Synthesis, Characterization, and Direct Intracellular Imaging of Ultrasmall and Uniform Glutathione‐Coated Gold Nanoparticles

Gold nanoparticles (AuNPs) with core sizes below 2 nm and compact ligand shells constitute versatile platforms for the development of novel reagents in nanomedicine. Due to their ultrasmall size, these AuNPs are especially attractive in applications requiring delivery to crowded intracellular spaces in the cytosol and nucleus. For eventual use in vivo, ultrasmall AuNPs should ideally be monodisperse, since small variations in size may affect how they interact with cells and how they behave in the body. Here we report the synthesis of ultrasmall, uniform 144‐atom AuNPs protected by p ‐mercaptobenzoic acid followed by ligand exchange with glutathione (GSH). Quantitative scanning transmission electron microscopy (STEM) reveals that the resulting GSH‐coated nanoparticles (Au(GSH)) have a uniform mass distribution with cores that contain 134 gold atoms on average. Particle size dispersity is analyzed by analytical ultracentrifugation, giving a narrow distribution of apparent hydrodynamic diameter of 4.0 ± 0.6 nm. To evaluate the nanoparticles’ intracellular fate, the cell‐penetrating peptide TAT is attached noncovalently to Au(GSH), which is confirmed by fluorescence quenching and isothermal titration calorimetry. HeLa cells are then incubated with both Au(GSH) and the Au(GSH)‐TAT complex, and imaged without silver enhancement of the AuNPs in unstained thin sections by STEM. This imaging approach enables unbiased detection and quantification of individual ultrasmall nanoparticles and aggregates in the cytoplasm and nucleus of the cells. The synthesis and characterization of an ultrasmall and uniform glutathione‐coated gold nanoparticle is reported. It is also shown that scanning transmission electron microscopy (STEM) enables the visualization and quantification of individual gold nanoparticles as well as small aggregates in the cytoplasm and nucleus of HeLa cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92372/1/2277_ftp.pd

Pyrimidine Nucleotides with 4-Alkyloxyimino and Terminal Tetraphosphate δ-Ester Modifications as Selective Agonists of the P2Y 4 Receptor

P2Y2 and P2Y4 receptors are G protein-coupled receptors, activated by UTP and dinucleoside tetraphosphates, which are difficult to distinguish pharmacologically for lack of potent and selective ligands. We varied structurally phosphate and uracil moieties in analogues of pyrimidine nucleoside 5′-triphosphates and 5′-tetraphosphate esters. P2Y4 receptor potency in phospholipase C stimulation in transfected 1321N1 human astrocytoma cells was enhanced in N4-alkyloxycytidine derivatives. OH groups on a terminal δ-glucose phosphoester of uridine 5′-tetraphosphate were inverted or substituted with H or F to probe H-bonding effects. N4-(Phenylpropoxy)-CTP 16 (MRS4062), Up4-[1]3′-deoxy-3′-fluoroglucose 34 (MRS2927) and N4-(phenylethoxy)-CTP 15 exhibit ≥10-fold selectivity for human P2Y4 over P2Y2 and P2Y6 receptors (EC50 values 23, 62 and 73 nM, respectively). δ-3-Chlorophenyl phosphoester 21 of Up4 activated P2Y2 but not P2Y4 receptor. Selected nucleotides tested for chemical and enzymatic stability were much more stable than UTP. Agonist docking at CXCR4-based P2Y2 and P2Y4 receptor models indicated greater steric tolerance of N4-phenylpropoxy group at P2Y4. Thus, distal structural changes modulate potency, selectivity, and stability of extended uridine tetraphosphate derivatives, and we report the first P2Y4 receptor-selective agonists

Structure-Based Design of Reactive Nucleosides for Site-Specific Modification of the A_2A Adenosine Receptor

