Cyanine Dyes

Introduction

Cyanine dyes are highly conjugated, fluorescent molecules with absorption and emission wavelengths in the near infra-red region (700–900 nm). The simplest synthetic route to heptamethine cyanine dyes 1 (so-called because of the seven carbons in the conjugated backbone) was first described by Narayanan and Patonay who heated N-alkylated indolium salts 2 with 2-chloro-1-formyl-3-(hydroxyl methylene) (3) in a Vilsmeier-type reaction.[1] These heptamethine cyanine scaffolds can be readily modified through displacement of the labile chloride group by nucleophiles,[2] [3] [4] resulting in fluorescent molecules with varying quantum yields, extinction coefficients, and fluorescence maxima. Conjugation to biomolecules is achieved through chlorine substitution by 3-(4-hydroxyphenyl) propionic acid.

The resulting cyanine dye has a carboxylic acid moiety which can be coupled to an amine-containing compound via amide-bond formation. Enhanced aqueous solubility is typically achieved through sulfonation of the indole 2. As biological tissue does not absorb strongly within the near infra-red window, cyanine fluorophores are ideal for in vivo optical imaging application,[5] [6] [7] while clinically, indocyanine green has been used for over 25 years in fluorescence angiography and opthalmology (mouse LD₅₀ = 60 mg/kg).[8,9]

Scheme 1 Synthesis of heptamethine cyanine dyes 1

Abstract

(A) Necrosis Detection Necrotic tissue is found in a variety of disease states including cancer and sepsis[10] where levels of extracellular DNA are increased due to dead or dying cells. Murthy et al. described a hybrid heptamethine (IR-786)–bisbenzimidazole (Hoechst 33258)[11] probe that accumulates in necrotic tissue by binding to extracellular DNA.[2] In vivo analysis in mice ischemia–reperfusion models confirmed probe ­accumulation in necrotic tissue.[2]
(B) pH Sensor Nagano and co-workers synthesised a ratiometric, NIR heptamethine pH sensor. By using two excitation wavelengths (670 nm and 750 nm), the relative fluorescence intensities (λem = 780 nm) ­allowed pH values between 6 and 10 to be readily measured. Incubation of HeLa cells with the sensor resulted in staining of lysosomes and mitochondria with a demonstrable ability to monitor intracellular pH changes.[4]
(C) Reactive Oxygen Species Detection Uncontrolled reactive oxygen species (ROS) are implicated in several inflammatory disease states.[12] Nagano and co-workers reported the real-time analysis of ROS by linking two NIR cyanine dyes with different oxidation potentials.[13] A turn on fluorescence signal was observed upon oxidation of the more susceptible cyanine dye as this removed the static quenching effect. A strong fluorescence signal was found after incubation with a variety of ROS such as the hydroxyl radical (·OH) using Fenton’s reagent and superoxide (O2 ·–) generated from xanthene oxidase.[13]
(D) H2S Molecule Sensor Hydrogen sulfide is known to be an important gaseous signaling molecule and is key in the regulation of blood pressure.[14] Zhang and co-workers developed a real-time NIR sensor for H2S by incorporating 3-nitrophenol onto the heptamethine dye scaffold which resulted in photo-induced electron transfer (PET)[15] and quenching of the cyanine dye fluorescence.[3] This was liberated by nitro group reduction with hydrogen sulfide. Incubation with other reactive sulfide species such as glutathione and cysteine gave a far weaker fluorescence increase.
(E) Silver Sensor Bioaccumulation of metal ions such as silver can demonstrate adverse biological effects due to binding to functional groups such as thiols.[16] Zheng, Jiang and co-workers developed a Ag+ sensor based on a heptamethine cyanine motif that contained an adenine moiety.[17] Aggregation[18] of the cyanine dye with increasing concentrations of Ag+ ions resulted in a fluorescence shift of 185 nm with a detection limit of 34 nM. High selectivity over other metal ions such as copper and iron was demonstrated.

• References

• 1 Narayanan N, Lee S, Sy J, Patonay G. J. Org. Chem. 1995; 5: 2391
• 2 Dasari M, Kim D, Lee S, Brown M, Davis M, Murthy N. Org. Lett. 2010; 12: 3300
• 3 Wang R, Yu F, Chen L, Chen H, Wang L, Zhang W. Chem. Commun. 2012; 48: 11757
• 4 Myochin T, Kiyose K, Hanaoka K, Kojima H, Terai T, Nagano T. J. Am. Chem. Soc. 2011; 133: 3401
• 5 Fry ES. Appl. Opt. 2000; 39: 2743
• 6 Linder KE, Metcalfe E, Nanjappan P, Arunachalam T, Ramos K, Skedzielewski TM, Marinelli ER, Tweedle MF, Nunn AD, Swenson RE. Biocon. Chem. 2011; 22: 1287
• 7 Thielbeer F, Chankeshwara SV, Johansson EM. V, Norouzi N, Bradley M. Chem. Sci. 2013; 4: 425
• 8 Taichman GC, Ph D, Hendry P, Keon W. Tex. Heart Inst. J. 1987; 14: 133
• 9 Kodjikian L, Richter T, Halberstadt M, Beby F, Flueckiger F, Boehnke M, Garweg JG. Graefe‘s Arch. Clin. Exp. Ophthalmol. 2005; 243: 917
• 10 Amaravadi RK, Thompson CB. Clin. Cancer Res. 2007; 13: 7271
• 11 Latt SA, Stetten G. J. Histochem. Cytochem. 1976; 24: 24
• 12 Valko M, Leibfritz D, Moncol J, Cronin MT. D, Mazur M, Telser J. Int. J. Biochem. Cell Biol. 2007; 39: 44
• 13 Oushiki D, Kojima H, Terai T, Arita M, Hanaoka K, Urano Y, Nagano T. J. Am. Chem. Soc. 2010; 132: 2795
• 14 Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, Mustafa AK, Mu W, Zhang S, Snyder SH, Wang R. Science 2008; 322: 587
• 15 Griesbeck AG, Hoffmann N, Warzecha K.-D. Acc. Chem. Res. 2007; 40: 128
• 16 Navarro E, Piccapietra F, Wagner B, Marconi F, Kaegi R, Odzak N, Sigg L, Behra R. Environ. Sci. Technol. 2008; 42: 8959
• 17 Zheng H, Yan M, Fan X.-X, Sun D, Yang S.-Y, Yang L.-J, Li J.-D, Jiang Y.-B. Chem. Commun. 2012; 48: 2243
• 18 Tan C, Atas E, Müller JG, Pinto MR, Kleiman VD, Schanze KS. J. Am. Chem. Soc. 2004; 126: 13685