Letter pubs.acs.org/OrgLett
Decorated BODIPY Fluorophores and Thiol-Reactive Fluorescence Probes by an Aldol Addition Lukas J. Patalag,† Jan A. Ulrichs,† Peter G. Jones,‡ and Daniel B. Werz*,† †
Institut für Organische Chemie and ‡Institut für Anorganische und Analytische Chemie, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany S Supporting Information *
ABSTRACT: A novel entry to meso-decorated BODIPY motifs on the basis of an unusual aldol-type addition with diethyl ketomalonate is reported. The evolving β-hydroxyl group can be optionally eliminated, which suppresses the fluorescence of the BODIPY core by attachment of a π-electronically coupled methylidene malonate unit. This unit serves as a versatile, highly electrophilic acceptor platform for various nucleophilic additions. Corresponding products benefit from a fully restored fluorescence.
D
products instead of the anticipated elimination species. Scheme 1 depicts an overview of the key fluorophores and dyes of the
uring the last few decades, the BODIPY motif has developed into a reliable and competitive fluorescent scaffold with convincing spectroscopic properties.1 Its one-step preparation and reasonable chemical robustness, with high, generally solvent-independent fluorescence efficiencies, are accompanied by various convenient modes of postfunctionalization that finally transform the motif into a functional molecule.2 Facile halogenations,3 subsequent cross-coupling reactions,4 and Knoevenagel condensations5 are typical modifications allowed by the electronically ambivalent π-system. Depending on the reactive environment chosen, the dipyrromethene core can either be addressed by its tamed pyrrole-like nucleophilicity6 or employed as an electrophilic aza-Michael system.7 The latter quality accounts for the acidity of vicinal C−H groups, especially at positions 3,5 and the meso position 8 (Figure 1).
Scheme 1. Principle of Observed Fluorescence Modulation on the Basis of Aldol Addition, Elimination, and Nucleophilic Addition
investigated derivatization steps. Products of the aldol addition benefit from an increased hydrophilicity and equip the BODIPY motif with three new polar functionalities. Upon elimination of the β-hydroxyl group in a one-step procedure, fluorescence is suppressed by an ICT mechanism, but it can be reactivated by addition of a suitable nucleophile that electronically decouples the former two π-systems. Diethyl ketomalonate reacts smoothly with meso-methylderivatized BODIPY cores under typical Knoevenagel conditions but at much lower temperatures (Scheme 2). The high, in some cases quantitative, reaction yields are unaffected by the more easily accessible methyl groups at positions 3 and 5 (R1 = Me) or by steric hindrance in the vicinity of the meso position (R2 = Me).
Figure 1. BODIPY scaffold, showing typical positions for derivatization.
Ortiz et al.8 found that a meso-methyl substituent is the most liable to deprotonate and can act as an anchor for diverse nucleophilic substitutions. Furthermore, the meso position serves as an excellent structural starting point for numerous sensorically active moieties9 on the basis of ICT (intramolecular charge transfer), PeT (photoelectron transfer), or Förster resonance energy transfer10 processes and for the extension of the π-system in general.11 Synthetic contributions to access the meso position promise to have a great impact on the development of novel fluorescent indicators and labels. Herein, we show that the replacement of aromatic aldehydes by highly electrophilic keto functionalities during Knoevenagel condensations furnishes selectively meso-substituted addition © 2017 American Chemical Society
Received: March 8, 2017 Published: April 7, 2017 2090
DOI: 10.1021/acs.orglett.7b00693 Org. Lett. 2017, 19, 2090−2093
Letter
Organic Letters Scheme 2. Aldol Addition with Diethyl Ketomalonate and an Exemplary Esterification Step
Figure 2. Absorption spectra of 2c, 4a, 7, and 8 (DCM), emission spectra of 2c and 4a (DCM), and crystal structure of 4c. Emission of 7 and 8 is explained in the Supporting Information.
The tertiary hydroxyl group thus formed can be employed as a nucleophilic anchor to esterify aliphatic acid chlorides at elevated temperatures, as demonstrated for 3. Despite the basic conditions, no retro-aldol reaction was observed. A one-pot procedure was devised to convert hydroxylfunctionalized species 2a−c into their unsaturated counterparts (Scheme 3). TFAA transforms the hydroxyl group into a
torsion angle of about 60°, as shown by single-crystal X-ray diffraction analysis of 4c.12 DFT calculations (B3LYP/6-311G(d,p)) reveal the cause of the depleted fluorescence of 4a, 4c, and 5 (Figure 3). While the
Scheme 3. TFAA-Mediated Elimination To Obtain Methylidene Malonate Derivatized BODIPYs and an Exemplary Sulfonation Step
Figure 3. Frontier orbital energies of 4c (DFT) and their pictorial representations.
sufficiently effective leaving group, which can then be eliminated by excess 2-methylpyridine. Interestingly, 2b with methyl groups at positions 1 and 7 does not undergo the desired elimination but furnishes the trifluoroacetate intermediate instead. The steric demand of the methyl residues seems to prevent an efficient conjugation between the BODIPY core and the methylidene malonate moiety, which might be a significant thermodynamic driving force of the reaction pathway. A further sulfonation step was tested on 4c to give a water-soluble species 5 in excellent yield. Methylidene malonate-equipped BODIPYs 4a, 4c, and 5 show no significant fluorescence. The electron-withdrawing strength of the new meso substituent accounts for a slight bathochromic shift of ∼7 nm (4c) but a great broadening of the absorption peak (Figure 2). Varying dihedral angles between the π-system of the BODIPY and the methylidene malonate unit in solution influence the degree of conjugation and thus the required excitation energy. The broadened absorption peak implies that the Michael system is quite flexible, with an energy minimum at a
HOMO resembles the typical patterns calculated for mesounsubstituted BODIPYs, the LUMO has only a limited presence at the dipyrromethene core. Much electron density is shifted toward the methylidene malonate moiety upon excitation, which inhibits a fluorescent relaxation mechanism from a considerably reorganized framework geometry. In contrast to a nonconjugated adjacent maleimide acceptor, it is not an oxidative PeT that governs the suppression of fluorescence but a distinctly intramolecular charge transfer process. The marked deviation of the meso residue from an orthogonal conformation ensures enough conjugation for a highly efficient transfer of charge, rendering the corresponding species virtually nonfluorescent (Φfl ≈ 0.003). To study the electrophilic acceptor quality of the methylidene malonate equipped BODIPYs, we chose 4a as the prototypical Michael system (Scheme 4). With KOtBu as base, diethylmal2091
DOI: 10.1021/acs.orglett.7b00693 Org. Lett. 2017, 19, 2090−2093
Letter
Organic Letters
dipole in asymmetric [3 + 2] cycloadditions.13 Spectroscopic data of all prepared compounds are presented in the Supporting Information. As we found that most nucleophiles need either more strongly basic or rather harsh neutral conditions to attack the Michael system of 4a, we examined whether the Michael acceptors 4a, 4c, 7, 8, and the water-soluble species 5 might be selective thiol probes under mild and aqueous conditions.14 Except for 5, a PBS buffer/EtOH solvent system was found to be suitable for a kinetic study applying a pseudo-first-order approximation with excess of N-acetylcysteine as thiol reagent. Evolvement of fluorescence was recorded on a spectrofluorometer at 5 μM concentration (Figure 4) for several hours.
Scheme 4. Procedures for a Range of Nucleophilic Additions to the Michael System of BODIPY 4a
Figure 4. Fluorescence enhancement of 4a (5 μM) upon reaction with N-acetylcysteine (200-fold excess) in a PBS buffer/EtOH solvent system (pH ∼7.4) at 25 °C. Excitation wavelength was 470 nm. Maximum of the detected background noise is centered at 513 nm.
