How varying planar aromatic ring systems generate a rainbow of luminescence

Understanding the dye properties of fluorescent probes is key to their successful application in various in vitro and in vivo settings. Fluorescence labeling applications are designed to interact with a variety of molecules including proteins, lipids, and nucleic acids. Fluorophores are available with a number of different reactive groups designed to couple to specific functional groups on target molecules. These probes can be functionalized to react with amines, ketones, sulfhydryls, etc. (see side note on common conjugation groups). Various substituents can be added to these probes with a goal to increase quantum yield or extinction coefficient, to improve sensitivity, to augment the ability to cross specific biological membranes,² or to reduce dye-dye quenching effects. The most often used fluorescent core structures are water-soluble derivatives of cyanine, fluorescein, rhodamine, and coumarin. Other well-known core structures include boron dipyrromethene (BODIPY), naphthalimide, and phycobiliprotein derivatives. Here we will discuss the physical properties and key advantages of the major core structures of fluorescent labels and highlight the spectral characteristics of Cayman’s fluorescent probes in each of these categories.

Fluorescein Derivatives

Figure 1. Basic structure of reactive fluorescein derivatives.

Fluorescein fluoresces due to its multi-ring, aromatic xanthene core. This structure produces the rigid planar nature of its upper, fused three-ring system (Figure 1). Derivatives with substitutions off the C-5 or C-6 position of the lower aromatic ring provide reactivity toward particular functional groups in proteins and nucleic acids, allowing rapid labeling of biomolecules. Fluorescein has an excitation range of 488 to 495 nm and an emission range of 518 to 525 nm. Its quantum yield can be as high as 0.75 under ideal conditions, but its fluorescent intensity fades when dissolved in aqueous buffers, exposed to light, or stored over an extended timeframe. The quantum yield is quenched significantly when pH drops below 7.0.

Fluorescein derivatives typically contain either an amine-reactive group or a sulfhydryl-reactive group off the lower ring structure (Figure 2). These modifications target lysines or cysteines, respectively. The most well-known example is fluorescein isothiocyanate (FITC). Carboxyfluorescein derivatives, which bear an added carboxyl group, produce even more stable bonds compared to isothiocyanates and are commonly used as tracer agents, to sequence nucleic acids, or to label nucleotides. Aldehyde-reactive or ketone-reactive fluorescein derivatives contain a hydrazide group off the C-5 carbon on the lower ring. Carbohydrates, glycoproteins, RNA, and other molecules containing sugar residues can be oxidized to produce reactive formyl groups that this type of probe can target. DNA and RNA can also be modified with hydrazide-reactive probes by first exposing their cytosine residues to bisulfite to form reactive sulfone intermediates.

Figure 2. Fluorescein-5-maleimide conjugates with sulfhydryl-containing proteins.

Fluorescein-based probes available from Cayman.

Rhodamine Derivatives

Figure 3. Rhodamine core structure.

The fluorescent character of rhodamine derivatives comes from the presence of a planar, multi-ring aromatic xanthene core. This structure is quite similar to fluorescein with nitrogen atoms replacing the oxygens on the outer rings (Figure 3). Derivations of this structure occur by adding substitutions off the C-5 or C-6 atoms of its lower ring that are reactive with certain functional groups of proteins and nucleic acids (Figure 4). Additional alterations to the basic rhodamine structure can provide improved stability or fluorescent intensity or even cause red shifting. Most rhodamine derivatives have excitation wavelengths ranging from low- to high-500 nm range and emission wavelengths at mid- to high-500 nm within the orange-red visible spectrum. Sulforhodamine 101 emits notably higher at over 600 nm. With the exception of sulforhodamine 101, the typical quantum yield for most rhodamine derivatives is 0.25, which is lower than that of fluorescein. However, the fluorescent intensity of rhodamine derivatives fades less rapidly than that of fluorescein when dissolved in aqueous buffers, exposed to light, or stored for a long time. Since the orange-red luminescence of most rhodamine derivatives unambiguously contrasts the green of fluorescein, these probes pair nicely in double staining techniques.

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The fluorescent intensity of rhodamine derivatives fades less rapidly than that of fluorescein

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The most commonly used derivative of rhodamine is tetramethylrhodamine, which has two methyl groups attached to each nitrogen on its outer rings. Other useful derivatives include rhodamine B, which contains two ethyl groups on each nitrogen and a carboxylate group at the C-3 position of the lower ring; rhodamine 110, which contains only the carboxylate on the lower ring; and sulforhodamine 101, which contains dual aliphatic rings off the upper-ring nitrogens and sulfonate groups on the C-3 and C-5 positions of its lower ring. Additional rhodamine derivatives contain either amine-, sulfhydryl-, aldehyde-/ketone-, or cytosine-reactive groups on the C-5 or C-6 position of the lower ring to permit coupling to target molecules. Tetramethylrhodamine isothiocyanate (TRITC), the most popular amine-reactive derivative, is synthesized by modifying either the C-5 or C-6 positions with isothiocyanate. The two resulting isomers are nearly identical in reactivity yet display slightly different intensities and excitation/emission wavelengths. Note that Cayman offers TRITC as mixed isomers. TRITC is more stable to photobleaching than FITC, and its excitation and emission spectra are less sensitive to pH. However, self-quenching is possible through dye-dye interaction when used at high concentrations.

Figure 4. Tetramethylrhodamine isothiocyanate (mixed isomers) conjugates with amine-containing proteins.

Rhodamine-based probes available from Cayman.

Coumarin Derivatives

Figure 5. Basic structure of coumarin (left), AMC (middle), and AFC (right).

Coumarin occurs naturally in certain plants and is a member of the benzopyrone chemical class (Figure 5). Though colorless in its natural state, many of its derivatives possess fluorescent properties within the blue region of the visible spectrum with a typical quantum yield ranging from 0.54-0.78. Coumarin derivatives are especially useful for double-labeling techniques, since its emission spectra does not overlap with other major fluorophores. Derivatives of 7-amino-4-methyl coumarin (AMC), including the popular 3-acetic acid derivative AMCA, may be coupled to amine-containing molecules via its carboxylate group (Figure 5). With the addition of spacer arms or key functional groups, it can be made reactive to sulfhydryl- or aldehyde-/ketone-containing molecules.

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Coumarins are especially useful for double-labeling techniques, since emission spectras do not overlap with other major fluorophores

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AMCA is highly stable against photobleaching with an ability to retain full fluorescence three-times longer than fluorescein-based probes, and its fluorescent intensity is not affected by changes in pH in the range of 3 to 10. AMC derivatives can be detected at an intensely blue fluorescence (440-460 nm) using an excitation wavelength of 340-380 nm. 7-amino-4-trifluoromethylcoumarin (AFC) derivatives have the same basic structure as AMC with the methyl group replaced by a trifluoromethyl group (Figure 5). AFC absorbs and emits at higher wavelengths than AMC with an excitation range of 395 to 400 nm and an emission range of 495 to 505 nm. Substrates with either AMC or AFC attached to the C-terminal carboxyl group are typically used for assaying carboxypeptidases or amidases, whereas molecules consisting of coumarin attached via an ester linkage to a fatty acid can be used as substrates for lipases. Cleavage of the amide or ester bond between the substrate and coumarin/AMC/AFC group results in the release of the fluorophore (Figure 6). Enzyme activity can be quantified by fluorescent detection of the free fluorophore.

Figure 6. Exposure of the AMC arachidonoyl amide conjugate to FAAH activity results in FAAH accepting the amide head group of arachidonoyl amide and releasing the fluorescent AMC.

Coumarin-based probes available from Cayman.

BODIPY Derivatives

Figure 7. Basic structure of BODIPY fluorophores.

Fluorescent compounds with a core structure consisting of a boron-dipyrromethene (BODIPY) tricyclic ring system are characterized by having a bound boron atom in their center (Figure 7). The fused, multi-ring structure can be modified at the C-1, C-3, C-5, C-7, and C-8 positions to produce compounds with widely varying fluorescent properties, including spectral shifts in its excitation and emission wavelengths. Most BODIPY derivatives offer high extinction coefficients, quantum yields greater than 0.8, and little sensitivity to changes in pH. Due to narrow bandwidths and a very slight range for Stokes shifts, BODIPY probes are usually activated at sub-optimal wavelengths to prevent interferences such as light scattering or cross-over from the wide bandwidth of the excitation source.

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BODIPY derivatives offer high extinction coefficients, high quantum yields, and are not sensitive to pH changes

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If used at high concentrations, these probes are also highly sensitive to dye-dye quenching effects during energy transfer from an excited-state fluorophore to a ground-state fluorophore. BODIPY derivatives can be synthesized to couple with amine-containing molecules and are used for tagging DNA probes at the 5’ end, lipid molecules on their head groups, or oligonucleotides containing an amine on their 5’ phosphate group (Figure 8). BODIPY probes can also be modified to contain reactivity toward sulfhydryls, carboxylic acids, or aldehyde/ketone groups. BODIPY compounds with a designation of FL in their name mimic the green luminescence of fluorescein.

Figure 8. 3-BODIPY-propanoylaminocaproic acid N-hydroxysuccinimide ester conjugates with amine-containing proteins.

BODIPY-based probe available from Cayman.

Naphthalimide Derivatives

Figure 9. Core structure of naphthalimide.

Fluorescent probes with a core structure based on naphthalimide can be modified at their amide nitrogen to contain reactive groups suitable for conjugation with biomolecules (Figure 9). N-(2-hydroxyethyl)-Naphthalimide is an N-substituted 1,8-naphthalimide used as a fluorescent probe and as a precursor for protection of amine groups. It can be used to detect nucleic acids and their precursors, which quench the fluorescence of fluorophore groups (Figure 10). N-(2-hydroxyethyl)-Naphthalimide displays spectral shifts in its excitation and emission wavelengths depending on solvent polarity, with a higher Stokes shift in less polar solvents. The quantum yield of most naphthalimide derivatives falls between 0.23 and 0.57. Lucifer Yellow is a well-known naphthalimide derivative whose spectral characteristics fall within the yellow region of the spectrum. It is most often used for cell staining applications, especially for neurophysiology.

Figure 10. N-(2-hydroxyethyl)-Naphthalimide intercalates within the base pairs of DNA, which quenches the fluorescence of fluorophore groups.

Naphthalimide-based probe available from Cayman.

Phycobiliprotein Derivatives

Figure 11. Structure of phycoerythrobilin, a red phycobilin present in phycoerythrin.

Eukaryotic blue-green algae and cyanobacteria contain phycobiliproteins that capture light energy, which is passed via fluorescence resonance energy transfer to chlorophylls during photosynthesis. Phycobiliproteins are located in the cytoplasm or stroma of the chloroplasts of these organisms and do not fluoresce in this native state. Once purified, however, their excitation energy is released as intense luminosity with a quantum yield as high as 0.98. They are composed of multiple chromophoric bilin (tetra-pyrrole) groups, which confers extremely high absorbance coefficients (up to 30-fold higher than synthetic fluorophores) to each molecule and generates an extraordinary luminescence (Figure 11). Four main phycobiliproteins: R-phycoerythrin (R-PE), B-phycoerythrin (B-PE), C-phycocyanin (C-PC), and allophycocyanin (APC) serve as valuable fluorescent tags by offering slightly different fluorescent properties related to their bilin content. The fluorescent yield of the brightest phycobiliproteins is comparable to that of 30 fluorescein or 100 rhodamine molecules. R-PE and B-PE are isolated from G. coulteri and P. cruentum red algae, respectively, and C-PC and APC are both isolated from A. variabilis.

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The fluorescent yield of the brightest phycobiliproteins is comparable to that of 30 fluorescein or 100 rhodamine molecules

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The ability of these proteins to generate fluorescence in the red to far-red region of the spectrum helps to avoid interference from other biological materials during analysis. The bilin groups are also highly protected by covalent binding to the protein backbone, so they are not easily quenched by conjugation to antibodies, avidin, biotin, etc. or experimental reagents (Figure 12). Phycobiliproteins possess excellent water solubility, allowing for easy chemical modification, and offer low opportunity for nonspecific binding in fluorescent detection applications. The use of tandem phycobiliprotein conjugates (e.g., C-PC crosslinked to APC as for SureLight^® P3) creates an energy donor-acceptor pair. The C-PC component is excited at 614 nm, emitting energy at 643 nm that can be accepted by APC, which in turn emits light at 662 nm. This creates a large shift in spectral characteristics, increasing the effective Stokes shift dramatically. Numerous tandem R-PE/red-shifted cyanine dye pairs have been generated to create superior fluorescent reagents with extended emission characteristics. These tandem dyes are particularly useful during multiplexed analysis of diverse targets in a sample.

Figure 12. Example phycoerythrin-streptavidin conjugate recognizing biotin-tagged proteins.

Phycobiliprotein-based probes available from Cayman.

Cayman offers a range of probes to detect intracellular events, protein interactions, a host of enzyme substrates, and many other significant targets. Download this at-a-glance guide to select the most appropriate fluorochromes for your experiment (PDF).

Conjugation Chemistry Services

Need a small molecule labeled and can’t find it available commercially? Our chemists are experts in conjugation chemistry. Contact us by phone (888) 526-5351 or email contractresearch@caymanchem.com to discuss your research needs.

Common Conjugation Groups¹

Isothiocyanates (ITCs): Form thioureas upon reaction with primary amines, being almost entirely selective for modifying ε- (e.g., lysine residues) and N-terminal amines in proteins. This reaction involves attack of the nucleophile on the central, electrophilic carbon of the isothiocyanate group. This shift in electrons creates a thiourea linkage between ITC and the protein with no leaving group.

Succinimidyl esters (NHS esters): React with aliphatic amines and show very low reactivity with aromatic amines, alcohols, phenols (including tyrosine), and histidine. The NHS ester reaction proceeds at slightly alkaline pH values resulting in a highly stable amide-linked derivative.

Iodoacetamides: React with thiols (also called mercaptans or sulfhydryls) primarily targeting cysteine residues found in peptides, proteins, and thiolated polynucleotides. Since some proteins only have a single cysteine residue, thiol-reactive probes are useful for site-specific labeling. Thiol reaction sites are naturally available on free cysteine side chains, or they can be generated by reduction of indigenous disulfides or using various thiolation reagents. The thioether bond formed when an iodoacetamide reacts with a protein thiol is very stable. However, there will be cleavage of this bond under the conditions necessary for complete protein hydrolysis for amino acid analysis. Iodoacetamides are unstable in light.

Maleimides: React with free thiols yielding stable thioether bonds similar to that as described for iodoacetamides above.

Hydrazines: React with ketone groups to form stable, covalent, hydrazone linkages and with aldehydes to yield hydrazone linkages that are relatively less stable, though they may be formed faster. Hydrazine derivatives include hydrazides, semicarbazides, and carbohydrazides.

References

1. Hermanson, G.T. Bioconjugate Techniques (Third edition) Chapter 10 – Fluorescent Probes. Elsevier 395–463 (2013). https://doi.org/10.1016/B978-0-12-382239-0.00010-8

2. Chen, J., Jiang, X., Zhang, C., et al. ACS Sens.2(9), 1257-1261 (2017).