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Approach to a Substituted Heptamethine Cyanine Chain by the Ring Opening of Zincke Salts Lenka Š tackova,́ † Peter Š tacko,*,† and Petr Klań * Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

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S Supporting Information *

ABSTRACT: 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.

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INTRODUCTION Since their first synthesis in 1856, cyanine dyes have become invaluable fluorophores in contemporary chemistry and biology.1 The defining feature of the cyanines is an oddnumbered polyene linking two nitrogen-containing heterocycles, related to both their intriguing photophysical properties and unique chemical reactivities.2 Within this family, heptamethine cyanines dyes (Cy7, Figure 1a), containing seven carbon atoms in the linker, are particularly valued for their absorption and emission maxima located in the center of the near-infrared (NIR) window (∼800 nm).3 In biology, the presence of endogenous chromophores, such as hemoglobin and water, and optical scattering limit the depth of light penetration into tissues. Light absorption in the phototherapeutic window (600−1000 nm), where these effects are minimized, thus ensures a broad scope of biological applications. The expanding medical significance of Cy7 dyes is demonstrated by FDA-approved indocyanine green (ICG) with hundreds of clinical trials emerging even after 50 years of its clinical use (Figure 1b).4 Another promising derivative, IR800CW, has been explored for a number of fluorescence-guided surgery applications (Figure 1b).5−7 Furthermore, cyanines have been used in a number of other applications, including pH sensing,8,9 analyte-responsive sensing,2,10 glutathione11 or reactive oxygen species12−14 visualization, single-molecule fluorescence and super-resolution imaging,15−17 photodynamic therapy,18,19 NIR photouncaging,20,21 and others.22 These applications take advantage not only of their excellent photophysical properties but also the distinct chemical reactivity of the cyanine polyene itself.23 The nature of heptamethine chain substituents has a profound influence on both the photophysical and photochemical properties of the Cy7 dyes as well as their chemical stability. © XXXX American Chemical Society

Figure 1. (a) Heptamethine cyanine dyes (Cy7). The numbering of the heptamethine chain is depicted in red. (b) Indocyanine green. (c) IR800CW.

The introduction of electron-withdrawing nitrile,24 amide,25 or triazole26 moieties in the chain meso position improves the dye photostability as a result of decreased reactivity toward singlet oxygen. The installation of the 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO)-bearing substituent at the C4′ position resulted Received: March 7, 2019

A

DOI: 10.1021/jacs.9b02537 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society Scheme 1. Chain Modification, Schiff Base Preparation, and Our Current Strategya

a

(a) Conventional approach to the heptamethine chain modification involving palladium-catalyzed coupling reactions (left) and SRN1 reactions with N, O, or S nucleophiles (right). (b) Custom Schiff base preparation via cyclohexene formylation. (c) Our current strategy involving the ring opening of pyridinium (Zincke) salts and subsequent incorporation of the original substituted pyridine residue (red) into the heptamethine chain.

Scheme 2. Typical Zincke Reaction (Route A) and Utilization of Its Intermediates in the Synthesis of Cy7 Dyes (Route B)a

a

The original substituted pyridinium residue (red) is transferred and incorporated into heptamethine scaffold 5.

in an ∼33-fold increase in the quantum yield of singlet oxygen formation compared to that of ICG sensitization.27 Bioconjugation of cyanines commonly achieved via the attachment of C4′-O-aryl or C4′-S-alkyl linkers often suffers from poor chemical stability of the conjugate toward biologically relevant nucleophiles, such as glutathione or cellular proteins.28−30 One way to circumvent this issue is to employ C4′-O-alkylsubstituted cyanines which have so far been accessible only by Smiles rearrangement of C4′-N-methylethanolamine precursors.31 In addition, an elegant design of the C4′-dialkylamine linker bearing a carbamate functionality allowed us to harness the undesired photooxidiation in a NIR photouncaging process.20,21 Despite the widespread utilization of cyanines and the evident importance of polyene substitution, the strategies used to modify the heptamethine chain remain surprisingly underdeveloped and limited. A majority of the current methods start from the C4′-chloro-substituted cyanine and rely on an electrontransfer-mediated SRN1 reaction with N, O, or S nucleo-

