (AsnH33, SerH95, and ArgL96) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (AsnH33Ala, SerH95Ala, and ArgL96Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry.

Meso-ester BODIPYs for the imaging of hypoxia in tumor cells

We report the p-nitrobenzyl ester of a meso-carboxyl BODIPY as a new fluorogenic probe for the monitoring of nitroreductase activity. It showed remarkable fluorescence enhancement (∼220-fold), high sensitivity (detection limit of 0.9ng/mL), and high selectivity for nitroreductase over other bio-related species. In vitro cell studies confirmed its utility for the rapid fluorescence imaging of hypoxic cancer cells.

ChemInform Abstract: Photochemical Dissociation of p-Nitrobenzyl Sulfonate Esters via an Intramolecular Electron Transfer.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Structural and spectroscopic studies of new o-, m- and p-nitrobenzyl esters of lasalocid acid

New o-, m- and p-nitrobenzyl esters of lasalocid ( o-ELAS, m-ELAS and p-ELAS) have been synthesised and studied by X-ray, 1H, 13C NMR, FT-IR, UV–Vis, fluorescence as well as PM5 semiempirical methods. The crystal structure of o-ELAS is stabilized by three intramolecular hydrogen bonds in which the ketone is not engaged. The FT-IR spectra of o-ELAS, m-ELAS and p-ELAS in the chloroform and acetonitrile solutions indicate that in these solvents other structures are realized in which the ketone group is hydrogen bonded. In the solution the hydrogen bonds in which the carbonyl ester groups are involved becomes slightly weaker and partially dissociated. The dissociation of these hydrogen bonds increases with increasing solvent polarity. The structures of the o-, m- and p-nitrobenzyl esters of lasalocid acid are discussed in detail.

Simultaneous Hydrogenolysis of p-Nitrobenzyl Esters and Carbamates Side-Chains in the THF 1β-Carbapenem OCA-983 in Biphasic Media

Deprotection of p-nitrobenzyl esters and valyl carbamates in carbapenem CL 192,276 produced the active compound OCA-983 in excellent yields. Straight chain alkanols such as 1-butanol, 1-pentanol and 1-hexanol in water at certain ratios were effective solvent systems. Alkyl acetates in water also resulted in simultaneous deprotection of PNB and PNZ side-chains albeit at slower rates. The deprotected carbapenem was isolated in excellent yield and purity after removal of the aqueous media. This procedure is applicable to sensitive compounds that are soluble in water without the need to use a buffer and allows for ease of isolation from the aqueous phase.

Semipermanent p-nitrobenzyloxycarbonyl ( pNZ) protection of Orn and Lys side chains: prevention of undesired α-Fmoc removal and application to the synthesis of cyclic peptides

Semipermanent side-chain protection of Orn and Lys with p-nitrobenzyloxycarbonyl ( pNZ) for Fmoc/ t Bu chemistry does not result in the unwanted removal of α-Fmoc that occurs when groups such as Alloc are used for the same application. Furthermore, pNZ can be used in conjuction with p-nitrobenzyl ester ( pNB) to prepare cyclic peptides.

Practical Synthesis of the New Carbapenem Antibiotic Ertapenem Sodium

[reaction: see text] A practical synthesis for the large-scale production of the new carbapenem antibiotic, [4R,5S,6S]-3-[[(3S,5S)-5-[[(3-Carboxyphenyl)amino]carbonyl]-3-pyrrolidinyl]thio]-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid monosodium salt (ertapenem sodium, 1), has been developed. The synthesis features the novel use of 1,1,3,3-tetramethylguanidine as base for the low-temperature reaction of a thiol, derived from trans-4-hydroxy-L-proline, with the carbapenem nucleus activated as the enol phosphate. Hydrogenolysis of a p-nitrobenzyl ester is effected using a palladium on carbon catalyst to give an overall yield for the two steps of 90%. The use of bicarbonate in the hydrogenolysis was key in providing protection of the pyrrolidine amine as the sodium carbamate improving both the performance of the reaction and the stability of the product. This discovery made processing at manufacturing scale possible. Experimental evidence for the formation of the sodium carbamate is provided. A remarkably expedient process for the simultaneous purification and concentration of the aqueous product stream relies on ion-pairing extraction for the removal of the water-soluble 1,1,3,3-tetramethylguanidine. Crystallization then affords 59-64% overall yield of the monosodium salt form of the product.

Polymer-supported O-alkylisoureas: useful reagents for the O-alkylation of carboxylic acids.

