This section includes dyes that have absorption maxima beyond about 520 nm, extending to nearly 800 nm. Significant exceptions, however, are the long-wavelength Alexa Fluor dyes, which are all discussed in Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3, the long-wavelength BODIPY dyes—BODIPY TMR, BODIPY TR, BODIPY 630/650 and BODIPY 650/665—which are described in BODIPY Dye Series—Section 1.4 and the 2',4',5',7'-tetrabromofluorescein (eosin) and JOE dyes, which also absorb maximally beyond 520 nm but are discussed with other fluoresceins in Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5. In many applications, the versatile Alexa Fluor and BODIPY dyes provide demonstrably superior performance relative to the dyes in this section.
Rhodamine dyes (Amine-reactive, orange- and red-fluorescent fluorophores in this section—Table 1.9) are among the most photostable fluorescent labeling reagents available. Moreover, spectra of most of these dyes are not affected by changes in pH between 4 and 10, an important advantage over the fluoresceins for many biological applications. The most common members of this group have been the tetramethylrhodamines—including the reactive isothiocyanate (TRITC) and carboxylic acid (TAMRA) derivatives—as well as the X-rhodamines. The X prefix of the X-rhodamines, which include Texas Red derivatives, refers to the fluorophore's extra julolidine rings (Figure 1.6.1). These rings prevent rotation about the nitrogen atoms, resulting in a shift in the fluorophore's spectra to longer wavelengths and usually an increase in its fluorescence quantum yield.
QSY 7, QSY 9 and QSY 21 dyes are essentially nonfluorescent diarylrhodamine chromophores with strong absorption in the visible wavelength region, and they have proven to be extremely effective fluorescence quenchers. QSY 7, QSY 9 and QSY 21 dyes complement the QSY 35 dye, a nonfluorescent quencher based on the NBD fluorophore that absorbs maximally near 475 nm, and the dabcyl quencher, both of which are described in Reagents for Analysis of Low Molecular Weight Amines—Section 1.8.
Figure 1.6.1 The amine substituents of X-rhodamine, sulforhodamine 101 and Texas Red dyes are rigidified in a julolidine ring structure.
Tetramethylrhodamine (TMR) has been an important fluorophore for preparing protein conjugates, especially the fluorescent antibody and avidin derivatives used in immunochemistry. Under the name TAMRA, the carboxylic acid of TMR has also achieved prominence as a dye for oligonucleotide labeling (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Amine-reactive dyes for nucleic acid sequencing—Table 8.7) and single-molecule detection applications. Because it can be prepared in high purity, the 5-isomer of TAMRA (C6121) is one of the five dyes in our Reference Dye Sampler Kit (R14782, Fluorescence Microscopy Accessories and Reference Standards—Section 23.1). TMR is efficiently excited by the 543 nm spectral line of the green He-Ne laser, which is increasingly being used for analytical instrumentation; diode lasers with 561 nm output are slightly suboptimal but still effective.
TMR dyes such as TAMRA and TRITC are quite hydrophobic () when compared with their fluorescein counterparts FAM and FITC. As a result, they have a tendency to aggregate in aqueous solutions under conditions where the labeling density is sufficient to permit dye–dye interactions. A further consequence of these interactions is fluorescence self-quenching, which reduces the fluorescence output of the conjugate. Dye–dye interactions and self-quenching are much less prevalent with the more polar and water-soluble Alexa Fluor dyes. Another indication of intermolecular interactions of TMR dyes is that the absorption spectrum of TMR-labeled proteins is frequently complex (Figure 1.6.2), usually splitting into two absorption peaks at about 520 and 550 nm, so that the actual degree of labeling is difficult to determine. Excitation at wavelengths in the range of the short-wavelength peak fails to yield the expected amount of fluorescence, indicating that it arises from a nonfluorescent dye aggregate. Furthermore, when the TMR-labeled protein conjugate is denatured by guanidine hydrochloride, the long-wavelength absorption increases, the short-wavelength peak mostly disappears and the fluorescence yield almost doubles (Figure 1.6.2). The absorption spectra of TMR-labeled nucleotides and of other probes such as our rhodamine phalloidin (R415, Probes for Actin—Section 11.1) do not split into two peaks, indicating a labeling ratio of one dye molecule per biomolecule. The emission spectrum of TMR conjugates does not vary much with the degree of labeling. An improved method for estimating the degree of substitution of TRITC conjugates has been described.
