Facile synthesis of thiol-reactive Cy3 and Cy5 derivatives with enhanced water solubility

The cyanine dyes Cy3 and Cy5 have proven valuable in numerous applications involving conjugation with proteins. Practical syntheses of lysine-selective, succinimidyl ester derivatives of these dyes have been published, and succinimidyl esters are commercially available. However, the published syntheses of cysteine-selective derivatives produce relatively low yields from expensive starting materials, or produce molecules with marginal water solubility for protein labeling. We report here facile syntheses (four steps, >50% overall yield) of iodoacetamide, sulfhydryl-reactive derivatives of the Cy3 and Cy5 fluorophores. These novel derivatives have good water solubility (>2.5 mM) and bear only one reactive side chain, reducing possible protein cross-linking encountered with previous derivatives.     

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 相似文献   

Specific labeling of polypeptides at amino-terminal cysteine residues using Cy5-benzyl thioester

Even for moderately sized proteins, the multiple occurrence of cysteine and lysine residues often prevents the specific labeling of polypeptides with a single probe. To increase specificity, a method was developed to convert the commonly available succinimidyl esters of fluorescent dyes into benzyl thioesters via trimethyl aluminum-activated benzyl mercaptan. The thioester can then be reacted very specifically with polypeptides containing an N-terminal cysteine residue, forming a stable amide bond, analogous to the native chemical ligation of peptide fragments. Both reaction steps are easy to perform and proceed to high yields. The practicability of the approach was demonstrated using the popular cyanine dye Cy5 and a soluble peptide, and it is expected to be applicable to a wide range of succinimidyl esters and both chemically and recombinantly synthesized proteins. The method should dramatically facilitate the preparation of proteins for experiments requiring exact positioning of labels, for instance, F?rster resonance energy transfer studies.     

Cy5 total protein normalization in Western blot analysis

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0 μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence.     

Comparison of the Sequence-Dependent Fluorescence of the Cyanine Dyes Cy3, Cy5, DyLight DY547 and DyLight DY647 on Single-Stranded DNA

Cyanine dyes are commonly used for fluorescent labeling of DNA and RNA oligonucleotides in applications including qPCR, sequencing, fluorescence in situ hybridization, Förster resonance energy transfer, and labeling for microarray hybridization. Previous research has shown that the fluorescence efficiency of Cy3 and Cy5, covalently attached to the 5′ end of single-stranded DNA, is strongly sequence dependent. Here, we show that DY547 and DY647, two alternative cyanine dyes that are becoming widely used for nucleic acid labeling, have a similar pattern of sequence-dependence, with adjacent purines resulting in higher intensity and adjacent cytosines resulting in lower intensity. Investigated over the range of all 1024 possible DNA 5mers, the intensities of Cy3 and Cy5 drop by ∼50% and ∼65% with respect to their maxima, respectively, whereas the intensities of DY547 and DY647 fall by ∼45% and ∼40%, respectively. The reduced magnitude of change of the fluorescence intensity of the DyLight dyes, particularly of DY647 in comparison with Cy5, suggests that these dyes are less likely to introduce sequence-dependent bias into experiments based on fluorescent labeling of nucleic acids.     

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.     

Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.     

A highly fluorescent DNA toolkit: synthesis and properties of oligonucleotides containing new Cy3, Cy5 and Cy3B monomers

Cy3B is an extremely bright and stable fluorescent dye, which is only available for coupling to nucleic acids post-synthetically. This severely limits its use in the fields of genomics, biology and nanotechnology. We have optimized the synthesis of Cy3B, and for the first time produced a diverse range of Cy3B monomers for use in solid-phase oligonucleotide synthesis. This molecular toolkit includes phosphoramidite monomers with Cy3B linked to deoxyribose, to the 5-position of thymine, and to a hexynyl linker, in addition to an oligonucleotide synthesis resin in which Cy3B is linked to deoxyribose. These monomers have been used to incorporate single and multiple Cy3B units into oligonucleotides internally and at both termini. Cy3B Taqman probes, Scorpions and HyBeacons have been synthesized and used successfully in mutation detection, and a dual Cy3B Molecular Beacon was synthesized and found to be superior to the corresponding Cy3B/DABCYL Beacon. Attachment of Cy3, Cy3B and Cy5 to the 5-position of thymidine by an ethynyl linker enabled the synthesis of an oligonucleotide FRET system. The rigid linker between the dye and nucleobase minimizes dye-dye and dye-DNA interactions and reduces fluorescence quenching. These reagents open up new future applications of Cy3B, including more sensitive single-molecule and cell-imaging studies.     