Polymer-supported O-alkylisoureas were prepared by reaction of an alcohol with a polymer-supported carbodiimide under copper(II) catalysis. These reagents were used to transform carboxylic acids into the corresponding methyl, benzyl, allyl, and p-nitrobenzyl esters in a highly chemoselective manner in high yields and in very high purity after simple resin filtration and solvent evaporation. The reactions could be carried out using both conventional or microwave heating, with reaction times as short as 3-5 min in the latter case, without compromising yield, purity, or chemoselectivity. Unfortunately, the corresponding solid-supported tert-butyl isoureas could not be prepared.

HPLC/UV determination of organic acids in fruit juices and nectars

A reversed phase HPLC method for separation and determination of organic acids in fruit juices and nectars is presented. The method is based on the reaction of free organic acids with O-(4 nitrobenzyl)-N,N′-diisopropylisourea (PNBDI) in presence of dioxane. Excess of reagent was removed with a strong cation-exchange resin. The p-nitrobenzyl esters were separated on a C18 reversed phase column using gradient elution with water and acetonitrile and UV detection at 265 nm. Benzylmalonic acid was used as internal standard. The determinations were performed in the linear range of 0.05–2 g/l for lactic, acetic and succinic acids, 0.1–12 g/l for tartaric, malic and citric acids. The detection limits were 0.025 g/l, 0.017 g/l, 0.050 g/l, 0.039 g/l, 0.025 g/l and 0.060 g/l, respectively for lactic, acetic, tartaric, malic, succinic and citric acids. Validation of the method was carried out by the standard additions method, the recoveries ranged from 91.4 to 103.0%. The precision of the method was also evaluated and reported a CV% as less than 3.1%. Organic acid profiles of citrus fruit, pineapple and apple natural juices (home-made), commercial juices and nectars were established.

Antibody-catalyzed removal of the p-nitrobenzyl ester protecting group: the molecular basis of broad substrate specificity.

Antibody catalysts for the removal of the p-nitrobenzyl ester protecting group have been generated to accommodate a broad range of substrates. Antibody 7B9, which was elicited against p-nitrobenzyl phosphonate 1, catalyzed the hydrolyses of p-nitrobenzyl monoesters of nonsubstituted, and beta- and gamma-substituted glutaric acids with almost identical Km and kcat values. In addition, 7B9 displayed substrate tolerance towards the a-substituents and accepted the p-nitrobenzyl esters of Leu, Norleu, and Phe. To define the molecular basis of the broad substrate tolerance, we have cloned and sequenced the antibody and constructed a model of the active-site-hapten complex. The model showed a relatively shallow pocket of the antigen-combining site that accommodates the p-nitrobenzyl moiety, and this is consistent with the observed substrate specificity. Thus, in the antibody-catalyzed reaction, the alpha-, beta-, and gamma-substituents of the substrates should be outside the combining site and ignored by the antibody recognition. A structural comparison of 7B9 with antibody D2.3, elicited against the structurally similar haptenic phosphonate, suggests the significance of the linker moiety in hapten design, which endows antibody catalysts with broad substrate specificity. These investigations provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates for practical applications in organic synthetic chemistry.

Antibody-Catalyzed Removal of the p-Nitrobenzyl Ester Protecting Group: The Molecular Basis of Broad Substrate Specificity

Antibody catalysts for the removal of the p-nitrobenzyl ester protecting group have been generated to accommodate a broad range of substrates. Antibody 7B9, which was elicited against p-nitrobenzyl phosphonate 1, catalyzed the hydrolyses of p-nitrobenzyl monoesters of nonsubstituted, and β- and γ-substituted glutaric acids with almost identical Km and kcat values. In addition, 7B9 displayed substrate tolerance towards the α-substituents and accepted the p-nitrobenzyl esters of Leu, Norleu, and Phe. To define the molecular basis of the broad substrate tolerance, we have cloned and sequenced the antibody and constructed a model of the active-site-hapten complex. The model showed a relatively shallow pocket of the antigen-combining site that accommodates the p-nitrobenzyl moiety, and this is consistent with the observed substrate specificity. Thus, in the antibody-catalyzed reaction, the α-, β-, and γ-substituents of the substrates should be outside the combining site and ignored by the antibody recognition. A structural comparison of 7B9 with antibody D2.3, elicited against the structurally similar haptenic phosphonate, suggests the significance of the linker moiety in hapten design, which endows antibody catalysts with broad substrate specificity. These investigations provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates for practical applications in organic synthetic chemistry.

Synthesis, reactivity and biochemical evaluation of 1,3-substituted azetidin-2-ones as enzyme inhibitors

A series of monocyclic azetidinones were prepared, bearing, at position C-3, an acetylamino or a bromo substituent, at position N-1, a carboxymethyl group protected as p-nitrobenzyl ester (PNB) and α-functionalized with a potential leaving group (LG). These structures were designed as potential suicide-inhibitors of enzymes containing a serine nucleophile in their active site. The β-lactam ring of these molecules was found to be stable in phosphate buffer (pH 7.5), but the PNB ester was rapidly cleaved. This constitutes a practical method of in situ deprotection. Depending on the nature of the LG group on the carboxymethyl chain, substitution of this group (LG = F) or decarboxylation (LG = SO 2Ph) was observed under hydrolytic conditions. The 1,3-disubstituted azetidinones were inactive against β-lactamases of classes A, B, C, and D. Three compounds behaved as weak reversible inhibitors of porcine pancreatic elastase (PPE).

Preparation of crystalline p-nitrobenzyl 2-hydroxymethyl carbapenem as a key intermediate for the anti-MRS carbapenem L-786,392

Crystalline p-nitrobenzyl esters of 2-hydroxymethyl carbapenem derivatives were prepared in one pot from the corresponding diazo compounds and Bu 3SnCH 2OH. The TES protected 2-hydroxymethyl carbapenem was successfully converted to an anti-MRS carbapenem L-786,392.

Empirical equation for the accurate prediction of retention in planar chromatography

Either a second order or a third polynomial equation accurately predicts R F in a mobile phase which is a binary mixture of a strong and weak solvent. The mole fraction of the strong solvent is used as the independent variable. The p-nitrobenzyl esters of fifteen dansyl amino acids were used as model solutes for planar chromatography, using each of five different mobile phases on silica gel layers. For either of the polynomial equations, a minimum of 98% of the predicted R F values, at mole fractions not used for establishing the equations, are within 0.05 or less of an R F unit (when compared to the experimental R F ), and 90% of the predictions are within 0.03 or less of an R F unit.

Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents.

Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibiotic p-nitrobenzyl ester in aqueous-organic solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleus p-nitrobenzyl ester was not feasible, the p-nitrophenyl ester was employed. Catalytic performance on the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. One p-nitrobenzyl esterase variant performs as well in 30% dimethylformamide as the wildtype enzyme in water, reflecting a 16-fold increase in esterase activity. Random pairwise gene recombination of two positive variants led to a further two-fold improvement in activity. Considering also the increased expression level achieved during these experiments, the net result of four sequential generations of random mutagenesis and the one recombination step is a 50-60-fold increase in total activity. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model developed based on its homology to acetylcholinesterase and triacylglycerol lipase. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.

Solvent-Dependent Regression Equations for the Prediction of Retention in Planar Chromatography

Both first- and second-order regression models are presented that relate retention, as the R f of individual solutes, the log k' of individual solutes, or the average R f of a mixture of solutes, to the properties of the weak solvent in each of a series of 25 binary mobile phases consisting of a specified concentration of ethyl acetate as a common strong solvent. The stepwise procedure is used for constructing these models, which are for either simulated or experimental separations on silica gel. Similar regression models are used to predict separation quality, defined by a suitable metric. A comparison of the forward and backward stepwise procedures finds that the former is the more reliable method for constructing these models. The solutes are either steroids or the p-nitrobenzyl esters of dansyl amino acids, and the solvent descriptors are density, dipole moment, molar volume, polarizability, saturated surface area, and unsaturated surface area. The quality of regression fits obtained with models using computed dipole moment is comparable to that obtained with models using experimental (literature) dipole moment. Both nonstandardized and standardized regression models are presented. The relative contribution of each descriptor to the variability in retention may be estimated from the latter models. A set of three descriptors-dipole moment, polarizability, and saturated surface area-predicts R f for each of the amino acid derivatives at an ethyl acetate mole fraction of 0.30. A set of two descriptors-dipole moment and saturated surface area-predicts log k' for each of these compounds at an ethyl acetate mole fraction of 0.20. Such concordance in descriptors is not found in models predicting retention of individual steroids.

The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli

p-Nitrobenzyl esters serve as protecting groups on intermediates in the manufacture of clinically important oral β-lactam antibiotics; de-esterification of the intermediates is required for synthesis of the final product. A Bacillus subtilis PNB carboxy-esterase (PNBCE) catalyzes hydrolysis of several β-lactam antibiotic PNB esters to the corresponding free acid and PNB alcohol. This communication (i) describes cloning the pnbA gene, which encodes PNBCE, (ii) provides the nucleotide sequence of the pnbA open reading frame (ORF) and (iii) describes a method for efficiently expressing the ORF in Escherichia coli. The amino acid (aa) sequence, deduced from the nucleotide sequence of the pnbA ORF, matched an experimentally determined N-terminal aa sequence of B. subtilis PNBCE and also matched an active site sequence previously identified by biochemical analyses. Specific activity of PNBCE in crude extracts was more than 90-fold greater in recombinant E. coli, as compared to B. subtilis. This increase in expression led to more than a 500-fold improvement in the efficiency of purification of PNBCE.