Synthesis of Boron-Containing Nucleoside Analogs.

Over the last century, nucleoside-based therapeutics have demonstrated remarkable effectiveness in the treatment of a wide variety of diseases from cancer to HIV. In addition, boron-containing drugs have recently emerged as an exciting and fruitful avenue for medicinal therapies. However, borononucleosides have largely been unexplored in the context of medicinal applications. Herein, we report the synthesis, isolation, and characterization of two novel boron-containing nucleoside compound libraries which may find utility as therapeutic agents. Our synthetic strategy employs efficient one-step substitution reactions between a diverse variety of nucleoside scaffolds and an assortment of n-alkyl potassium trifluoroborate-containing electrophiles. We demonstrated that these alkylation reactions are compatible with cyclic and acyclic nucleoside substrates, as well as increasing alkyl chain lengths. Furthermore, regioselective control of product formation can be readily achieved through manipulation of base identity and reaction temperature conditions.

Synthesis and Antimicrobial Evaluation of γ-Borono Phosphonate Compounds in Escherichia coli and Mycobacterium smegmatis.

Drug resistance in bacteria is a serious threat, and drugs with novel modes of action are constantly needed. Fosmidomycin is a naturally occurring antibiotic that inhibits the nonmevalonate pathway via inhibition of the enzyme 1-deoxylulose-5-phosphate reductoisomerase (DXR). This work is the first report in which a boronic acid is evaluated as an isostere of the retrohydroxamate moiety of fosmidomycin. We report the novel synthesis of a γ-borono phosphonate analog of fosmidomycin and its corresponding prodrugs. We evaluate the inhibition of DXR and the antimicrobial activity of γ-borono phosphonate compounds against Escherichia coli wild type, E. coli Δglycerol-3-phosphate transporter, and Mycobacterium smegmatis. Despite its structural similarities, the γ-borono phosphonate compound shows antimicrobial activity against E. coli with a mechanism of action that is different from fosmidomycin. This was proven with an underutilized method for studying in vitro inhibition of the MEP pathway in E. coli via isopentenyl pyrophosphate chemical rescue. These results indicate that these compounds may serve as a promising scaffold for developing a new class of antimicrobial agents.

Vacuum Matrix-Assisted Ionization Source Offering Simplicity, Sensitivity, and Exceptional Robustness in Mass Spectrometry.

Vacuum matrix-assisted ionization (vMAI) uses select matrix compounds which when exposed to the vacuum of a mass spectrometer produce gas-phase ions from associated volatile or nonvolatile analyte without external energy input. Here, a vMAI source was constructed to replace the commercial inlet of a Thermo Orbitrap mass spectrometer. This allowed for rapid introduction of the matrix/analyte sample by a probe, contrary to vacuum matrix-assisted laser desorption/ionization (MALDI) sources. The matrix/analyte sample is inserted into a region of the "S-lens" entrance, where the spontaneously formed ions can be effectively transferred to the mass analyzer. This specifically designed ion source requires no laser, high voltage, heat, or nebulizing gases. A low voltage is used to transmit the ions through the commercial "S-lens" assembly and airflow can be used to modulate the ionization event. A few picograms of the drug erythromycin, assisted by the 3-nitrobenzonitrile vMAI matrix, is sufficient to produce mass spectra for over 1 min with the MH+ ion as the base peak in each mass spectrum. There is minimal carryover when loading high concentration samples and complex mixtures, contrary to direct infusion electrospray ionization, providing the probe is thoroughly cleaned between each new sample acquisition. Analyses of biological fluids, bacterial extracts, tissue, and high concentration samples have so far shown no indication of inlet or instrument contamination with these samples. The typical ultrahigh resolution and mass accuracy of the mass spectrometer are achieved, and a path forward to potential high throughput acquisitions demonstrated. It is expected that robustness can be introduced to any mass spectrometer through implementation of such a simple source.

Protection of the Benzoxaborole Moiety: Synthesis and Functionalization of Zwitterionic Benzoxaborole Complexes.

The synthesis and utility of three benzoxaborole protecting groups are reported. These protecting groups improve organic solubility and allow otherwise incompatible reactions (oxidations, substitutions, and mild reductions) to be achieved in the presence of the benzoxaborole moiety. 3-( N, N-Dimethylamino)-1-propanol was determined to be useful in one-step sequences and is readily cleaved upon workup. Two other groups, N-methylsalicylidenimine and 2-[1-(methylimino)ethyl]phenol, are suitable for multistep syntheses. Deprotection with mild aqueous acid allows for chromatography-free isolation of the benzoxaborole in high yields.

