A unique approach to development of near-infrared fluorescent sensors for in vivo imaging

Near-infrared (NIR) fluorescent sensors have emerged as promising molecular tools for imaging biomolecules in living systems. However, NIR fluorescent sensors are very challenging to be developed. Herein, we describe the discovery of a new class of NIR fluorescent dyes represented by 1a/1c/1e, which are superior to the traditional 7-hydroxycoumarin and fluorescein with both absorption and emission in the NIR region while retaining an optically tunable hydroxyl group. Quantum chemical calculations with the B3LYP exchange functional employing 6-31G(d) basis sets provide insights into the optical property distinctions between 1a/1c/1e and their alkoxy derivatives. The unique optical properties of the new type of fluorescent dyes can be exploited as a useful strategy for development of NIR fluorescent sensors. Employing this strategy, two different types of NIR fluorescent sensors, NIR-H(2)O(2) and NIR-thiol, for H(2)O(2) and thiols, respectively, were constructed. These novel sensors respond to H(2)O(2) or thiols with a large turn-on NIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H(2)O(2) and NIR-thiol are capable of imaging endogenously produced H(2)O(2) and thiols, respectively, not only in living cells but also in living mice, demonstrating the value of the new NIR fluorescent sensor design strategy. The new type of NIR dyes presented herein may open up new opportunities for the development of NIR fluorescent sensors based on the hydroxyl functionalized reactive sites for biological imaging applications in living animals.     

A unique class of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF switching: rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living animals, as NIR light leads to minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have introduced a new strategy to design NIR functional dyes with the carboxylic-acid-controlled fluorescence on-off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a series of Changsha (CS1-6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness, good photostability, and sufficient chemical stability. Significantly, the new CS1-6 NIR dyes are superior to the traditional rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1-6 NIR dyes. Furthermore, using CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable fluorescence-controlled mechanism for many useful applications in biological studies.     

A General Strategy for Development of Near‐Infrared Fluorescent Probes for Bioimaging

Near‐infrared (NIR) fluorescent dyes with favorable photophysical properties are highly useful for bioimaging, but such dyes are still rare. The development of a unique class of NIR dyes via modifying the rhodol scaffold with fused tetrahydroquinoxaline rings is described. These new dyes showed large Stokes shifts (>110 nm). Among them, WR3, WR4, WR5, and WR6 displayed high fluorescence quantum yields and excellent photostability in aqueous solutions. Moreover, their fluorescence properties were tunable by easy modifications on the phenolic hydroxy group. Based on WR6, two NIR fluorescent turn‐on probes, WSP‐NIR and SeSP‐NIR, were devised for the detection of H₂S. The probe SeSP‐NIR was applied in visualizing intracellular H₂S. These dyes are expected to be useful fluorophore scaffolds in the development of new NIR probes for bioimaging.     

Bright,Color-Tunable Fluorescent Dyes in the Vis/NIR Region: Establishment of New “Tailor-Made” Multicolor Fluorophores Based on Borondipyrromethene

A new series of high-performance fluorophores named Keio Fluors (KFL), which are based on borondipyrromethene (BODIPY), are reported. The KFL dyes cover a wide spectral range from the yellow (547 nm) to the near-infrared (NIR, 738 nm) region, and their emission wavelength could be easily and subtly controlled based on simple molecular modifications only, without losing their optical properties. This “tailor-made” synthetic strategy for tuning the emission wavelength enabled the creation of fourteen KFL dyes with well-controlled emission colors (yellow, orange, red, far-red, and NIR). Moreover, these KFL dyes also retain their excellent optical properties, such as spectral bands sharper than quantum dots, high extinction coefficients (140 000–316 000 M ⁻¹ cm⁻¹), and high quantum yields (0.56–0.98), without any critical solvent polarity dependent decrease of their brightness. These advantageous characteristics make the KFL dyes potentially useful as new candidates of fluorescent standard dyes to substitute or to complement existing long-wavelength fluorescent dyes, such as cyanines, oxazines, rhodamines, or other BODIPY dyes.     

Stable,Wavelength‐Tunable Fluorescent Dyes in the NIR‐II Region for In Vivo High‐Contrast Bioimaging and Multiplexed Biosensing

Small‐molecule organic fluorophores, spectrally active in the 900–1700 nm region, with tunable wavelength and sensing properties are sought‐after for in vivo optical imaging and biosensing. A panel of fluorescent dyes ( CX ) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo‐ and photo‐stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high‐contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug‐induced hepatotoxicity by detection of OONO^?. This report presents a series of NIR‐II dyes with promising spectroscopic properties for high‐contrast bioimaging and multiplexed biosensing.     

Stable,Wavelength‐Tunable Fluorescent Dyes in the NIR‐II Region for In Vivo High‐Contrast Bioimaging and Multiplexed Biosensing

Small‐molecule organic fluorophores, spectrally active in the 900–1700 nm region, with tunable wavelength and sensing properties are sought‐after for in vivo optical imaging and biosensing. A panel of fluorescent dyes ( CX ) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo‐ and photo‐stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high‐contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug‐induced hepatotoxicity by detection of OONO^?. This report presents a series of NIR‐II dyes with promising spectroscopic properties for high‐contrast bioimaging and multiplexed biosensing.     

Keto‐benzo[h]‐Coumarin‐Based Near‐Infrared Dyes with Large Stokes Shifts for Bioimaging Applications

Fluorescence imaging is a promising tool for the visualization of biomolecules in living systems and there is great demand for new fluorescent dyes that absorb and emit in the near‐infrared (NIR) region. Herein, we constructed three new fluorescent dyes ( NBC dyes) based on keto‐benzo[h]coumarin ( k‐BC ) and benzopyrilium salts. These dyes showed large Stokes shifts (>100 nm) and NIR emission (>800 nm). The relationship between the structures and optical properties of these dyes was further investigated by using density functional theory calculations at the B3LYP/6‐3G level of theory. Fluorescence images indicated that the fabricated dyes exhibited good photostability and low cytotoxicity and, thus, have potential applications as imaging agents in living cells and animals.     

Synthesis and Photophysics of BF2‐Rigidified Partially Closed Chain Bromotetrapyrroles: Near Infrared Emitters and Photosensitizers

We report the synthesis, crystallographic, optical, and triplet and singlet oxygen generation properties of a series of BF₂‐rigidified partially closed chain bromotetrapyrroles as near infrared emitters and photosensitizers. These novel dyes were efficiently synthesized from a nucleophilic substitution reaction between pyrroles and the 3,5‐bromo‐substituents on the tetra‐ and hexabromoBODIPYs and absorb in the near‐infrared region (681–754 nm) with high molar extinction coefficients. Their fluorescent emission (708–818 nm) and singlet oxygen generation properties are significantly affected by alkyl substitutions on the two uncoordinated pyrrole units of these dyes and the polarity of solvents. Among them, dyes 4 ca and 4 da show good singlet oxygen generation efficiency and good NIR fluorescence emission (fluorescence quantum yields of 0.14–0.43 in different solvents studied).     

Preparation of fluorescent polystyrene microspheres by gradual solvent evaporation method

Highly cross-linked polystyrene beads of 9.2 μm were synthesized by seed polymerization with styrene as monomer and divinylbenzene as cross linker. Other sized monodisperse PS microspheres were also prepared by varying seed particle diameter and proportion of swelling agents. Furthermore, the polystyrene beads were stained by gradual solvent evaporation method using dyes such as rhodamine 101 and acridine orange. Gradual solvent evaporation method facilitates a high concentration of fluorescent dyes on beads. This is the key to obtain fluorescent beads with high intensity. The results showed that the fabricated fluorescent microspheres could be excited to various wavelengths (such as yellow, green, red and scarlet). Our synthesized microspheres offer high fluorescence emission efficiency compared to commercial fluorescent microspheres in the mean time have other properties in common.     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

Ratiometric analysis of fluorescence lifetime for probing binding sites in albumin with near-infrared fluorescent molecular probes

A number of diseases have been linked to abnormal conformation of albumin, a major extracellular protein in blood. Current protein structural analysis requires pure isolated samples, thereby limiting their use for albumin analysis in blood. In this study, we report a new approach for high-throughput structure-related analysis of albumin by using the fluorescence lifetime properties of near-infrared (NIR) polymethine dyes. Based on molecular modeling, polymethine dyes are bound to two binding sites with different polarities on albumin. As a result, an NIR molecular probe exhibits two distinct lifetimes with two corresponding fluorescent fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture by ratiometric analysis. After screening a small library of NIR polymethine dyes, we identified and used a polymethine dye with optimal fluorescence lifetime properties to assess structure-related differences in commercially available bovine serum albumin as model systems. The results show that changes in the lifetime of NIR dyes reflect the perturbation of the tertiary structures of albumin and that albumin prepared by different methods has slightly altered tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes in albumin in whole blood without prior isolation of the pure protein.     

Thiophene-functionalized isoindigo dyes bearing electron donor substituents with absorptions approaching the near infrared region

A series of six new, highly soluble N,N'-dialkylated isoindigo derivatives bearing different electron donating thiophene units at the 6,6'-positions were synthesized by Stille cross-coupling reaction. The optical and electrochemical properties of these dyes were studied by UV-vis spectroscopy and cyclic voltammetry, revealing a good tunability of their electronic properties by peripheral substituents with amino groups leading to strong absorption reaching the NIR region. The DFT calculations of the frontier molecular orbitals of these dyes corroborate the observed substituent effect on absorption and redox properties.     

Near-infrared small molecular fluorescent dyes for photothermal therapy

This review aims to provide a summary of the progress in fluorescent dyes for photothermal therapy in recent years and it is classified according to the structure of organic molecules including cyanines, phthalocyanines, rhodamine analogues and BODIPYs.     

Combining aminocyanine dyes with polyamide dendrons: a promising strategy for imaging in the near-infrared region

Cyanine dyes are known for their fluorescence in the near-IR (NIR) region, which is desirable for biological applications. We report the synthesis of a series of aminocyanine dyes containing terminal functional groups such as acid, azide, and cyclooctyne groups for further functionalization through, for example, click chemistry. These aminocyanine dyes can be attached to polyfunctional dendrons by copper-catalyzed azide alkyne cycloaddition (CuAAC), strain-promoted azide alkyne cycloaddition (SPAAC), peptide coupling, or direct S(NR)1 reactions. The resulting dendron-dye conjugates were obtained in high yields and displayed high chemical stability and photostability. The optical properties of the new compounds were studied by UV/Vis and fluorescence spectroscopy. All compounds show large Stokes shifts and strong fluorescence in the NIR region with high quantum yields, which are optimal properties for in vivo optical imaging.     

A pH Sensitive Fluorescence Probe Based on Tricarbocyanine

A pH sensitive near-infrared (NIR) fluorescent probe, IR-DO3A, was synthesized. It was based on combination of tricarbocyanine that had typical NIR absorption and fluorescent emission spectrum, and DO3A (1,4,7,10-tetra azacyclododecane -1,4,7-triyl)triacetic acid) that had three side chain of carboxylic acids. Phenyl sulfonamide that was used to maintain the density of electronic cloud of tricarbocyanine backbone and improve the solubility of the whole molecule was selected to connect tricarbocyanine and DO3A by several substitution reactions under some certain conditions. The final product, IR-DO3A was confirmed by NMR and MS spectrums, and had a better solubility in polar solvent than tricarbocyanine. The optical properties of IR-DO3A in two mixture solvents were studied. It had maximum Uv-vis absorption at 750nm and 805nm, fluorescent emission at 808nm, all these wavelengths were in NIR range and there was a 58nm Stokes-Shift. Also its optical properties at different pH in water were studied and it was sensitive in pH 6~9, which related with the protonation and deprotonation processes of IR-DO3A at different pH. These pH sensitive and NIR-fluorescent properties may result in an application for revealing the pH changes of physiological diseases.     

Efficient NIR‐Light Emission from Solid‐State Complexes of Boron Difluoride with 2′‐Hydroxychalcone Derivatives

This article describes a series of nine complexes of boron difluoride with 2′‐hydroxychacone derivatives. These dyes were synthesized very simply and exhibited intense NIR emission in the solid state. Complexation with boron was shown to impart very strong donor–acceptor character into the excited state of these dyes, which further shifted their emission towards the NIR region (up to 855 nm for dye 5 b , which contained the strongly donating triphenylamine group). Strikingly, these optical features were obtained for crystalline solids, which are characterized by high molecular order and tight packing, two features that are conventionally believed to be detrimental to luminescence in organic crystals. Remarkably, the emission of light from the π‐stacked molecules did not occur at the expense of the emission quantum yield. Indeed, in the case of pyrene‐containing dye 4 , for example, a fluorescence quantum yield of about 15 % with a fluorescence emission maximum at 755 nm were obtained in the solid state. Moreover, dye 3 a and acetonaphthone‐based compounds 1 b , 2 b , and 3 b showed no evidence of degradation as solutions in CH₂Cl₂ that contained EtOH. In particular, solutions of brightly fluorescent compound 3 a (brightness: ε×Φ_f=45 000 M ^?1 cm^?1) could be stored for long periods without any detectable changes in its optical properties. All together, these new dyes possess a set of very interesting properties that make them promising solid‐state NIR fluorophores for applications in materials science.     

Red/NIR Thermally Activated Delayed Fluorescence from Aza-BODIPYs

The search for long-lived red and NIR fluorescent dyes is challenging and hitherto scarcely reported. Herein, the viability of aza-BODIPY skeleton as a promising system for achieving thermal activated delayed fluorescent (TADF) probes emitting in this target region is demonstrated for the first time. The synthetic versatility of this scaffold allows the design of energy and charge transfer cassettes modulating the stereoelectronic properties of the energy donors, the spacer moieties and the linkage positions. Delayed emission from these architectures is recorded in the red spectral region (695–735 nm) with lifetimes longer than 100 μs in aerated solutions at room temperature. The computational-aided photophysical study under mild and hard irradiation regimes disclose the interplay between molecular structure and photonic performance to develop long-lived fluorescence red emitters through thermally activated reverse intersystem crossing. The efficient and long-lasting NIR emission of the newly synthesized aza-BODIPY systems provides a basis to develop advanced optical materials with exciting and appealing photonic response.     

Conformationally restricted aza-BODIPY: highly fluorescent, stable near-infrared absorbing dyes

Novel NIR fluorescent, conformational restricted aza-dipyrromethene boron difluoride (aza-BODIPY) dyes were prepared by an efficient process. Such conformational restricted aza-BODIPY dyes possess intense absorption, strong fluorescence, high chemical and photostability. Additionally, the sharp fluorescence of non-amine containing aza-BODIPY dyes is insensitive to solvent polarity.     

Design of a Near Infrared Fluorescent Ureter Imaging Agent for Prevention of Ureter Damage during Abdominal Surgeries

The inadvertent severing of a ureter during surgery occurs in as many as 4.5% of colorectal surgeries. To help prevent this issue, several near-infrared (NIR) dyes have been developed to assist surgeons with identifying ureter location. However, the majority of these dyes exhibit at least some issue that precludes their widespread usage such as high levels of uptake in other tissues, overlapping emission wavelengths with other NIR dyes used for other fluorescence-guided surgeries, and/or rapid excretion times through the ureters. To overcome these limitations, we have synthesized and characterized the spectral properties and biodistribution of a new series of PEGylated UreterGlow derivatives. The most promising dye, UreterGlow-11 was shown to almost exclusively excrete through the kidneys/ureters with detectable fluorescence observed for at least 12 h. Additionally, while the excitation wavelength is similar to that of other NIR dyes used for cancer resections, the emission is shifted by ~30 nm allowing for discrimination between the different fluorescence-guided surgery probes. In conclusion, these new UreterGlow dyes show promising optical and biodistribution characteristics and are good candidates for translation into the clinic.     

The Aggregation Regularity Effect of Multiarylpyrroles on Their Near-Infrared Aggregation-Enhanced Emission Property

Increasing the quantum yield of near-infrared (NIR) emissive dyes is critical for biological applications because these fluorescent dyes generally show decreased emission efficiency under aqueous conditions. In this work, we designed and synthesized several multiarylpyrrole (MAP) derivatives, in which a furanylidene (FE) group at the 3-position of the pyrrole forms donor-π-acceptor molecules, MAP-FE, with a NIR emissive wavelength and aggregation-enhanced emission (AEE) features. Different alkyl chains of MAP-FEs linked to phenyl groups at the 2,5-position of the pyrrole ring resulted in different emissive wavelengths and quantum yields in aggregated states, such as powders or single crystals. Powder XRD data and single crystal analysis elucidated that the different lengths of alkyl chains had a significant impact on the regularity of MAP-FEs when they were forced to aggregate or precipitate, which affected the intermolecular interaction and the restriction degree of the rotating parts, which are essential components. Therefore, an increasing number of NIR dyes could be developed by this design strategy to produce efficient NIR dyes with AEE. Moreover, this method can provide general guidance for other related fields, such as organic solar cells and organic light-emitting materials, because they are all applied in the aggregated state.