Tailoring B-doped silicon nanocrystal surface chemistry via phosphorus pentachloride - mediated surface alkoxylation.

Doped silicon nanocrystals (SiNCs) are promising materials that could find use in a wide variety of applications. Realizing methods to tailor the surface chemistry of these particles offers greater tunability of the material properties as well as broader solvent compatibility. Herein, we report organic-soluble B-doped SiNCs prepared via a thermal processing method followed by phosphorus pentachloride etching induced functionalization with alkoxy ligands of varied chain lengths. This approach provides a scalable route to solution processable B-doped SiNCs and establishes a potential avenue for the functionalization of other doped SiNCs.

A Potent Fluorescent Reversible-Covalent Inhibitor of Cardiac Muscle Contraction.

Compounds that directly modulate the response of the cardiac sarcomere have potential in the treatment of cardiac disease. While a number of sarcomere activators have been discovered and extensively studied, very few inhibitors have been identified. We report a potent cardiac sarcomere inhibitor, DN-F01, targeting the cardiac muscle thin filament protein troponin complex. Functional studies show that DN-F01 has a strong inhibitory calcium-dependent effect on cardiac myofibrillar ATPase activity with an IC50 value of 11 ± 4 nmol/L. DN-F01 is shown to bind to a cardiac troponin C-troponin I chimera (cChimera) with a K D of ∼50 nM using fluorescence spectroscopy, indicating that troponin is the likely target for DN-F01. NMR titrations of DN-F01 to C35S and A-Cys cChimera show covalent and noncovalent binding of DN-F01 bound to the calcium-saturated cChimera.

The Role of Electrostatics in the Mechanism of Cardiac Thin Filament Based Sensitizers.

Heart muscle contraction is regulated by calcium binding to cardiac troponin C. This induces troponin I (cTnI) switch region binding to the regulatory domain of troponin C (cNTnC), pulling the cTnI inhibitory region off actin and triggering muscle contraction. Small molecules targeting this cNTnC-cTnI interface have potential in the treatment of heart disease. Most of these have an aromatic core which binds to the hydrophobic core of cNTnC, and a polar and often charged 'tail'. The calmodulin antagonist W7 is unique in that it acts as calcium desensitizer. W7 binds to the interface of cNTnC and cTnI switch region and weakens cTnI binding, possibly by electrostatic repulsion between the positively charged terminal amino group of W7 and the positively charged RRVR144-147 region of cTnI. To evaluate the role of electrostatics, we synthesized A7, where the amino group of W7 was replaced with a carboxyl group. We determined the high-resolution solution NMR structure of A7 bound to a cNTnC-cTnI chimera. The structure shows that A7 does not change the overall conformation of the cNTnC-cTnI interface, and the naphthalene ring of A7 sits in the same hydrophobic pocket as that of W7, but the charged tail takes a different route to the surface of the complex, especially with respect to the position of the switch region of cTnI. We measured the affinities of A7 for cNTnC and the cNTnC-cTnI complex and that of the cTnI switch peptide for the cNTnC-A7 complex. We also compared the binding of W7 and A7 for two cNTnC-cTnI chimeras, differing in the presence or absence of the RRVR region of cTnI. A7 decreased the binding affinity of cTnI to cNTnC substantially less than W7 and bound more tightly to the more positively charged chimera. We tested the effects of W7 and A7 on the force-calcium relation of demembranated rat right ventricular trabeculae and demonstrated that A7 has a much weaker desensitization effect than W7. We also synthesized A6, which has one less methylene group on the hydrocarbon chain than A7. A6 did not affect binding of cTnI switch peptide nor change the calcium sensitivity of ventricular trabeculae. These results suggest that the negative inotropic effect of W7 may result from a combination of electrostatic repulsion and steric hindrance with cTnI.

Optimizing fluorine labelling for 19F solid-state NMR in oriented biological systems.

When planning a fluorine labeling strategy for 19F solid state NMR (ssNMR) studies of the structure and/or mobility of fluorine labeled compounds in situ in an oriented biological system, it is important to characterize the NMR properties of the label. This manuscript focuses on the characterization of a selection of aromatic fluorine compounds in dimyristoylphosphatidylcholine bilayers using 19F ssNMR from the standpoint of determining the optimum arrangement of fluorine nuclei on a pendant aromatic ring before incorporation into more complex biological systems.

Feasibility of trifluoromethyl TROSY NMR at high magnetic fields.

Insights into the structure and dynamics of large biological systems has been greatly improved by two concurrent NMR approaches: the application of transverse relaxation-optimized spectroscopy (TROSY) techniques in multi-dimensional NMR, especially the methyl-TROSY, and the resurgence of 19F NMR using trifluoromethyl (CF3) probes. Herein we investigate the feasibility of combining these approaches into a trifluoromethyl-TROSY experiment. Using a CF3-labelled parvalbumin, we have evaluated the natural abundance 13C-19F correlation spectra and find no indication of a CF3 TROSY at high magnetic fields.

Thioimidate Bond Formation between Cardiac Troponin C and Nitrile-containing Compounds.

We have investigated the mechanism and reactivity of covalent bond formation between cysteine-84 of the regulatory domain of cardiac troponin C and compounds containing a nitrile moiety similar to the calcium sensitizer levosimendan. The results of modifications to the levosimendan framework ranged from a large increase in covalent bond formation to complete inactivity. We present the biological activity of one of the most potent compounds. Limitations, including compound solubility and degradation at acidic pH, have prevented thorough investigation of the potential of these compounds. Our studies reveal the efficacious nature of the malononitrile moiety in targeting cNTnC and its potential in future cardiotonic drug design.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.