Selective Halogenation of Pyridines Using Designed Phosphine Reagents.

Halopyridines are key building blocks for synthesizing pharmaceuticals, agrochemicals, and ligands for metal complexes, but strategies to selectively halogenate pyridine C-H precursors are lacking. We designed a set of heterocyclic phosphines that are installed at the 4-position of pyridines as phosphonium salts and then displaced with halide nucleophiles. A broad range of unactivated pyridines can be halogenated, and the method is viable for late-stage halogenation of complex pharmaceuticals. Computational studies indicate that C-halogen bond formation occurs via an SNAr pathway, and phosphine elimination is the rate-determining step. Steric interactions during C-P bond cleavage account for differences in reactivity between 2- and 3-substituted pyridines.

Diazo Reagent Labeling with Mass Spectrometry Analysis for Sensitive Determination of Ribonucleotides in Living Organisms.

Ribonucleotide analogues and their related phosphorylated metabolites play critical roles in tumor metabolism. However, determination of the endogenous ribonucleotides from the complex biological matrix is still a challenge due to their high structural similarity and high polarity that will lead to the low retention and low detection sensitivities by liquid chromatogram mass spectrometry analysis. In this study, we developed the diazo reagent labeling strategy with mass spectrometry analysis for sensitive determination of ribonucleotides in the living organism. A pair of light and heavy stable isotope labeling reagents, 2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (d5-2-DMBA), were synthesized to label ribonucleotides. 2-DMBA showed high specificity and high efficiency for the labeling of ribonucleotides. Our results demonstrated that the detection sensitivities of 12 ribonucleotides increased by 17-174-fold upon 2-DMBA labeling. The obtained limits of detection (LODs) of ribonucleotides ranged from 0.07 fmol to 0.41 fmol. Using this method, we achieved the sensitive and accurate detection of ribonucleotides from only a few cells (8 cells). To the best of our knowledge, this is the highest detection sensitivity for ribonucleotides ever reported. In addition, we found that the contents of almost all of these ribonucleotides were significantly increased in human breast carcinoma tissues compared to tumor-adjacent normal tissues, suggesting that endogenous ribonucleotides may play certain functional roles in the regulation of cancer development and formation. This method also can be potentially applied in the analysis of phosphorylated compounds.

An effective reagent to functionalize alcohols with phosphocholine.

Phosphocholine is a small haptenic molecule that is both a precursor and degradation product of choline. Phosphocholine decorates a number of biologics such as lipids and oligosaccharides. In this study, an air and bench stable phosphocholine donor has been developed and evaluated with a number of alcohol acceptors. Using a one-pot, three-step sequence, (phosphitylation, oxidation, and phosphate deprotection) phosphocholine derivatives are synthesized in high yields. Of particular interest is the synthesis of miltefosine, the lone oral drug approved to treat leishmaniasis. Due to its prohibitive expense ($1500 per g), miltefosine is not accesable for the majority of the world's patients. Based on the described reaction sequence, this drug can be produced for $25 per g.

Synthetic Strategies, Reactivity and Applications of 1,5-Naphthyridines.

This review covers the synthesis and reactivity of 1,5-naphthyridine derivatives published in the last 18 years. These heterocycles present a significant importance in the field of medicinal chemistry because many of them exhibit a great variety of biological activities. First, the published strategies related to the synthesis of 1,5-naphthyridines are presented followed by the reactivity of these compounds with electrophilic or nucleophilic reagents, in oxidations, reductions, cross-coupling reactions, modification of side chains or formation of metal complexes. Finally, some properties and applications of these heterocycles studied during this period are examined.

Electronically-tuned triarylmethine scaffolds for fast and continuous monitoring of H2S levels in biological samples.