Adenosine receptors (ARs) are members of the G protein-coupled receptor (GPCR) superfamily and have shown much promise as therapeutic targets. We have used an agonist-bound A_2AAR X-ray crystallographic structure to design a chemically reactive agonist for site-specific chemical modification of the receptor. To further explore and chemically engineer its binding cavity, a 2-nitrophenyl active ester was attached through an elongated chain at adenine C2 position. This general structure was designed for irreversible transfer of a terminal acyl group to a nucleophilic amino group on the A_2AAR. Preincubation with several <i>O</i>-acyl derivatives prevented radioligand binding that was not regenerated upon extensive washing. In silico receptor docking suggested two lysine residues (second extracellular loop) as potential target sites for an <i>O</i>-acetyl derivative (MRS5854, <b>3a</b>), and site-directed mutagenesis indicated that K153 but not K150 is essential. Similarly, a butyl azide for click reaction was incorporated in the active ester moiety (<b>3b</b>). These promising results indicate a stable, covalent modification of the receptor by several reactive adenosine derivatives, which could be chemical tools for future imaging, structural probing, and drug discovery. Thus, structure-based ligand design has guided the site-specific modification of a GPCR

Adenine nucleotide control of coronary blood flow during exercise

The adenine nucleotide hypothesis postulates that the ATP released from red blood cells is broken down to ADP and AMP in coronary capillaries and that ATP, ADP, and AMP act on purinergic receptors on the surface of capillary endothelial cells. Purinergic receptor activation initiates a retrograde conducted vasodilator signal to the upstream arteriole that controls coronary blood flow in a negative feedback manner. A previous study (M. Farias 3rd, M. W. Gorman, M. V. Savage, and E. O. Feigl, Am J Physiol Heart Circ Physiol 288: H1586–H1590, 2005) demonstrated that coronary venous plasma ATP concentration increased during exercise and correlated with coronary blood flow. The present experiments test the adenine nucleotide hypothesis by examining the balance between oxygen delivery (via coronary blood flow) and myocardial oxygen consumption during exercise before and after purinergic receptor blockade. Dogs (n = 7) were chronically instrumented with catheters in the aorta and coronary sinus and a flow transducer around the circumflex coronary artery. During control treadmill exercise, myocardial oxygen consumption increased and the balance between oxygen delivery and myocardial oxygen consumption fell as indicated by a declining coronary venous oxygen tension. Blockade of P1 and P2Y1 purinergic receptors combined with inhibition of nitric oxide synthesis significantly decreased the balance between oxygen delivery and myocardial oxygen consumption compared with control. The results support the hypothesis that ATP and its breakdown products ADP and AMP are part of a negative feedback control mechanism that matches coronary blood flow to myocardial oxygen consumption at rest and during exercise

Pyrimidine Nucleotides with 4-Alkyloxyimino and Terminal Tetraphosphate δ-Ester Modifications as Selective Agonists of the P2Y 4

P2Y(2) and P2Y(4) receptors are G protein-coupled receptors, activated by UTP and dinucleoside tetraphosphates, which are difficult to distinguish pharmacologically for lack of potent and selective ligands. We varied structurally phosphate and uracil moieties in analogues of pyrimidine nucleoside 5′-triphosphates and 5′-tetraphosphate esters. P2Y(4) receptor potency in phospholipase C stimulation in transfected 1321N1 human astrocytoma cells was enhanced in N(4)-alkyloxycytidine derivatives. OH groups on a terminal δ-glucose phosphoester of uridine 5′-tetraphosphate were inverted or substituted with H or F to probe H-bonding effects. N(4)-(Phenylpropoxy)-CTP 16 (MRS4062), Up(4)-[1]3′-deoxy-3′-fluoroglucose 34 (MRS2927) and N(4)-(phenylethoxy)-CTP 15 exhibit ≥10-fold selectivity for human P2Y(4) over P2Y(2) and P2Y(6) receptors (EC(50) values 23, 62 and 73 nM, respectively). δ-3-Chlorophenyl phosphoester 21 of Up(4) activated P2Y(2) but not P2Y(4) receptor. Selected nucleotides tested for chemical and enzymatic stability were much more stable than UTP. Agonist docking at CXCR4-based P2Y(2) and P2Y(4) receptor models indicated greater steric tolerance of N(4)-phenylpropoxy group at P2Y(4). Thus, distal structural changes modulate potency, selectivity, and stability of extended uridine tetraphosphate derivatives, and we report the first P2Y(4) receptor-selective agonists