View source for Cyanine

{{short description|Class of dyes}}
{{distinguish|Cyanide}}
'''Cyanines,''' also referred to as '''tetramethylindo(di)-carbocyanines'''<ref>{{Cite journal|last1=Kvach|first1=Maksim V.|last2=Ustinov|first2=Alexey V.|last3=Stepanova|first3=Irina A.|last4=Malakhov|first4=Andrei D.|last5=Skorobogatyi|first5=Mikhail V.|last6=Shmanai|first6=Vadim V.|last7=Korshun|first7=Vladimir A.|date=2008|title=A Convenient Synthesis of Cyanine Dyes: Reagents for the Labeling of Biomolecules|url=https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/ejoc.200701190|journal=European Journal of Organic Chemistry|language=en|volume=2008|issue=12|pages=2107–2117|doi=10.1002/ejoc.200701190|issn=1099-0690}}</ref> are a synthetic dye family belonging to the [[polymethine]] group.  Although the name derives etymologically from terms for shades of [[blue]], the cyanine family covers the [[electromagnetic spectrum]] from [[infrared|near IR]] to [[ultraviolet|UV]].
Chemically, cyanines are a [[Conjugated system|conjugated]] system between two [[nitrogen]] atoms; in each [[resonance structure]], exactly one nitrogen atom is oxidized to an [[iminium]].  Typically, they form part of a nitrogenous [[heterocycle|heterocyclic system]].<ref>{{cite book |chapter=Cyanine dyes |doi=10.1351/goldbook.C01487 |chapter-url=https://goldbook.iupac.org/terms/view/C01487|title=The IUPAC Compendium of Chemical Terminology |year=2014 }}</ref>
The main application for cyanine dyes is in [[Fluorescence microscopy|biological labeling]].  Nevertheless, there is a wide literature on both their synthesis and uses, and cyanines are common in some [[CD]] and [[DVD]] media.
== Structure ==
[[Image:Cyanine.png|thumb|right|300px|The Cyanines:<br>I = Streptocyanines,<br>II = Hemicyanines,<br>III = Closed cyanines]]
Cyanines have been classified in many ways:<ref>{{cite book|last1=Kim|first1=Eunha|last2=Park|first2=Seung Bum|editor1-last=Demchenko|editor1-first=Alexander P.|title=Advanced Fluorescence Reporters in Chemistry and Biology I: Fundamentals and Molecular Design Volume 8 of Springer Series on Fluorescence|date=2010|publisher=Springer|location=Berlin|isbn=9783642047022|page=172|chapter-url=https://books.google.com/books?id=oaA8Msj61JEC&pg=PA172|chapter=Discovery of New Synthetic Dyes: Targeted Synthesis or Combinatorial Approach?}}</ref>
* ''Streptocyanines'' or ''open chain cyanines'':
: R<sub>2</sub>N<sup>+</sup>=CH[CH=CH]''<sub>n</sub>''-NR<sub>2</sub> <big>'''(I)'''</big>
* ''Hemicyanines'':
: Aryl=N<sup>+</sup>=CH[CH=CH]''<sub>n</sub>''-NR<sub>2</sub> <big>'''(II)'''</big>
* ''Closed chain cyanines'':
:Aryl=N<sup>+</sup>=CH[CH=CH]''<sub>n</sub>''-N=Aryl <big>'''(III)'''</big>
Additionally, these classes are recognized:<ref name=Ullmann>{{Ullmann |doi=10.1002/14356007.a16_487.pub2|title=Methine Dyes and Pigments|year=2008|last1=Berneth|first1=Horst|isbn=978-3527306732}}</ref>
*''Neutrocyanines'':
: R<sub>2</sub>N<sup>+</sup>=CH[CH=CH]''<sub>n</sub>''-CN and  R<sub>2</sub>N<sup>+</sup>=CH[CH=CH]''<sub>n</sub>''-CHO
* ''Merocyanines'' including [[spiropyran]]s and [[quinophthalone]]s.
* ''Apocyanines''
where two quaternary [[nitrogen]]s are joined by a [[polymethine]] chain.<ref name=ernst>{{cite journal | vauthors = Ernst LA, Gupta RK, Mujumdar RB, Waggoner AS | title = Cyanine dye labeling reagents for sulfhydryl groups | journal = Cytometry | volume = 10 | issue = 1 | pages = 3–10 | date = Jan 1989 | pmid = 2917472 | doi = 10.1002/cyto.990100103 | doi-access = free }}</ref> Both nitrogens may each be independently part of a [[aromaticity#Heterocyclics|heteroaromatic]] [[Moiety (chemistry)|moiety]], 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 [[photo emulsion|photographic emulsions]], i.e., to increase the range of [[wavelength]]s which will form an image on the film, making the film [[panchromatic]].<ref name=Ullmann/> Cyanines are also used in [[CD-R]] 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,<ref>{{cite web |title=Archival Lifespan of TDK CD-R |url=https://www.cdrom2go.com/archival-lifespan-of-tdk-cd-r |website=cdrom2go.com |publisher=US Digital Media |access-date=3 April 2022}}</ref> 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 compound|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 acid]]s or [[protein]] molecules. Labeling is done for visualization and quantification purposes. Biological applications include [[comparative genomic hybridization]] and [[gene chip technology|gene chips]], which are used in [[transcriptomics]], and various studies in [[proteomics]] such as RNA localization,<ref>{{cite journal | vauthors = Blower MD, Feric E, Weis K, Heald R | title = Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules | journal = The Journal of Cell Biology | volume = 179 | issue = 7 | pages = 1365–73 | date = Dec 2007 | pmid = 18166649 | pmc = 2373496 | doi = 10.1083/jcb.200705163 }}</ref> molecular interaction studies by [[Förster resonance energy transfer]] (FRET) and fluorescent [[immunoassay]]s.