A sensor for the detection and quantification of H2S in biological samples should ideally meet a set of criteria such as fast detection, high sensitivity in the desired concentration range, high selectivity, non-interference from biomolecules like proteins, ease of synthesis, long-term stability and water solubility. Although a number of H2S probes are known, none of them possess all the above attributes that are relevant for practical applications. As part of a program to develop reliable chemical probes for continuous monitoring of this gasotransmitter in the blood plasma of sepsis-prone individuals in post-operative wards, we have looked at the possibility of improving the reactivity and selectivity profile of triarylmethine dyes towards different nucleophiles. After achieving high sensitivity through electronic control, the interference from sulfite, thiosulfate and metabisulfite was addressed by introducing a metal salt-mediated desulfuration step that results in dye regeneration selectively from its H2S adduct. Typically, if the analyte contains only H2S, the loss of absorbance in the first step gets completely reinstated after the second step; absorbance changes in both steps vary linearly with sulfide concentration and either of these two steps can be used for the quantification of H2S with the help of standard plots. In the presence of interfering ions, the first step will show decolourization due to the presence of all of them whereas only the H2S-adduct will undergo desulfuration in the second step which can be used for quantification. The decolourization step is instantaneous while the desulfuration requires only about 50 s, making the entire protocol complete in less than a minute. The methodology optimized here also meets the requirements mentioned above for real-life applications.

Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry.

Low-molecular-weight (LMW) thiols are important small molecules that regulate or maintain redox homeostasis in physiological and pathological processes. Assessing the concentrations of LMW thiols in biological systems may provide valuable information regarding physiological processes and the early diagnosis of some diseases. Here, we developed a method to simultaneously determine the concentrations of multiple LWM thiols in single cells by chemical derivatization assisted liquid chromatography-mass spectrometry (LC-MS). In this method, we synthesized a pair of stable isotope reagents, N-(acridin-9-yl)-2-bromoacetamide (AYBA) and N-(1,2,3,4-[2H4]-acridin-9-yl)-2-bromoacetamide ([2H4]AYBA). AYBA was used to derivatize LWM thiols in human cervical cancer (HeLa) cells, while [2H4]AYBA was used to derivatize standard LWM thiols to prepare internal standards for the LC-MS method development. The proposed AYBA derivatization greatly enhanced the detection sensitivity of LWM thiols by LC-MS, and thereby achieved the simultaneous detection of multiple LWM thiols by LC-MS in ∼1000 HeLa cells. Finally, the developed method was successfully utilized for the quantitative analysis of multiple LWM thiols in a single HeLa cell and the content changes of LWM thiols in a single HeLa cell before and after oxidative stress treatment. Accordingly, six LMW thiols were detected, including cysteamine, cysteine, glutathione, homocysteine, hydrogen sulfide, and pantetheine.

A dual-responsive colorimetric probe for the detection of Cu2+ and Ni2+ species in real water samples and human serum.

The monitoring of heavy transition metals has increasingly attracted great attention because they pollute the environment and have unique physiological functions. Chemosensors are useful tools for monitoring heavy transition metals due to their simple visualization, excellent sensitivity and high selectivity. Herein, we have developed a novel chemosensor for the detection of water-soluble Cu2+ and Ni2+ species with different mechanisms, and low detection limits of 2.1 nM for Cu2+ and 1.2 nM for Ni2+ were obtained. The colorimetric probe CPH has been applied to qualitative and quantitative detection of Cu2+ and Ni2+ species in real samples.

Synthesis of water-soluble hypervalent iodine reagents for fluoroalkylation of biological thiols.

New open-chain and water-soluble hypervalent iodine reagents were synthesized and used for the transfer of fluoroalkyl groups to sulfur atoms of cysteine and cysteine-containing peptides under biocompatible conditions. Some of the reagents displayed excellent reactivity despite their limited stability in aqueous media. In reactions with a short cysteine-containing peptide, in addition to the expected S-fluoroalkylated product, a range of side-products were obtained. The amount of side-products depended on the conditions used (type of reagent, concentration, and pH). With highly activated hypervalent iodine reagents, a new reactive mode was observed - reaction with disulfides to form fluoroalkyl thiols.

Streamlined Total Synthesis of Shishijimicin A and Its Application to the Design, Synthesis, and Biological Evaluation of Analogues thereof and Practical Syntheses of PhthNSSMe and Related Sulfenylating Reagents.

Shishijimicin A is a scarce marine natural product with highly potent cytotoxicities, making it a potential payload or a lead compound for designed antibody-drug conjugates. Herein, we describe an improved total synthesis of shishijimicin A and the design, synthesis, and biological evaluation of a series of analogues. Equipped with appropriate functionalities for linker attachment, a number of these analogues exhibited extremely potent cytotoxicities for the intended purposes. The synthetic strategies and tactics developed and employed in these studies included improved preparation of previously known and new sulfenylating reagents such as PhthNSSMe and related compounds.

Rational design of a high-affinity, fast, red calcium indicator R-CaMP2.

Fluorescent Ca(2+) reporters are widely used as readouts of neuronal activities. Here we designed R-CaMP2, a high-affinity red genetically encoded calcium indicator (GECI) with a Hill coefficient near 1. Use of the calmodulin-binding sequence of CaMKK-α and CaMKK-ß in lieu of an M13 sequence resulted in threefold faster rise and decay times of Ca(2+) transients than R-CaMP1.07. These features allowed resolving single action potentials (APs) and recording fast AP trains up to 20-40 Hz in cortical slices. Somatic and synaptic activities of a cortical neuronal ensemble in vivo were imaged with similar efficacy as with previously reported sensitive green GECIs. Combining green and red GECIs, we successfully achieved dual-color monitoring of neuronal activities of distinct cell types, both in the mouse cortex and in freely moving Caenorhabditis elegans. Dual imaging using R-CaMP2 and green GECIs provides a powerful means to interrogate orthogonal and hierarchical neuronal ensembles in vivo.

A bisubstrate reagent orchestrating adenosine triphosphate and l-tyrosine and making tyrosyl adenylate: partial mimicking of tyrosyl-tRNA synthetase.

We report the development of a bisubstrate reagent that, similar to tyrosyl t-RNA synthetase (TyrTS), provides a surface for ATP and l-Tyr to render a pseudo-intramolecular reaction forming 5'-tyrosyl adenylate (tyrAd). The presence of the reagent in solution with TyrTS marred the enzymatic reaction and, noticeably, tyrAd formed under the catalytic mode of the biomodel reagent was not picked up by TyrTS and hence was not transferred to tRNA. A potential application of this reagent, which doesn't allow the formation of tyrosyl tRNA, may lie in an emerging therapeutic targeting the translation machinery of cells without inhibiting the normal workings of enzymes.

Optical pH Sensor Covering the Range from pH 0-14 Compatible with Mobile-Device Readout and Based on a Set of Rationally Designed Indicator Dyes.

In this work, a family of pH-responsive fluorescent probes has been designed in a rational manner with the aid of quantum chemistry tools, covering the entire pH range from 0-14. Relying on the boron-dipyrromethene (BODIPY) core, all the probes as well as selected reference dyes display very similar spectroscopic properties with ON-OFF fluorescence switching responses, facilitating optical readout in simple devices used for detection and analysis. Embedding of the probes and reference dyes into hydrogel spots on a plastic strip yielded a test strip that reversibly indicates pH with a considerably small uncertainty of ∼0.1 pH units. These strips are not only reusable but, combined with a 3D-printed case that can be attached to a smartphone, the USB port of which drives the integrated LED used for excitation, allows for autonomous operation in on-site or in-the-field applications; the developed Android application software ("app") further simplifies operation for unskilled users.

Synthesis and characterization of photoaffinity labelling reagents towards the Hsp90 C-terminal domain.

Glucosyl-novobiocin-based diazirine photoaffinity labelling reagents (PALs) were designed and synthesized to probe the Hsp90 C-terminal domain unknown binding pocket and the structure-activity relationship. Five PALs were successfully synthesized from novobiocin in six consecutive steps employing phase transfer catalytic glycosylation. Reactions were monitored and guided by analytical LC/MS which led to different strategies of adding either a PAL precursor or a sugar moiety first. The structures and bonding linkages of these compounds were characterised by various 2D-NMR spectroscopy and MS techniques. Synthetic techniques provide powerful probes for unknown protein binding pockets.

Dichlorotrifluoromethoxyacetic Acid: Preparation and Reactivity.

We describe the first gram scale preparation of the reagent dichlorotrifluoromethoxyacetic acid. This stable compound is obtained in five steps starting from the cheap diethylene glycol. The reactivity of the sodium salt of this fluorinated acid was also tested and allowed the preparation of new amides.

Stereospecific Electrophilic Fluorination of Alkylcarbastannatrane Reagents.

We report the use of isolable primary and secondary alkylcarbastannatrane nucleophiles in site-specific fluorination reactions. These reactions occur without the need for transition metal catalysis or in situ activation of the nucleophile. In the absence of the carbastannatrane backbone, alkyltin nucleophiles exhibit no activity towards fluorination. When enantioenriched alkylcarbastannatranes are employed, fluorination occurs predominately via a stereoinvertive mechanism to generate highly enantioenriched alkyl fluoride compounds. These conditions can also be extended to stereospecific chlorination, bromination, and iodination reactions.

An Updated Synthesis of the Diazo-Transfer Reagent Imidazole-1-sulfonyl Azide Hydrogen Sulfate.

Imidazole-1-sulfonyl azide and salts thereof are valuable reagents for diazo-transfer reactions, most particularly conversion of primary amines to azides. The parent reagent and its HCl salt present stability and detonation risks, but the hydrogen sulfate salt is significantly more stable. An updated procedure for the large-scale synthesis of this salt avoids isolation or concentration of the parent compound or HCl salt and will facilitate the use of hydrogen sulfate salt as the reagent of choice for diazo transfer.

Direct aqueous synthesis of cyanomethyl thioglycosides from reducing sugars; ready access to reagents for protein glycosylation.

Unprotected carbohydrates can be directly converted into cyanomethyl thioglycosides in a completely stereoselective manner by reaction with 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and mercaptoacetonitrile in aqueous solution in the presence of triethylamine. Reaction with methoxide provides 2-imino-2-methoxyethyl (IME) thioglycosides, which may be used directly for the glycosylation of surface protein lysine residues.

Alternative reagents for methotrexate as immobilizing anchor moieties in the optimization of MASPIT: synthesis and biological evaluation.

We report the evaluation of two alternative chemical dimerizer approaches aimed at increasing the sensitivity of MASPIT, a three-hybrid system that enables small-molecule target protein profiling in intact human cells. To circumvent the potential limitations related to the binding of methotrexate (MTX) to endogenous human dihydrofolate reductase (DHFR), we explored trimethoprim (TMP) as an alternative prokaryote-specific DHFR ligand. MASPIT evaluation of TMP fusion compounds with tamoxifen, reversine, and simvastatin as model baits, resulted in dose-response curves shifted towards lower EC50 values than those of their MTX congeners. Furthermore, a scalable azido-TMP reagent was synthesized that displayed a similar improvement in sensitivity, possibly owing to increased membrane permeability relative to the MTX anchor. Applying the SNAP-tag approach to introduce a covalent bond into the system, on the other hand, produced an inferior readout than in the MTX- or TMP-tag based assay.

Safer one-pot synthesis of the 'SHAPE' reagent 1-methyl-7-nitroisatoic anhydride (1m7).

Estimating the reactivity of 2'-hydroxyl groups along an RNA chain of interest aids in the modeling of the folded RNA structure; flexible loops tend to be reactive, whereas duplex regions are generally not. Among the most useful reagents for probing 2'-hydroxyl reactivity is 1-methyl-7-nitroisatoic anhydride (1m7), but the absence of a reliable, inexpensive source has prevented widespread adoption. An existing protocol for the conversion of an inexpensive precursor 4-nitroisatoic anhydride (4NIA) recommends the use of NaH in dimethylformamide (DMF), a reagent combination that most molecular biology labs are not equipped to handle, and that does not scale safely in any case. Here we describe a safer, one-pot method for bulk conversion of 4NIA to 1m7 that reduces costs and bypasses the use of NaH. We show that 1m7 produced by this method is free of side products and can be used to probe RNA structure in vitro.

Design, Synthesis, and Characterization of Small-Molecule Reagents That Cooperatively Provide Dual Readouts for Triaging and, When Necessary, Quantifying Point-of-Need Enzyme Assays.

A newly designed small molecule reagent provides both qualitative and quantitative readouts in assays that detect enzyme biomarkers. The qualitative readout enables rapid triaging of samples so that only samples that contain relevant concentrations of the target analyte must be quantified. The reagent is accessible in essentially three steps and 34% overall yield, is stable as a solid when heated to 44 °C for >1 month, and does not produce background signal when used in an assay. This paper describes the design and synthesis of the reagent, characterizes its response properties, and establishes the scope of its reactivity.