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EP1165693A2 - Colorants a base de squaraine - Google Patents

Colorants a base de squaraine

Info

Publication number
EP1165693A2
EP1165693A2 EP00914284A EP00914284A EP1165693A2 EP 1165693 A2 EP1165693 A2 EP 1165693A2 EP 00914284 A EP00914284 A EP 00914284A EP 00914284 A EP00914284 A EP 00914284A EP 1165693 A2 EP1165693 A2 EP 1165693A2
Authority
EP
European Patent Office
Prior art keywords
group
compounds
meoh
dye
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00914284A
Other languages
German (de)
English (en)
Inventor
Richard Martin West
William Jonathan Cummins
Robert James Domett Nairne
Matthew Graham Bull
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GE Healthcare UK Ltd
Original Assignee
Amersham Pharmacia Biotech UK Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amersham Pharmacia Biotech UK Ltd filed Critical Amersham Pharmacia Biotech UK Ltd
Priority to EP00914284A priority Critical patent/EP1165693A2/fr
Publication of EP1165693A2 publication Critical patent/EP1165693A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/007—Squaraine dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label

Definitions

  • Berger et al EP 0 214 847 has described the use of other cyanine dyes some of which contain squarate groups in assays which involve a specific binding partner.
  • Other squarate dyes are described by Pease et al USP 4,830,786, by A J G Mank et al in Anal. Chem. 1995, 67, 1742-8, and by Amersham International in WO 97/40104.
  • Cushman et al WO 93/09172 and Krutak et al WO 94/19387 have described cyanine dyes containing squarate groups for use in thermoplastics and inks.
  • fluorescein (as its reactive derivatives) is one of the original fluorescent labels for biomolecules and continues to be widely used with a 488nm laser as an excitation light source. It is far from ideal, however, for it has a low extinction coefficient, it is fluorescent only in a certain pH range, and it has poor chemical stability and photostability and solubility characteristics.
  • the present invention provides squaraine compounds having the structure
  • n 1 , 2 or 3
  • W represents a wing moiety having the structure
  • L is a linker chain of 0 to 60 moieties, branched or unbranched which optionally contains one or more arylene groups, or O or N or N + or S or S + or P or P + or Se atoms
  • G is a functional or a reactive group by means of which the compound may be covalently linked to a biomolecule, other small molecule e.g. a dye or a group which enhances or reduces water solubility or provides electron donating or withdrawing properties to modify the spectral characteristics of the compound, and homo-dimers and -oligomers and hetero-dimers and -oligomers of the compounds.
  • R 1 and R 2 may be Ci - C 2 o hydrocarbon, for example alkyl or aryl or aralkyl. If one or both of R 1 and R 2 is hydrogen, then the compound will be pH-sensitive e.g. having fluorescent properties at one pH and not at another. Or R 1 and R 2 may together form a ring system, which is saturated or unsaturated, for example pyrrolidine, piperidine, pyrrole, piperazine, or morpholine, or a fused ring system. Alternatively R 1 and R 2 may together with the N atom to which they are joined form an aza crown or other metal- binding group such as EDTA or other metal chelating moiety containing various combinations of N, O and S ligand atoms. R 1 or both of R 1 and R 2 may be a group -L-G which is discussed below. Examples of amine moieties A are:
  • the wing moiety W is preferably
  • R 3 and R 4 are H or Ci - C 2 o or a group -L-G, and n is 0 - 3.
  • R 3 may be H, in which case the compound may be pH sensitive, for example exhibiting fluorescent properties at one pH and not at another.
  • R 3 may be Ci - C 2 o hydrocarbon as discussed above for R 1 and R 2 .
  • R 3 may be -L-G as discussed below.
  • the dotted line preferably represents a heterocyclic ring system, either single ring or fused ring and generally unsaturated, for example pyrrole or pyridine or indole or benzindole or benzoxazole or benzothiazole or quinolyl.
  • the ring system represented by the dotted line has the structure:
  • X is O or S or -NR 4 or -CR 10 R 11 where each of R 10 and R 11 is Ci - C 2 o hydrocarbon or -L -G, or R 10 and R 11 together form a single ring of fused ring or heterocyclic or cycloaliphatic group.
  • the integer m may be 1 or 2 or 3.
  • m 1
  • these are squaric compounds; when n is 2 they are croconic compounds; when m is 3 they are rhodizonic compounds.
  • L is a linker chain of 0 to 30 or 60 moieties, preferably selected form alkylene, alkenylene and alkynylene or a branched or straight chain of up to 30 carbon atoms optionally incorporating one to six O or N or N + or S or S + or P or P + or Se atoms or arylene groups.
  • G may be a functional or a reactive group by means of which the squaraine compound may be covalently linked to a biomolecule or other molecule.
  • G as a functional group are such nucleophiles as NH2, OH, SH.
  • G as a reactive group are -COOH, activated carboxyl such as acid halide or anhydride, CO active ester, -NCS, O phosphoramidite, -NC(0)CH 2 l and maleimide
  • biomolecules are nucleosides, nucleotides and analogues thereof, oligonucleotides and nucleic acids, and also amino acids, peptides, proteins, antibodies, polysaccharides, lipids, sugars and other small molecules.
  • Another example of a biomolecule in this context is a cyclodextrin. Cyclodextrin - fluorophore conjugates have been shown to possess greatly enhanced photostability in aqueous solution (Tetrahedron Letters, Volume 38, No 35, pages 6167-6170, 1997).
  • G may be a group which enhances water solubility such as sulphonate, phosphate, quaternary ammonium, sugar and polyether; or that reduces water solubility e.g. alkyl.
  • G may be a group which provides electron donating or withdrawal properties to modify the spectral characteristics of the squaraine compound; for example halogen, alkoxy, nitro or cyano. See the Chemistry of Synthetic Dyes, Venkataraman, Academic Press, New York, 1971 , 4, Chapter 5 Part iiic, pages 228-240 particularly Table 1 on page 230.
  • the compounds of this invention have interesting fluorescent properties, they are also in general coloured and can be used as conventional dyes e.g. in colorimetric assays.
  • dimers and oligomers of the compounds defined above are also envisaged according to the invention.
  • a dimer may have the structure W 1 -Sq-A 1 -Sq-W 2 or alternatively the structure
  • a 1 -Sq-W 1 -Sq-A 2 where A 1 and A 2 each represent an amine moiety A, and W 1 and W 2 each represent a wing moiety W.
  • R 1 and R 2 are part of a substituted fused ring system this ring system may contain one or more amino groups capable of forming a dye species. In such cases the substituted ring system is acting as a scaffold upon which dye molecules can be formed. If all the dye species are the same then an enhancement of the fluorescent signal would result. If different dye species were present energy transfer could take place.
  • a 1 and A 2 are the same, the compounds are expected to have more intense dye properties than the monomers.
  • An example of such a dimer is:
  • an energy-transfer cassette comprising a donor dye and an acceptor dye, to provide increased separation between the absorption wavelength and the emission wavelength of the compound. Trimers and higher oligomers may be made and used in similar fashion.
  • the dyes of the invention can be used to form energy transfer cassettes with known dyes such as cyanine, rhodamine, etc.
  • the donor dye is a dye of the invention and replaces fluorescein the lack of pH sensitivity, greater photostability and increased extinction coefficient could be advantageous. Examples are
  • the energy transfer cassettes are but one means of providing an energy transfer system.
  • the dyes of the invention can also be used in simple FRET assays where energy transfer is facilitated by bringing the donor and acceptor to a distance where energy transfer becomes viable. Such systems can be based solely on the dyes described above or use known dyes as one of the donor acceptor pair.
  • Another aspect of a FRET assay is the use of quencher dyes which initially result in no signal when the donor is in close proximity to the quencher. When an event occurs that results in a greater physical separation between the two dyes the quencher dye is no longer able to quench the fluorescence of the second dye and thus a signal is produced. It is reported that N0 2 groups attached to squarate dyes (Dye and Pigment, Vol 35, 331 , 1997) greatly reduce the fluorescence of squarate based dyes. By such means quencher dyes from the invention dyes can be produced to enable dyes to be produced for such FRET assays.
  • the compounds when one of R 1 and R 3 is H, the compounds may be pH sensors.
  • A comprises a metal chelating moiety, the compounds may act as metal ion sensors.
  • A comprises a NADH system, the compounds may act as redox sensors. Examples are:
  • Redox Sensor e.g. NADH
  • the invention also includes a method of making the compounds as defined, which method comprises the steps of i) reacting the wing moiety W with a dialkyl squarate or analogue to give an intermediate a)
  • Reacting intermediate a) with R 1 R 2 NH gives a 1 ,2 squarate or analogue.
  • Reacting intermediate b) with R 1 R 2 NH gives a 1 ,3 squarate or analogue.
  • the invention also includes an assay or labelling method which comprises contacting a sample containing an amine with a compound a) or a compound b)
  • the amine may be in solution or on a solid phase, and may be for example a peptide or a 5'-amino derivatised oligonucleotide.
  • the assay may be qualitative or quantitative and may be designed to identify a particular amine by reference to the spectral characteristics of the resulting dye.
  • the groups -NR 1 R 2 may comprise an amino-sugar, or amine with additional quaternary ammonium groups.
  • the amine function may be part of a metal chelating system e.g. azacrown:
  • ⁇ max (MeOH) 440nm ⁇ H (300MHz, CDCI 3 ) 1.37 (3H, t), 1.88 (6H, s), 3.98 (2H, q), 4.52 (3H, s), 5.41 (1 H, s), 7.20 (1 H, d), 7.36 (1 H, m), 7.52 (1 H, m), 7.84 (2H, m) and 8.09 (1 H, m).
  • R1-R2 -(CH 2 ) 5 -
  • R1-R2 -(CH 2 ) 2 -0-(CH 2 ) 2 -
  • R1 -R2 -(CH 2 ) 2 -CH(C0 2 H)-(CH 2 ) 2
  • This new product was expected to be the 1 ,2-adduct, 3-(1-piperidino)-4-(1 -ethyl-3,3-dimethyl- 2-benzinolinylidenemethyl)-cyclobut-3-en-1 ,2-dione:
  • ⁇ max (MeOH) 492nm ⁇ H (300MHz, CD 3 OD) 1 .373 (3H, t), 1 .80-2.03 (10H, m), 2.491 (1 H, m), 3.36-3.47 (4H, m), 3.63 (2H, m), 4.13 (2H, broad q), 4.76 (2H, m), 5.734 (1 H, s), 6.822 (2H, s), 7.38 (1 H, t), 7.45 (1 H, d), 7.54 (1 H, m), 7.91 (2H, app. d) and 8.16 (1 H, d).
  • ⁇ H (300MHz, CD3OD) 1 .28 (3H, t), 1 .48 (9H, s), 1.99 (6H, s), 4.29 (2H, q), 6.04 (1 H, s), 6.73 (2H, d) 7.22 (1 H, app. t), 7.47-7.76 (2H, m), 7.82 (2H, d) and 7.98-8.27 (3H, m).
  • ⁇ m ax (MeOH) 532nm ⁇ H (300MHz, CDCI3) 1 .25 (3H, s), 1 .43 (3H, t), 1 .57 (4H, broad s), 2.01 (6H, s), 4.19 (2H, q), 5.92 (1 H, s), 7.17-7.85 (8H, m), 7.89- 7.94 (2H, m) and 8.19 (1H,d).
  • R1-R2 -(CH 2 ) 5 -
  • R1-R2 -(CH 2 ) 2 -0-(CH 2 ) 2 -
  • the product squarate dye has a nitrogen which is incompletely alkylated, having a hydrogen atom attached instead of a third carbon atom.
  • This hydrogen can be removed under basic conditions, causing a marked change in visible absorption and fluorescence properties; the dye becomes pH-sensitive.
  • the nature of the original amine provides a means of controlling the pKa value of this change, i.e. the dye can be "tuned" to change from one form to the other over a controlled pH range. The following examples illustrate this effect.
  • a LOmMol solution in DMF was prepared; this was diluted 1 :100 with 0.02Mol bis-tris propane/HCI aqueous buffer to give a 10 ⁇ Mol working solution.
  • any of the dyes in example 5 can be acylated on the nitrogen with acyl halide reagents, as demonstrated by the following examples:
  • ⁇ max (MeOH) 440nm ⁇ H (300MHz, CDCI 3 ) 1.372 (3H, t), 4.04 (2H, q), 4.451 (3H, s), 5.445 (1 H, s), 7.06 (1 H, d), 7.15 (1 H, app. t), 7.33 (app. t) and 7.48 (1 H, dd).
  • the dried reaction mixture was used to prepare dyes without further purification.
  • ⁇ max (MeOH) 590+554nm ⁇ H (300MHz, CD 3 OD) 1.435 (3H, t), 1.76 (6H, broad s), 3.94 (4H, broad s), 4.27 (2H, q), 6.217 (1 H, s), 7.40 (1 H, m), 7.53 (1 H, d), 7.63- 7.68 (2H, m), 8.17 (1 H, d) and 8.26 (1 H, d).
  • E. coli lysate was prepared such that there was 25 ⁇ g of protein in a final volume of 9 ⁇ l lysate buffer (8M Urea, 4% (w/v) CHAPS, 40mM Tris (pH8)). To this was added 10mM TCEP (1 ⁇ l of an aqueous solution) and the mixture incubated at 37°C for one hour. The resulting reduced protein was then labelled by the addition of a DMF solution of dye 3j (2 ⁇ l of a 10nmol/1 ⁇ l DMF solution). Labelling was effected by incubation at 37°C for 30mins.
  • sample buffer (12 ⁇ l of a buffer comprising of 8M Urea, 4% (w/v) CHAPS, 20mg/ml DTT, 4% IPG buffer) and the protein subjected to a 2D gel analysis i.e. IEF page followed by SDS page.
  • sample buffer (12 ⁇ l of a buffer comprising of 8M Urea, 4% (w/v) CHAPS, 20mg/ml DTT, 4% IPG buffer
  • 2D gel analysis i.e. IEF page followed by SDS page.
  • the resultant gel was the visualised by a fluorescent scanner for protein positions on the gel as indicated by the dye labelled proteins.
  • Labelling of cDNA with dye 10a was carried out as follows. Eight cDNA reactions were set up each containing 2 ⁇ g of human skeletal muscle mRNA, 2 ⁇ l of random primers (Nonamers, Amersham Pharmacia RPK0158), and 12 ⁇ l of water. For the no reverse transcriptase reactions 14 ⁇ l of water was added. The reactions were heated to 70 °C for 10 mins and then transferred to room temperature for 15 mins.
  • the free aminoallyl-dUTP was removed by adding 450 ⁇ l of water, placing in a Microcon 30 column (Millipore cat no 42410). The column was spun for 8 mins at 13krpm in a microfuge (MicroCentaur, MSE). 450 ⁇ l of water was added again to the column and spun through for 8 minutes as before. The 450 ⁇ l water was added once again and the column spun as previously. The purified cDNA was then eluted from the column by a 1 minute spin at 13krpm into a fresh tube.
  • the cDNA was dried down in a Speedvac.
  • the cDNA was resuspended in 4.5 ⁇ l of water.
  • 3.2x10 "7 moles of ( ⁇ 0.25mgs) of Squaramide NHS ester and Cy3-NHS ester (Amersham Pharmacia PA23001) were each resuspended in 72 ⁇ l 0.1 M Sodium bicarbonate pH9.
  • 4.5 ⁇ l of the appropriate dye solutions were then added to the cDNA solution.
  • the reactions were incubated at room temperature in the dark for 1.5 hours.
  • 4.5 ⁇ l of 4M hydroxylamine (Sigma H2391 ) was then added and incubation continued for a further l Ominutes.
  • the next step was to remove the free dye from the dye incorporated into the cDNA.
  • 500 ⁇ l of buffer PB (Qiagen, Qiaquick kit cat28106) was then added and the probes added to a Qiaquick column (Qiagen cat 28106).
  • the columns were spun in a MicroCentaur microfuge for 1 minute at 13krpm.
  • 500 ⁇ l of buffer PE (Qiagen cat28106) was added to wash the column and the spin repeated. The wash and spin step was repeated twice more. A further 1 minute spin was carried out before elution with 100 ⁇ l of elution buffer (Qiagen cat28106).
  • the dye incorporation into the probes was then measured using a Cary UV/visible spectrophotometry.

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Abstract

L'invention concerne des composés représentés par la structure: W = Sq A, dans laquelle A est représenté par la formule -NR?1R2; R1 et R2¿ sont identiques ou différents et chacun représente H ou un hydrocarbure en C¿1?-C20 ou un groupe -L-G, ou R?1 ou R2¿ représente -OR?5 ou -NR6R7¿ ou -COR?7 ou NR6 COR7¿ ou -N = R?8, ou R1 et R2¿ forment ensemble un noyau unique ou un système cyclique à un noyaux condensés, un groupe azacouronne ou un groupe se liant avec les métaux saturé ou insaturé, et substitué ou non substitué ; Sq est représenté par la formule (A) ou (B), dans lesquelles m vaut 1, 2 ou 3, W représente une fraction latérale présentant la structure (C), L' est un lieur constitué de 0-3 fraction(s) sélectionnée(s) dans le groupe comprenant des atomes de carbone et des groupes arylène, la ligne pointillée représente un noyau unique ou un système de noyaux condensés, aromatique ou hétérocyclique, non substitué ou substitué, contenant ou joint à un atome N tertiaire ou quaternaire, et présentant une insaturation coordonnée à celle de L' = Sq ; R?5, R6, R7 et R8¿ représentent chacun H ou un hydrocarbure en C¿1?-C20 ou un groupe -L-G ; R?9¿ représente une charge négative ou un groupe -L-G ; L représente une chaîne de liaison constituée de 0 à 30 fractions, ramifiée ou non ramifiée, qui contient éventuellement un ou plusieurs groupes arylène ou des atomes O, N, N?+, S, S+, P, P+¿ ou Se, et G est un groupe fonctionnel ou réactif grâce auquel le composé peut être lié de manière covalente à une biomolécule, ou un groupe qui améliore ou réduit la solubilité dans l'eau ou confère des propriétés de donneur ou de retrait d'électron qui modifient les caractéristiques spectrales du composé. L'invention concerne également des homodimères, des homo-oligomères, des hétérodimères et oligomères de ces composés.
EP00914284A 1999-03-31 2000-03-30 Colorants a base de squaraine Withdrawn EP1165693A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP00914284A EP1165693A2 (fr) 1999-03-31 2000-03-30 Colorants a base de squaraine

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP99302510 1999-03-31
EP99302510 1999-03-31
EP00914284A EP1165693A2 (fr) 1999-03-31 2000-03-30 Colorants a base de squaraine
PCT/GB2000/001223 WO2000058405A2 (fr) 1999-03-31 2000-03-30 Colorants a base de squaraine

Publications (1)

Publication Number Publication Date
EP1165693A2 true EP1165693A2 (fr) 2002-01-02

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EP00914284A Withdrawn EP1165693A2 (fr) 1999-03-31 2000-03-30 Colorants a base de squaraine

Country Status (5)

Country Link
EP (1) EP1165693A2 (fr)
JP (1) JP2002540279A (fr)
AU (1) AU3568000A (fr)
CA (1) CA2366263A1 (fr)
WO (1) WO2000058405A2 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7563891B2 (en) 2004-05-21 2009-07-21 Becton, Dickinson & Company Long wavelength thiol-reactive fluorophores
JP4999334B2 (ja) * 2005-02-28 2012-08-15 住友化学株式会社 色素化合物、該化合物を用いた光電変換素子及び光電気化学電池
JP4737599B2 (ja) * 2005-04-01 2011-08-03 日本カーリット株式会社 含金属スクアリリウム化合物及び該化合物を用いた光学記録媒体
JP5090181B2 (ja) * 2005-12-09 2012-12-05 株式会社林原 遮光剤
WO2018105269A1 (fr) * 2016-12-07 2018-06-14 富士フイルム株式会社 Élément de conversion photoélectrique, capteur optique et élément d'imagerie
CN107245061B (zh) * 2017-06-13 2020-09-08 常州大学 一种基于苯并噻唑的1,2位对称方酸菁染料探针及其制备方法和应用
US11091646B2 (en) 2017-08-14 2021-08-17 Seta Biomedicals, Llc Luminescent squaraine rotaxane compounds

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Publication number Priority date Publication date Assignee Title
WO1993009956A1 (fr) * 1991-11-20 1993-05-27 Polaroid Corporation Colorants au squarylium
GB9209047D0 (en) * 1992-04-27 1992-06-10 Minnesota Mining & Mfg Thermal transfer materials
DK0898596T3 (da) * 1996-04-19 2001-12-17 Amersham Pharm Biotech Uk Ltd Farvestoffer af kvadrattypen og deres anvendelse ved en fremgangsmåde til sekvensbestemmelse

Non-Patent Citations (1)

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Title
See references of WO0058405A3 *

Also Published As

Publication number Publication date
CA2366263A1 (fr) 2000-10-05
AU3568000A (en) 2000-10-16
WO2000058405A3 (fr) 2001-02-01
WO2000058405A2 (fr) 2000-10-05
JP2002540279A (ja) 2002-11-26

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