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Article http://pubs.acs.org/journal/acsodf

PyrrolylBODIPYs: Syntheses, Properties, and Application as Environment-Sensitive Fluorescence Probes Changjiang Yu,* Wei Miao, Jun Wang, Erhong Hao, and Lijuan Jiao* The Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Laboratory of Molecule-Based Materials (State Key Laboratory Cultivation Base), School of Chemistry and Materials Science, Anhui Normal University, No. 1 East Beijing Road, Wuhu, Anhui 241000, China S Supporting Information *

ABSTRACT: Four pyrrole B-ring-functionalized pyrrolylBODIPYs and their B-ring unsubstituted analogues were synthesized from easily accessible starting 5-halo-2-formylpyrroles and were characterized by nuclear magnetic resonance, high-resolution mass spectrometry, X-ray analysis, and optical/ electronic properties. In great contrast to the substitution(s) at the other two pyrrolic units, electron-donating substituent(s) at pyrrole B-ring bring significant blue shift of the absorption and emission bands. Cyclic voltammetry and density functional theory calculations indicate that this blue shift may be attributed to the increased highest occupied molecular orbital and the lowest unoccupied molecular orbital energy levels and the overall increase in the energy band gaps. These pyrrolylBODIPYs generally show intense absorption (centered at 570−624 nm) and fluorescence emission (582−654 nm) in nonpolar solvents. A gradual decrease in the fluorescence intensity was observed for these dyes with the increase in solvent dipolar moment, which combines with the red to far-red absorption/emission, rendering these pyrrolylBODIPYs potential applications as environment-sensitive fluorescence probes as demonstrated in this work for bovine serum albumin.

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INTRODUCTION Polarity-responsive fluorescent dyes have attracted wide research interests in biology and medicine research areas, as fluorescent probes for the monitoring of a variety of biological and biochemical processes or as “smart” imaging and therapeutic agents because the type of fluorescent dyes generally can be activated by the hydrophobicity of a specific microenvironment.1 For example, probes such as 8-anilino-1naphthalenesulfonic acid and thioflavin T generally exhibit low fluorescence in aqueous solution but become highly fluorescent in nonpolar solution,2 whereas Prodan and Nile red exhibit emission at longer wavelength in aqueous solution but at shorter wavelength in nonpolar solution. Nagano and coworkers reported a novel environment-sensitive BODIPY (boron dipyrromethene) probe library in which the fluorescence properties are well-controlled through different mesoaryl groups.2a However, these BODIPYs and many other reported probes often absorb and emit at shorter wavelength (less than 550 nm). Development of environment-sensitive fluorescence probes absorbing and emitting in the far-red to the NIR region is highly desirable. BODIPYs (Figure 1a)3−5 have attracted wide research interests in a variety of research fields, for example, as laser dyes,6 fluorescent sensors,7 fluorescent switches,8 fluorescence labeling,9 and photosensitizers,10,11 owing to their tunable outstanding photophysical properties, including high absorption coefficients, intense fluorescence, and rich chemistry, © 2017 American Chemical Society

which render the facile functionalization and modification of the chromophore. Installation of the pyrrole unit at the αposition of the chromophore to form the so-called pyrrolylBODIPY (Figure 1a) is an efficient way to extend the π-conjugation of the chromophore and brings the desired red shift of the spectra. For example, BODIPY 576/589 as a commercialized long-wavelength fluorescent pyrrolylBODIPY derivative has been widely applied for bioimaging.12,13 As part of our continuous research efforts toward longwavelength BODIPY dyes, our group and Ravikanth et al. have recently developed a set of facile synthetic routes toward pyrrolylBODIPY chromophores to address the synthetic difficulties of this type of chromophore (Figure 1b,c).14−21 For example, we have synthesized pyrrolic unsubstituted pyrrolylBODIPY I from the oxidative addition of pyrrole to pyrrolic-free meso-arylBODIPYs15 or the “one-pot” condensation of acyl chloride16 and the halogenated pyrrolylBODIPY II from the aromatic substitution of the α-haloBODIPY with pyrrole17−19 (Figure 1b). However, all of those pyrrolylBODIPYs did not have substituents on the pyrrolic rings, and most of them showed few variations in the fluorescence intensities in different solvents upon changing the polarity. Received: April 12, 2017 Accepted: June 28, 2017 Published: July 13, 2017 3551

DOI: 10.1021/acsomega.7b00444 ACS Omega 2017, 2, 3551−3561

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applications as an environmental sensitive “turn-on” fluorescence probe for the detection of local polarity changes of biological samples, as demonstrated in this work for bovine serum albumin (BSA).