Figure 1.6.2 Effect of protein conjugation on the absorption spectrum of tetramethylrhodamine. The absorption spectrum of tetramethylrhodamine conjugated to goat anti–mouse IgG antibody (TMR-GAM, T2762) shows an additional peak at about 520 nm when compared with the spectrum of the same concentration of the free dye (TMR). Partial unfolding of the protein in the presence of 4.8 M guanidine hydrochloride (TMR-GAM + GuHCl) results in a spectrum more similar to that of the free dye.
Mixed-Isomer and Single-Isomer TRITC Preparations
Our tetramethylrhodamine isothiocyanate (TRITC) is of the highest quality available from any commercial source. Both the mixed-isomer (T490) and single-isomer (T1480, T1481) TRITC preparations typically have extinction coefficients above 80,000 cm-1M-1, whereas some competitive sources of TRITC have extinction coefficients reported to be below 50,000 cm-1M-1. TRITC is widely used by other companies to prepare most of their so-called "rhodamine" immunoconjugates; however, they also often employ reactive versions of rhodamine B or Lissamine rhodamine B, which have somewhat different spectra, resulting in some confusion in matching the product name to the correct fluorophore.
Succinimidyl Esters of Carboxytetramethylrhodamine
Almost all Molecular Probes TMR conjugates are prepared using succinimidyl esters of carboxytetramethylrhodamine (TAMRA dye), rather than TRITC, because bioconjugates from succinimidyl esters are more stable and often more fluorescent. We offer the mixed-isomer (C300) and single-isomer (C6121, C6122) preparations of carboxytetramethylrhodamine, as well as the corresponding mixed-isomer (C1171) and single-isomer (C2211, C6123) succinimidyl esters. The single-isomer preparations are most important for high-resolution techniques such as DNA sequencing and separation of labeled carbohydrates by capillary electrophoresis. 6-TAMRA dye is one of the traditional fluorophores (5-FAM, 6-JOE, 6-TET, 6-HEX, 6-TAMRA and 6-ROX dyes) used in first-generation electrophoresis-based DNA sequencing (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Amine-reactive dyes for nucleic acid sequencing—Table 8.7).
We have also prepared the mixed-isomer TAMRA-X succinimidyl ester (5(6)-TAMRA-X, SE; T6105), which contains a seven-atom aminohexanoyl spacer ("X") between the reactive group and the fluorophore (). This spacer helps to separate the fluorophore from its point of attachment, reducing the interaction of the fluorophore with the biomolecule to which it is conjugated, making it more accessible to secondary detection reagents and facilitating orientational averaging in fluorescence resonance energy transfer (FRET) applications (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Polyclonal anti-tetramethylrhodamine and anti–Texas Red dye antibodies that recognize the tetramethylrhodamine, Rhodamine Red-X, X-rhodamine and Texas Red fluorophores are available (Anti-Dye and Anti-Hapten Antibodies—Section 7.4).
Lissamine Rhodamine B Sulfonyl Chloride
Lissamine rhodamine B sulfonyl chloride (L20, L1908; ) is much less expensive than Texas Red sulfonyl chloride (see below), and the fluorescence emission spectrum of its protein conjugates lies between those of tetramethylrhodamine and Texas Red conjugates (Figure 1.6.3). It is more frequently employed as a synthetic precursor for preparing affinity labeling reagents than as a labeling reagent for protein conjugation.
Figure 1.6.3 Normalized fluorescence emission spectra of goat anti–mouse IgG antibody conjugates of 1) fluorescein, 2) rhodamine 6G, 3) tetramethylrhodamine, 4) Lissamine rhodamine B and 5) Texas Red dyes.