Cyanine dyes are available with different modifications such as methyl, ethyl or butyl substituents,  carboxyl, acetylmethoxy, and sulfo groups which alter their hydrophilicity.<ref name=CYanine>[http://www.interchim.fr/ft/B/BB7493.pdf CYanine] dyes</ref>
{| class="wikitable sortable" border="1"
|-
! Probe
! Ex (nm)
! Em (nm)
! MW
! Quantum yield
|-
| Cy2
| 489
| 506
|  714
| QY 0.12
|-
| Cy3
| (512);550
| 570;(615)
|  767
| QY 0.15  <ref name = "Mujumdar_1993"/>*
|-
| Cy3B
| 558
| 572;(620)
|  658
| QY 0.67
|-
| Cy3.5
| 581
| 594;(640)
|  1102
| QY 0.15
|-
| Cy5
| (625);650
| 670
|  792
| QY 0.27<ref name = "Mujumdar_1993" >{{cite journal | vauthors = Mujumdar B, Ernst A, Mujumdar SR, Lewis CJ, Waggoner AS | title = Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters | journal = Bioconjugate Chemistry | date = Mar 1993 | volume = 4 | issue = 2 | pages = 105–111 | doi = 10.1021/bc00020a001 | pmid = 7873641 }}</ref>
|-
| Cy5.5
| 675
| 694
|  1128
| QY 0.28<ref>{{cite journal | vauthors = Umezawa K, Matsui A, Nakamura Y, Citterio D, Suzuki K | title = Bright, color-tunable fluorescent dyes in the Vis/NIR region: establishment of new "tailor-made" multicolor fluorophores based on borondipyrromethene | journal = Chemistry: A European Journal | volume = 15 | issue = 5 | pages = 1096–106 | year = 2009 | pmid = 19117043 | doi = 10.1002/chem.200801906 }}</ref>
|-
| Cy7
| 743
| 767
|  818
| QY 0.28
|}
Ex (nm): Excitation wavelength in [[nanometers]] <br>
Em (nm): Emission wavelength in [[nanometers]] <br>
MW: [[Molecular weight]]<br>
QY: [[Quantum yield]]
<nowiki>*</nowiki> Depends strongly on viscosity, temperature, and biomolecular interactions.<ref>{{Cite journal|last1=Levitus|first1=Marcia|last2=Ranjit|first2=Suman|date=2011|title=Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments|url=https://www.cambridge.org/core/journals/quarterly-reviews-of-biophysics/article/cyanine-dyes-in-biophysical-research-the-photophysics-of-polymethine-fluorescent-dyes-in-biomolecular-environments/9DC53FC6C19B19D508A1E03E21C4ADD1|journal=Quarterly Reviews of Biophysics|language=en|volume=44|issue=1|pages=123–151|doi=10.1017/S0033583510000247|pmid=21108866|s2cid=7345293 |issn=1469-8994}}</ref>
== Common cyanine dyes and their uses ==
Because they yield brighter and more stable fluorescence, cyanines can advantageously replace conventional dyes such as [[fluorescein]] and [[rhodamine]]s.
* Cy3 and Cy5 are the most popular, typically used combined for 2 color detection.
[[File:Cy3 Cy5 dyes.gif|right|300px]]
Cy3 fluoresces greenish yellow (~550 [[Nanometer|nm]] excitation, ~570&nbsp;nm emission), while Cy5 is fluorescent in the far-red region (~650 excitation, 670&nbsp;nm emission).<ref>{{cite web |url=http://www.jacksonimmuno.com/technical/f-cy3-5.asp |author=Jackson ImmunoResearch |title=Cyanine Dyes (Cy2, Cy3, and Cy5) |access-date=2008-10-31}}</ref> Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for tetramethylrhodamine (TRITC). Due to its high [[molar extinction coefficient]], this dye is also easily detected by naked eye on [[electrophoresis gel]]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 [[Charge-coupled device|CCD detectors]] have maximum sensitivity and biological objects give low background interference.
The scanners {{Which|date=June 2024}} actually use diverse [[laser]] emission wavelengths (typically 532 [[Metre#SI prefixed forms of metre|nm]] and 635 [[Metre#SI prefixed forms of metre|nm]]) and filter wavelengths (550-600 [[Metre#SI prefixed forms of metre|nm]] and 655-695 [[Metre#SI prefixed forms of metre|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).{{Citation needed|date=June 2024}}
* 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&nbsp;nm).
Cy7 is a near-IR fluor that is invisible to the naked eye (excitation/emission maximum 750/776&nbsp;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.<ref name=CYanine /> [[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.<ref name= ernst/> 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]]s (as shown), and the [[side chains]] were unspecified. Due to this ambiguity various structures are designated Cy3 and Cy5 in the literature. The [[side chain|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 (chemistry)|moiety]] such as [[succinimide|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|Alexa Fluor dyes]], [[DyLight Fluor|Dylight]], [[FluoProbes|FluoProbes dyes]], Sulfo Cy dyes,<ref>{{Cite web |url=http://www.cyandye.us/ |title=Cyandye, LLC |access-date=2013-11-01 |archive-url=https://web.archive.org/web/20181003073347/http://www.cyandye.us/ |archive-date=2018-10-03 |url-status=dead }}</ref> Seta dyes,<ref>[http://www.setabiomedicals.com/ SETA BioMedicals]</ref> IRIS dyes from Cyanine Technologies <ref>{{cite web |url=http://www.cyanines.eu/life-science/products/iris-dyes |title=Iris Dyes &#124; Cyanine Technologies |access-date=2015-01-26 |url-status=dead |archive-url=https://archive.today/20150126174359/http://www.cyanines.eu/life-science/products/iris-dyes |archive-date=2015-01-26 }}</ref> and others can be used interchangeably with Cy dyes in most biochemical applications, with claimed improvements in solubility, fluorescence, or photostability.<ref>[http://www.interchim.fr/ft/F/FP488c.pdf FluoProbes488 comparison to FITC, Cyanine2]</ref><ref>[http://www.interchim.fr/ft/F/FP547c.pdf FluoProbes547H comparison in Confocal Microscopy]</ref>
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 ===
[[File:Gloeotrichia in Sytox.jpg|thumb|right|A [[Cyanobacteria|cyanobacterium]] stained green with cyanine dye]]
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#Fluorescence|microscopy]] (mainly the visible range, but also [[Microscopy#Ultraviolet microscopy|UV]] and [[Microscopy#Infrared microscopy|IR]]), microplate assays, [[microarray]]s, as well as "light-up probes," and in vivo imaging.<ref>{{cite book |last1=Armitage |first1=Bruce A. |title=DNA Binders and Related Subjects |chapter=Cyanine Dye–DNA Interactions: Intercalation, Groove Binding, and Aggregation |journal=Topics in Current Chemistry |volume=253 |date=27 January 2005 |pages=55–76 |doi=10.1007/b100442 |language=en|isbn=978-3-540-22835-6 }}</ref>
==== Nucleic acid labeling ====
In micorarray experiments [[DNA]] or [[RNA]] is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester ([[N-Hydroxysuccinimide|NHS]]-ester) reactive group. Since NHS-esters react readily only with [[aliphatic]] [[amine]] groups,  which nucleic acids lack, [[nucleotide]]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 (fluorescence)|quenching]] effects.
==== Protein labeling ====
For protein labeling, Cy3 and Cy5 dyes sometimes bear a [[N-Hydroxysuccinimide|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.<ref>{{cite journal | vauthors = Unlü M, Morgan ME, Minden JS | title = Difference gel electrophoresis: a single gel method for detecting changes in protein extracts | journal = Electrophoresis | volume = 18 | issue = 11 | pages = 2071–7 | date = Oct 1997 | pmid = 9420172 | doi = 10.1002/elps.1150181133 | s2cid = 604007 }}</ref><ref>{{cite journal | vauthors = Osterman IA, Ustinov AV, Evdokimov DV, Korshun VA, Sergiev PV, Serebryakova MV, Demina IA, Galyamina MA, Govorun VM, Dontsova OA | title = A nascent proteome study combining click chemistry with 2DE | journal = Proteomics | volume = 13 | issue = 1 | pages = 17–21 | date = Jan 2013 | pmid = 23161590 | doi = 10.1002/pmic.201200393 | s2cid = 9002232 | url = https://www.cyandye.com/A_nascent_proteome_study_combining_click_chemistry_with_2DE.pdf | access-date = 2015-06-27 | archive-url = https://web.archive.org/web/20150630075256/https://www.cyandye.com/A_nascent_proteome_study_combining_click_chemistry_with_2DE.pdf | archive-date = 2015-06-30 | url-status = dead }}</ref> 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]] (close to "aqua") and is derived from the [[Ancient Greek|Greek]] {{lang|grc|κυάνεος}}/{{lang|grc|κυανοῦς}} ''kyaneos/kyanous'' which means a somewhat different color: "dark blue".
== References ==
{{reflist|2}}
== External links ==
{{wiktionary}}
{{Authority control}}
[[Category:Cyanine dyes| ]]
[[Category:Quaternary ammonium compounds]]