philes13,25,32−38 or palladium-catalyzed Suzuki39 or Sonogashira40 coupling reactions (Scheme 1a). The other option involves the preparation of a custom Schiff base intermediate from a substituted cyclohexene in the Vilsmeier−Haack reaction (Scheme 1b). However, this procedure gives moderate yields only for methyl- and phenyl-substituted cyclohexenes presumably because of harsh conditions employed in the synthesis.41,42 Besides a cyclopentyl or cyclohexyl ring embedded within the heptamethine scaffold, no modifications at the C3′ and C5′ positions have been reported to date. Therefore, essentially only the C4′-position modifications are available for modification to date, imposing fundamental restrictions on the design of new Cy7 fluorophores. Herein, we report the first general synthesis method to access Cy7 dyes possessing diverse substituents and substitution patterns at the C3′−C5′ positions of a heptamethine chain under mild conditions (Scheme 1c). B

DOI: 10.1021/jacs.9b02537 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION Typically, the Zincke reaction is used to convert pyridines to Npyridinium salts by the reaction of N-2,4-dinitrophenylpyridinium (Zincke salt) with primary amines at elevated temperatures (Scheme 2, route A).43 We envisioned that, under specific conditions, intermediate 2 produced by the ring opening of pyridinium salt 1, as suggested in the synthesis of benzothiazole Cy7 derivatives with an unsubstituted heptamethine chain,44 could instead be intercepted by another equivalent of aniline as a nucleophile, and resulting intermediate 3 would undergo a condensation with quaternized heterocycle 4 to give the final cyanine 5 in a one-pot tandem fashion (Scheme 2, route B). In such a manner, the substituents on the pyridine residue would be transferred and incorporated into the cyanine as a part of the heptamethine scaffold. We also hypothesized that the ring opening of very electron-deficient pyridinium salts could also be performed directly with 4 without the need for any auxiliary nucleophile. We synthesized a number of pyridinium salts 1a−v on a multigram scale by the reaction of 2,4-dinitrophenyl (DNP) tosylate or triflate with substituted pyridines at an elevated temperature (Supporting Information). The salts were isolated in high purity simply by filtration and proved to be benchtopstable for months. Subjecting 1a to a reaction with heterocycle 4a in the presence of sodium acetate (6 equiv) and in the absence of aniline at room temperature overnight provided corresponding cyanine 5a in 24% yield (Table 1, entry 1). The

confirmed in a reaction of 1a and aniline using HPLC and UV− vis spectroscopy by comparison with an authentic sample of 3 (Figures S154 and S155). Conversely, the use of a strong electron-withdrawing (EWG) 4-cyano substituent led to only a marginal increase in the yield compared to that of an unanalyzed reaction (34%, entry 5). On the contrary, weakly electron accepting 4- or 3-bromo substituents dramatically improved the yield to 72 and 73%, respectively (entries 4 and 7). Decreasing the aniline amount to 1 equiv had no negative effect on the yield, whereas its further decrease to substoichiometric amounts or reducing the number of equivalents of sodium acetate led to lower yields of 5a (entries 6−10). N-Phenylpyridinium was observed as a major side product upon the temperature increase as a result of intramolecular Schiff base cyclization.43 The dependence of cyanine formation yields on the nature of aniline substituents implies a delicate interplay of electronic effects. An aniline reagent is required to be electron-rich enough to facilitate pyridinium ring opening and, at the same time, be sufficiently electron-poor to activate the resulting imine for a facile condensation with the heterocycle. Thus, while 4methoxyaniline expedites a rapid cleavage of the pyridinium ring, corresponding electron-rich imine 3 reacts too slowly in the subsequent condensation (and vice versa for 4-cyanoaniline). Established empirically, 3- and 4-bromoaniline facilitated both steps with a desirable efficiency and thus the highest yields. Following the optimization, we sought to investigate the scope of this method and its tolerance toward various substituents and substitution patterns. A variety of substituted pyridinium salts 1a−t were examined in the reaction with 4a, and the results are summarized in Table 2. In most cases, the pure products were isolated directly by filtration of the precipitate from the reaction mixture. The products were, in some cases, contaminated with 2,4-dinitroaniline, which was removed by rapid column chromatography. The method is versatile, and it tolerates both electronwithdrawing and electron-donating groups on the pyridinium ring. The pyridinium salts with EWGs reacted faster and usually with higher yields (vide infra) than those with EDGs. A decreased reactivity of pyridines with mediocre EDGs (1b, 1m) under the optimized conditions was compensated for by a higher loading of 4-bromoaniline (3 equiv). On the other hand, 1n did not undergo the ring-opening reaction under these conditions, presumably because of a strong resonance electron-donating effect of the methoxy substituent. We reasoned that employing a less polar solvent, such as ethanol or pyridine, would lead to less favorable solvation of the pyridinium salt, thus promoting the pyridinium ring opening. Indeed, when the reaction was carried out in pyridine, cyanine 5n was successfully isolated in 40% yield. On the contrary, strong EWGs (1d, 1g, and 1k) did not actually demand the presence of aniline as an auxiliary nucleophile, and the pyridinium ring opening could be achieved efficiently in its absence. Our observations are consistent with the mechanism of the Zincke reaction and the subsequent condensation, both of which involve a nucleophilic attack and therefore require a sufficiently electron-deficient substrate (Scheme 2). Both C3′-alkyl- and C3′-aryl-substituted pyridinium salts gave cyanines 5b and 5c in very good yields. Substrates bearing halogens also efficiently afforded products 5d−5f in yields of 49−90%, increasing in the order of increasing halogen electronegativity. Various functional derivatives of carboxylic acids, such as esters (5g and 5h), carboxylic acid (5i), amide (5j), and nitrile (5k), as well as an acyl derivative (5l) were well

