Cyanines are defined as "synthetic dyes with the general formula R2NnCH=N+R2↔R2N+=CHnNR2 in which the nitrogen and part of the conjugated chain usually form part of a heterocyclic system, such as imidazole, pyridine, pyrrole, quinoline and thiazole." Cyanines are used in industry biotechnology.
Merocyanines including spiropyrans and quinophthalones.
Apocyanines
where two quaternary nitrogens are joined by a polymethine chain. Both nitrogens may each be independently part of a heteroaromaticmoiety, such as pyrrole, imidazole, thiazole, pyridine, quinoline, indole, benzothiazole, etc.
History and use in industry
Cyanines were first synthesized over a century ago. They were originally used, and still are, to increase the sensitivity range of photographic emulsions, i.e., to increase the range of wavelengths which will form an image on the film, making the film panchromatic. Cyanines are also used in CD-R and DVD-R media. The ones used are mostly green or light blue in color, and are chemically unstable. For that reason, unstabilized cyanine discs are unsuitable for archival CD and DVD use. Recent cyanine discs contain stabilizers that slow the deterioration significantly. These discs are often rated with an archival life of 75 years or more. The other dyes used in CD-Rs are phthalocyanine and azo.
Use in biotechnology
For applications to biotechnology, special cyanine dyes are synthesized from 2, 3, 5 or 7-methine structures with reactive groups on either one or both of the nitrogen ends so that they can be chemically linked to either nucleic acids or protein molecules. Labeling is done for visualization and quantification purposes. Biological applications include comparative genomic hybridization and gene chips, which are used in transcriptomics, and various studies in proteomics such as RNA localization, molecular interaction studies by fluorescence energy transfer and fluorescent immunoassays. Cyanine dyes are available with different modifications such as methyl, ethyl or butyl substituents, carboxyl, acetylmethoxy, and sulfo groups which alter their hydrophilicity.
Because they yield brighter and more stable fluorescence, cyanines can advantageously replace conventional dyes such as fluorescein and rhodamines.
Cy3 and Cy5 are the most popular, used typically combined for 2 colors detection.
Cy3 fluoresces greenish yellow, while Cy5 is fluorescent in the red region. Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for Tetramethylrhodamine. Due to its high molar extinction coefficient, this dye is also easily detected by naked eye on electrophoresis gels, and in solution. Cy5 became a popular replacement for far redfluorescent dyes because of its high extinction coefficient and its fluorophore emission maximum in the red region, where many CCD detectors have maximum sensitivity and biological objects give low background interference. The scanners actually use diverse laser emission wavelengths and filter wavelengths to avoid background contamination. They are thus able to easily distinguish colors from Cy3 and from Cy5, and also able to quantify the amount of Cy3 and Cy5 labeling in one sample.
Other cyanine dyes are useful:
Cy3.5 can replace sulfoRhodamine 101. Cy5.5 is a near-infrared fluorescence-emitting dye. Cy7 is a near-IR fluor that is invisible to the naked eye. It is used in in vivo imaging applications, as well as the Cy7.5 dye. Sulfo–Cyanine dyes bear one or two sulfo groups, rendering the Cy dye water-soluble, but tri- and quadri-sulfonated forms are available for even higher water solubility. PEGylation is another modification that confers hydrophilicity, not only to the dye but also to the labeled conjugate.
Nomenclature and structure
The Cy3 and Cy5 nomenclature was first proposed by Ernst, et al. in 1989, and is non-standard since it gives no hint of their chemical structures. In the original paper the number designated the count of the methines, and the side chains were unspecified. Due to this ambiguity various structures are designated Cy3 and Cy5 in the literature. The R groups do not have to be identical. In the dyes as used they are short aliphatic chains one or both of which ends in a highly reactive moiety such as N-hydroxysuccinimide or maleimide.
Alternatives
Many analogs of standard Cy 2 / 3 / 3.5 / 5 / 5.5 / 7 / 7.5 dyes were developed, using diverse modification: Alexa Fluor dyes, Dylight, FluoProbes dyes, Sulfo Cy dyes, Seta dyes, IRIS dyes from Cyanine Technologies and others can be used interchangeably with Cy dyes in most biochemical applications, with claimed improvements in solubility, fluorescence, or photostability. While patent protection for the standard Cy series of dyes has lapsed, the trademarked Cy naming remains in place. Consequently, dyes that are identical to Cy dyes, but called different names, are now sold.
In microarray experiments DNA or RNA is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester reactive group. Since NHS-esters react readily only with aliphatic amine groups, which nucleic acids lack, nucleotides have to be modified with aminoallyl groups. This is done through incorporating aminoallyl-modified nucleotides during synthesis reactions. A good ratio is a label every 60 bases such that the labels are not too close to each other, which would result in quenching effects.
Protein labeling
For protein labeling, Cy3 and Cy5 dyes sometimes bear a succinimidyl group to react with amines, or a maleimide group to react with a sulfhydryl group of cysteine residues. Cy5 is sensitive to its electronic environment. Changes in the conformation of the protein it is attached to will produce either enhancement or quenching of the emission. The rate of this change can be measured to determine enzyme kinetic parameters. The dyes can be used for similar purposes in FRET experiments. Cy3 and Cy5 are used in proteomics experiments so that samples from two sources can be mixed and run together through the separation process. This eliminates variations due to differing experimental conditions that are inevitable if the samples were run separately. These variations make it extremely difficult, if not impossible, to use computers to automate the acquisition of the data after the separation is complete. Using these dyes makes the automation trivial.
Etymology
The word cyanin is from the English word "cyan", which conventionally means a shade of blue-green and is derived from the Greek κυάνεος/κυανοῦς kyaneos/kyanous which means a somewhat different color: "dark blue".