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RESULTS AND DISCUSSION Syntheses and Characterizations. The key synthetic precursors, 5-halo-2-formylpyrroles 1a−c, were synthesized using the literature-reported procedures22−28 (Scheme 1) via Scheme 1. Syntheses and Yields of 5-Halogenated 2Formylpyrrole 1a−c

chlorination and subsequent formylation reaction on pyrrole (Scheme 1a), the oxidation and subsequent decarboxylic iodination reaction of 3b (Scheme 1b), and the Vilsmeier− Haack and the subsequent hydrolysis of the resultant enamine intermediate 2c (Scheme 1c). These resultant 5-halo-2-formylpyrroles 1a−c smoothly condensed with 2,4-dimethylpyrrole or 2,4-dimethyl-3-ethylpyrrole in the presence of POCl3 and generated the desired pyrrolylBODIPYs 4 upon further BF2 complexation in one-pot with rationable isolated yields (18−30%) (Scheme 2). In comparison with α-halogenated formylisoindoles, these 5-halo2-formylpyrroles show reduced electrophilicity in this condensation reaction. It generally requires extended reaction time and higher temperature for the completion of the reaction. The yields are acceptable when considering the fact that most of

Figure 1. (a) Chemical structure of the core structure of BODIPY and pyrrolylBODIPY and the commercial pyrrolylBODIPY 576/589 marketed by Invitrogen; (b) our recently reported synthetic strategies to pyrrolic unsubstituted or halogenated pyrrolylBODIPYs I and II; and (c) our lately developed synthetic strategies to A- and C-ring alkyl-substituted isoindoleBODIPYs III and the concept of our synthesis of A-, B-, C-ring alkyl-substituted pyrrolylBODIPYs 4 presented in this work.

Our group lately has developed a facile synthetic route to pyrrolic alkyl-substituted isoindoleBODIPY III from a condensation of 3-halo-1-formylisoindole with excess amounts of pyrrole.20,21 This synthetic route allows the installation of various substituents at both A and C pyrrolic units of the chromophore via simple variation in the starting pyrrole derivatives. Installation of alkyl substituents at these two pyrrolic units of the chromophore brings a dramatic red shift of the spectra.21 In great contrast to 3-halo-1-formylisoindole, which is hard to functionalize at the fused benzene moiety, there are a variety of readily available 5-halo-2-formylpyrroles containing various electron-rich (alkyl or alkoxy) substituents at the pyrrolic positions. Herein, considering that pyrrolic alkyl substituents may greatly affect the optical and electronic properties and the solubility of pyrrolylBODIPY dyes, we have replaced 3-halo-1formylisoindole with readily available alkyl or alkoxy-substituted 5-halo-2-formylpyrroles and reported the efficient synthesis of a set of A-, B-, and C-ring-functionalized pyrrolylBODIPYs 4 from the condensation of highly diverse 5-halo-2-formylpyrroles with readily available pyrrole derivatives (Scheme 2), such as 2,4-dimethyl or 2,4-dimethyl-3-ethylpyrroles, their characterizations via X-ray analysis, photophysical and electronic properties, and the preliminary investigation of their

Scheme 2. Synthesis and Yields of PyrrolylBODIPYs 4a−f from 5-Halo-2-formylpyrroles 1a−c and Pyrrole Derivatives

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larger dihedral angles observed for 4b with respect to 4c may be attributed to the steric hindrance effect of methyl substitution in the coordinated pyrrole B unit and the adjacent methyl substituent on the uncoordinated pyrrole A unit. Various intramolecular hydrogen bondings were observed among the hydrogen in the NH moiety of the uncoordinated pyrrole A unit, the fluorine atoms in the BF2 unit, and the hydrogen of methyl substituent in the 5-position of the uncoordinated pyrrole A unit. The F−H distance between the hydrogen atom of the 5-methyl group and the fluorine atoms is in the range of 2.581−2.846 Å. These hydrogen bondings restrict the rotations of the uncoordinated pyrrole A unit. Spectroscopic Properties. PyrrolylBODIPYs 4a−f all show bright red fluorescence under 365 nm handheld UV lamp irradiation condition in solvents including hexane, toluene, and dichloromethane. Their photophysical spectra were analyzed in dichloromethane as shown in Figures 3 and S2−S7, and the corresponding data are summarized in Tables 1 and 2. These pyrrolylBODIPYs show strong absorption (centered at around 570−624 nm) and emission (centered at around 591−654 nm) in dichloromethane, which are comparable to the commercialized BODIPY 576/589 (Figure 1). By fixing the substituent patterns on both A- and C-rings, the installation of electron-donating substituent(s) at the B-ring causes significant blue shift of the absorption/emission bands of these pyrrolylBODIPYs. The more electron-rich substituents on B-ring, the greater the blue shift of the spectra. As shown in Figure 3, a gradual blue shift of the absorption/emission bands was observed from 4a (607/632 nm) to 4b (588/630 nm) and 4c (570/591 nm). Similar trend is also observed from 4d (624/ 654 nm) to 4e (606/646 nm) and 4f (586/609 nm). These pyrrolylBODIPYs 4a−f exhibit intense fluorescence (ϕ = 0.41− 0.99) in hexane. Their fluorescence decay profiles fit a singleexponential decay with lifetimes in the range of 2.62 to 6.11 ns in anhydrous dichloromethane (Table 1, Figures S8−S13), which are comparable to the most reported BODIPYs.29 As summarized in Table 2, the solvatochromic effect on λmax abs and λmax em for each compound was studied in seven different organic solvents (Figures S2−S7). These results indicated that max λmax abs and λem of the six pyrrolylBODIPYs are positioned within relative narrow wavelength ranges (13−26 nm for absorption and 9−35 nm for emission of the six pyrrolylBODIPYs). max Bathochromical shifts were also observed for λmax abs and λem with increasing solvent polarizability (from acetonitrile to toluene), similar to many reported BODIPYs.22,30

these resultant B-ring alkyl-substituted pyrrolylBODIPYs are barely accessible from the previous synthetic strategies. These resultant pyrrolylBODIPYs and their key synthetic precursors were fully characterized with proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), and high-resolution mass spectrometry (HRMS). The structure of 4b and 4c were further confirmed by X-ray analysis (Figure 2).

Figure 2. X-ray structures of 4b and 4c. Front view (a) and side view (b) of 4b and front view (c) and side view (d) of 4c. C, light gray; H, gray; N, blue; B, dark yellow; and F, bright green.

X-ray Structure Analysis. Single crystals suitable for X-ray analysis of 4b and 4c were obtained by the slow diffusion of hexane into their dichloromethane solutions at room temperature. Both of these two dyes crystallized in the monoclinic system [P21/c for 4b, and P21/n for 4c, (Tables S1 and S2)]. The BODIPY subunit in these two dyes is planar, with the central boron atoms coordinated with two neighboring nitrogen atoms in a tetrahedral geometry (Figures S1 and 2). The plane defined by the two fluorine atoms and the center boron atom is perpendicular to that of the BODIPY fragment, characteristic for most previously reported BODIPY dyes.20,21 Alkyl substituents on the coordinated pyrrole B unit greatly affect the dihedral angle between the BODIPY fragment and the uncoordinated pyrrole A moiety. The dihedral angles between the uncoordinated pyrrole A unit and the plane of the BODIPY fragment are 48.9(2)° for 4b and 25.3(1)° for 4c. The

Figure 3. Overlaid normalized absorption (a) and fluorescence emission (b) spectra of pyrrolylBODIPYs 4a−f in dichloromethane. 3553


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Table 1. Photophysical Properties of 4a−f in Dichloromethane dyes

a λmax abs (nm)

ε (cm−1 M−1)b

λmax em (nm)

ϕc

Stokes shiftsd(cm−1)

τe (ns)

Kff (×109 s−1)

Knrg (×109 s−1)

4a 4b 4c 4d 4e 4f

607 588 570 624 606 586

5.04 4.69 4.85 4.76 4.72 4.78

632 630 591 654 646 609

0.31 0.27 0.48 0.22 0.23 0.44

652 1134 623 735 1022 644

4.50 3.30 6.11 3.80 2.62 5.45

0.07 0.08 0.08 0.06 0.09 0.08

0.15 0.22 0.09 0.21 0.29 0.10

a All of the values correspond to the strongest absorption peaks. bMolar absorption coefficients are in the maximum of the highest peak. cThe fluorescence quantum yields were obtained by using Cresyl violet perchlorate (ϕ = 0.54 in methanol) as the reference compound. Standard errors are less than 5%. dStokes-shifts for all dyes refer to the difference between the absorption and the emission maximum. eFluorescence lifetime; standard deviations are less than 0.1 ns. fKf = ϕ/τ. gKnr= (1 − ϕ)/τ.

A gradual decrease in the fluorescence quantum yields was observed for these pyrrolylBODIPYs with the increase in solvent dipolar moments from hexane to toluene, dichloromethane, THF, methanol, acetonitrile, DMSO, and PBS (Table 2, Figure S14b). For example, a gradual decrease in the fluorescence quantum yield for 4f was observed with the increase in solvent dipolar moments from hexane (0.70) to toluene (0.51), dichloromethane (0.44), THF (0.31), methanol (0.04), acetonitrile (0.08), DMSO (0.06), and PBS (0.03). This solvent-dependent fluorescence emission can be observed by naked eye under 365 nm handheld UV lamp irradiation condition (Figure 4). The quantum yields and fluorescence lifetimes of 4c and 4f having the electron-rich methoxy group are much higher than those of other pyrrolylBODIPYs in almost all solvents, for example, in CH2Cl2 and THF, as summarized in Table 1, which might result from that their nonradiative rate constants are much smaller than those of others. Electrochemical Properties. Cyclic voltammetry (CV) of pyrrolylBODIPYs 4a−f measured in anhydrous dichloromethane containing 0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6) as the supporting electrolyte is shown in Figure 5 and is summarized in Table 3. Among these, 4a and 4d each exhibit an irreversible reduction wave with the half-wave potential at 0.68 and 0.53 V, respectively, and a reversible oxidation wave (with Epc at −1.21 and −1.25 V, respectively) [vs saturated calomel electrode (SCE)]. By contrast, 4b−c and 4e−f each show one reversible reduction wave and one reversible oxidation wave. For example, the half-wave potentials for the reduction/oxidation waves are at −1.23/0.71 V for 4b and −1.31/0.65 V for 4c (vs SCE). On the basis of the onset potential of their first oxidation and reduction waves, the highest occupied molecular orbital (HOMO)−lowest unoccupied molecular orbital (LUMO) energy levels for 4a, 4b, and 4c were estimated to be at −4.99/−3.31, −5.02/−3.25, and −4.96/−3.17 eV, respectively. The corresponding energy band gaps for 4a, 4b, and 4c are 1.68, 1.77, and 1.79, respectively. The installation of electron-rich alkyl and alkoxy substituents on the coordinated pyrrole B-ring unit causes the decrease in the energy band gaps of these dyes. Similar results were observed in pyrrolylBODIPYs 4d−f, which are in good agreement with their spectra. The redox potentials for 4d measured via CV in various solvents with different polarities, such as DMSO, acetonitrile, THF, and dichloromethane, are summarized in Table 4. The decrease in the reduction potential for 4d was observed from CH3CN (−0.91 V) to THF (−1.11 V) and CH2Cl2 (−1.25 V) as shown in Figures 5 and S15. This indicates that the pyrrolylBODIPY is relatively easier to be reduced in solvents

with solvent polarizability increased, and these pyrrolylBODIPYs might undergo photoinduced electron-transfer processes from uncoordinated pyrrole A-ring to the BODIPY fragment. Density Functional Theory Calculations. For the further understanding of the photophysical properties of these pyrrolylBODIPYs, the ground-state geometries of pyrrolylBODIPYs 4a, 4b, and 4c were optimized using density functional theory (DFT) calculations at B3LYP/6-31G (d) level based on their crystal data. The HOMO and LUMO of these pyrrolylBODIPYs were mainly distributed at the pyrrolylBODIPY chromophores (Figure 6), which is similar to their isoindole-derived pyrrolylBODIPY analogues.21 The installation of alkyl substituents on the coordinated pyrrole B-ring causes the increase in the HOMO−LUMO energy band gaps from 4a (2.39 eV) to 4b (2.44 eV). Replacing these alkylsubstituents with a more electron-rich methoxy group further increases the HOMO−LUMO energy band gap to 2.56 eV (for 4c). This increased HOMO−LUMO energy band gap is in good agreement with the observed blue shift of the spectra and the CV measurement. We further performed time-dependent DFT (TD-DFT) calculations on 4a−c in dichloromethane using the selfconsistent reaction field method and the polarizable continuum model (Table S3). The calculated absorption wavelength maximum (in the range of 502−534 nm) is blue-shifted with respect to the observed experimental spectra, which is similar to that disclosed in some recent publications.31 The lowest energy absorption bands for these pyrrolylBODIPY dyes are mainly contributed by their HOMO → LUMO transitions with respect to their electronic transition from the ground states to the first excited ones (S0 → S1). Biological Applications of These PyrrolylBODIPYs. To demonstrate the potential application of these pyrrolylBODIPYs as environmental-sensitive probes in biological systems, the application of these pyrrolylBODIPYs for sensing BSA in PBS was investigated. As shown in Figures 7 and S16, each of these pyrrolylBODIPYs shows relatively broad absorption with a weak fluorescence emission in the PBS buffer solution. The addition of BSA caused a red shift of the absorption and a significant enhancement of the absorption coefficients. More importantly, all of these pyrrolylBODIPYs showed a dramatic fluorescence increase in the presence of BSA as shown in Figure 7 (very weak fluorescence without BSA and strong fluorescence with BSA). Obvious spectral red shifts of the fluorescence emission bands for these pyrrolylBODIPYs 4a−c and almost no change for 4d−f were also observed, and the data are summarized in Table 5. For example, the addition of BSA to 4d in PBS, which has the longest maximum emission wavelength among these six pyrrolylBODIPYs, brings a gradual 3554


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Table 2. Photophysical Properties of PyrrolylBODIPYs 4a−f in Different Solventsa

4a

4b

4c

4d

4e

4f

solvents

λmax abs (nm)

log εmaxb

λmax em (nm)

ϕc

Stokes shift (cm−1)

hexane toluene dichloromethane THFd methanol acetonitrile DMSOe PBSf hexane toluene dichloromethane THF methanol acetonitrile DMSO PBS hexane toluene dichloromethane THF methanol acetonitrile DMSO PBS hexane toluene dichloromethane THF methanol acetonitrile DMSO PBS hexane toluene dichloromethane THF methanol acetonitrile DMSO PBS hexane toluene dichloromethane THF methanol acetonitrile DMSO PBS

610 614 607 606 600 598 606 583 601 601 588 589 580 576 580 561 571 576 570 570 564 563 570 561 628 632 624 622 615 614 616 620 617 619 606 606 594 593 596 604 587 592 586 586 581 579 588 591

5.09 5.09 5.04 5.03 4.91 4.96 4.39 3.42 4.84 4.74 4.69 4.69 4.65 4.65 3.87 2.91 4.93 4.82 4.85 4.77 4.69 4.80 3.80 3.67 4.88 4.78 4.76 4.78 4.78 4.73 4.47 3.88 4.99 4.88 4.72 4.85 4.79 4.79 4.25 3.23 4.95 4.88 4.78 4.82 4.81 4.77 4.50 3.59

623 632 632 616 604 606 613 604 618 632 630 617 596 610 597 592 582 590 591 588 583 585 592 586 641 653 654 650 635 638 637 645 637 649 646 642 638 641 639 644 600 608 609 604 597 602 605 610

0.54 0.37 0.31 0.07 0.06 0.05 0.04 0.04 0.61 0.50 0.27 0.02 0.01 0.02 0.02 0.02 0.99 0.99 0.48 0.39 0.07 0.11 0.04 0.05 0.52 0.27 0.22 0.05 0.03 0.03 0.03 0.02 0.41 0.29 0.23 0.02 0.03 0.02 0.03 0.02 0.70 0.51 0.44 0.31 0.04 0.08 0.06 0.03

342 464 652 268 110 221 188 596 458 816 1134 770 463 968 491 933 311 412 623 537 578 668 652 760 323 509 735 597 512 613 562 625 509 747 1022 925 1161 1263 1129 1028 369 445 644 509 461 660 478 527

Figure 4. Photographs of 4a−c in different solutions under handheld UV lamp irradiation (365 nm) conditions.

Figure 5. CVs of 4a−f measured in dichloromethane containing 0.1 M TBAPF6 at room temperature.

Table 3. Electrochemical Data Acquired and HOMO− LUMO Gaps Determined from Spectroscopy of 4a−fa dyes

Eox (V)

Ered (V)

Eonset red (V)

Eonset ox (V)

LOMO (eV)

HOMO (eV)

Eg (eV)

4a 4b 4c 4d 4e 4f

0.62 0.65 0.59 0.49 0.54 0.48

−1.21 −1.28 −1.36 −1.25 −1.33 −1.40

−1.09 −1.15 −1.23 −1.13 −1.22 −1.29

0.59 0.62 0.56 0.45 0.50 0.46

−3.31 −3.25 −3.17 −3.27 −3.18 −3.11

−4.99 −5.02 −4.96 −4.85 −4.90 −4.86

1.68 1.77 1.79 1.58 1.72 1.75

a

All of the values are corrected for changes in refractive indexes of different solvents. bMolar absorption coefficients are in the maximum of the highest peak. cFluorescence quantum yields (ϕ) were obtained by using Cresyl violet perchlorate (ϕ = 0.54 in methanol) as the reference compound. Standard errors are less than 5%. dTetrahydrofuran (THF). eDimethyl sulfoxide (DMSO). fSodium phosphate buffer solution (PBS, pH 7.4).

Eox = the first oxidation peak potential; Ered = irreversible reduction = the onset oxidative potentials; Eonset peak potentials; Eonset ox red = the + 4.4); EHOMO = onset reduction potentials; ELUMO = −e(Eonset red + 4.4); and Eg = ELUMO − EHOMO. −e(Eonset ox a

reported BSA probes.1 Less extent but similar fluorescence response was also observed for binding 4d with other proteins (chicken serum protein, casein, and thrombin) under the same condition (Figure S18).

increase in the fluorescence intensity (up to 40-fold) (Figure 8). PyrrolylBODIPY 4d interacts with BSA with a binding constant of 5.7 × 104 M−1 (Figure S17), which is similar to the 3555


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substituents on the B-ring brings a dramatic blue shift of the absorption and emission spectra. This is in great contrast to those substituents on pyrrole A- and C-rings, which generally cause red shift of the spectra. CV and DFT calculations confirm that these substituents on the B-ring cause a significant increase in the HOMO−LUMO energy band gap for the pyrrolylBODIPY chromophore. These pyrrolylBODIPYs show an intense absorption (570−632 nm) and fluorescence emission (582− 654 nm) in nonpolar solvents (such as hexane, toluene, and dichloromethane), which changes dramatically with the variation in the polarity of the solvents. This polarity dependency and the deep red/NIR emission may find potential applications of these pyrrolylBODIPYs as environmentsensitive fluorescence probes for biological samples as demonstrated in this work for BSA.

Table 4. Reduction Potential of 4d in Different Polarity Solvents, Containing 0.1 M TBAPF6 with a Scan Rate of 50 mV/s at Room Temperature DMSO Ered (V vs SCE) a

n.d.

a

CH3CN

THF

CH2Cl2

−0.91

−1.11

−1.25

n.d. = not detectable.

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EXPERIMENTAL SECTION General. Reagents and solvents were used as received from commercial suppliers unless noted otherwise. All reactions were performed in oven-dried or flame-dried glassware and were monitored by thin-layer chromatography. Flash column chromatography was performed using silica gel (300−400 mesh). 1H NMR and 13C NMR (75 MHz) spectra were recorded at 300 or 500 MHz by a NMR spectrometer in DMSO-d6 or CDCl3. Chemical shifts (δ) were given in ppm relative to CDCl3 (7.26 ppm for 1H and 77 ppm for 13C) or internal tetramethyl silane (δ = 0 ppm) as internal standard. Data were reported as follows: chemical shift, multiplicity, coupling constants, and integration. HRMS spectra were obtained using APCI-TOF in the positive mode. UV−vis absorption spectra were recorded on a Shimadzu UV-4100 spectrophotometer, and fluorescence spectra were recorded on a Hitachi F-4500 fluorescence spectrophotometer, with a quartz cuvette (path length = 1 cm). Photophysical Measurements. The absorption and fluorescence emission spectra for these pyrrolylBODIPYs 4a− f were measured in hexane, toluene, dichloromethane, THF, acetonitrile, methanol, DMSO, and PBS (pH 7.4) at room temperature. The relative fluorescence quantum yields for 4a−f were determined by comparing the areas under the corrected emission spectrum of the test sample in various organic solvents Cresyl violet perchlorate (ϕ = 0.54 in anhydrous methanol)33 as the standards. Non-degassed, spectroscopic grade solvents and a 10 mm quartz cuvette were used. Dilute solutions (0.01 < A < 0.05) were used to minimize the reabsorption effects. Quantum yields were determined using the following equation34

Figure 6. Relative energies of the frontier molecular orbitals of 4a−c. Calculations are based on ground-state geometry by DFT at the B3LYP/6-31G* level.

It should be noted here that the relative broad absorption and low extinction coefficient for our pyrrolylBODIPYs 4 indicated their possible aggregations in aqueous PBS media owing to their hydrophobic character. Thus, our turn on response of pyrrolylBODIPYs 4 in the presence of BSA may also be ascribed to the disaggregation-induced fluorescence enhancement, which has been reported as a powerful method to develop interesting fluorescent probes.32a For example, BODIPY-based fluorescent probe for selectively imaging tau protein aggregates32b and RecA inteins32c has been reported recently. We thus measured the UV−vis titration of 4d in PBS buffer with the addition of various equivalents of BSA. The absorption only showed slight increase when increasing the concentration of BSA from 0 to 30 μM (as shown in Figure S19). Large increase in absorption intensity was only observed when further increasing the concentration of BSA from 30 to 50 μM. By contrast, the fluorescence intensity of 4d at 645 nm exhibited almost 40-fold increase with the addition of 30 μM BSA (Figure 8). These results indicated that this turn on response of pyrrolylBODIPYs 4 in the presence of BSA may be at least in part due to the changes in polarity, although pyrrolylBODIPYs may bind with the surface of BSA as aggregates in aqueous PBS.

ϕX = ϕS(IX /IS)(AS /AX )(nX /nS)2

where ϕS is the reported quantum yield of the standard, I is the integrated emission spectra, A is the absorbance at the excitation wavelength, and n is the refractive index of the solvent being used. X subscript is the test sample, and S subscript is the standard. Fluorescence lifetimes (τ) of the S1 state were measured by time-correlated single-photon counting method on an Edinburgh FLS920 steady-state lifetime fluorescence spectrometer with corresponding excitation and emission being monitored at the peak maximum. The fluorescence lifetime values were obtained from deconvolution and distribution lifetime analysis. When the fluorescence decays were single exponential, the rate constants of radiative (Kf) and non-

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CONCLUSIONS We have synthesized six novel A-, B-, C-ring-functionalized pyrrolylBODIPYs 4 from a POCl3-promoted condensation of various readily available (alkyl or methoxyl substituted) 5-halo2-formylpyrrole with pyrroles. X-ray analysis shows that the installation of alkyl or methoxy substituents at the pyrrole Bring of these pyrrolylBODIPYs greatly affects the dihedral angle between the BODIPY chromophore and the uncoordinated Aring in the solid state. Installation of these electron-rich 3556


