Dynamic Nitroxide Functional Materials.

A substrate-independent and versatile coating platform for (spatially resolved) surface functionalization, based on nitroxide radical coupling (NRC) reactions and the formation of thermo-labile alkoxyamine functional groups, was introduced. Nitroxide-decorated poly(glycidyl methacrylate) (PGMA) microspheres, obtained through bioinspired copolymer surface deposition using dopamine and a nitroxide functional dopamine derivative as monomers, were conjugated with small functional groups in a rewritable process. Reversible coding of the nitroxide functional microspheres by NRC and decoding through thermal alkoxyamine fission were monitored and characterized by electron paramagnetic resonance (EPR) spectroscopy and X-ray photoelectron spectroscopy (XPS). In addition, this nitroxide coating system was exploited in "grafting-to" polymer surface ligations of poly(methyl methacrylate) (PMMA) and poly(2,2,2-trifluoroethyl methacrylate) (PTFEMA) in spatially confined areas. Polymer strands terminated with an Irgacure 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) photoinitiator were obtained through chain-transfer polymerization, and subsequently coupled to nitroxide-immobilized poly(dopamine) (PDA)-coated silicon substrates by using rapid photoclick NRC reactions. Light-driven polymer surface coding was visualized by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and XPS imaging.

Wavelength Dependence of Light-Induced Cycloadditions.

The wavelength-dependent conversion of two rapid photoinduced ligation reactions, i.e., the light activation of o-methylbenzaldehydes, leading to the formation of reactive o-quinodimethanes (photoenols), and the photolysis of 2,5-diphenyltetrazoles, affording highly reactive nitrile imines, is probed via a monochromatic wavelength scan at constant photon count. The transient species are trapped by cycloaddition with N-ethylmaleimide, and the reactions are traced by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. The resulting action plots are assessed in the context of Beer-Lambert's law and provide combined with time-dependent density functional theory and multireference calculations an in-depth understanding of the underpinning mechanistic processes, including conical intersections. The π → π* transition of the carbonyl group of the o-methylbenzaldehyde correlates with a highly efficient conversion to the cycloadduct, showing no significant wavelength dependence, while conversion following the n → π* transition proceeds markedly less efficient at longer wavelengths. The influence of absorbance and reactivity has critical consequences for an effective reaction design: At high concentrations of o-methylbenzaldehydes (c = 8 mmol L-1), photoligations with N-ethylmaleimide (possible for λ ≤ 390 nm) are ideally performed at 330 nm, whereas at high light penetration regimes at lower concentrations (c = 0.3 mmol L-1), 315 nm irradiation leads to the highest conversion. Activation and trapping of 2,5-diphenyltetrazoles (possible for λ ≤ 322 nm) proceeds best at a wavelength shorter than 295 nm, irrespective of concentration.

Wavelength-Dependent Photochemical Stability of Photoinitiator-Derived Macromolecular Chain Termini.

Herein, we report the unique-and first time-wavelength-dependent investigation with strictly monochromatic light of 305-405 nm wavelength into the stability of photoinitiator-derived chain termini of poly(methyl methacrylate) using a tunable laser system fused with pulsed-laser irradiation and size exclusion chromatography hyphenated to high-resolution electrospray mass spectrometry (PLI-SEC-ESI-MS). We assess several substitution patterns of methyl groups on the common benzoyl-type radical fragment. Critically, methyl substitution in the 2- and 6-positions of the benzoyl moiety, i.e., in both ortho-positions, resulted in stable chain ends up to approximately 350 nm. The stability can be attributed to a blue-shift of the n-π* transitions (relevant for the end group reactivity) as predicted by earlier density functional theory (DFT) calculations on model species. In sharp contrast, our experiments show a far reduced stability of the end groups commencing from 400 nm onwards, when the dual ortho-methyl substitution in the benzoyl fragment is missing. Thus, we demonstrate that the substitution pattern on the phenyl ring of the benzoyl group dictates the chain end stability as a function of wavelength in excellent agreement with the quantum chemical predictions. Our study thus provides critical insights into selecting suitable photoinitiation systems for specific wavelength regimes.

Imaging Single-Chain Nanoparticle Folding via High-Resolution Mass Spectrometry.

Herein, we introduce the first approach to map single-chain nanoparticle (SCNP) folding via high-resolution electrospray ionization mass spectrometry (ESI MS) coupled with size exclusion chromatography. For the first time, the successful collapse of polymeric chains into SCNPs is imaged by characteristic mass changes, providing detailed mechanistic information regarding the folding mechanism. As SCNP system we employed methyl methacrylate (MMA) statistically copolymerized with glycidyl methacrylate (GMA), resulting in p(MMA-stat-GMA), subsequently collapsed by using B(C6F5)3 as catalyst. Both the precursor polymer and the SCNPs can be well ionized via ESI MS, and the strong covalent cross-links are stable during ionization. Our high-resolution mass spectrometric approach can unambiguously differentiate between two mechanistic modes of chain collapse for every chain constituting the SCNP sample.

Two-in-One: λ-Orthogonal Photochemistry on a Radical Photoinitiating System.

An alkyne functional radical photoinitiator, 2-(4-(2-hydroxy-2-methylpropanoyl)phenoxy)ethyl hex-5-ynoate, and evidence that both reactive moieties - the alkyne and the photoinitiator terminus - can be independently addressed with light of disparate wavelength (λ-orthogonality) are introduced. The alkyne functionality is subjected to a visible light (420 nm) induced copper-catalyzed Huisgen reaction, which is employed for the selective functionalization of the initiator with a poly(ethylene glycol) (PEG) chain. This reaction proceeds completely λ-orthogonal in the presence of the UV-reactive photoinitiating moiety. Conversely, it is demonstrated that the alkyne functionality of the photoinitiator is quantitatively orthogonal to UV irradiation emitted by the pulsing action of an excimer laser (351 nm, pulsed-laser polymerization, PLP) and the generated radical species. In turn, the PEGylated initiator can readily be employed as a macrophotoinitiator during PLP. The introduced λ-orthogonally addressable dual functional initiator can be used in a wide range of applications, including surface lithography and post-synthetic modification of photocured materials.

Bis(mesitoyl)phosphinic acid: photo-triggered release of metaphosphorous acid in solution.

Bis(mesitoyl)phosphinic acid and its sodium salt display a unique photo-induced reactivity: both derivatives stepwise release two mesitoyl radicals and, remarkably, metaphosphorous acid (previously postulated as transient species in the gas phase), providing a new phosphorus-based reagent.

Cleaning the Click: A Simple Electrochemical Avenue for Copper Removal from Strongly Coordinating Macromolecules.

We introduce a novel electrochemical method for the purification of complex water-soluble functional polymers contaminated with copper salts originating from copper-catalyzed azide/alkyne ligation chemistry, for which no standard purification protocol is suitable. A triethylene glycol methyl ether methacrylate (TEGMA) star polymer with 2-ureido-4H-pyrimidone (UPy) end groups was prepared via an activator generated by electron transfer atom transfer radical polymerization (AGET ATRP) and copper-catalyzed azide/alkyne cycloaddition (CuAAc) and selected as a model system for electrolysis of an aqueous polymer solution. We systematically investigate the influence of sample concentration, voltage, and time of electrolysis on the quality of the purification. Atom emission spectroscopy (AES) reveals almost quantitative removal of copper, and size exclusion chromatography (SEC) as well as proton nuclear magnetic resonance spectroscopy (1H NMR) ensure the full integrity of the polymer under all selected conditions.

Cell Penetrating Peptoids (CPPos): Synthesis of a Small Combinatorial Library by Using IRORI MiniKans.

Cell penetrating peptoids (CPPos) are potent mimics of the corresponding cell penetrating peptides (CPPs). The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a "proof of principle" peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines)] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.

Atrial septal defects versus ventricular septal defects in BREATHE-5, a placebo-controlled study of pulmonary arterial hypertension related to Eisenmenger's syndrome: a subgroup analysis.

BACKGROUND: Eisenmenger's syndrome (ES) is the most advanced form of pulmonary arterial hypertension related to congenital heart disease. Evolution of pulmonary vascular disease differs markedly between patients with atrial septal defects (ASD) versus ventricular septal defects (VSD), potentially affecting response to treatment. We compared the effects of bosentan and placebo in patients with isolated ASD (ASD subgroup) versus patients with isolated VSD or both defects (VSD subgroup). METHODS: Post-hoc analysis of a 16-week, multicenter, randomized, double-blind, placebo-controlled trial was performed. Fifty-four patients (13: ASDs, 36: VSDs, 5: VSD+ASD) were randomized to bosentan 62.5 mg bid for four weeks (uptitrated to 125 mg bid thereafter) or placebo. Main outcome measures were: indexed pulmonary vascular resistance (PVRi), exercise capacity, mean pulmonary artery pressure (mPAP), pulmonary blood flow index (Qpi), and changes in oxygen saturation (SpO2). RESULTS: Placebo-corrected median (95% CI) treatment effects on PVRi were -544.0 dyn·s·cm⁻5 (-1593.8, 344.7) and -436.4 dyn·s·cm⁻5 (-960.0, 167.0) in the ASD and VSD subgroups, respectively. Effects of bosentan on exercise capacity and mPAP were similar in both subgroups. No changes in SpO2 or Qpi were observed in either bosentan or placebo subgroups. CONCLUSIONS: Improvements in exercise capacity and cardiopulmonary hemodynamics, without desaturation, were observed in ES patients with both ASDs and VSDs. Although not reaching statistical significance, improvements were similar to those in the BREATHE-5 analyses, suggesting that the location of septal defects is not a key determinant of treatment response. These data further support the use of bosentan for the treatment of ES, independent of shunt location.