Two-photon induced fluorescence of Cy5-DNA in buffer solution and on silver island films

We report the observation of a strong two-photon induced fluorescence emission of Cy5-DNA within the tunable range of a Ti:Sapphire laser. The estimated two-photon cross-section for Cy5-DNA of 400GM is about 3.5-fold higher than it was reported for rhodamine B. The fundamental anisotropies of Cy5-DNA are close to the theoretical limits of 2/5 and 4/7 for one- and two-photon excitation, respectively. We also observed an enhanced two-photon induced fluorescence (TPIF) of Cy5-DNA deposited on silver island films (SIFs). In the presence of SIFs, the TPIF is about 100-fold brighter. The brightness increase of Cy5-DNA TPIF near SIFs is mostly due to enhanced local field.     

Synthesis of fluorescent analogs of alpha-conotoxin MII

Alpha-conotoxins (alpha-CTxs) are small peptides that are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) and have been used to study the kinetics of nAChRs. Alpha-CTx MII, from the venom of Conus magus, has been shown to potently block both rat alpha3beta2 and rat chimeric alpha6/alpha3beta2beta3 cloned nAChRs expressed in Xenopus oocytes. Tetramethylrhodamine (TMR), Bodipy FL, Alexa Fluor 488, and terbium chelates (TbCh) are fluorescent molecules that can be reacted with the N-terminus of the conopeptide to produce fluorescent conjugates. TMR and Bodipy FL were individually conjugated to alpha-CTx MII using different succinimidyl ester amine labeling reactions resulting in the formation of carboxamide conjugates. Alexa Fluor 488 succinimidyl ester conjugation reaction yielded low amounts of conjugate. TbCh was also individually reacted with the N-terminus of MII using the isothiocyanate conjugation reaction resulting in the formation of a thiourea conjugate. The conjugates were purified using reverse-phase high-pressure liquid chromatography (RP-HPLC) and their masses verified by matrix-assisted laser desorption-ionization with time-of-flight mass spectroscopy (MALDI-TOF MS). When tested on target nAChRs expressed in Xenopus oocytes, TMR-MII, Bodipy FL-MII, and TbCh-MII potently blocked the response to acetylcholine with slow off-rate kinetics. These fluorescent conjugates can be used to localize specific subtypes of neuronal nAChRs or ligand-binding sites within receptors in various tissue preparations; additionally, they may also be used to study conformational changes in receptors using fluorescence or lanthanide-based resonance energy transfer.     

Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.     

Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.     

In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)(4)

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)(4 )presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin alpha(V)beta(3) with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a "stealth" molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)(4)-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)(4)-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)(4)-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)(4)-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.     

Synthesis and properties of fluorescent NF-kappa B-recognizing hairpin oligodeoxyribonucleotide decoys

