Derivatives molecular probes

Other lipid compositions with synthetic lipids, hydrogenated SPC (HSPC) and PEG-modified phospholipids are often used, especially for liposome formulations intended for parenteral applications use (long circulating or stealth liposomes) (41). Several analytical methods to follow loss of lipids during the preparation steps are available. Radioactively labeled lipids ( H-DPPC, C-DPPC) or cholesterol ( H-cholesterol) or H-cholesteryl hexadecyl ether (NEN Life Science Products, Boston, MA, USA) or lipophilic fluorescence dyes (e.g. lipophilic BODIPy derivatives. Molecular Probes) are added at appropriate amounts to the initial lipid mixtures. 

The bacterial CAT enzyme catalyzes the transfer of acetyl groups to chloramphenicol from acetyl coenzyme A (acetyl CoA). In a typical assay, this reaction is monitored with relabeled chloramphenicol After separation by thin-layer chromatography (TLC), the acetylated and nonacetylated forms can be distinguished by autoradiography, and quantitation is achieved by isolating the forms and measuring their radioactivity in a scintillation counter. Quantitative CAT assays have been performed on Drosophila tissue culture and dissociated cell extracts (Di Nocera and Dawid 1983 Benyajati and Dray 1984 Thummel et al. 1988 Krasnow et al. 1989 Ye et al. 1997). CAT can also be detected with commercially available antibodies. In addition, a nonradioactive CAT assay exists that utilizes a fluorescent chloramphenicol derivative (Molecular Probes). 

Studies on covalent hydration of N-heterocycles (67AG(E)919,76AHC(20)117) have revealed the diagnostic value of alkyl substituents in structural assignments due to their steric hindrance effects in addition reactions. C-Methyl substituents are therefore also considered as molecular probes to solve fine-structural problems in the pteridine field. The derivatives... 

If the photoequilibrium concentrations of the cis and trans isomers of the photoswitchable ionophore in the membrane bulk and their complexation stability constants for primary cations are known, the photoinduced change in the concentration of the complex cation in the membrane bulk can be estimated. If the same amount of change is assumed to occur for the concentration of the complex cation at the very surface of the membrane, the photoinduced change in the phase boundary potential may be correlated quantitatively to the amount of the primary cation permeated to or released from the membrane side of the interface under otherwise identical conditions. In such a manner, this type of photoswitchable ionophore may serve as a molecular probe to quantitatively correlate between the photoinduced changes in the phase boundary potential and the number of the primary cations permselectively extracted into the membrane side of the interface. Highly lipophilic derivatives of azobis(benzo-15-crown-5), 1 and 2, as well as reference compound 3 were used for this purpose (see Fig. 9 for the structures) [43]. Compared to azobenzene-modified crown ethers reported earlier [39 2], more distinct structural difference between the cis... 

The disassembly pathway of molecular probe 39 is initiated by catalytic cleavage of phenylacetic acid by PGA, elimination of azaquinone methide, decarboxylation, and cyclization to release dimethylurea derivative and phenol 40 (Fig. 5.38). The latter rapidly undergoes double quinone methide elimination to release the two reporter units and by-product 41. The output of these cascade... 

BODIPY is a short for 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, the basic structure of this type of fluorophore (see Fig. 6.5). Derivatives of this dye have been created by modification of positions 1, 3, 5, 7, and 8, generating an array of fluorophores with very distinct excitation and emission properties [38]. Molecular Probes has synthesized a wide number of BODIPY dyes whose excitation... 

In the literature, fluorescence spectroscopy in OFD has been limited to the use of ultraviolet (UV) or visible dyes as molecular probes.(1) The most common fluorescent dye used in OFD is fluorescein and its derivatives/21 23) Fluorescein possesses a good fluorescence quantum yield and is commercially available with an isothiocyanate functionality for linking to the polymeric support/24-26 Additionally, selective laser excitation can be performed because the absorbance maximum of fluorescein coincides with the 499-nm laser line emitted from an argon laser. Unfortunately, argon lasers are costly and bulky, thus limiting the practicality of their use. Similar difficulties exist with other popular commercial dyes. 

It is at present still difficult to correlate the absolute intensity of the SHG with the number of cationic complexes at the membrane surface. Therefore, a quantitative discussion, showing how the permselective uptake of primary cations forming SHG active complexes into the membrane side of the phase boundary corresponds to the increase in the membrane potential, is not possible yet. Lipophilic derivatives of photoswitchable azobis(benzo-15-crown-5) were recently shown as a molecular probe to determine photoinduced changes in the amount of the primary cation uptake into the membrane phase boundary in relation to the photoinduced EMF changes under otherwise identical conditions. 

Studies by Photoswitchable Azobis(benzo-15-crown-5) Derivatives as a Molecular Probe... 