Longer-term bosentan therapy improves functional capacity in Eisenmenger syndrome: results of the BREATHE-5 open-label extension study.

BACKGROUND: Bosentan, an oral endothelin ET(A)/ET(B) receptor antagonist, improves hemodynamics and exercise capacity in patients with Eisenmenger syndrome but longer-term effects are unknown. This study investigated the efficacy and safety of bosentan up to 40 weeks in these patients. METHODS: Following the 16-week, double blind, placebo-controlled BREATHE-5 study of bosentan in patients with Eisenmenger syndrome, an open-label extension (OLE) was performed. Patients who completed BREATHE-5 received bosentan for an additional 24 weeks (62.5 mg b.i.d. for 4 weeks, then 125 mg b.i.d.) and were analyzed in two groups; ex-placebo and ex-bosentan, according to BREATHE-5 treatment. RESULTS: Thirty-seven patients with Eisenmenger syndrome who participated in BREATHE-5 were included in the OLE. At week 24, the 6-minute walk distance (mean+/-SE) increased from OLE baseline for the ex-placebo (+33.2+/-23.9 m) and ex-bosentan group (+6.7+/-10.0 m). The overall improvement from baseline of BREATHE-5 was +61.3+/-8.1 m (95% confidence interval: [44.7, 78.0]) for the ex-bosentan group. WHO functional class was improved in both groups. Bosentan did not reduce systemic arterial blood oxygen saturation; safety profile was comparable to previous trials. CONCLUSIONS: In conclusion, these longer follow-up data support the efficacy and safety profile reported in the preceding BREATHE-5 study of bosentan treatment of Eisenmenger syndrome, challenging the notion that pulmonary vascular disease and severe functional impairment in these patients are not amenable to therapy.

Bosentan therapy in patients with Eisenmenger syndrome: a multicenter, double-blind, randomized, placebo-controlled study.

BACKGROUND: Eisenmenger syndrome is characterized by the development of pulmonary arterial hypertension with consequent intracardiac right-to-left shunt and hypoxemia in patients with preexisting congenital heart disease. Because Eisenmenger syndrome is associated with increased endothelin expression, patients may benefit from endothelin receptor antagonism. Theoretically, interventions that have some effect on the systemic vascular bed could worsen the shunt and increase hypoxemia. METHODS AND RESULTS: The Bosentan Randomized Trial of Endothelin Antagonist Therapy-5 (BREATHE-5) was a 16-week, multicenter, randomized, double-blind, placebo-controlled study evaluating the effect of bosentan, a dual endothelin receptor antagonist, on systemic pulse oximetry (primary safety end point) and pulmonary vascular resistance (primary efficacy end point) in patients with World Health Organization functional class III Eisenmenger syndrome. Hemodynamics were assessed by right- and left-heart catheterization. Secondary end points included exercise capacity assessed by 6-minute walk distance, additional hemodynamic parameters, functional capacity, and safety. Fifty-four patients were randomized 2:1 to bosentan (n=37) or placebo (n=17) for 16 weeks. The placebo-corrected effect on systemic pulse oximetry was 1.0% (95% confidence interval, -0.7 to 2.8), demonstrating that bosentan did not worsen oxygen saturation. Compared with placebo, bosentan reduced pulmonary vascular resistance index (-472.0 dyne.s.cm(-5); P=0.0383). The mean pulmonary arterial pressure decreased (-5.5 mm Hg; P=0.0363), and the exercise capacity increased (53.1 m; P=0.0079). Four patients discontinued as a result of adverse events, 2 (5%) in the bosentan group and 2 (12%) in the placebo group. CONCLUSIONS: In this first placebo-controlled trial in patients with Eisenmenger syndrome, bosentan was well tolerated and improved exercise capacity and hemodynamics without compromising peripheral oxygen saturation.