onate smoothly attacks at the β-methylidene carbon to furnish the symmetrical and fluorescent tetraester 6 in an equilibrium reaction. When more acidic methylene-active reactants such as malononitrile are used, the addition product forms only in small amounts as an intermediate. Instead, a second deprotonation leads to the elimination of diethylmalonate to form the even stronger nonfluorescent acceptors 7 and 8 (Scheme 4b). Here again, the equilibrium reaction allows the reisolation of significant amounts of the starting material. Attempts to involve 4a in Stetter-like reaction pathways with a thiazolium catalyst led to the reduction of the electron-poor double bond (Scheme 4c). Presumably, the electron-rich and sterically hindered Breslow intermediate triggers an electron transfer rather than forming a new C−C bond. All three components, diacetyl as an acetyl source, diisopropylamine as base, and the catalyst, are necessary to enable the very fast reduction. BODIPY 9 should allow typical reaction pathways for methylene-active compounds. A protocol with LiClO4 in dioxane proved to be a quite general procedure to allow the addition of a series of weak N-nucleophiles such as pyrazine or indazole (Scheme 4e). However, a similar elimination of diethylmalonate (Scheme 4b) was observed when benzhydrazide was employed as nucleophile. The resulting acylhydrazone 15 and the expected addition product 14 represent only weak emitters. The acylhydrazone moiety was reported as a versatile functionality for cyclizations to 1,3,4-oxadiazoles or as a 1,3-
While acceptors 7 and 8 proved to be too electrophilic and reacted even under slightly basic conditions, 4a and 4c showed complete and selective conversion after about 16 h. Integration of fluorescence intensity over the whole range (480−800 nm) results in a 150-fold increase for 4a, 120-fold for 4c, and 90-fold for water-soluble congener 5. Considering a 10 nm window (530−540 nm for 4a/c and 510−530 nm for 5), the signal-tonoise ratio is even increased because of the shift and narrowing of the emission maxima after thiol addition. This gives a 420-fold increase for 4a, a 250-fold increase for 4c, and a 170-fold increase for 5. These are substantially higher values than determined for the commercially available fluorescein-5-maleimide acceptor (10-fold increase).15 BODIPY 4a can even compete with the novel o-maleimide BODIPY thiol probe (350-fold increase) developed by Nagano et al. in a four-step synthesis.16 No increase of fluorescence intensity was observed when the addition of Nacetylcysteine was omitted. To gain more insights into the kinetics of the thiol addition and to compare the novel probes, we fitted the fluorescence enhancements of 4a, 4c, and 5 by applying the common equation for a pseudo-first-order reaction: [A] = [A]0 × e kobst with kobs = k[B]thiol
[A] and [A]0 correspond to the concentration of the BODIPY acceptor at t and t = 0, respectively. [B]thiol is the concentration of N-acetylcysteine at the beginning (200-fold excess), which is only diminished by 0.5% during the reaction course. Figure 5 2092
DOI: 10.1021/acs.orglett.7b00693 Org. Lett. 2017, 19, 2090−2093
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Letter
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Daniel B. Werz: 0000-0002-3973-2212 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS L.J.P. thanks Alexey Butkevich, Vladimir Belov, and Prof. Dr. Stefan Hell (MPI for Biophysical Chemistry, Göttingen) for the possibility to measure fluorescence quantum yields, the DFG (SFB 803, A05) for funding, and Oscar Arias and Katharina Bolsewig (TU Braunschweig) for kind support.
■ Figure 5. Exponential fittings of the corresponding integrated fluorescence emissions (480−800 nm) as a function of time after addition of N-acetylcysteine (200-fold excess) at 25 °C. 4a/4c in PBS buffer/EtOH = 2/1, 5 in pure PBS buffer.
demonstrates that the exponential fittings reflect the real scenario of the thiol addition. Even though more electrophilic, Michael acceptor 5 reacts considerably more slowly than the more hydrophobic 4a and 4c. This might be explained either by an unfavorable electrostatic interaction between the bisulfonate salt 5 and the acetate anion of N-acetylcysteine at pH ∼7.4 or by attractive van der Waals interactions between 4a/4c and Nacetylcysteine. Both reactants constitute rather hydrophobic compounds in the highly hydrophilic environment of the solvent system, which might compromise an undisturbed diffusion control. In conclusion, we introduced the first aldol-type addition to meso-methyl-substituted BODIPY motifs with diethyl ketomalonate as a carbonyl source. The highly efficient and selective reaction introduces an advantageous hydrophilicity, providing a basic functional group and useful anchor for cation binding or bioconjugation purposes. Upon one-step elimination of H2O, the scaffold can be transformed into a nonfluorescent Michael acceptor that can be employed as an electrophilic platform for the addition of various nucleophiles. If these are electronically innocent, the fluorescence of the BODIPY core is fully restored and can be used to visualize nucleophilicities in aqueous environments. This was demonstrated for N-acetylcysteine as thiol reagent, which readily and selectively adds to the acceptor moiety even at high dilutions. Enhancement of fluorescence is associated with an excellent signal-to-noise ratio that outperforms the commercially available fluorescein-5-maleimide−thiol acceptor by a factor of 42. This novel reaction mode presents a straightforward entry into a versatile functionalization of the BODIPY motif and might find broad use in synthetic, analytical, and biochemical laboratories.
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REFERENCES
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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/acs.orglett.7b00693. Synthetic procedures, experimental details, spectroscopic and crystallographic data (PDF) X-ray data for 4c (CIF) 2093
DOI: 10.1021/acs.orglett.7b00693 Org. Lett. 2017, 19, 2090−2093
