Using Peptide Arrays to Profile Phosphatase Activity in Cell Lysates

Phosphorylation is an important post-translational modification on proteins involved in many cellular processes; however, understanding of the regulation and mechanisms of global phosphorylation remains limited. Herein, we utilize self-assembled monolayers on gold for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) with three phosphorylated peptide arrays to profile global phosphatase activity in cell lysates derived from five mammalian cell lines. Our results reveal significant differences in the activities of protein phosphatases on phospho- serine, threonine, and tyrosine substrates and suggest that phosphatases play a much larger role in the regulation of global phosphorylation on proteins than previously understood.     

Development of an Enzyme-Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly

This paper presents an enzyme building block for the assembly of megamolecules. The system is based on the inhibition of the human-derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate group, which uses sulfur fluoride exchange click chemistry to covalently inhibit CRABP2. We conjugated both the inhibitor and a fluorescein tag to an oligo(ethylene glycol) backbone and measured a second-order rate constant for the protein inhibition reaction of approximately 3,600 M⁻¹s⁻¹. We used this new enzyme-inhibitor pair to assemble multi-protein structures in one-pot reactions using three orthogonal assembly chemistries to demonstrate exact control over the placement of protein domains within a single, homogeneous molecule. This work enables a new dimension of control over specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures.     

An Optimized Protocol for the Synthesis of Peptides Containing trans-Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes

Despite the great advances in solid-phase peptide synthesis (SPPS), the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), into different peptide sequences. The two groups enable fast and modular post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide and peptide-drug conjugates. Due to the excellent biocompatibility, the click-functionalization of the peptides can be performed directly in live cells. We further show that the introduction of both clickable groups into peptides enables construction of smart, multifunctional probes that can streamline complex chemical biology experiments such as visualization and pull-down of metabolically labeled glycoconjugates. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.     

Synthesis of bio-based poly(methacrylates) using SG1-containing amphiphilic macroinitiators by nitroxide mediated miniemulsion polymerization

SG1-based amphiphilic macroinitiators were synthesized from oligoethylene glycol methyl ether methacrylate and 10 mol% acrylonitrile or styrene (as the controlling comonomer) to conduct the nitroxide mediated polymerization of bio-based methacrylic monomers (isobornyl methacrylate (IBOMA) and C13 alkyl methacrylate (C13MA)) in miniemulsion. The effect of the addition of surfactant (DOWFAX 8390), co-stabilizer (n-hexadecane) and different reaction temperatures (80, 90 and 100°C) on polymerization kinetics was studied. We found that the NMP of IBOMA/C13MA using amphiphilic macroalkoxyamines were most effective during miniemulsion polymerization (linear trend of M_n versus conversion and high latex stability) in presence of 2 wt% surfactant and 0.8 wt% co-stabilizer (relative to monomer) at 90°C. The effect of surfactant, co-stabilizer and temperature on particle size during the polymerization was studied and suggested a decrease in initial particle size with the addition of surfactant and co-stabilizer. Finally, the thermal properties of IBOMA/C13MA polymers, prepared by amphiphilic macroinitiators, were examined thoroughly, indicating a T_g in the range of −44°C < T_g < 109°C.     

Molecular and Functional Imaging Studies of Psychedelic Drug Action in Animals and Humans

Hallucinogens are a loosely defined group of compounds including LSD, N,N-dimethyltryptamines, mescaline, psilocybin/psilocin, and 2,5-dimethoxy-4-methamphetamine (DOM), which can evoke intense visual and emotional experiences. We are witnessing a renaissance of research interest in hallucinogens, driven by increasing awareness of their psychotherapeutic potential. As such, we now present a narrative review of the literature on hallucinogen binding in vitro and ex vivo, and the various molecular imaging studies with positron emission tomography (PET) or single photon emission computer tomography (SPECT). In general, molecular imaging can depict the uptake and binding distribution of labelled hallucinogenic compounds or their congeners in the brain, as was shown in an early PET study with N¹-([¹¹C]-methyl)-2-bromo-LSD ([¹¹C]-MBL); displacement with the non-radioactive competitor ketanserin confirmed that the majority of [¹¹C]-MBL specific binding was to serotonin 5-HT_2A receptors. However, interactions at serotonin 5HT_1A and other classes of receptors and pleotropic effects on second messenger pathways may contribute to the particular experiential phenomenologies of LSD and other hallucinogenic compounds. Other salient aspects of hallucinogen action include permeability to the blood–brain barrier, the rates of metabolism and elimination, and the formation of active metabolites. Despite the maturation of radiochemistry and molecular imaging in recent years, there has been only a handful of PET or SPECT studies of radiolabeled hallucinogens, most recently using the 5-HT_2A/2C agonist N-(2[¹¹CH₃O]-methoxybenzyl)-2,5-dimethoxy- 4-bromophenethylamine ([¹¹C]Cimbi-36). In addition to PET studies of target engagement at neuroreceptors and transporters, there is a small number of studies on the effects of hallucinogenic compounds on cerebral perfusion ([¹⁵O]-water) or metabolism ([¹⁸F]-fluorodeoxyglucose/FDG). There remains considerable scope for basic imaging research on the sites of interaction of hallucinogens and their cerebrometabolic effects; we expect that hybrid imaging with PET in conjunction with functional magnetic resonance imaging (fMRI) should provide especially useful for the next phase of this research.     

