Dieter H. Klaubert

A novel acidotropic pH indicator and its potential application in labeling acidic organelles of live cells

BACKGROUND Ratio imaging has received intensive attention in the past few decades. The growing potential of ratio imaging is significantly limited, however, by the lack of appropriate fluorescent probes, for acidic organelles in particular. The classic fluorescent dyes (such as fluoresceins, rhodamines and coumarins) are not suitable for studying acidic organelles (such as lysosomes) because their fluorescence is significantly decreased under neutral or acidic conditions. This has motivated us to develop probes that can be used in ratio imaging that are strongly fluorescent even in acidic media. RESULTS The compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethyl-aminocarbamoyl) methoxy)phenyl)oxazole (PDMPO) was prepared and characterized as a new acidotropic dual-excitation and dual-emission pH indicator. It emits intense yellow fluorescence at lower pH and gives intense blue fluorescence at higher pH. This unique pH-dependent fluorescence property was readily explored to selectively stain lysosomes and to determine the pH of the organelle in an emission-ratio-imaging mode. PDMPO is selectively localized to lysosomes and exhibits a pH-dependent dual excitation and emission. CONCLUSIONS PDMPO selectively labels acidic organelles (such as lysosomes) of live cells and the two distinct emission peaks can be used to monitor the pH fluctuations of live cells in ratio measurements. Additionally, the very large Stokes shift and excellent photostability of PDMPO make the compound an ideal fluorescent acidotropic probe. The unique fluorescence properties of PDMPO might give researchers a new tool with which to study acidic organelles of live cells.

Fluorescent molecular probes V: A sensitive caspase-3 substrate for fluorometric assays

(Z-Asp-Glu-Val-Asp)-Rhodamine 110 [(Z-DEVD)2-Rh 110] was prepared and characterized as a sensitive fluorogenic substrate for the determination of caspase-3 activity.

Fluorescent Molecular Probes II. The Synthesis, Spectral Properties and Use of Fluorescent Solvatochromic Dapoxyl Dyes

Abstract— 2,5‐Diphenyloxazoles that embody a dimethylamino group at position 4 of the 5‐phenyl ring and a sulfonyl group at position 4 of the 2‐phenyl ring were prepared as new fluorescent solvatochromic dyes. In these molecules, there is a “push‐pull” electron transfer system from the 5‐phenyl moiety to the 2‐phenyl ring. These compounds show strong solvent‐dependent fluorescence that is well correlated with the empirical solvent polarity parameter ET (30). The solvent polarity dependence suggests that the fluorescence arises from an intramolecular charge transfer. The fluorescence‐environment dependence, long emission wavelength, large extinction coefficients, high fluorescence quantum yields and large Stokes shift of the fluorophores can be used to develop ultrasensitive fluorescent molecular probes to study a variety of biological events and processes.

A facile protocol for the convenient preparation of amino-substituted α-bromo- and α,α-dibromo arylmethylketones

Abstract Amino-substituted arylmethylketones are selectively brorninated in sulfuric acid to afford the corresponding dibromomethylarylketones that are then debrominated with diethylphosphite to give the desired bromomethylarylketones in excellent yield.

Addition of arylmetallics to azodicarboxylates: A novel synthesis of arylhydrazines by aromatic hydrazination

Abstract Aryllithiums and arylmagnesium bromides add to the NN bond of di-t-butyl azodicarboxylate, and subsequent acid deprotection provides the arylhydrazine.

Fluorescent molecular probes VI : The spectral properties and potential biological applications of water-soluble Dapoxyl sulfonic acid

Abstract Dapoxyl sulfonic acid was characterized as a new water-soluble fluorescent solvatochromic dye. This molecule contains a ‘push–pull’ electron transfer system from the 5-phenyl moiety to the 2-phenyl ring that generates the environment-sensitive fluorescence. The strong environment-sensitive fluorescence of Dapoxyl sulfonic acid can be explored to study a variety of biological events and processes, in addition to its long emission wavelength, large extinction coefficient, high fluorescence quantum yield and large Stokes shift and excellent photostability.

Fluorescent molecular probes I. The synthesis and biological properties of an ELF® β-glucuronidase substrate that yields fluorescent precipitates at the enzymatic activity sites

Abstract A novel quinazolinone-based fluorogenic β-glucuronidase substrate has been synthesized and evaluated. In this protocol, a partially protected salicyl glucuronide is oxidatively condensed with anthranilamide to afford the required quinazolinonyl glucuronic acid methyl ester that is readily deprotected to yield the desired glucuronide, known as ELF®-97 glucuronide. The glucuronide is a sensitive fluorogenic β-glucuronidase substrate that yields a bright yellow-green fluorescent precipitate upon enzymatic reaction.

Fluorescent molecular probes III. 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) : A new polar dual-excitation and dual-emission pH indicator with a pKa of 7.0

Abstract 2′,7′-Bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) was prepared and characterized as a new polar dual-excitation and dual-emission pH indicator with a pKa of 7.0.

Novel derivatization of protein thiols with fluorinated fluoresceins

Abstract Fluorescein diacetate analogs 3a–3c are shown to react selectively with protein sulfhydryl groups at neutral pH in aqueous solution, by means of substitution at the perfluorinated ring. The acetate moieties are cleaved by treatment with aqueous carbonate or by endogenous esterases, giving a fluorescent protein conjugate.

Lipophilic sulfophenylcarbocyanine dyes: Synthesis of a new class of fluorescent cell membrane probes

Abstract Two lipophilic sulfophenylcarbocyanine dyes, 3 and 4 , were synthesized as a new class of fluorescent cell membrane probes. Compared with other commonly used membrane probes, the new dyes have improved fluorescent quantum yield, retention in cell membranes and are easier to use in staining cells.