The Fe(II)- and alpha-ketoglutarate-dependent dioxygenases catalyze hydroxylation reactions of considerable biomedical and environmental significance. Recently, the first oxidized iron intermediate in the reaction of a member of this family, taurine:alpha-ketoglutarate dioxygenase (TauD), was detected and shown to be a high-spin, formally Fe(IV) complex. The demonstration in this study that decay of the Fe(IV) complex is approximately 30-fold slower when it is formed in the presence of 1-[2H]2-taurine provides evidence that the intermediate abstracts hydrogen from C1, the site of hydroxylation, and suggests that quantum-mechanical tunneling may contribute to C1-H cleavage.

[reaction: see text] A boronic acid-containing coumarin aldehyde was designed and synthesized. The sensor binds to catecholamines such as dopamine and norepinephrine by forming an iminium ion with the amine as well as a boronate ester with the catechol. An internal hydrogen bond produces a colorimetric response to these analytes with good selectivity for catecholamines over simple amines. The fluorescence of the sensor is quenched by the catechol.

Properly substituted coumarin aldehydes can be used to detect amines and amino acids under neutral, high ionic strength conditions by the formation of highly fluorescent iminium ions. The fluorescence of one sensor increases by 26-fold upon the addition of glycine. This strong florescent response is attributed to hydrogen bonding of the chromophore carbonyl by the acidic iminium proton.

In the preceding communication, 1 we described the synthesis of a potentially general precursor (2, Scheme 1) of the highly promising chemotherapeutic agent Taxol 2 (1, Scheme 2) and its analogues. Our strategy for the elaboration of this ABbicyclic precursor into the ABC-tricyclic core of the taxanes was predicated on the view 1b that epimerization of the C7 center of Taxol 3 proceeds through the intermediacy of the AB-bicyclic enolaldehyde or its ketone isomer, leading to the intriguing possibility that the C-ring of Taxol could self-assemble under exceptionally mild conditions from a considerably less complex AB-bicyclic ketoaldehyde precursor (e.g., 9). In this communication, the viability of this aldol cyclization strategy is demonstrated in a synthesis of Taxol (1), representing the shortest sequence yet reported for the preparation of this important natural product. 4,5 The elaboration of our general taxane precursor (2, Scheme 1) into Taxol started with its homologation with Ph 3 PC(H)-OMe (91%) 6 followed by a one-step hydrolysis of the enol ether and acetonide groups (HCl, NaI) to provide aldehyde 3 (94%). 7 Selective protection of the C9 hydroxyl was then accomplished in 92% yield with TESCl and pyridine. Dess-Martin periodinane oxidation 8 of the C10 alcohol and introduction of C20 with [Me 2 NCH 2 ]I (g0.1 M) 9 and Et 3 N (excess) was conducted in one operation to produce enal 4 in 97% yield. The remaining carbons of the taxane skeleton were then introduced through the addition of 4 to a solution of allylmagnesium bromide and ZnCl 2 (89%) 10 which after BOM (benzyloxymethyl) protection (N,N-diisopropylethylamine solvent) provided the ether 5 as a single diastereomer. 10c The presence of ZnCl 2 in the former reaction completely suppressed addition of the Grignard reagent to the cyclic carbonate. Removal of the C9 silyl group (NH 4 F, MeOH) 11 provided an unstable hydroxyketone (93% over two steps) which was reacted immediately with PhLi 12 to form the C2 benzoate providing, after in situ acetylation, the acetate 6 in 79% yield. Transposition of the acetoxyketone under kinetic 5a or equilibrating conditions (Et 2 NH, KOAc, DMF) 13 resulted in limited success. However, when the guanidinium base 7 14 was employed for this transposition, the desired acetoxyketone 8 and recyclable 6 were obtained in 80% as a 4:3 equilibrium mixture. The monosubstituted alkene in 8 was then cleaved through addition of an ozone solution to form aldehyde 9 in 86% yield.The viability of the key aldol cyclization was addressed at this point. Previous studies in our laboratory 1b,15 showed that ketoaldehydes similar to 9 but incorporating a C1-C2 cyclic carbonate did not undergo aldol cyclization, preferring instead

A method for the selective labeling and imaging of catecholamines in live and fixed secretory cells is reported. The method integrates a tailored approach using a novel fluorescence-based turn-on molecular sensor (NeuroSensor 521) that can exploit the high concentration of neurotransmitters and acidic environment within secretory vesicles for the selective recognition of norepinephrine and dopamine. The utility of the method was demonstrated by selectively labeling and imaging norepinephrine in secretory vesicles such that discrimination between norepinephrine-and epinephrine-enriched populations of chromaffin cells was observed. This method was validated in fixed cells by co-staining with an anti-PNMT antibody. KEYWORDS: Fluorescent sensor, catecholamine, norepinephrine, cell imaging, chromaffin T he catecholamines dopamine, norepinephrine, and epinephrine are the principal neurotransmitters in the sympathetic nervous system. 1 In particular, norepinephrine regulates many critical functions that include attention, memory, learning, emotion, and autonomic and cardiovascular function. In the periphery, norepinephrine increases heart rate, cardiac contractility, vascular tone, renin-angiotensin system activity, and renal sodium reabsorption. 2 Norepinephrine is secreted by chromaffin cells, which package catecholamines at high concentrations (0.5−1.0 M) and at low pH (5.0−5.5) in neurosecretory vesicles. 3 Chromaffin cells possess approximately 30 000 large dense-core vesicles (LDCV) with norepinephrine and epinephrine. 4 Chromaffin cells that store and release mainly epinephrine can be separated from those that utilize mainly norepinephrine through density-gradient centrifugation, though a third subpopulation which secrete both epinephrine and norepinephrine has been identified via cyclic voltametry. 5 Over the years, chromaffin cells have become a standard platform for the study of processes related to exocytosis. Thus, chromaffin cells appeared to be an ideal platform for the study of novel sensors for neurotransmitters.Currently, catecholamines can be studied via electrochemical and chromatographic techniques that provide characterization and quantification, although these techniques can only provide crude spatial information. 6 Recently, fluorescent false neurotransmitters (FFNs) have been developed which are selectively loaded into vesicles that express neuronal vesicular monoamine transporter (VMAT) and represent an optical approach for labeling vesicles containing catecholamines and imaging catecholamine release at the single-vesicle level. 7 However, FFNs are loaded into all secretory vesicles expressing the VMAT protein without discrimination to cell type and thus, the approach cannot distinguish distinct cell populations that secrete a particular neurotransmitter.Fluorescent sensors remain a compelling technology for approaching the general problem of selective neurotransmitter detection. In recent years, a number of catecholamine sensors have been reported including RNA aptamers, fluorescen...