The unique chemistry of benzoxaboroles: current and emerging applications in biotechnology and therapeutic treatments.

Benzoxaboroles have garnered much attention in recent years due to their diverse applications in bio-sensing technology, material science, and therapeutic intervention. Part of the reason arises from the benzoxaboroles' unique chemical properties, especially in comparison to their acyclic boronic acid counterparts. Furthermore, the low bio-toxicity combined with the high target specificity associated with benzoxaboroles make them very attractive as therapeutic agents. Herein, we provide an updated summary on the current knowledge of the fundamental chemical reactivity of benzoxaboroles, followed by highlighting their major applications reported to date.

Examination of the reactivity of benzoxaboroles and related compounds with a cis-diol.

Benzoxaboroles have been emerging as an interesting and useful scaffold in drug discovery due to their apparently unique reactivity toward diols under physiological conditions. In this work, the reaction of benzoxaborole with the diol-containing, fluorescent dye Alizarin Red S is probed. Steady-state and presteady-state experiments have been conducted for the characterization of the reactions over a wide range of pH. Results indicate that Alizarin Red S reacts with both the boronic (neutral, trigonal) form as well as the boronate (anionic, tetrahedral) form of benzoxaborole in a reaction largely analogous to that previously determined for the simple phenylboronic acid. However, in certain key aspects, the reactivity of the benzoxaborole was found to differ from that of simple phenylboronic acid. The structural origin of these differences has been explored by examination of compounds related to both benzoxaborole and phenylboronic acid. These results may be applied to rational drug discovery efforts aimed at expanding the use of benzoxaboroles in medicine.

Elucidation of the mechanism of the reaction between phenylboronic acid and a model diol, Alizarin Red S.

In this work, the reaction between phenylboronic acid and the diol-containing, fluorescent dye Alizarin Red S (ARS) was probed. Fluorescence titrations, (11)B NMR measurements, and both pre- and steady-state kinetic experiments were used for the characterization of this reaction over a large pH range (4-10.5). It was shown that ARS preferentially reacted with the boronic (neutral, trigonal) form of phenylboronic acid; however, the boronate (anionic, tetrahedral) form was also reactive. All in all, four reactant species were implicated in the formation of four different adduct species. The rate of a given adduct formation depended on the combination of the solution pH and the pK(a)'s of both ARS and the arylboronic acid. The reaction was found to proceed in two distinct kinetic steps with the products and starting materials in facile exchange. In addition, the elucidation of the mechanism indicated the presence of two fluorescent products with the structure of the major contributor differing from what had been cited in the literature.

Ring Structure and Aromatic Substituent Effects on the pK a of the Benzoxaborole Pharmacophore.

In this work, we present an investigation into the physical properties of a unique class of aromatic boronic acids, the benzoxaboroles. Using spectrophotometric methods, the ionization constants of a family of substituted benzoxaboroles are determined. Heterocyclic ring modifications are examined to determine their effects on the ionization of the boronic acid moiety. It is also shown that the substituent effects about the aromatic ring follow a Hammett relationship with the compounds' measured pK a values. Finally, these substituent effects are also shown to extend to the sugar binding properties of these compounds under physiologically relevant conditions. Combined, these data will inform medicinal chemists wishing to tailor the ionization and/or ability of this class of compound to bind diol-containing biomolecules.

Boron-containing inhibitors of synthetases.

The use of boron in small-molecule pharmaceuticals is increasing. Boron's ubiquitous occurrence in nature and the recent success of a boronic acid drug (Velcade®) in the clinic have alleviated many concerns over its use in pharmaceuticals. In addition, the unique physicochemical properties of boronic acids make them an attractive addition to the medicinal chemists toolbox. This tutorial review will discuss these properties and potential benefits for anyone interested in finding novel enzyme inhibitors. An exceptional class of boronic acids, the oxaboroles, will be highlighted and their properties and uses will be discussed in detail. Finally, the current paradigm for the reaction of boronic acids with enzyme nucleophiles will be summarized.

Microtubule-assisted mechanism for functional metabolic macromolecular complex formation.

Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cell-based assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells.

Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate.

