Glotz G., Polena J., Khan N. M., Mukherjee A., Kloz M., Slavíček P., Klán P. The First Microseconds of the Life of Excited Heptamethine Cyanine Revealed by Femtosecond Stimulated Raman Spectroscopy. Commun. Chem. 2026 (in print).
Heptamethine cyanines are a well-known class of organic near-infrared (NIR) fluorophores that play an indispensable role in chemistry and biology. Despite their ubiquity, the underlying photophysical and photochemical dynamics triggered by excitation remain surprisingly elusive. In this study, we investigated a prototypical heptamethine cyanine (Cy7) using femtosecond stimulated Raman spectroscopy. Combining transient Raman spectra with quantum chemical calculations allowed us to develop a comprehensive picture of the species produced during Cy7 excitation and their behavior on the fs to sub-ms time scale. We have unambiguously identified the excited singlet and triplet states and the resulting configurational photoisomers using clearly distinguishable Raman shifts. We also reveal solvent-mediated relaxation channels, in particular ultrafast photoinduced electron transfer to dissolved oxygen, generating cyanine radical dication in addition to superoxide. Together, these insights provide a coherent mechanistic framework for Cy7 photodynamics and provide design guidance for next-generation NIR probes.
Bedocchi O., Polena J., Okorončekova J., Slavíček P., Klán P. Engineering the Photophysics of Cyanines by Chain C1´ Substituents. J. Org. Chem. 2025, 90, 17797.
Cyanine dyes are widely used in bioimaging, sensing, optoelectronic, and medicinal applications due to their tunable photophysical properties. However, controlling their electronic structures and photophysical properties remains a challenge. Here we report a general synthetic route to pentamethine and heptamethine cyanines bearing C1′ chain substituents that allow substantial control of their electronic, photophysical, and photochemical properties. By varying the terminal heterocycle and introducing various substituents at the 1′-position, we investigated the role of symmetry breaking and its impact on bond length alternation (BLA) and out-of-plane rotation (OPR). Our analysis shows that OPR, coupled with BLA, suppresses or hypsochromically shifts the first absorption band, thereby significantly altering the absorption properties of the studied dyes. This effect is particularly pronounced in structures with different heterocyclic end groups and bulky or electron deficient substituents at the 1′-position. Through quantum chemical calculations and spectroscopic analyses, we demonstrate how these modifications can be used to tune optical properties of these dyes across the visible region, paving the way for their further customization.
Madea D., Peňáková J., Mehara J., Akisaka R., Martínek M., Roithová J., Klán P. Photooxidation of Dipyrrinones: Reaction with Singlet Oxygen and Characterization of Reaction Intermediates. J. Org. Chem. 2025, 90, 2403-2420.
Bilirubin (BR) is a water-insoluble product of heme catabolism in mammals. Elevated blood concentrations of BR, especially in the neonatal period, are treated with blue-green light phototherapy. The major mechanism of BR elimination during phototherapy is photoisomerization, while a minor, less studied mechanism of degradation is oxidation. In this work, we studied the oxidation of the bilirubin model tetramethyl-dipyrrinone (Z-13) by singlet oxygen in methanol using UV–vis and ESI-MS spectroscopy, resulting in propentdyopents as the main oxidation products. We also identified two additional intermediates that were formed during the reaction (hydroperoxide 21a and imine 17). The structure of the hydroperoxide was confirmed by helium-tagging IR spectroscopy. Such reaction intermediates formed during the oxidation of BR or bilirubin models have not been described so far. We believe that this work can be used as a first step in studying the complex oxidation mechanism of BR during phototherapy.
Okoročenkova J., Filgas J., Khan N. M., Slavíček P., Klán P. Thermal Truncation of Heptamethine Cyanine Dyes. J. Am. Chem. Soc. 2024, 146, 19768-19781.
Cyanine dyes are a class of organic, usually cationic molecules containing two nitrogen centers linked through conjugated polymethine chains. The synthesis and reactivity of cyanine derivatives have been extensively investigated for decades. Unlike the recently described phototruncation process, the thermal truncation (chain shortening) reaction is a phenomenon that has rarely been reported for these important fluorophores. Here, we present a systematic investigation of the truncation of heptamethine cyanines (Cy7) to pentamethine (Cy5) and trimethine (Cy3) cyanines via homogeneous, acid–base-catalyzed nucleophilic exchange reactions. We demonstrate how different substituents at the C3′ and C4′ positions of the chain and different heterocyclic end groups, the presence of bases, nucleophiles, and oxygen, solvent properties, and temperature affect the truncation process. The mechanism of chain shortening, studied by various analytical and spectroscopic techniques, was verified by extensive ab initio calculation, implying the necessity to model catalytic reactions by highly correlated wave function-based methods. In this study, we provide critical insight into the reactivity of cyanine polyene chains and elucidate the truncation mechanism and methods to mitigate side processes that can occur during the synthesis of cyanine derivatives. In addition, we offer alternative routes to the preparation of symmetrical and unsymmetrical meso-substituted Cy5 derivatives.
Ramundo A., Janoš J., Muchová L., Šranková M., Vítek L., Slavíček P., Klán P. Visible-Light-Activated Carbon Monoxide Release from Porphyrin-Flavonol Hybrids. J. Am. Chem. Soc. 2024, 146, 920-929.
We report on porphyrin–flavonol hybrids consisting of a porphyrin antenna and four covalently bound 3-hydroxyflavone (flavonol) groups, which act as highly efficient photoactivatable carbon monoxide (CO)-releasing molecules (photoCORMs). These bichromophoric systems enable activation of the UV-absorbing flavonol chromophore by visible light up to 650 nm and offer precise spatial and temporal control of CO administration. The physicochemical properties of the porphyrin antenna system can also be tuned by inserting a metal cation. Our computational study revealed that the process occurs via endergonic triplet–triplet energy transfer from porphyrin to flavonol and may become feasible thanks to flavonol energy stabilization upon intramolecular proton transfer. This mechanism was also indirectly supported by steady-state and transient absorption spectroscopy techniques. Additionally, the porphyrin–flavonol hybrids were found to be biologically benign. With four flavonol CO donors attached to a single porphyrin chromophore, high CO release yields, excellent uncaging cross sections, low toxicity, and CO therapeutic properties, these photoCORMs offer exceptional potential for their further development and future biological and medical applications.
Jézéquel Y. A., Svěrák F., Ramundo A., Orel V., Martínek M., Klán P. Structure-Photoreactivity Relationship Study of Substituted 3-Hydroxyflavones and 3-Hydroxyflavothiones for Improving Carbon Monoxide Photorelease. J. Org. Chem. 2024, 89, 4888-4903.
Carbon monoxide (CO) is notorious for its toxic effects but is also recognized as a gasotransmitter with considerable therapeutic potential. Due to the inherent challenges in its delivery, the utilization of organic CO photoreleasing molecules (photoCORMs) represents an interesting alternative to CO administration characterized by high spatial and temporal precision of release. This paper focused on the design, synthesis, and photophysical and photochemical studies of 20 3-hydroxyflavone (flavonol) and 3-hydroxyflavothione derivatives as photoCORMs. Newly synthesized compounds bearing various electron-donating and electron-withdrawing groups show bathochromically shifted absorption maxima and considerably enhanced CO release yields compared to the parent unsubstituted flavonol, exceeding 0.8 equiv of released CO in derivatives exhibiting excited states with a charge-transfer character. Until now, such outcomes have been limited to flavonol derivatives possessing a π-extended aromatic system. In addition, thione analogs of flavonols, 3-hydroxyflavothiones, show substantial bathochromic shifts of their absorption maxima and enhanced photosensitivity but provide lower yields of CO formation. Our study elucidates in detail the mechanism of CO photorelease from flavonols and flavothiones, utilizing steady-state and time-resolved spectroscopies and photoproduct analyses, with a particular emphasis on unraveling the structure–photoreactivity relationship and understanding competing side processes.
Ramundo A., Hurtová M., Bożek I., Osifová Z., Russo M., Ngoy B. P., Křen V., Klán P. Multimodal Carbon Monoxide Photorelease from Flavonoids. Org. Lett. 2024, 26, 708-712.
Photooxygenation of flavonoids leads to the release of carbon monoxide (CO). Our structure–photoreactivity study, employing several structurally different flavonoids, including their 13C-labeled analogs, revealed that CO can be produced via two completely orthogonal pathways, depending on their hydroxy group substitution pattern and the reaction conditions. While photooxygenation of the enol 3-OH group has previously been established as the CO liberation channel, we show that the catechol-type hydroxy groups of ring B can predominantly participate in photodecarbonylation.
Mujawar T., Ševelda P., Madea D., Klán P., Švenda J. A Platform for the Synthesis of Oxidation Products of Bilirubin. J. Am. Chem. Soc. 2024, 146, 1603-1611.
Bilirubin is the principal product of heme catabolism. High concentrations of the pigment are neurotoxic, yet slightly elevated levels are beneficial. Being a potent antioxidant, oxidative transformations of bilirubin occur in vivo and lead to various oxidized fragments. The mechanisms of their formation, intrinsic biological activities, and potential roles in human pathophysiology are poorly understood. Degradation methods have been used to obtain samples of bilirubin oxidation products for research. Here, we report a complementary, fully synthetic method of preparation. Our strategy leverages repeating substitution patterns in the parent tetracyclic pigment. Functionalized ready-to-couple γ-lactone, γ-lactam, and pyrrole monocyclic building blocks were designed and efficiently synthesized. Subsequent modular combinations, supported by metal-catalyzed borylation and cross-coupling chemistries, translated into the concise assembly of the structurally diverse bilirubin oxidation products (BOXes, propentdyopents, and biopyrrins). The discovery of a new photoisomer of biopyrrin A named lumipyrrin is reported. Synthetic bilirubin oxidation products made available in sufficient purity and quantity will support future in vitro and in vivo investigations.
Tovtik R., Muchová E., Štacková L., Slavíček P., Klán P. Spin-Vibronic Control of Intersystem Crossing in Iodine-Substituted Heptamethine Cyanines. J. Org. Chem. 2023, 88, 6716-6728.
Spin–orbit coupling between electronic states of different multiplicity can be strongly coupled to molecular vibrations, and this interaction is becoming recognized as an important mechanism for controlling the course of photochemical reactions. Here, we show that the involvement of spin–vibronic coupling is essential for understanding the photophysics and photochemistry of heptamethine cyanines (Cy7), bearing iodine as a heavy atom in the C3′ position of the chain and/or a 3H-indolium core, as potential triplet sensitizers and singlet oxygen producers in methanol and aqueous solutions. The sensitization efficiency was found to be an order of magnitude higher for the chain-substituted than the 3H-indolium core-substituted derivatives. Our ab initio calculations demonstrate that while all optimal structures of Cy7 are characterized by negligible spin–orbit coupling (tenths of cm–1) with no dependence on the position of the substituent, molecular vibrations lead to its significant increase (tens of cm–1 for the chain-substituted cyanines), which allowed us to interpret the observed position dependence.
Sekhar A. R., Chitose Y., Janoš J., Dangoor S. I., Ramundo A., Satchi-Fainaro R., Slavíček P., Klán P., Weinstain R. Porphyrin as a Versatile Visible-Light-Activatable Organic/Metal Hybrid Photoremovable Protecting Group. Nat. Commun. 2022, 13, 3614.
Photoremovable protecting groups (PPGs) represent one of the main contemporary implementations of photochemistry in diverse fields of research and practical applications. For the past half century, organic and metal-complex PPGs were considered mutually exclusive classes, each of which provided unique sets of physical and chemical properties thanks to their distinctive structures. Here, we introduce the meso-methylporphyrin group as a prototype hybrid-class PPG that unites traditionally exclusive elements of organic and metal-complex PPGs within a single structure. We show that the porphyrin scaffold allows extensive modularity by functional separation of the metal-binding chromophore and up to four sites of leaving group release. The insertion of metal ions can be used to tune their spectroscopic, photochemical, and biological properties. We provide a detailed description of the photoreaction mechanism studied by steady-state and transient absorption spectroscopies and quantum-chemical calculations. Our approach applied herein could facilitate access to a hitherto untapped chemical space of potential PPG scaffolds.
Fukushima H., Matikonda S. S., Usama S. M., Furusawa A., Kato T., Štacková L., Klán P., Kobayashi H., Schnermann M. J. Cell Labeling with Spatiotemporal Control Using Cyanine Phototruncation. J. Am. Chem. Soc. 2022, 144, 11075-11080.
Photoconvertible tracking strategies assess the dynamic migration of cell populations. Here we develop phototruncation-assisted cell tracking (PACT) and apply it to evaluate the migration of immune cells into tumor-draining lymphatics. This method is enabled by a recently discovered cyanine photoconversion reaction that leads to the two-carbon truncation and consequent blue-shift of these commonly used probes. By examining substituent effects on the heptamethine cyanine chromophore, we find that introduction of a single methoxy group increases the yield of the phototruncation reaction in neutral buffer by almost 8-fold. When converted to a membrane-bound cell-tracking variant, this probe can be applied in a series of in vitro and in vivo experiments. These include quantitative, time-dependent measurements of the migration of immune cells from tumors to tumor-draining lymph nodes. Unlike previously reported cellular photoconversion approaches, this method does not require genetic engineering and uses near-infrared (NIR) wavelengths. Overall, PACT provides a straightforward approach to label cell populations with spatiotemporal control.
Russo M., Orel V., Štacko P., Šranková M., Muchová L., Vítek L., Klán P. Structure-Photoreactivity Relationship of 3-Hydroxyflavone-Based CO-Releasing Molecules. J. Org. Chem. 2022, 87, 4750-4763.
Carbon monoxide (CO) is an endogenous signaling molecule that regulates diverse physiological processes. The therapeutic potential of CO is hampered by its intrinsic toxicity, and its administration poses a significant challenge. Photoactivatable CO-releasing molecules (photoCORMs) are an excellent tool to overcome the side effects of untargeted CO administration and provide precise spatial and temporal control over its release. Here, we studied the CO release mechanism of a small library of derivatives based on 3-hydroxy-2-phenyl-4H-benzo[g]chromen-4-one (flavonol), previously developed as an efficient photoCORM, by steady-state and femto/nanosecond transient absorption spectroscopies. The main objectives of the work were to explore in detail how to enhance the efficiency of CO photorelease from flavonols, bathochromically shift their absorption bands, control their acid–base properties and solubilities in aqueous solutions, and minimize primary or secondary photochemical side-reactions, such as self-photooxygenation. The best photoCORM performance was achieved by combining substituents, which simultaneously bathochromically shift the chromophore absorption spectrum, enhance the formation of the productive triplet state, and suppress the singlet oxygen production by shortening flavonol triplet-state lifetimes. In addition, the cell toxicity of selected flavonol compounds was analyzed using in vitro hepatic HepG2 cells.
Madea D., Mujawar T., Dvořák A., Pospíšilová K., Muchová L., Čubáková P., Kloz M., Švenda J., Vítek L., Klán P. Photochemistry of (Z)-Isovinylneoxanthobilirubic Acid Methyl Ester, a Bilirubin Dipyrrinone Subunit: Femtosecond Transient Absorption and Stimulated Raman Emission Spectroscopy. J. Org. Chem. 2022, 87, 3089-3103.
Bilirubin (BR) is an essential metabolite formed by the catabolism of heme. Phototherapy with blue-green light can be applied to reduce high concentrations of BR in blood and is used especially in the neonatal period. In this work, we studied the photochemistry of (Z)-isovinylneoxanthobilirubic acid methyl ester, a dipyrrinone subunit of BR, by steady-state absorption, femtosecond transient absorption, and stimulated Raman spectroscopies. Both the (Z)- and (E)-configurational isomers of isovinylneoxanthobilirubic acid undergo wavelength-dependent and reversible photoisomerization. The isomerization from the excited singlet state is ultrafast (the lifetimes of (Z)- and (E)-isomers were found to be ∼0.9 and 0.1 ps, respectively), and its efficiencies increase with increased photon energy. In addition, we studied sensitized photooxidation of the dipyrrinone subunit by singlet oxygen that leads to the formation of propentdyopents. Biological activities of these compounds, namely, effects on the superoxide production, lipoperoxidation, and tricarboxylic acid cycle metabolism, were also studied. Finally, different photochemical and biological properties of this BR subunit and its structural analogue, (Z)-vinylneoxanthobilirubic acid methyl ester, studied before, are discussed.
Janoš J., Madea D., Mahvidi S., Mujawar T., Švenda, Jakub; Suchan J., Slavíček P., Klán P. Conformational Control of Photodynamics of Bilirubin Dipyrrinone Subunit: Femtosecond Spectroscopy Combined with Nonadiabatic Simulations. J. Phys. Chem. A 2020, 124, 10457-10471
The photochemistry of bilirubin has been extensively studied due to its importance in the phototherapy of hyperbilirubinemia. In the present work, we investigated the ultrafast photodynamics of a bilirubin dipyrrinone subunit, vinylneoxanthobilirubic acid methyl ester. The photoisomerization and photocyclization reactions of its (E) and (Z) isomers were studied using femtosecond transient absorption spectroscopy and by multireference electronic structure theory, where the nonadiabatic dynamics was modeled with a Landau–Zener surface hopping technique. The following picture has emerged from the combined theoretical and experimental approach. Upon excitation, dipyrrinone undergoes a very fast vibrational relaxation, followed by an internal conversion on a picosecond time scale. The internal conversion leads either to photoisomerization or regeneration of the starting material. Further relaxation dynamics on the order of tens of picoseconds was observed in the ground state. The nonadiabatic simulations revealed a strong conformational control of the photodynamics. The ultrafast formation of a cyclic photochemical product from a less-populated conformer of the studied subunit was predicted by our calculations. We discuss the relevance of the present finding for the photochemistry of native bilirubin. The work has also pointed to the limits of semiclassical nonadiabatic simulations for simulating longer photochemical processes, probably due to the zero-point leakage issue.
Madea D., Mahvidi S., Chalupa D., Mujawar T., Dvořák A., Muchová L., Janoš J., Slavíček P., Švenda J., Vítek L., Klán P. Wavelength-Dependent Photochemistry and Biological Relevance of a Bilirubin Dipyrrinone Subunit. J. Org. Chem. 2020, 85, 13015-13028.
Phototherapy is a standard treatment for severe neonatal jaundice to remove toxic bilirubin from the blood. Here, the wavelength-dependent photochemistry of vinylneoxanthobilirubic acid methyl ester, a simplified model of a bilirubin dipyrrinone subunit responsible for a lumirubin-like structural rearrangement, was thoroughly investigated by liquid chromatography and mass and absorption spectroscopies, with the application of a multivariate curve resolution analysis method supplemented with quantum chemical calculations. Irradiation of the model chromophore leads to reversible Z → E photoisomerization followed by reversible photocyclization to a seven-membered ring system (formed as a mixture of diastereomers). Both the isomerization processes are efficient (ΦZE ∼ ΦEZ ∼ 0.16) when irradiated in the wavelength range of 360–410 nm, whereas the E-isomer cyclization (Φc = 0.006–0.008) and cycloreversion (Φ–c = 0.002–0.004) reactions are significantly less efficient. The quantum yields of all processes were found to depend strongly on the wavelength of irradiation, especially when lower energy photons were used. Upon irradiation in the tail of the absorption bands (490 nm), both the isomers exhibit more efficient photoisomerization (ΦZE ∼ ΦEZ ∼ 0.30) and cyclization (Φc = ∼0.07). In addition, the isomeric bilirubin dipyrrinone subunits were found to possess important antioxidant activities while being substantially less toxic than bilirubin.
Štacková L., Russo M., Muchová L., Orel V., Vítek L., Štacko P., Klán P. Cyanine-Flavonol Hybrids for Near-Infrared Light-Activated Delivery of Carbon Monoxide. Chem. Eur. J. 2020, 26, 13184-13190.
Carbon monoxide (CO) is an endogenous signaling molecule that controls a number of physiological processes. To circumvent the inherent toxicity of CO, light-activated CO-releasing molecules (photoCORMs) have emerged as an alternative for its administration. However, their wider application requires photoactivation using biologically benign visible and near-infrared (NIR) light. In this work, a strategy to access such photoCORMs by fusing two CO-releasing flavonol moieties with a NIR-absorbing cyanine dye is presented. These hybrids liberate two molecules of CO in high chemical yields upon activation with NIR light up to 820 nm and exhibit excellent uncaging cross-sections, which surpass the state-of-the-art by two orders of magnitude. Furthermore, the biocompatibility and applicability of the system in vitro and in vivo are demonstrated, and a mechanism of CO release is proposed. It is hoped that this strategy will stimulate the discovery of new classes of photoCORMs and accelerate the translation of CO-based phototherapy into practice.
Štacková L., Muchová E., Russo M., Slavíček P., Štacko P., Klán P. Deciphering the Structure–Property Relations in Substituted Heptamethine Cyanines. J. Org. Chem. 2020, 85, 9776-9790.
Heptamethine cyanines (Cy7) are fluorophores essential for modern bioimaging techniques and chemistry. Here, we systematically evaluated the photochemical and photophysical properties of a library of Cy7 derivatives containing diverse substituents in different positions of the heptamethine chain. A single substitution allows modulation of their absorption maxima in the range of 693–805 nm and photophysical properties, such as quantum yields of singlet-oxygen formation, decomposition, and fluorescence or affinity to singlet oxygen, within 2–3 orders of magnitude. The same substituent in different positions of the chain often exhibits distinctly contradictory effects, demonstrating that both the type and position of the substituent are pivotal for the design of Cy7-based applications. The combination of experimental results with quantum-chemical calculations provides insights into the structure–property relationship, the elucidation of which will accelerate the development of cyanines with properties tailored for specific applications, such as fluorescent probes and sensors, photouncaging, photodynamic therapy, or singlet-oxygen detection.
Russo M., Štacko P., Nachtigallová D., Klán P. Mechanisms of Orthogonal Photodecarbonylation Reactions of 3-Hydroxyflavone-Based Acid-Base Forms. J. Org. Chem. 2020, 85, 3527-3537.
Heptamethine cyanines (Cy7) are fluorophores essential for modern bioimaging techniques and chemistry. Here, we systematically evaluated the photochemical and photophysical properties of a library of Cy7 derivatives containing diverse substituents in different positions of the heptamethine chain. A single substitution allows modulation of their absorption maxima in the range of 693–805 nm and photophysical properties, such as quantum yields of singlet-oxygen formation, decomposition, and fluorescence or affinity to singlet oxygen, within 2–3 orders of magnitude. The same substituent in different positions of the chain often exhibits distinctly contradictory effects, demonstrating that both the type and position of the substituent are pivotal for the design of Cy7-based applications. The combination of experimental results with quantum-chemical calculations provides insights into the structure–property relationship, the elucidation of which will accelerate the development of cyanines with properties tailored for specific applications, such as fluorescent probes and sensors, photouncaging, photodynamic therapy, or singlet-oxygen detection.
Madea D., Martínek M., Muchová L., Vítek L., Klán P. Structural Modifications of Nile Red Carbon Monoxide Fluorescent Probe: Sensing Mechanism and Applications. J. Org. Chem. 2020, 85, 3473-3489.
Carbon monoxide (CO) is a cell-signaling molecule (gasotransmitter) produced endogenously by oxidative catabolism of heme, and the understanding of its spatial and temporal sensing at the cellular level is still an open challenge. Synthesis, optical properties, and study of the sensing mechanism of Nile red Pd-based CO chemosensors, structurally modified by core and bridge substituents, in methanol and aqueous solutions are reported in this work. The sensing fluorescence “off–on” response of palladacycle-based sensors possessing low-background fluorescence arises from their reaction with CO to release the corresponding highly fluorescent Nile red derivatives in the final step. Our mechanistic study showed that electron-withdrawing and electron-donating core substituents affect the rate-determining step of the reaction. More importantly, the substituents were found to have a substantial effect on the Nile red sensor fluorescence quantum yields, hereby defining the sensing detection limit. The highest overall fluorescence and sensing rate enhancements were found for a 2-hydroxy palladacycle derivative, which was used in subsequent biological studies on mouse hepatoma cells as it easily crosses the cell membrane and qualitatively traces the localization of CO within the intracellular compartment with the linear quantitative response to increasing CO concentrations.
Štacková L., Štacko P., Klán P. Approach to Substituted Heptamethine Cyanine Chain by Ring Opening of Zincke Salts. J. Am. Chem. Soc. 2019, 141, 7155-7162.
Cyanine dyes play an indispensable and central role in modern fluorescence-based biological techniques. Despite their importance and widespread use, the current synthesis methods of heptamethine chain modification are restricted to coupling reactions and nucleophilic substitution at the meso position in the chain. Herein, we report the direct transformation of Zincke salts to cyanine dyes under mild conditions, accompanied by the incorporation of a substituted pyridine residue into the heptamethine scaffold. This work represents the first general approach that allows the introduction of diverse substituents and different substitution patterns at the C3′–C5′ positions of the chain. High yields, functional tolerance, versatility toward the condensation partners, and scalability make this method a powerful tool for accessing a new generation of cyanine derivatives.