Chemically Separable Co(II) Spin-State Isomers.

The phenomenon of spin crossover involves coordination complexes with switchable spin states. This spin state change is accompanied by significant geometric changes such that low and high spin forms of a complex are distinct isomers that exist in equilibrium with one another. Typically, spin-state isomers interconvert rapidly and are similar enough in polarity to prevent their independent separation and isolation. We report here the first example, to our knowledge, of cobalt(II) spin-state isomers that can be physically separated. The reaction of Mo2(dpa)4 (dpa = 2,2'-dipyridylamide) with CoBr2 produces a mixture of two heterometallic compounds with a linear, metal-metal-bonded Mo[Formula: see text]Mo-Co chain. The complexes, SC-[BrMo2(dpa)4Co]Br (SC-2) and HS-[BrMo2(dpa)4CoBr] (HS-2), have identical compositions (Mo2Co(dpa)4Br2) but different ground spin states and coordination geometries of the Co(II) ion. In the solid state, SC-2 undergoes incomplete spin crossover from an S = 1/2 state to an S = 3/2 state, and HS-2 has a high spin, S = 3/2, ground state, as confirmed by SQUID magnetometry and EPR spectroscopy. Crystallographic analyses of SC-2 and HS-2 show that SC-2 has an elongated Co-Br distance relative to HS-2 and is best described as the salt [BrMo2(dpa)4Co]Br. This limits SC-2's solubility in nonpolar solvents and allows for the physical separation of the two isomers. Solution studies of SC-2 and HS-2 indicate that SC-2 and HS-2 interconvert slowly relative to the NMR time scale. Additional solution-state EPR and UV-vis absorption measurements demonstrate that the choice of solvent polarity determines the predominant isomer present in solution.

Magnetic-Field Dependence of LC-Photo-CIDNP in the Presence of Target Molecules Carrying a Quasi-Isolated Spin Pair.

NMR spectroscopy is well known for its superb resolution, especially at high applied magnetic field. However, the sensitivity of this technique is very low. Liquid-state low-concentration photo-chemically-induced dynamic nuclear polarization (LC-photo-CIDNP) is a promising emerging methodology capable of enhancing NMR sensitivity in solution. LC-photo-CIDNP works well on solvent-exposed Trp and Tyr residues, either in isolation or within proteins. This study explores the magnetic-field dependence of the LC-photo-CIDNP experienced by two tryptophan isotopologs in solution upon in situ LED-mediated optical irradiation. Out of the two uniformly 13C,15N-labeled Trp (Trp-U-13C,15N) and Trp-α-13C-ß,ß,2,4,5,6,7-d7 species employed here, only the latter bears a quasi-isolated 1Hα-13Cα spin pair. Computer simulations of the predicted polarization due to geminate recombination of both species display a roughly bell-shaped field dependence. However, while Trp-U-13C,15N is predicted to show a maximum at ca. 500 MHz (11.7 T) and a fairly weak field dependence, Trp-α-13C-ß,ß,2,4,5,6,7-d7 is expected to display a much sharper field dependence accompanied by a dramatic polarization increase at lower field (ca. 200 MHz, 4.7 T). Experimental LC-photo-CIDNP studies on both Trp isotopologs at 1µM concentration, performed at selected fields, are consistent with the theoretical predictions. In summary, this study highlights the prominent field-dependence of LC-photo-CIDNP enhancements (ε) experienced by Trp isotopologs bearing a quasi-isolated spin pair.

Variable Temperature LED-NMR: Rapid Insights into a Photocatalytic Mechanism from Reaction Progress Kinetic Analysis.

A multitude of techniques are available to obtain a useful understanding of photocatalytic mechanisms. The combination of LED illumination with nuclear magnetic resonance spectroscopy (LED-NMR) provides a rapid, convenient means to directly monitor a photocatalytic reaction in situ. Herein, we describe a study of the mechanism of an enantioselective intermolecular [2 + 2] photocycloaddition catalyzed by a chiral Ir photocatalyst using LED-NMR. The data-rich output of this experiment is suitable for same-excess and variable time normalization analyses (VTNA). Together, these identified an unexpected change in mechanism between reactions conducted at ambient and cryogenic temperatures. At -78 °C, the kinetic data are consistent with the triplet rebound mechanism we previously proposed for this reaction, involving sensitization of maleimide and rapid reaction with a hydrogen-bound quinoline within the solvent cage. At room temperature, the cycloaddition instead proceeds through intracomplex energy transfer to the hydrogen-bound quinolone. These results highlight the potential sensitivity of photocatalytic reaction mechanisms to the precise reaction conditions and the further utility of LED-NMR as a fast, data-rich tool for their interrogation that compares favorably to conventional ex situ kinetic analyses.

Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A.

Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) comprise biosynthetic pathways that provide access to diverse, often bioactive natural products. Metabolic engineering can improve production metrics to support characterization and drug-development studies, but often native hosts are difficult to genetically manipulate and/or culture. For this reason, heterologous expression is a common strategy for natural product discovery and characterization. Many bacteria have been developed to express heterologous biosynthetic gene clusters (BGCs) for producing polyketides and nonribosomal peptides. In this article, we describe tools for using Pseudomonas putida, a Gram-negative soil bacterium, as a heterologous host for producing natural products. Pseudomonads are known to produce many natural products, but P. putida production titers have been inconsistent in the literature and often low compared to other hosts. In recent years, synthetic biology tools for engineering P. putida have greatly improved, but their application towards production of natural products is limited. To demonstrate the potential of P. putida as a heterologous host, we introduced BGCs encoding the synthesis of prodigiosin and glidobactin A, two bioactive natural products synthesized from a combination of PKS and NRPS enzymology. Engineered strains exhibited robust production of both compounds after a single chromosomal integration of the corresponding BGC. Next, we took advantage of a set of genome-editing tools to increase titers by modifying transcription and translation of the BGCs and increasing the availability of auxiliary proteins required for PKS and NRPS activity. Lastly, we discovered genetic modifications to P. putida that affect natural product synthesis, including a strategy for removing a carbon sink that improves product titers. These efforts resulted in production strains capable of producing 1.1 g/L prodigiosin and 470 mg/L glidobactin A.

Structural and biophysical characterization of the Burkholderia pseudomallei IspF inhibitor L-tryptophan hydroxamate.

The enzyme 2-methylerythritol 2,4-cyclodiphosphate synthase, IspF, is essential for the biosynthesis of isoprenoids in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The development of inhibitors that target IspF may lead to novel classes of anti-infective agents or herbicides. Enantiomers of tryptophan hydroxamate were synthesized and evaluated for binding to Burkholderia pseudomallei (Bp) IspF. The L-isomer possessed the highest potency, binding BpIspF with a KD of 36 µM and inhibited BpIspF activity 55% at 120 µM. The high-resolution crystal structure of the L-tryptophan hydroxamate (3)/BpIspF complex revealed a non-traditional mode of hydroxamate binding where the ligand interacts with the active site zinc ion through the primary amine. In addition, two hydrogen bonds are formed with active site groups, and the indole group is buried within the hydrophobic pocket composed of side chains from the 60 s/70 s loop. Along with the co-crystal structure, STD NMR studies suggest the methylene group and indole ring are potential positions for optimization to enhance binding potency.

Fast-pulsing LED-enhanced NMR: A convenient and inexpensive approach to increase NMR sensitivity.

Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) has recently emerged as a powerful technology for the detection of aromatic amino acids and proteins in solution in the low-micromolar to nanomolar concentration range. LC-photo-CIDNP is typically carried out in the presence of high-power lasers, which are costly and maintenance-heavy. Here, we show that LC-photo-CIDNP can be performed with light-emitting diodes (LEDs), which are inexpensive and much less cumbersome than lasers, laser diodes, flash lamps, or other light sources. When nuclear magnetic resonance (NMR) sample concentration is within the low-micromolar to nanomolar range, as in LC-photo-CIDNP, replacement of lasers with LEDs leads to no losses in sensitivity. We also investigate the effect of optical-fiber thickness and compare excitation rate constants of an Ar ion laser (488 nm) and a 466 nm LED, taking LED emission bandwidths into account. In addition, importantly, we develop a novel pulse sequence (13C RASPRINT) to perform ultrarapid LC-photo-CIDNP data collection. Remarkably, 13C RASPRINT leads to 4-fold savings in data collection time. The latter advance relies on the fact that photo-CID nuclear hyperpolarization does not suffer from the longitudinal-relaxation recovery requirements of conventional NMR. Finally, we combine both the above improvements, resulting in facile and rapid (≈16 s-2.5 min) collection of 1 and 2D NMR data on aromatic amino acids and proteins in solution at nanomolar to low micromolar concentration.

High Permeation Rates in Liposome Systems Explain Rapid Glyphosate Biodegradation Associated with Strong Isotope Fractionation.

Bacterial uptake of charged organic pollutants such as the widely used herbicide glyphosate is typically attributed to active transporters, whereas passive membrane permeation as an uptake pathway is usually neglected. For 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) liposomes, the pH-dependent apparent membrane permeation coefficients ( Papp) of glyphosate, determined by nuclear magnetic resonance (NMR) spectroscopy, varied from Papp (pH 7.0) = 3.7 (±0.3) × 10-7 m·s-1 to Papp (pH 4.1) = 4.2 (±0.1) × 10-6 m·s-1. The magnitude of this surprisingly rapid membrane permeation depended on glyphosate speciation and was, at circumneutral pH, in the range of polar, noncharged molecules. These findings point to passive membrane permeation as a potential uptake pathway during glyphosate biodegradation. To test this hypothesis, a Gram-negative glyphosate degrader, Ochrobactrum sp. FrEM, was isolated from glyphosate-treated soil and glyphosate permeation rates inferred from the liposome model system were compared to bacterial degradation rates. Estimated maximum permeation rates were, indeed, 2 orders of magnitude higher than degradation rates of glyphosate. In addition, biodegradation of millimolar glyphosate concentrations gave rise to pronounced carbon isotope fractionation with an apparent kinetic isotope effect, AKIEcarbon, of 1.014 ± 0.003. This value lies in the range typical of non-masked enzymatic isotope fractionation demonstrating that glyphosate biodegradation was not subject to mass transfer limitations and glyphosate exchange across the cell membrane was rapid relative to enzymatic turnover.

Expression and characterization of an enantioselective antigen-binding fragment directed against α-amino acids.

This work describes the design and expression of a stereoselective Fab that possesses binding properties comparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridoma clones that secrete a stereoselective anti-l-amino acid antibody, a corresponding biotechnologically produced Fab was generated. For that, appropriate primers were designed based on extensive literature and databank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL and CH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences were separately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells. Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that was demonstrated to bind to l-amino acids but not to recognize the corresponding d-enantiomers.

Antibody-based multiplex analysis of structurally closely related chiral molecules.

Four class-specific and stereoselective antibodies were labeled with different fluorophores to enable simultaneous quantification of the enantiomers of phenylalanine and phenyllactic acid. Using fluorescence immunoassays and microarrays, sensitive detection of each analyte was possible even in the presence of a large excess of the other structurally similar molecules.

Non-DNA-binding platinum anticancer agents: Cytotoxic activities of platinum-phosphato complexes towards human ovarian cancer cells.

DNA is believed to be the molecular target for the cytotoxic activities of platinum (Pt) anticancer drugs. We report here a class of platinum(II)- and platinum(IV)-pyrophosphato complexes that exhibit cytotoxicity comparable with and, in some cases, better than cisplatin in ovarian cell lines (A2780, A2780/C30, and CHO), yet they do not show any evidence of covalent binding to DNA. Moreover, some of these compounds are quite effective in cisplatin- and carboplatin-resistant cell line A2780/C30. The lack of DNA binding was demonstrated by the absence of a detectable Pt signal by atomic absorption spectroscopy using isolated DNA from human ovarian cells treated with a platinum(II)-pyrophosphato complex, (trans-1,2-cyclohexanediamine)(dihydrogen pyrophosphato) platinum(II), (pyrodach-2) and from NMR experiments using a variety of nucleotides including single- and double-stranded DNA. Furthermore, pyrodach-2 exhibited reduced cellular accumulations compared with cisplatin in cisplatin- and carboplatin-resistant human ovarian cells, yet the IC(50) value for the pyrophosphato complex was much less than that of cisplatin. Moreover, unlike cisplatin, pyrodach-2 treated cells overexpressed fas and fas-related transcription factors and some proapoptotic genes such as Bak and Bax. Data presented in this report collectively indicate that pyrodach-2 follows different cytotoxic mechanisms than does cisplatin. Unlike cisplatin, pyrodach-2 does not undergo aquation during 1 week and is quite soluble and stable in aqueous solutions. Results presented in this article represent a clear paradigm shift not only in expanding the molecular targets for Pt anticancer drugs but also in strategic development for more effective anticancer drugs.

LED-NMR Monitoring of an Enantioselective Catalytic [2+2] Photocycloaddition.

We report that an NMR spectrometer equipped with a high-power LED light source can be used to study a fast enantioselective photocatalytic [2+2] cycloaddition. While traditional ex situ applications of NMR provide considerable information on reaction mechanisms, they are often ineffective for observing fast reactions. Recently, motivated by renewed interest in organic photochemistry, several approaches have been reported for in situ monitoring of photochemical reactions. These previously disclosed methods, however, have rarely been applied to rapid (<5 min) photochemical reactions. Furthermore, these approaches have not previously been used to interrogate the mechanisms of photocatalytic energy-transfer reactions. In the present work, we describe our experimental setup and demonstrate its utility by determining a phenomenological rate law for a model photocatalytic energy-transfer cycloaddition reaction.

Computational structural analysis of an anti-L-amino acid antibody and inversion of its stereoselectivity.

The binding site of a monoclonal anti-L-amino acid antibody (anti-L-AA) was modeled using the program SWISS-MODEL. Docking experiments with the enantiomers of phenylalanine revealed that the antibody interacts with L-phenylalanine via hydrogen bonds and hydrophobic contacts, whereas the D-enantiomer is rejected due to steric hindrance. Comparison of the sequences of this antibody and an anti-D-amino acid antibody (anti-D-AA) indicates that both immunoglobulins derived from the same germline progenitor. Substitution of four amino acids residues, three in the framework and one in the complementarity determining regions (CDRs), allowed in silico conversion of the anti-L-AA into an antibody that stereoselectively binds D-phenylalanine.

Geminal Diol of Dihydrolevoglucosenone as a Switchable Hydrotrope: A Continuum of Green Nanostructured Solvents.

The addition of water to dihydrolevoglucosenone (Cyrene) creates a solvent mixture with highly unusual properties and the ability to specifically and efficiently solubilize a wide range of organic compounds, notably, aspirin, ibuprofen, salicylic acid, ferulic acid, caffeine, and mandelic acid. The observed solubility enhancement (up to 100-fold) can be explained only by the existence of microenvironments mainly centered on Cyrene's geminal diol. Surprisingly, the latter acts as a reversible hydrotrope and regulates the polarity of the created complex mixture. The possibility to tune the polarity of the solvent mixture through the addition of water, and the subsequent generation of variable amounts of Cyrene's geminal diol, creates a continuum of green solvents with controllable solubilization properties. The effective presence of microheterogenieties in the Cyrene/water mixture was adequately proven by (1) Fourier transform infrared/density functional theory showing Cyrene dimerization, (2) electrospray mass-spectrometry demonstrating the existence of dimers of Cyrene's geminal diol, and (3) the variable presence of single or multiple tetramethylsilane peaks in the 1H NMR spectra of a range of Cyrene/water mixtures. The Cyrene-water solvent mixture is importantly not mutagenic, barely ecotoxic, bioderived, and endowed with tunable hydrophilic/hydrophobic properties.

Enantiomer separation of alpha-hydroxy acids in high-performance immunoaffinity chromatography.

In this study, a monoclonal anti-d-hydroxy acid antibody was immobilized onto a synthetic high-flow-through chromatographic support material to produce a chiral stationary phase suitable for enantiomer separation of free alpha-hydroxy acids. Chiral separation of several aliphatic and aromatic members of this class of compounds was achieved in HPLC under mild isocratic buffer conditions using phosphate buffered saline, pH 7.4, as mobile phase. Due to the high degree of stereoselectivity exhibited by the immobilized antibody, in all cases the l-enantiomer eluted with the void volume, while the d-enantiomer was retained and eluted second. The effect of the mobile phase parameters flow rate, temperature, pH, and ionic strength on the enantiomer separation of the model analyte mandelic acid was investigated. While it was found that variations in the flow rate did not change the retention factor k2, dramatic effects on the interaction between the immobilized antibody and d-mandelic acid were observed when any of the other mobile phase parameters were modulated.

Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12µM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX.

Detection of enantiomeric impurities in a simple membrane-based optical immunosensor.

Currently available methods for the detection of enantiomeric impurities generally require expensive and sophisticated instrumentation. Here, we describe a simple and inexpensive membrane-based chiral immunosensor that allows quantitative determination of chiral analytes up to an enantiomer excess of 99.9%. The experimental setup is based on a competitive reaction between the analyte and a biotin-derivatized analog for the binding sites of a stereoselective antibody, which is immobilized onto a membrane. The antibody-bound analog is detected with peroxidase-conjugated avidin that converts a colorless substrate into an insoluble dye on the membrane surface. The color intensity, which is inversely related to the concentration of analyte in a sample, can be evaluated with standard image analysis programs.

Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography.

The effect of the mobile phase parameters flow rate, temperature, pH and ionic strength, as well as the addition of various organic modifiers on the enantiomer separation of various aromatic alpha-amino acids was investigated using two antibody-based chiral stationary phases that have opposing stereoselectivity. On both columns, a decrease in flow rate or temperature resulted in increased interaction with the retained enantiomer. It was found that the retention factor k2 depends on the affinity between the analyte and the immobilized antibody and is not independent of the flow rate. Optimum separations of all amino acids investigated were obtained at pH 7.4 on both columns. While increased k2 values were obtained at low ionic strength on the anti-D-amino acid antibody column, no such effect was observed on the anti-L-amino acid antibody column. The addition of organic modifiers did not improve separations. In all studies, the unretained enantiomer eluted with the void volume.

Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography.

This work demonstrates the feasibility of using a camelid single domain antibody for immunoaffinity chromatographic separation of small molecules. An anti-caffeine VHH antibody was produced by grafting the complementarity determining sequences of a previously generated antibody onto an anti-RNase A antibody scaffold, followed by expression in E. coli. Analysis of the binding properties of the antibody by ELISA and fluorescence-based thermal shift assays showed that it recognizes not only caffeine, but also theophylline, theobromine, and paraxanthine, albeit with lower affinity. Further investigation of the effect of environmental conditions, i.e., temperature, pH, and ionic strength, on the antibody using these methods provided useful information about potential elution conditions to be used in chromatographic applications. Immobilization of the VHH onto a high flow-through synthetic support material resulted in a stationary phase capable of separating caffeine and its metabolites.

Determination of lactic acid enantiomers in human urine by high-performance immunoaffinity LC-MS.

In this study, a monoclonal anti-d-hydroxy acid antibody was used as chiral selector for chromatographic enantiomer separation and quantification of lactic acid contained in human urine samples. The immunoaffinity column was directly coupled to an electrospray ionization mass spectrometer for detection. Separations were performed at room temperature and under isocratic conditions using ammonium bicarbonate buffer (pH 7.8; 10 mM) as mobile phase. No elaborate sample preparation or analyte derivatization was required and individual runs were completed in less than 10 min.