Phosphorus-containing pseudopeptides, racemic at the C-terminal alpha-carbon, are potent mechanism-based inhibitors of folylpolyglutamate synthetase (FPGS). They are mimics of the tetrahedral intermediate postulated to form during FPGS-catalyzed biosynthesis of poly(gamma-l-glutamates). In the present paper, the FPGS inhibitory activity of each diastereomer coupled to three heterocycles is reported. The high R(f) pseudopeptide containing the 5,10-dideazatetrahydropteroyl (DDAH(4)Pte) heterocycle is most potent (K(is) = 1.7 nM). While the heterocyclic portion affects absolute FPGS inhibitory potency, the high R(f) species is more potent in each pair containing the same heterocycle. This species presumably has the same stereochemistry as the natural folate polyglutamate, i.e., (l-Glu-gamma-l-Glu). Unexpectedly, the low R(f) (presumed l-Glu-gamma-d-Glu) species are only slightly less potent (<30-fold) than their diastereomers. Further study of this phenomenon comparing l-Glu-gamma-l-Glu and l-Glu-gamma-d-Glu dipeptide-containing FPGS substrates shows that <1% contamination of commercial d-Glu precursors by l-Glu may give misleading information if l-Glu-gamma-l-Glu substrates have low K(m) values.

Concentration-dependent processivity of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase.

Folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-gamma-glutamate derivatives of reduced folates and antifolates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH 4PteGlu 1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high-performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH 4PteGlu 1 catalyzed by FPGS to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to assess folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH 4PteGlu 1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells.

Unnatural translation initiation.

Protein translation in nature always begins with an initiator transfer RNA (tRNA) carrying the amino acid methionine. This was circumvented in vitro with a reconstituted translation system utilizing initiator tRNA synthetically mischarged with the other natural amino acids. In addition, it was determined that this system could accommodate these non-methionine amino acids containing various N-alpha-acyl groups, many of which are useful for post-translational modification such as peptide cyclization.

Discovery of antibacterial cyclic peptides that inhibit the ClpXP protease.

A method to rapidly screen libraries of cyclic peptides in vivo for molecules with biological activity has been developed and used to isolate cyclic peptide inhibitors of the ClpXP protease. Fluorescence activated cell sorting was used in conjunction with a fluorescent reporter to isolate cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in Escherichia coli. Inhibitors shared little sequence similarity and interfered with unexpected steps in the ClpXP mechanism in vitro. One cyclic peptide, IXP1, inhibited the degradation of unrelated ClpXP substrates and has bactericidal activity when added to growing cultures of Caulobacter crescentus, a model organism that requires ClpXP activity for viability. The screen used here could be adapted to identify cyclic peptide inhibitors of any enzyme that can be expressed in E. coli in conjunction with a fluorescent reporter.

Synthesis of (6R)- and (6S)-5,10-dideazatetrahydrofolate oligo-gamma-glutamates: kinetics of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase.

Folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) catalyzes the ATP-dependent ligation of glutamic acid to reduced folates including (6S)-5,6,7,8-tetrahydrofolate (H4PteGlu), as well as to anticancer drugs such as 5,10-dideaza-5,6,7,8-tetrahydrofolate ((6R)-DDAH4PteGlu1, (6R)-DDATHF, Lometrexol). Synthesis of unlabeled mono- and polyglutamates, DDAH4PteGlu(n) (6R, n = 1-6; 6S, n = 1-2), as well as (6R)-DDAH4Pte[14C]Glu1, was effected from (6R)- or (6S)-5,10-dideazatetrahydropteroyl azide and glutamic acid, H-Glu-gamma-Glu(n)-gamma-Glu-OH (n = 0-4), or [14C]glutamic acid, respectively. These compounds were evaluated as FPGS substrates to determine steady-state kinetic constants. Michaelis-Menten kinetics were observed for (6R)-DDAH4PteGlu1, the isomer corresponding to H(4)PteGlu, whereas marked substrate inhibition was observed for (6S)-DDAH4PteGlu(n) (n = 1-2) and (6R)-DDAH4PteGlu(n) (n = 2-5), but not (6R)-DDAH4PteGlu6. Multiple ligation of glutamate renders a quantitative analysis of these data difficult. However, approximate values of K(M) = 0.65-1.6 microM and K(I) = 144-417 microM for DDAH4PteGlu(n) were obtained using a simple kinetic model.