Rhodamine Red-X Succinimidyl Ester
Lissamine rhodamine B sulfonyl chloride is unstable, particularly in aqueous solution, making it somewhat difficult to achieve reproducible conjugations using this dye. Unlike Lissamine rhodamine B sulfonyl chloride, which is a mixture of isomeric sulfonyl chlorides (), Rhodamine Red-X succinimidyl ester (R6160, ) is isomerically pure and is hydrolytically stable for practical purposes at the mild alkaline pH levels typically used for amine-reactive protein conjugation. Rhodamine Red-X succinimidyl ester incorporates a spacer between the fluorophore and the reactive site, resulting in minimized perturbation of the conjugation partner's functional properties. Moreover, we have found that protein conjugates of Rhodamine Red-X dye are frequently brighter than those of Lissamine rhodamine B (Figure 1.6.4), and less likely to precipitate during storage. Rhodamine Red-X succinimidyl ester is used in the FluoReporter Rhodamine Red-X Protein Labeling Kit (F6161); see Kits for Labeling Proteins and Nucleic Acids—Section 1.2 for further information on preparing red-fluorescent protein conjugates with this kit.
Figure 1.6.4 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates of Rhodamine Red-X succinimidyl ester (R6160, ) and Lissamine rhodamine B sulfonyl chloride (L20, L1908, ). Conjugate fluorescence is determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein. Higher numbers of fluorophores attached per protein are attainable with Rhodamine Red-X dye due to the lesser tendency of this dye to induce protein precipitation.
The derivatives of carboxy-X-rhodamine (ROX dye)—a dye originally developed in our laboratories in 1986—are widely used for oligonucleotide labeling and DNA sequencing applications (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Amine-reactive dyes for nucleic acid sequencing—Table 8.7). Conjugates of this dye and of the similar isothiocyanate (5(6)-XRITC, X491; ) have longer-wavelength spectra () than the spectra of Lissamine rhodamine B, but somewhat shorter-wavelength spectra than those of Texas Red conjugates. Both the pure 5-isomer (C6124) and 6-isomer (C6156) of carboxy-X-rhodamine are available, as are mixed-isomer (C1309, ) and single-isomer (C6125, C6126) preparations of the succinimidyl ester.
The Texas Red fluorophore emits at a longer wavelength than do either tetramethylrhodamine or Lissamine rhodamine B (Figure 1.6.3), making Texas Red conjugates among the most commonly used long-wavelength "third labels" in fluorescence microscopy (, ). Unlike the other rhodamines, the Texas Red fluorophore exhibits very little spectral overlap with fluorescein (Figure 1.6.3), and its fluorescence can be distinguished from that of phycoerythrins. When the correct optical filter sets are used, Texas Red conjugates are brighter and have lower background than conjugates of the other commonly used red-fluorescent dyes, with the exception of the Alexa Fluor 594 dye. Texas Red conjugates are particularly well suited for excitation by the 594 nm spectral line of the orange He-Ne laser; diode laser excitation at 561 nm is also efficient.
Texas Red Sulfonyl Chloride
Texas Red sulfonyl chloride is our trademarked mixture of isomeric sulfonyl chlorides () of sulforhodamine 101. This reagent is quite unstable in water, especially at the higher pH required for reaction with aliphatic amines. For example, dilute solutions of Texas Red sulfonyl chloride are totally hydrolyzed within 2–3 minutes in pH 8.3 aqueous solution at room temperature. Protein modification by this reagent is best done at low temperature. Once conjugated, however, the sulfonamides that are formed (Figure 1.6.5) are extremely stable; they even survive complete protein hydrolysis.
Because Texas Red sulfonyl chloride rapidly degrades upon exposure to moisture, we offer this reactive dye specially packaged as a set of 10 vials (T1905), each containing approximately 1 mg of Texas Red sulfonyl chloride for small-scale conjugations. We also offer the 10 mg unit size packaged in a single vial (T353) for larger-scale conjugations. Each milligram of Texas Red sulfonyl chloride modifies approximately 8–10 mg of protein. Note that sulfonyl chlorides are unstable in dimethylsulfoxide (DMSO) and should never be used in that solvent. Polyclonal anti-tetramethylrhodamine and anti–Texas Red antibodies that recognize tetramethylrhodamine, Rhodamine Red, X-rhodamine and Texas Red fluorophores are available (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Selected haptenylation reagents and their anti-hapten antibodies—Table 4.2).
Figure 1.6.5 Reaction of a primary amine with a sulfonyl chloride.
Texas Red-X Succinimidyl Ester
Texas Red sulfonyl chloride's susceptibility to hydrolysis and low solubility in water may complicate its conjugation to some biomolecules. To overcome this difficulty, we have developed Texas Red-X succinimidyl ester, which contains an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and its reactive group. The single-isomer preparation of Texas Red-X succinimidyl ester (T20175, ) is preferred over the mixed-isomer product (T6134) when the dye is used to prepare conjugates of low molecular weight peptides, oligonucleotides and receptor ligands that are to be purified by high-resolution techniques. Also, because isomers of a reactive dye may differ in their binding geometry, certain applications such as fluorescence resonance energy transfer (FRET) may benefit from the use of single-isomer reactive dyes (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Thiol-reactive Texas Red derivatives that are based on a similar synthetic approach are described in Thiol-Reactive Probes Excited with Visible Light—Section 2.2. Texas Red-X succinimidyl ester offers significant advantages over Texas Red sulfonyl chloride for the preparation of bioconjugates:
- In the absence of amines, greater than 80% of Texas Red-X succinimidyl ester's reactivity is retained in pH 8.3 solution after one hour at room temperature.
- Much less Texas Red-X succinimidyl ester (usually half or less of the amount of Texas Red sulfonyl chloride) is required to yield the same degree of labeling, making the effective costs of these two reagents about the same.
- Conjugations with Texas Red-X succinimidyl ester are more reproducible.
- Unlike Texas Red sulfonyl chloride, which can form unstable products with tyrosine, histidine, cysteine and other residues in proteins, the Texas Red-X succinimidyl ester reacts almost exclusively with amines.
- Protein conjugates prepared with Texas Red-X succinimidyl ester have a higher fluorescence yield than those with the same labeling ratio prepared with Texas Red sulfonyl chloride (Figure 1.6.6).
- Texas Red-X protein conjugates show a decreased tendency to precipitate during the reaction or upon storage.
Figure 1.6.6 Comparison of the relative fluorescence of goat anti–mouse IgG antibody conjugates of Texas Red-X succinimidyl ester (T6134, ) and Texas Red sulfonyl chloride (T353, ). Conjugate fluorescence was determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of the free dye and multiplying by the number of fluorophores per protein. Higher numbers of fluorophores attached per protein are attainable with the Texas Red-X dye due to the lesser tendency of this dye to induce protein precipitation.
Texas Red C2-Dichlorotriazine
Texas Red C2-dichlorotriazine (T30200) is a reactive dye with absorption/emission maxima of ~588/601 nm. Dichlorotriazines readily modify amines in proteins, and they are among the few reactive groups that are reported to react directly with polysaccharides and other alcohols in aqueous solution, provided that the pH is >9 and that other nucleophiles are absent.
Texas Red-X Conjugates and Texas Red-X Labeling Kits
Because of the advantages of Texas Red-X succinimidyl ester, we have converted some of our Texas Red conjugates to the Texas Red-X conjugates. We have prepared Texas Red-X conjugates of:
- Antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1)
- Streptavidin (S6370, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9)
- Phalloidin (T7471, Probes for Actin—Section 11.1, Spectral characteristics of Molecular Probes actin-selective probes—Table 11.2)
- Wheat germ agglutinin (W21405, Lectins and Other Carbohydrate-Binding Proteins—Section 7.7)
- dUTP (C7631, Labeling Oligonucleotides and Nucleic Acids—Section 8.2)
Protein conjugates of the Texas Red-X dye are readily prepared using our FluoReporter Texas Red-X Protein Labeling Kit (F6162) and Texas Red-X Protein Labeling Kit (T10244); see Kits for Labeling Proteins and Nucleic Acids—Section 1.2 for further information on preparing fluorescent protein conjugates with these kits. Zenon Texas Red-X Antibody Labeling Kit for mouse IgG1 antibodies (Z25045, Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) permits the rapid and quantitative labeling of antibodies from a purified antibody fraction or from a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant with the Texas Red-X dye. Polyclonal anti-tetramethylrhodamine and anti–Texas Red antibodies that recognize tetramethylrhodamine, Rhodamine Red, X-rhodamine and Texas Red fluorophores are available (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Anti-fluorophore antibodies and their conjugates—Table 7.8).
Naphthofluorescein carboxylic acid and its succinimidyl ester (C652, C653; ) have emission maxima of approximately 670 nm in aqueous solution at pH 10 (). However, the fluorescence of naphthofluorescein is pH dependent (pKa ~7.6), requiring a relatively alkaline pH for maximal fluorescence.
The excitation and emission spectra of carboxyrhodamine 6G (CR 6G) fall between those of fluorescein and tetramethylrhodamine (Figure 1.6.3). With a peak absorption at ~520 nm, conjugates prepared from the mixed-isomer (C6157) or single-isomer (C6127, C6128) preparations of CR 6G succinimidyl esters are an excellent match to the 514 nm spectral line of the argon-ion laser. They also tend to exhibit a higher fluorescence quantum yield than tetramethylrhodamine conjugates, as well as excellent photostability. As with Rhodamine Green dyes, carboxyrhodamine 6G dyes are more suitable for preparing nucleotide and oligonucleotide conjugates than for preparing protein conjugates. Oligonucleotide conjugates of CR 6G have spectroscopic and electrophoretic properties that are superior to the JOE dye (C6171MP, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) that is often used for DNA sequencing (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Amine-reactive dyes for nucleic acid sequencing—Table 8.7).
Dyes that quench the fluorescence of visible light–excited fluorophores are increasingly important for use in fluorescence resonance energy transfer (FRET) proximity assays (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2), such as those based on DNA hybridization. Nonfluorescent acceptors are advantageous in FRET assays because they avoid the complications of proximity-independent signals resulting from direct excitation of fluorescent acceptors. Our QSY 7, QSY 9 and QSY 21 dyes (Molecular Probes nonfluorescent quenchers and photosensitizers—Table 1.10) are diarylrhodamine derivatives that have several properties that make them superior to the commonly used dabcyl chromophore (Reagents for Analysis of Low Molecular Weight Amines—Section 1.8) when preparing bioconjugates for use in FRET-based assays:
- Broad absorption in the visible-light spectrum, with an absorption maximum near 560 nm for both the QSY 7 and QSY 9 dyes and near 660 nm for the QSY 21 dye (Figure 1.6.7)
- Extinction coefficients that are typically in excess of 90,000 cm-1M-1
- Absorption spectra of the conjugates that are insensitive to pH between 4 and 10
- Fluorescence quantum yields typically <0.001 in aqueous solution (In a few isolated cases, we have observed that some QSY dyes can exhibit fluorescence when placed in a rigidifying environment such as glycerol.)
- Efficient quenching of the fluorescence emission of donor dyes by the QSY 7 and QSY 9 dyes, including blue-fluorescent coumarins, green- or orange-fluorescent dyes, and red-fluorescent Texas Red and Alexa Fluor 594 conjugates
- Quenching of red-fluorescent dyes, including Alexa Fluor 647 dye, by the long-wavelength light–absorbing QSY 21 dye (R<0> values for QSY and dabcyl quenchers—Table 1.11)
- Quenching of most green and red fluorophores that is more effective at far greater distances than is possible with dabcyl quenchers (R<0> values for QSY and dabcyl quenchers—Table 1.11, Figure 1.6.8)
- Residual fluorescence of the conjugates, at close spatial separations, that is typically lower than in conjugates that use dabcyl as the quencher
- High chemical stability of the conjugates and very good resistance to photobleaching
A particularly frequent and effective application of QSY dyes is as components of fluorogenic protease and peptidase substrates consisting of a fluorescent dye and a QSY quencher attached to opposite ends of a peptide sequence that is specifically recognized and cleaved by the enzyme. QSY 7 dye has been used to create activatable targeted probes comprising a fluorophore–quencher pair and a targeting protein moiety (avidin, which targets the D-galactose receptor, or trastuzumab, a monoclonal antibody that recognizes the human epithelial growth factor receptor type 2 or HER2/neu) for use in in vivo tumor imaging. The fluorophore–quencher interaction, in this case between tetramethylrhodamine and QSY 7 dyes, is disrupted when the probe is internalized in a tumor by receptor-mediated uptake, thereby activating the fluorescence. In this imaging application, QSY 7 was reported to be superior to azobenzene quenchers (e.g., dabcyl derivatives) because azobenzene dyes are vulnerable to reductive cleavage in vivo, producing a false positive signal. Both the amine-reactive and thiol-reactive QSY 7 derivatives (Q10193, Q10257) have been used to create molecular beacon probes for following the transport of mRNAs in Drosophila melanogaster oocytes.