Intramolecular fluorescence quenching of cyanine dyes was investigated using a model hairpin oligonucleotide decoy encoding a NF-kappaB p50 subunit binding site. Two types of hairpin oligonucleotides were synthesized: (1) 5'-(6-aminohexyl)- and 3'-(3-aminopropyl)-linked (I); (2) 5'-(6-aminohexyl)- and 3'-[3-(3-hydroxypropyldithio)propyl]-linked (II). Oligonucleotide I was covalently modified using monofunctional either Cy3- or Cy5.5-N-hydroxysuccinimide esters. Using reverse-phase HPLC, mono-and dicyanineamide derivatives of I were isolated. Mono-Cy3-modified derivatives of I, but not the mono-Cy5.5-modified derivatives, showed a 2-fold higher Cy3 fluorescence intensity compared to the free dye. There was no detectable difference in fluorescence between the di-Cy3 derivative of I and the free dye at the same concentration. However, there was a 4-fold quenching of fluorescence in the case of the di-Cy5.5 derivative of the same hairpin oligonucleotide. The quenching of Cy5.5 fluorescence could not be explained by the interaction of Cy5.5 with nucleotide bases as demonstrated by incubating free Cy5.5 dye with oligonuclotides. The quenching effect was further investigated using an oligonucleotide bearing a cleavable 3'-amino-terminated linker bearing an S-S bond (III). After modification of the 5'- and 3'-end of oligonucleotide III with a Cy5.5 monofunctional hydroxysuccinimide ester, a 70-75% quenching of fluorescence was observed. Fluorescence was 100% dequenched after the reduction of S-S bond. The obtained result unequivocally demonstrates that the formation of intramolecular Cy5.5 dimers is the dominant mechanism of fluorescence quenching in symmetric dye-dye hairpin decoy beacons.     

Measurement of the intracellular pH in Escherichia coli with the internally conjugated fluorescent probe 5- (and 6-)carboxyfluorescein succinimidyl ester

The 5- (and 6-)carboxyfluorescein succinimidyl ester (cFDASE) is a pH-dependent fluorescent probe which conjugates with the aliphatic amine functions of the cells. Because of this property, the fluorescence efflux rate is low, 10 to 40% after 30 min of fermentable carbon source incubation. Thus, this technique allows a continuous high-time measurement (>30 min) without correction of the signal, but also a dynamic study phenomenon of few seconds. Due to the advantages presented by this technique, the method was adapted for Escherichia coli. The internal pH measurement obtained under various conditions are in accuracy with those from radiolabelled technique and from other fluorescence probe, with the advantages mentioned previously.     

Design consideration and probes for fluorescence resonance energy transfer studies

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 A in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.     

Synthesis and biological applications of two novel fluorescent proteins-labeling probes

Two novel chlorinated fluoresceins 2′,4′,5′,7′-tetrachloro-6-(5-carboxypentyl)-4,7-dichloro fluorescein succinimidyl ester (1G) and 2′,4′,5′,7′-tetrachloro-6-(3-carboxypropyl)-4,7-dichlorofluorescein succinimidyl ester (2G) were synthesized as fluorescent probes for labeling proteins. Structures of target compounds and intermediates were determined via IR, MS, ¹H NMR and element analysis. The investigation in immunofluorescence histochemistry showed them had strong fluorescence, high photostability and good biocompatibility.     

Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, (125)I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with (125)I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).     

Conjugation of the photoluminescent silicon nanoparticles to streptavidin

We have covalently attached multiple photoluminescent silicon nanoparticles (SNs) to streptavidin molecules. Conjugation of SNs to a target protein is achieved using the multistage photoassisted procedure. In a first step, the terminal hydrogen in the freshly prepared SNs is substituted with an alkane monolayer that serves as a platform for chemical linkage to a heterobifunctional cross-linker: 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester. A resulting surface coating stabilizes nanoparticles against oxidation and aggregation. Next, an open end of bifunctional cross-linker-diazirine succinimidyl ester is reacted with carboxyl moieties of streptavidin and forms an amide bond. Gel and capillary electrophoresis of the SN-streptavidin complex demonstrated separate elution of the conjugation product and unreacted protein. Then, the number of SNs per protein molecule was determined by measuring complex charge variation by capillary electrophoresis. Conjugate functionality was tested by allowing it to interact with biotinylated polystyrene microbeads. Intense photoluminescence at carefully washed microbeads demonstrated selective binding of silicon nanoparticle bearing streptavidin to biotinylated microbeads. The high quantum yield of streptavidin-SN conjugate in combination with the small size and biocompatibility of silicon nanoparticles presents an attractive platform for the fluorescence labeling in diverse bioassays.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.

In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.