Bengali women to symbolize marriage. Only Bengali women wear the red spot at the center of the forehead. The decorative spot worn in the middle of the forehead by other Indian women is—today—pasted on. Much of the history of Rose Bengal has been reviewed elsewhere [241], Citations to Rose Bengal have increased in the last three or so years, and the diversity of its application has greatly expanded. Many of the Rose Bengal derivatives listed are now commercially available from either Molecular Probes or Aldrich. 

Another hydrazine derivative of fluorescein, 5-(((2-(Carbohydrazino)methyl)thio)-acetyl)-aminofluorescein, contains a longer spacer arm off its No. 5 carbon atom of its lower ring than fluorescein-5-thiosemicarbazide, described previously (Molecular Probes). The reagent can be used to react spontaneously with aldehyde- or ketone-containing molecules forming a hydrazone linkage (Fig. 209). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits a maximal excitation at 490 nm and a maximal luminescence emission peak at 516 nm when dissolved in buffer at pH 8. In the same buffered environment, the compound has an extinction coefficient of approximately 75,000 M-1cm 1 at 490 nm. 

The iodoacetamido derivatives of tetramethylrhodamine possess a sulfhydryl-reactive iodoacetyl group (Chapter 1, Section 4.2, and Chapter 2, Section 2.1) at either the 5-or 6-carbon position on their lower ring. The isomers are commercially available only in mixed form, but some reactivity and specificity differences between the purified 5 and 6 derivatives toward various sulfhydryl sites in proteins may be observed (Ajtai, 1992) (Molecular Probes, Pierce). 

Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with hydrazine (Molecular Probes, Pierce). The result is a sulfonyl hydrazine group on the No. 5 carbon position of the lower ring structure of sulforhodamine 101. The intense Texas Red fluorophore has a quantum yield that is inherently higher than either the tetramethylrhodamine or the Lissamine rhodamine B derivatives of the basic rhodamine molecule. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes derivatives of this fluorescent probe among the best choices of labels for use in double staining techniques. 

Diethylamino-3-[(4 -(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA) is a derivative of the basic aminomethylcoumarin structure that contains a sulfhydryl-reactive iodoacetyl group and diethyl substitutions on its amine. This particular cou-marin derivative is among the most fluorescent UV-excitable iodoacetamide probes available (Sippel, 1981) (Molecular Probes). 

BODIPY 530/550 C3 is 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (Molecular Probes). This derivative of the basic BODIPY structure contains two phenyl rings off the No. 5 and 7 carbon atoms and a propionic acid group on the No. 3 carbon atom. The carboxylate group may be used to attach the fluo-rophore to amine-containing molecules via a carbodiimide reaction to create an amide bond. The substituents on this BODIPY fluorophore result in alterations to its spectral properties, pushing its excitation and emission maximums up to higher wavelengths. 

BODIPY 593/503 C3 hydrazide is 4,4-difluoro-l,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionyl hydrazide (Molecular Probes). Unlike BODIPY 530/550 C3 hydrazide, this BODIPY derivative contains substituents that shift to lower wavelengths the spectral characteristics of its fluorescent properties. The molecule is highly reactive toward aldehyde-containing compounds, including glycoproteins that have been oxidized with sodium periodate to create the requisite groups (Fig. 230). 

One Lucifer Yellow derivative is available for labeling sulfhydryl-containing molecules. Lucifer Yellow iodoacetamide is a 4-ethyliodoacetamide derivative of the basic disulfonate aminonaphthalimide fluorophore structure (Molecular Probes). The io-doacetyl groups react with —SH groups in proteins and other molecules to form stable thioether linkages (Fig. 240). 

The absorption and emission maxima from this table will provide clues to the spectral ranges that are useful for excitation and for fluorescence detection with a particular fluorochrome. However, the absorption and emission spectra have breadth, with slopes and shoulders and secondary peaks (see Fig. 5.6). With efficient fluorochromes, excitation and fluorescence detection at wavelengths distant from the maxima may be possible. Therefore, inspection of the full, detailed spectra is necessary to get the full story. In addition, spectra may shift in different chemical environments (this will explain why maxima vary in different reference tables from different sources). Values in this table are derived primarily from the Molecular Probes Handbook and the article by Alan Waggoner (Chapter 12) in Melamed et al. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

Recently, BOCILLIN FL, a fluorescent penicillin, was synthesized for the detection and characterization of PBPs [34]. BOCILLIN FL, a derivative of penicillin V, is an orange solid with extinction coefficient of 68,000 M 1 cm1 and a maximal absorption at 504 nm (Molecular Probes, Inc.). It fluoresces at 511 nm upon excitation at 504 nm. BOCILLIN FL has been used to detect PBPs from the membrane preparations of several bacterial species, including S. pneumoniae, E. coli, and P. aeruginosa (Fig. 3). A typical procedure of using BOCILLIN FL for the detection of PBPs involves preparation of the bacterial cytoplasmic membranes, incubation of the membrane preparations with BOCILLIN FL, and visualization of PBPs by a UV transilluminator or with the aid of a... 

---