Controlling the Degree of Functionalization: In-Depth Quantification and Side-Product Analysis of Diazonium Chemistry on SWCNTs

We present an in-depth qualitative and quantitative analysis of a reaction between 4-iodobenzenediazonium tetrafluoroborate and single-walled carbon nanotubes (SWCNTs) via thermogravimetric analysis coupled with mass spectrometry (TG-MS) or a gas chromatography and mass spectrometry (TG-GC-MS) as well as Raman spectroscopy. We propose a method for precise determination of the degree of functionalization and quantification of physisorbed aromates, detaching around their boiling point, alongside covalently bonded ones (cleavage over 200 °C). While the presence of some side products like phenol- or biphenyl species could be excluded, residual surfactant and minor amounts of benzene could be identified. A concentration-dependent experiment shows that the degree of functionalization increases with the logarithm of the concentration of applied diazonium salt, which can be exploited to precisely adjust the amount of aryl addends on the nanotube sidewall, up to 1 moiety per 100 carbon atoms.     

“Smart” poly(2‐(dimethylamino)ethyl methacrylate‐ran‐9‐(4‐vinylbenzyl)‐9H‐carbazole) copolymers synthesized by nitroxide mediated radical polymerization

A series of poly(2‐(dimethylamino)ethyl methacrylate‐ran‐9‐(4‐vinylbenzyl)‐9H‐carbazole) (poly(DMAEMA‐ran‐VBK)) random copolymers, with VBK molar feed compositions f_VBK,0 = 0.02–0.09, were synthesized using 10 mol % [tert‐butyl[1‐(diethoxyphosphoryl)‐2,2‐dimethylpropyl]amino] nitroxide (SG1) relative to 2‐([tert‐butyl[1‐(diethoxyphosphoryl)‐2,2‐dimethylpropyl]amino]oxy)‐2‐methylpropionic acid (BlocBuilder) at 80 °C and 90 °C. Controlled polymerizations were observed, even with f_VBK,0 = 0.02, as reflected by a linear increase in number average molecular weight (M_n) versus conversion X ≤ 0.6 with final copolymers characterized by relatively narrow, monomodal molecular weight distributions (M_w/M_n ≈ 1.5). Poly(DMAEMA‐ran‐VBK) copolymers were deemed sufficiently pseudo‐“living” to reinitiate a second batch of N,N‐dimethylacrylamide (DMAA), with very few apparent dead chains, as indicated by the monomodal shift in the gel permeation chromatography chromatograms. Poly(DMAEMA‐ran‐VBK) random copolymers exhibited tuneable lower critical solution temperature (LCST), in aqueous solution, by modifying copolymer composition, solution pH and by the addition of the water‐soluble poly(DMAA) segment. ¹H NMR analysis determined that, in water, the VBK units of the poly(DMAEMA‐ran‐VBK) random copolymer were segregated to the interior of the copolymer aggregate regardless of solution temperature and that poly(DMAEMA‐ran‐VBK)‐b‐poly(DMAA) block copolymers formed micelles above the LCST. In addition, the final random copolymer and block copolymer exhibited temperature dependent fluorescence due to the VBK units. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Nitroxide‐mediated radical copolymerization of methyl methacrylate controlled with a minimal amount of 9‐(4‐vinylbenzyl)‐9H‐carbazole

The controlled nitroxide‐mediated homopolymerization of 9‐(4‐vinylbenzyl)‐9H‐carbazole (VBK) and the copolymerization of methyl methacrylate (MMA) with varying amounts of VBK were accomplished by using 10 mol % {tert‐butyl[1‐(diethoxyphosphoryl)‐2,2‐dimethylpropyl]amino} nitroxide relative to 2‐({tert‐butyl[1‐(diethoxyphosphoryl)‐2,2‐dimethylpropyl]amino}oxy)‐2‐methylpropionic acid (BlocBuilder?) in dimethylformamide at temperatures from 80 to 125 °C. As little as 1 mol % of VBK in the feed was required to obtain a controlled copolymerization of an MMA/VBK mixture, resulting in a linear increase in molecular weight versus conversion with a narrow molecular weight distribution (M_w /M_n ≈ 1.3). Preferential incorporation of VBK into the copolymer was indicated by the MMA/VBK reactivity ratios determined: r_VBK = 2.7 ± 1.5 and r_MMA = 0.24 ± 0.14. The copolymers were found significantly “living” by performing subsequent chain extensions with a fresh batch of VBK and by ³¹P NMR spectroscopy analysis. VBK was found to be an effective controlling comonomer for NMP of MMA, and such low levels of VBK comonomer ensured transparency in the final copolymer. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Direct and iterative methods for interval parametric algebraic systems producing parametric solutions

This paper deals with interval parametric linear systems with general dependencies. Motivated by the so‐called parameterized solution introduced by Kolev, we consider the enclosures of the solution set in a revised affine form. This form is advantageous to a classical interval solution because it enables us to obtain both outer and inner bounds for the parametric solution set and, thus, intervals containing the endpoints of the hull solution, among others. We propose two solution methods, a direct method called the generalized expansion method and an iterative method based on interval‐affine Krawczyk iterations. For the iterative method, we discuss its convergence and show the respective sufficient criterion. For both methods, we perform theoretical and numerical comparisons with some other approaches. The numerical experiments, including also interval parametric linear systems arising in practical problems of structural and electrical engineering, indicate the great usefulness of the proposed methodology and its superiority over most of the existing approaches to solving interval parametric linear systems.     

Stability of the linear complementarity problem properties under interval uncertainty

Central European Journal of Operations Research - We consider the linear complementarity problem with uncertain data modeled by intervals, representing the range of possible values. Many properties...