Table 1. Optimization of the Preparation of Cyanine 5a from Zincke Salt 1aa

entry

R-PhNH2

aniline

AcONa

yieldb

1 2 3 4 5 6 7 8 9 10

4-H 4-MeO 4-Br 4-CN 4-Br 3-Br 3-Br 4-Br 4-Br

3 equiv 3 equiv 3 equiv 3 equiv 1.2 equiv 1.2 equiv 1.2 equiv 0.2 equiv 0.2 equiv

6 equiv 6 equiv 6 equiv 6 equiv 6 equiv 6 equiv 6 equiv 4 equiv 6 equiv 3 equiv

24% 47% CH3 > Br (1.7, 1.27, and 0.38 kcal mol−1, respectively).47,48 The accommodation of various sensitive functional groups, including ester, amide, nitrile, azide, alkyne, and unprotected hydroxyl and carboxylic moieties at different positions of the Cy7 chain, highlights the mildness and synthetic utility of this protocol. In addition, a number of the described cyanines can be employed in further reactions including coupling chemistry,39,40 “click” chemistry,49 and Staudinger ligation.50 To further demonstrate the applicability of this method, the scalability of

the reaction was assessed by performing the preparation of 5g on a multigram scale. Cyanine 5g was successfully prepared in a 2.2 g amount in a single batch without any detrimental effects on the purity or yield of the product. In fact, this large-scale reaction was accomplished with a higher yield (92%) than that obtained on a smaller scale (75%, Table 2). Furthermore, we probed the generality of the reaction toward various heterocycles (Table 3). Using N-methyl-2-methylquinolinium 4b as the condensation partner resulted in the formation of cyanine 10 in 47% yield, thus offering easy access to an underexplored class of quinolinium-based Cy7 dyes. In the case of benzoxazolium-based heterocycles, the formation of the corresponding cyanine was initially observed; however, the product decomposed in the course of the reaction. On the other hand, cyanines 8 and 11 were formed from a benzothiazoliumbased heterocycle in yields comparable to those of their indolium-derived analogues (5a and 5e). Employing indoliumcontaining N-propyl sulfonate, which is routinely used to improve the solubility of cyanines in aqueous media, provided cyanine 7 in 45% yield, comparable to that of 5i. Heterocycles with an additional benzene ring fused in different positions of 4a were also very well tolerated and afforded corresponding cyanines 9 and 12 in 87 and 47% yields, respectively. The photophysical properties, absorption (λmax(abs)) and emission (λmax(em)) maxima, and molar absorption coefficients (εmax) were determined in methanol as a solvent for all of the synthesized derivatives (Table 4). The absorption spectra exhibit one major absorption band with maxima in the range of 707−815 nm, relatively small Stokes shifts, and large molar absorption coefficients, typical for Cy7 dyes (Figures S4 and S5). EWGs in the C4′ position (5g, λmax(abs) = 778 nm; 5k, λmax(abs) = 805 nm; and 5l, λmax(abs) = 771 nm) are responsible for a bathochromic shift of the maxima compared to that of the unsubstituted derivative 5a (λmax(abs) = 740 nm). This is in E

DOI: 10.1021/jacs.9b02537 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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CONCLUSIONS The utilization of the Zincke salts ring opening in the cyanine synthesis reported herein represents the first general method for the derivatization of the heptamethine chain. Using this approach, the substituted pyridine backbone is transferred and incorporated into a cyanine dye as a part of the heptamethine chain. A wide variety of substituents can thus be installed at different positions of the chain, and different heterocycles can be employed as condensation partners under mild conditions and in high yields and purities. This method constitutes a very powerful addition to the limited arsenal of tools available for the synthesis of substituted cyanines and promises the emergence of more complex cyanine-based molecules tailored for specific applications.

Table 4. Photophysical Properties of the Prepared Cyanine Dyes in Methanol cyanine

λmax(abs)/nm

λmax(em)/nm

ϵmaxa

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 7 8 9 10 11 12

740 736 738 761 736 733 778 707 748 735 805 771 728 753 735 735 770 799 815 735 754 758 782 815 749 792

769 760 765 788 762 760

237 000 273 000 152 000 209 000 205 000 233 000 150 000 139 000 240 000 205 000 215 000 137 000 190 000 180 000 224 000 269 000 202 000 288 000 148 000 269 000 172 000c 212 000 183 000 204 000 161 000 141 000c

b

750 779 763 833 804 753 782 764 766 798 828 847 766 784 786 814 848 774 823

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.9b02537.

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Materials and methods; synthesis; and NMR, absorption, and emission spectra (PDF)

AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Petr Klán: 0000-0001-6287-2742

ϵmax/mol−1 dm3 cm−1. bNot determined. cPBS (10 mM, pH 7.4, I = 100 mM).

a

Author Contributions †

These authors contributed equally.

Notes

accordance with previously reported 4′-bromo and 4′-iodosubstituted Cy7 dyes, which also possess absorption maxima (λmax(abs) = 770 nm) bathochromically shifted by 30 nm compared to that of 5a.51 On the other hand, the presence of the electron-donating methoxy group on C4′ exhibits a hypsochromic shift of the maximum to 693 nm.51 A more electrondonating substituent in this position, such as the alkylamino moiety, has been shown to exhibit an even larger hypsochromic shift of the maximum to ∼626−670 nm, respectively.25,37 Acylation of the 4-amino (EDG) derivative to its acetamido (EWG) analogue results in a significant reverse bathochromic shift from ∼650 to ∼800 nm.25 The effects of the substituents at the C3′ position are much more subtle and rather unambiguous (Table 4). The presence of an EWG can induce both hypsochromic (5h, λmax(abs) = 707 nm) and bathochromic shifts (5d, λmax(abs) = 761 nm). Interestingly, the identical group exhibits either a bathochromic or a hypsochromic effect on the absorption maximum in different positions on the heptamethine chain. For instance, derivatives 5g and 5h bearing an ester functionality at the C4′ and C3′ positions showed absorption maxima at 778 and 708 nm, respectively. In general, EWGs in the C4′ position and EDGs in the C3′ position result in a bathochromic shift of the absorption maxima, whereas substitution in the C4′ position exhibits a much stronger influence on the photophysical properties. The Stokes shifts of ∼25−35 nm seem to be unaffected by the nature or position of the substituents. A thorough investigation to understand the substituent effects on the photophysical properties of the cyanine dyes is currently being carried out in our laboratory.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project has received funding from the European Union’s Horizon 2020 Research and Innovation Program under Marie Skłodowska-Curie, and it is cofinanced by the South Moravian Region under grant agreement no. 665860 (SoMoPro III Program - Project VLAMBA, Nr. 6SA17811, P.Š .). This publication reflects only the authors’ views, and the EU is not liable for any use that may be made of the information contained herein. Support for this work was provided by the Czech Science Foundation (GA18-12477S, P.K.) and RECETOX Research Infrastructure (LM2015051 and CZ.02.1.01/0.0/0.0/16_013/ 0001761). We acknowledge Lukás ̌ Maier (Masaryk University) for assistance with NMR analysis and Miroslava Bittová (Masaryk University) for HRMS analysis

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DOI: 10.1021/jacs.9b02537 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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DOI: 10.1021/jacs.9b02537 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX