Mechanistic Investigation of the Nickel-Catalyzed Suzuki Reaction of <i>N</i>,<i>O</i>‑Acetals: Evidence for Boronic Acid Assisted Oxidative Addition and an Iminium Activation Pathway

The mechanism of a recently reported Suzuki coupling reaction of quinoline-derived allylic <i>N</i>,<i>O</i>-acetals has been studied using a combination of structural, stereochemical, and kinetic isotope effect experiments. The data indicate that C–O activation is facilitated by Lewis acid assistance from the boronic acid coupling partner and an ionic S_N1-like mechanism accounts for oxidative addition. In this context, we demonstrate the first direct observation of oxidative addition to a quinolinium salt. Notably, this mechanism is distinct from the more commonly described S_N2­(′)-type oxidative addition of low-valent transition metals to most allylic electrophiles

Mechanistic Investigation of the Nickel-Catalyzed Suzuki Reaction of <i>N</i>,<i>O</i>‑Acetals: Evidence for Boronic Acid Assisted Oxidative Addition and an Iminium Activation Pathway

The mechanism of a recently reported Suzuki coupling reaction of quinoline-derived allylic <i>N</i>,<i>O</i>-acetals has been studied using a combination of structural, stereochemical, and kinetic isotope effect experiments. The data indicate that C–O activation is facilitated by Lewis acid assistance from the boronic acid coupling partner and an ionic S_N1-like mechanism accounts for oxidative addition. In this context, we demonstrate the first direct observation of oxidative addition to a quinolinium salt. Notably, this mechanism is distinct from the more commonly described S_N2­(′)-type oxidative addition of low-valent transition metals to most allylic electrophiles

Mechanistic Investigation of the Nickel-Catalyzed Suzuki Reaction of <i>N</i>,<i>O</i>‑Acetals: Evidence for Boronic Acid Assisted Oxidative Addition and an Iminium Activation Pathway

The mechanism of a recently reported Suzuki coupling reaction of quinoline-derived allylic <i>N</i>,<i>O</i>-acetals has been studied using a combination of structural, stereochemical, and kinetic isotope effect experiments. The data indicate that C–O activation is facilitated by Lewis acid assistance from the boronic acid coupling partner and an ionic S_N1-like mechanism accounts for oxidative addition. In this context, we demonstrate the first direct observation of oxidative addition to a quinolinium salt. Notably, this mechanism is distinct from the more commonly described S_N2­(′)-type oxidative addition of low-valent transition metals to most allylic electrophiles

Mechanistic Investigation of the Nickel-Catalyzed Suzuki Reaction of <i>N</i>,<i>O</i>‑Acetals: Evidence for Boronic Acid Assisted Oxidative Addition and an Iminium Activation Pathway

The mechanism of a recently reported Suzuki coupling reaction of quinoline-derived allylic <i>N</i>,<i>O</i>-acetals has been studied using a combination of structural, stereochemical, and kinetic isotope effect experiments. The data indicate that C–O activation is facilitated by Lewis acid assistance from the boronic acid coupling partner and an ionic S_N1-like mechanism accounts for oxidative addition. In this context, we demonstrate the first direct observation of oxidative addition to a quinolinium salt. Notably, this mechanism is distinct from the more commonly described S_N2­(′)-type oxidative addition of low-valent transition metals to most allylic electrophiles

Characterization of pSer40/41MCM2 antibody.

<p>A) HeLa whole cell extract was incubated with Lambda phosphatase in the presence or absence of phosphatase inhibitors. Proteins were analyzed by western blot with the indicated antibodies. B) Equal amounts of protein extract were separated on a single SDS-PAGE gel and transferred onto membranes. Vertical slices of membrane (each with identical protein content) were then incubated with anti-pSer40/41MCM2 antibody in the presence of the indicated competitor peptides (upper panels). Membranes were then re-probed with an anti-MCM2 antibody (lower panels). Identical exposures are shown. C) HeLa cells growing on coverslips were fixed and stained with anti-pSer40/41MCM2 antibody in the presence of the indicated competitor peptides. DNA was counterstained with DAPI. D) Serial sections of normal and cancerous colon tissue were used in IHC with the pSer40/41MCM2 antibody in the presence or absence of competitor peptide. Nuclear positivity is shown by brown color. E) HeLa cells were treated with PHA-767491 for the indicated time. Protein extracts were prepared and analysed by western blot with the indicated antibodies.</p

Ryuvidine elicits a DNA damage response.

<p>A) Protein extracts prepared from HeLa cells treated with either PHA-767491, Ryuvidine, Mitoxantrone or hydroxyurea (HU) for the indicated time were analyzed by western blot with indicated antibodies. B) Formation of γ-H2AXnuclear foci in cells treated with ATM inhibitor KU55933, Mitoxantrone, Ryuvidine alone or in combination was assessed by fluorescence microscopy. Nuclei were stained with DAPI. Representative fields are shown.</p

Confirmation and <i>in vitro</i> characterization of Hit compounds.

<p>A) HeLa cells were treated with the ten compounds that produced the strongest reduction in pSer40/41MCM2 levels in the primary screen. Protein extracts were prepared and levels of residual pSer40/41MCM2 phosphorylation were measured by western blot analysis with the indicated antibodies. B) The same compounds were tested for their ability to inhibit CDC7 kinase activity in <i>in vitro</i> kinase assay. Kinase reactions were performed on a synthetic MCM2 substrate in the absence or presence of the indicated drug and reactions were run on SDS-PAGE gels. CDC7 activity on the synthetic MCM2 substrate was monitored by Western blotting with an anti-pSer108MCM2 antibody. As a control the levels of CDC7 kinase and synthetic MCM2 substrate present in each reaction were also assessed and shown to be similar in all reactions.</p

Ryuvidine and Mitoxantrone reduce CDC7 levels and pSer40/41 MCM2 phosphorylation.

<p>A) Molecular structure of PHA-767491, Ryuvidine and Mitoxantrone. B) HeLa cells were incubated with either CDC7 inhibitor PHA-767491, Ryuvidine or Mitoxantrone at 10 micromolar each for the indicated time. Protein extracts were prepared and analyzed by western blot with the indicated antibodies. C) Levels of Cdc7-independent pSer41MCM2 and Cdc7-dependent pSer40/41MCM2 phosphorylation assessed in HeLa cells treated with the indicated concentration of PHA-767491, Ryuvidine or Mitoxantrone. D) Cells were incubated with either PHA-767491, Ryuvidine or Mitoxantrone at the indicated concentration for nine hours. Protein extracts were then prepared and analyzed by western blot with the indicated antibodies. E) HeLa cells incubated with either PHA-767491, Ryuvidine or Mitoxantrone at 10 micromolar each for nine hours in the presence or absence of 20 micromolar of caspase inhibitor Boc-D-fmk. Levels of Ser40/41MCM2 phosphorylation, CDC7 and MCM2 abundance as well as PARP cleavage, were assessed by western blotting.</p

Screening for modulators of Cdc7-dependent phosphorylation of Ser40/41 MCM2.

<p>Results of the screening of the Johns Hopkins Clinical Compound Library and Tocris kinase inhibitor library using In Cell Western Technology. Each dot represents a compound and the normalized pSer40/41 MCM2 intensity is reported - See text for experimental details. The names of the hits producing the strongest reduction in phosphorylation at Ser40/41MCM2 are indicated.</p

A Rationally Designed Agonist Defines Subfamily IIIA Abscisic Acid Receptors As Critical Targets for Manipulating Transpiration

Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates <i>Pyrabactin Resistance 1</i> (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin’s arylnitrile mimics ABA’s cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA’s carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin’s compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce <i>Arabidopsis</i> whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its <i>in vitro</i> potency translates to ABA-like activity <i>in vivo</i>. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration