Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the
polymethine
Polymethines are compounds made up from an ''odd'' number of methine groups (CH) bound together by alternating single and double bonds.Kachovski and Dekhtyar, ''Dyes and Pigments'', 22 (1983) 83-97. Compounds made up from an ''even'' number of meth ...
group. Although the name derives etymologically from terms for shades of
blue
Blue is one of the three primary colours in the RYB colour model (traditional colour theory), as well as in the RGB (additive) colour model. It lies between violet and cyan on the spectrum of visible light. The eye perceives blue when ...
, the cyanine family covers the
electromagnetic spectrum
The electromagnetic spectrum is the range of frequencies (the spectrum) of electromagnetic radiation and their respective wavelengths and photon energies.
The electromagnetic spectrum covers electromagnetic waves with frequencies ranging fro ...
from
near IR to
UV.
Chemically, cyanines are a
conjugated system between two
nitrogen
Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
atoms; in each
resonance structure
In chemistry, resonance, also called mesomerism, is a way of describing bonding in certain molecules or polyatomic ions by the combination of several contributing structures (or ''forms'', also variously known as ''resonance structures'' or '' ...
, exactly one nitrogen atom is oxidized to an
iminium
In organic chemistry, an iminium cation is a polyatomic ion with the general structure . They are common in synthetic chemistry and biology.
Structure
Iminium cations adopt alkene-like geometries. The central C=N unit is nearly coplanar with al ...
. Typically, they form part of a nitrogenous
heterocyclic system.
The main application for cyanine dyes is in
biological labeling. Nevertheless, there is a wide literature on both their synthesis and uses, and cyanines are common in some
CD and
DVD
The DVD (common abbreviation for Digital Video Disc or Digital Versatile Disc) is a digital optical disc data storage format. It was invented and developed in 1995 and first released on November 1, 1996, in Japan. The medium can store any kind ...
media.
Structure
Cyanines have been classified in many ways:
* ''Streptocyanines'' or ''open chain cyanines'':
: R
2N
+=CH
H=CH'
n''-NR
2 (I)
* ''Hemicyanines'':
: Aryl=N
+=CH
H=CH'
n''-NR
2 (II)
* ''Closed chain cyanines'':
:Aryl=N
+=CH
H=CH'
n''-N=Aryl
(III)
Additionally, these classes are recognized:
*''Neutrocyanines'':
: R
2N
+=CH
H=CH'
n''-CN and R
2N
+=CH
H=CH'
n''-CHO
* ''Merocyanines'' including
spiropyran A spiropyran is a type of organic chemical compound, known for photochromic properties that provide this molecule with the ability of being used in medical and technological areas. Spiropyrans were discovered in the early twentieth century. However ...
s and
quinophthalones.
* ''Apocyanines''
where two quaternary
nitrogen
Nitrogen is the chemical element with the symbol N and atomic number 7. Nitrogen is a nonmetal and the lightest member of group 15 of the periodic table, often called the pnictogens. It is a common element in the universe, estimated at se ...
s are joined by a
polymethine
Polymethines are compounds made up from an ''odd'' number of methine groups (CH) bound together by alternating single and double bonds.Kachovski and Dekhtyar, ''Dyes and Pigments'', 22 (1983) 83-97. Compounds made up from an ''even'' number of meth ...
chain.
Both nitrogens may each be independently part of a
heteroaromatic
In chemistry, aromaticity is a chemical property of cyclic compound, cyclic (ring (chemistry), ring-shaped), ''typically'' plane (geometry), planar (flat) molecular structures with pi bonds in Resonance (chemistry), resonance (those containing ...
moiety
Moiety may refer to:
Chemistry
* Moiety (chemistry), a part or functional group of a molecule
** Moiety conservation, conservation of a subgroup in a chemical species
Anthropology
* Moiety (kinship), either of two groups into which a society is ...
, such as
pyrrole,
imidazole
Imidazole (ImH) is an organic compound with the formula C3N2H4. It is a white or colourless solid that is soluble in water, producing a mildly alkaline solution. In chemistry, it is an aromatic heterocycle, classified as a diazole, and has non-a ...
,
thiazole
Thiazole, or 1,3-thiazole, is a heterocyclic compound that contains both sulfur and nitrogen. The term 'thiazole' also refers to a large family of derivatives. Thiazole itself is a pale yellow liquid with a pyridine-like odor and the molecular fo ...
,
pyridine
Pyridine is a basic heterocyclic organic compound with the chemical formula . It is structurally related to benzene, with one methine group replaced by a nitrogen atom. It is a highly flammable, weakly alkaline, water-miscible liquid with a ...
,
quinoline
Quinoline is a heterocyclic aromatic organic compound with the chemical formula C9H7N. It is a colorless hygroscopic liquid with a strong odor. Aged samples, especially if exposed to light, become yellow and later brown. Quinoline is only sli ...
,
indole
Indole is an aromatic heterocyclic organic compound
In chemistry, organic compounds are generally any chemical compounds that contain carbon-hydrogen or carbon-carbon bonds. Due to carbon's ability to catenate (form chains with other c ...
,
benzothiazole
Benzothiazole is an aromatic heterocyclic compound with the chemical formula . It is colorless, slightly viscous liquid. Although the parent compound, benzothiazole is not widely used, many of its derivatives are found in commercial products or ...
, 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
wavelength
In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats.
It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, t ...
s which will form an image on the film, making the film
panchromatic.
[ Cyanines are also used in ]CD-R
CD-R (Compact disc-recordable) is a digital optical disc storage format. A CD-R disc is a compact disc that can be written once and read arbitrarily many times.
CD-R discs (CD-Rs) are readable by most CD readers manufactured prior to the i ...
and DVD-R media. The ones used are mostly green or light blue colour, and are chemically unstable. For that reason, unstabilized cyanine discs are unsuitable for archival CD and DVD use. Recent cyanine discs contain stabilizers, typically a metal atom bonded to the cyanine molecule, 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
Phthalocyanine () is a large, aromatic, macrocyclic, organic compound with the formula and is of theoretical or specialized interest in chemical dyes and photoelectricity.
It is composed of four isoindole units linked by a ring of nitrogen ato ...
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
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
molecules. Labeling is done for visualization and quantification purposes. Biological applications include comparative genomic hybridization Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The ...
and gene chips, which are used in transcriptomics
Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. H ...
, and various studies in proteomics such as RNA localization, molecular interaction studies by fluorescence resonance energy transfer ( FRET) and fluorescent immunoassay
An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoass ...
s.
Cyanine dyes are available with different modifications such as methyl, ethyl or butyl substituents, carboxyl, acetylmethoxy, and sulfo groups which alter their hydrophilicity.[CYanine]
dyes
Ex (nm): Excitation wavelength in nanometers
330px, Different lengths as in respect to the molecular scale.
The nanometre (international spelling as used by the International Bureau of Weights and Measures; SI symbol: nm) or nanometer (American and British English spelling differences#-re ...
Em (nm): Emission wavelength in nanometers
330px, Different lengths as in respect to the molecular scale.
The nanometre (international spelling as used by the International Bureau of Weights and Measures; SI symbol: nm) or nanometer (American and British English spelling differences#-re ...
MW: Molecular weight
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...
QY: Quantum yield The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
Applications
Fluorescence spectroscopy
The fluorescence quantum yield is defined as the ratio of the numb ...
* Depends strongly on viscosity, temperature, and biomolecular interactions.
Common cyanine dyes and their uses
Because they yield brighter and more stable fluorescence, cyanines can advantageously replace conventional dyes such as fluorescein
Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
and rhodamine
Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and inks ...
s.
* Cy3 and Cy5 are the most popular, used typically combined for 2 colors detection.
Cy3 fluoresces greenish yellow (~550 nm excitation, ~570 nm emission), while Cy5 is fluorescent in the far-red region (~650 excitation, 670 nm emission). Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for Tetramethylrhodamine (TRITC). Due to its high molar extinction coefficient
In chemistry, the molar absorption coefficient or molar attenuation coefficient is a measurement of how strongly a chemical species absorbs, and thereby attenuates, light at a given wavelength. It is an intrinsic property of the species. The SI u ...
, this dye is also easily detected by naked eye on electrophoresis gel
Gel electrophoresis is a method for separation and analysis of biomolecule, biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or ...
s, and in solution.
Cy5 became a popular replacement for far red fluorescent dyes because of its high extinction coefficient (as small as 1 nanomol can be detected in gel electrophoresis by naked eye) 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
A laser is a device that emits light through a process of optical amplification based on the stimulated emission of electromagnetic radiation. The word "laser" is an acronym for "light amplification by stimulated emission of radiation". The fi ...
emission wavelengths (typically 532 nm and 635 nm) and filter wavelengths (550-600 nm and 655-695 nm) 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 (multiparametric detection).
* Other cyanine dyes are useful:
Cy3.5 can replace sulfoRhodamine 101.
Cy5.5 is a near-infrared (IR) fluorescence-emitting dye (excitation/emission maximum 678/694 nm).
Cy7 is a near-IR fluor that is invisible to the naked eye (excitation/emission maximum 750/776 nm). 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
PEGylation (or pegylation) is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG, in pharmacy called macrogol) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein ...
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 ]methine
In organic chemistry, a methine group or methine bridge is a trivalent functional group , derived formally from methane. It consists of a carbon atom bound by two single bonds and one double bond, where one of the single bonds is to a hydrogen. ...
s (as shown), and the side chains
In organic chemistry and biochemistry, a side chain is a chemical group that is attached to a core part of the molecule called the "main chain" or backbone. The side chain is a hydrocarbon branching element of a molecule that is attached to a ...
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
In organic chemistry, hydrocarbons ( compounds composed solely of carbon and hydrogen) are divided into two classes: aromatic compounds and aliphatic compounds (; G. ''aleiphar'', fat, oil). Aliphatic compounds can be saturated, like hexane, ...
chains one or both of which ends in a highly reactive moiety
Moiety may refer to:
Chemistry
* Moiety (chemistry), a part or functional group of a molecule
** Moiety conservation, conservation of a subgroup in a chemical species
Anthropology
* Moiety (kinship), either of two groups into which a society is ...
such as N-hydroxysuccinimide or maleimide
Maleimide is a chemical compound with the formula H2C2(CO)2NH (see diagram). This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -C(O)NHC(O)- functional group. Malei ...
.
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.
Applications
Cyanine dyes are used to label proteins, antibodies, peptides, nucleic acid probes, and any kind of other biomolecules to be used in a variety of fluorescence detection techniques: Flow cytometry, Microscopy (mainly Visible range, but also UV, IR), Microplate assays, Microarrays, as well as "light-up Probes," and in vivo imaging.
Nucleic acid labeling
In microarray experiments DNA or RNA is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester (NHS
The National Health Service (NHS) is the umbrella term for the publicly funded healthcare systems of the United Kingdom (UK). Since 1948, they have been funded out of general taxation. There are three systems which are referred to using the " ...
-ester) reactive group. Since NHS-esters react readily only with aliphatic
In organic chemistry, hydrocarbons ( compounds composed solely of carbon and hydrogen) are divided into two classes: aromatic compounds and aliphatic compounds (; G. ''aleiphar'', fat, oil). Aliphatic compounds can be saturated, like hexane, ...
amine
In chemistry, amines (, ) are compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are formally derivatives of ammonia (), wherein one or more hydrogen
Hydrogen is the chemical element wi ...
groups, which nucleic acids lack, nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecule ...
s 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
In materials science, quenching is the rapid cooling of a workpiece in water, oil, polymer, air, or other fluids to obtain certain material properties. A type of heat treating, quenching prevents undesired low-temperature processes, such as pha ...
effects.
Protein labeling
For protein labeling, Cy3 and Cy5 dyes sometimes bear a succinimidyl group to react with amines, or a maleimide
Maleimide is a chemical compound with the formula H2C2(CO)2NH (see diagram). This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -C(O)NHC(O)- functional group. Malei ...
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
Blue-green is the color that is between green and blue. It belongs to the cyan family of colors.
Variations Cyan (aqua)
Cyan, also called aqua, is the blue-green color that is between blue and green on a modern RGB color wheel.
The ...
(close to "aqua") and is derived from the Greek
Greek may refer to:
Greece
Anything of, from, or related to Greece, a country in Southern Europe:
*Greeks, an ethnic group.
*Greek language, a branch of the Indo-European language family.
**Proto-Greek language, the assumed last common ancestor ...
/ ''kyaneos/kyanous'' which means a somewhat different color: "dark blue".
References
External links
{{Authority control
Quaternary ammonium compounds