The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by fluorescence resonance energy transfer) is defined by the Förster radius (R0). The magnitude of R0 is dependent on the spectral properties of the donor and acceptor dyes. R0 values calculated for energy transfer from various Alexa Fluor dyes to QSY and dabcyl quenchers are listed in R<0> values for QSY and dabcyl quenchers—Table 1.11. FRET efficiencies from several donor dyes to the QSY 7 quencher in molecular beacon hybridization probes have also been calculated.
Figure 1.6.7 Normalized absorption spectra of the QSY 35 (blue), QSY 7 (red) and QSY 21 (orange) dyes. The QSY 7 and QSY 9 dyes have essentially identical spectra.
Figure 1.6.8 Fluorescence quenching of 5'-tetramethylrhodamine–labeled M13 primers by nonfluorescent dyes attached at the 3'-end. The comparison represents equal concentrations of oligonucleotides with 1) no 3'-quencher (control), 2) 3'-dabcyl quencer, and 3) 3'-QSY 7 quencher.
For preparing bioconjugates, we offer several reactive versions of these QSY dyes:
- Amine-reactive QSY 7 (), QSY 9 and QSY 21 succinimidyl esters (Q10193, Q20131, Q20132)
- Thiol-reactive QSY 7 C5-maleimide and QSY 9 C5-maleimide (Q10257, Q30457; Thiol-Reactive Probes Excited with Visible Light—Section 2.2)
- QSY 7 aliphatic amine (Q10464, Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4) , which can be coupled to carbodiimide-activated carboxylic acids and other functional groups
- α-FMOC-ε-QSY 7-L-lysine (Q21930, Peptide Analysis, Sequencing and Synthesis—Section 9.5), for automated synthesis of peptides containing the QSY 7 quencher
In addition to the QSY 7, QSY 9 and QSY 21 dyes, we offer other quenchers that absorb maximally below 500 nm, including the QSY 35 and dabcyl dyes (Molecular Probes nonfluorescent quenchers and photosensitizers—Table 1.10). These products are described in Reagents for Analysis of Low Molecular Weight Amines—Section 1.8.
Malachite green is a nonfluorescent photosensitizer that absorbs at long wavelengths (~630 nm, ). Its photosensitizing action can be targeted to particular cellular sites by conjugating malachite green isothiocyanate (M689, ) to specific antibodies. Enzymes and other proteins within ~10 Å of the binding site of the malachite green–labeled antibody can then be selectively destroyed upon irradiation with long-wavelength light. Studies by Jay and colleagues have demonstrated that this photoinduced destruction of enzymes in the immediate vicinity of the chromophore is apparently the result of localized production of hydroxyl radicals, which have short lifetimes that limit their diffusion from the site of their generation. Earlier studies had supported a thermal mechanism of action.
|C652||476.44||L||pH >6, DMF||598||49,000||668||pH 10||2|
|C653||573.51||F,D,L||DMF, DMSO||602||42,000||672||pH 10||2|
|C1171||527.53||F,D,L||DMF, DMSO||546||95,000||576||MeOH||1, 3|
|C2211||527.53||F,D,L||DMF, DMSO||546||95,000||579||MeOH||1, 3|
|C6121||430.46||L||pH >6, DMF||542||91,000||568||MeOH||1|
|C6122||430.46||L||pH >6, DMF||540||103,000||564||MeOH||1|
|C6123||527.53||F,D,L||DMF, DMSO||547||91,000||573||MeOH||1, 3|
|C6124||635.80||F,L||pH >6, DMF||567||92,000||591||MeOH||1|
|C6127||555.59||F,D,L||pH >6, DMF||524||108,000||557||MeOH|
|C6156||534.61||F,L||pH >6, DMF||570||113,000||590||MeOH||1|
|T490||443.52||F,DD,L||DMF, DMSO||544||84,000||572||MeOH||3, 5|
|T1480||443.52||F,DD,L||DMF, DMSO||543||99,000||571||MeOH||3, 5|
|T1481||443.52||F,DD,L||DMF, DMSO||544||90,000||572||MeOH||3, 5|
|T6105||640.69||F,D,L||DMF, DMSO||543||92,000||571||MeOH||1, 3|