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Figure 7. Fluorescence changes of pyrrolylBODIPYs 4 (5 μM) with or without BSA (40 μM) in PBS buffer containing 1% DMSO as a cosolvent; photographs of solution before and after the addition of BSA under natural light (i) and under 365 nm handheld UV lamp irradiation condition (ii); and fluorescence spectra of the solutions of 4a (a), 4b (b), 4a (c), 4d (d), 4e (e), and 4f (f) without BSA (black lines) or with BSA (red lines).

Table 5. Comparison of Spectroscopic Properties of PyrrolylBODIPYs 4a−f with or without BSA (40 μM) in PBS Containing 1% DMSO as a Cosolvent dyes BSA λmax abs λmax em ϕ

4a (−) 583 604 0.04

4b (+) 607 634 0.14

(−) 561 592 0.02

4c (+) 586 620 0.11

(−) 561 586 0.05

4d (+) 569 593 0.30

(−) 620 614 0.02

4e (+) 629 645 0.08

(−) 604 637 0.02

4f (+) 605 641 0.07

(−) 578 597 0.03

(+) 590 610 0.32

standard (∼1 mM potassium ferricyanide) and corrected for external cell resistance using the BASi Epsilon software. Crystallography. Crystals of pyrrolylBODIPYs 4b and 4c suitable for X-ray analysis were obtained by slow diffusion of hexane into their dichloromethane solutions. Diffraction was performed on a Bruker SMART APEXII CCD area detector diffractometer using graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at 293(2) K, with ϕ and ω scan techniques. An empirical absorption correction was applied using the SADABS program.35 All structures were solved by direct methods, completed by subsequent difference Fourier syntheses, and refined anisotropically for all non-hydrogen atoms by fullmatrix least-squares calculations based on F2 using the SHELXTL program package.36 The hydrogen atom coor-

radiative (Knr) deactivation were calculated from the measured fluorescence quantum yield and fluorescence lifetime in dichloromethane using the following equation: Kf = ϕ/τ and Knr = (1 − ϕ)/τ. CVs. CV experiments were performed with a Bioanalytical Systems Inc. (BASi) Epsilon potentiostat and analyzed using BASi Epsilon software. Electrochemical cells are consisted of a three-electrode setup including a glassy carbon as the working electrode, platinum wire as the counter electrode, and SCE as the pseudo reference electrode. Experiments were run at 20 mV s−1 scan rates in degassed dichloromethane solutions of the analytes (∼1 mM) and supporting electrolyte (0.1 M TBAPF6) at room temperature. CVs were referenced against an external 3557


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aqueous Na2CO3. The reaction mixture was further stirred for an additional 20 min. The organic layer was collected, and the aqueous layer (pH 7−8) was extracted with dichloromethane (60 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and removed under vacuum. The residue was purified through silica using 20% ethyl acetate/ hexane as eluant to generate 2c27 as a brown oil in 76% yield (0.86 g). 1H NMR (300 MHz, CDCl3): δ 7.00 (s, 1H), 5.59 (s, 1H), 4.12 (q, J = 7.2 Hz, 2H), 3.76 (s, 3H), 3.38 (q, J = 7.2 Hz, 2H), 1.32−1.26 (m, 6H). 5-Chloro-2-formylpyrrole 1a.22 To pyrrole (1.34 g, 1.4 mL) in anhydrous THF (100 mL) under nitrogen was added sulfuryl chloride (2.70 g) in a dropwise manner at −78 °C. The reaction mixture was stirred at −78 °C for 3 h before dropwise addition (over 10 min) of the Vilsmeier acylating agent generated from the reaction of DMF (1.7 mL, 22 mmol) with POCl3 (2.0 mL, 22 mmol) in 50 mL of dichloromethane at 0 °C. The reaction was further stirred at room temperature for 14 h, and the temperature was increased to reflux for an additional 15 min. An aqueous solution of saturated Na2CO3 was added to the reaction mixture and stirred for 30 min under refluxing condition. The reaction mixture was extracted with ether (80 mL × 3). Organic solvents were combined, dried over anhydrous MgSO4, and filtered, and the solvent was removed under vacuum. The residue was purified by flash chromatography (silica, CH2Cl2/ethyl acetate = 10/1, v/v) to afford 1a22 as a colorless solid in 30% yield (0.77 g). 1H NMR (300 MHz, CDCl3): δ 10.20 (br, s, 1H), 9.39 (s, 1H), 6.94 (q, J = 3.0 Hz, 1H), 6.23 (q, J = 3.0 Hz, 1H). 5-Iodo-2-formylpyrrole 1b.25 To a solution of 2b (418 mg, 2 mmol) in THF/H2O (v/v = 5/1, 25 mL) was added LiOH· H2O solution (202 mg, 4.8 mmol). The mixture was stirred at 70 °C for 4 h. Aqueous KHSO4 solution was added to the reaction mixture to adjust the pH value to around 3. The solution was extracted by dichloromethane (50 mL × 3), dried over anhydrous Na2SO4, and filtered, and the solvent was removed under vacuum. The residue as the reaction intermediate was eluted with methanol. 1H NMR (300 MHz, DMSO-d6): δ 12.98 (s, 1H), 12.28 (s, 1H), 9.78 (s, 1H), 2.70 (q, J = 7.5 Hz, 2H), 2.22 (s, 3H), 1.05 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6): δ 182.0, 162.6, 133.6, 130.3, 125.5, 125.0, 17.2, 15.6, 9.7. The resultant solid in 1,2dichloroethane (25 mL) was directly added to NaHCO3 (454 mg, 5.4 mmol) in water (25 mL) at 70 °C. To this mixture was added I2 (411 mg, 1.6 mmol) and KI (488 mg, 2.9 mmol) in 15 mL of water in one portion. The mixture was refluxed for 1 h and cooled down to room temperature. Excess amount of saturated aqueous solution of sodium thiosulfate was added into the reaction mixture to remove the remaining iodine. The reaction mixture was extracted, dried over anhydrous Na2SO4, and filtered, and the solvent was removed under vacuum. After recrystallization from MeOH, a slightly yellow powder of 1b25 was obtained in 75% total yield (388 mg). 1H NMR (300 MHz, CDCl3): δ 10.11 (s, 1H), 9.40 (s, 1H), 2.75 (q, J = 6.0 Hz, 2H), 1.99 (s, 3H), 1.19 (t, J = 6.0 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 176.0, 137.4, 133.0, 125.9, 17.7, 16.4, 11.5. 5-Bromo-2-formylpyrrole 1c.28 To NaOH (4 M, 7 mL) in ethanol (80 mL) was added 2c (0.86 g, 3.33 mmol). The reaction mixture was heated at reflux for 3 h before cooling down to room temperature. The reaction mixture was concentrated, neutralized with HCl (3 M), extracted with dichloromethane (80 mL × 4), filtered, and dried over anhydrous Na2SO4, and the solvent was removed under

Figure 8. Fluorescence variation in 4d (5.0 μM) with the addition of BSA (up to 45 μM) in PBS buffer containing 1% DMSO as a cosolvent, excited at 570 nm. The molar concentrations of BSA were 0, 1.5, 3.0, 6.0, 7.5, 10, 15, 20, 30.0, and 45.0 μM.

dinates were calculated with SHELXTL by using an appropriate riding model with varied thermal parameters. The residual electron densities were of no chemical significance. Crystal structures of 4b (CCDC 1055369) and 4c (CCDC 978649) are provided in the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac. uk/data_request/cif. Computational Details. All of the calculations were carried out by the methods implemented in Gaussian 09 package37 on the ground-state geometry, which were optimized by using the DFT method at the B3LYP/6-31G (d) level.38 The same method was used for vibrational analysis to verify that the optimized structures correspond to local minima on the energy surface. At the optimized geometries, TD DFT computations were used to obtain the vertical excitation energies and oscillator strengths under the B3LYP/6-31+G (d) theoretical level. Synthesis. The key synthetic precursors 5-chloro-2formylpyrrole 1a,22 2b,25 5-iodo-2-formylpyrrole 1b,25 2c,27 and 5-bromo-2-formylpyrrole 1c28 were prepared using a literature or a modified literature procedure.22−28 2b.25 To 3b (780 mg, 4 mmol) in THF/acetic acid/H2O (90 mL, v/v/v = 4/1/4) was added CAN (ceric ammonium nitrate, 10 g, 18 mmol). The reaction mixture was stirred at room temperature for 3 h, poured into water, and extracted with dichloromethane (60 mL × 3). Organic layers were combined, washed with saturated aqueous Na2CO3, and dried over anhydrous sodium sulfate, and the solvent was removed under vacuum to give 2b25 in 86% yield (720 mg). 1H NMR (300 MHz, CDCl3): δ 9.77 (s, 1H), 9.54 (s, 1H), 4.36 (q, J = 6.0 Hz, 2H), 2.75 (q, J = 9.0 Hz, 2H), 2.30 (s, 3H), 1.38 (t, J = 6.0 Hz, 3H), 1.20 (t, J = 9.0 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 179.3, 161.0, 136.8, 129.5, 126.2, 124.7, 60.8, 29.6, 16.8, 16.3, 14.3, 9.5. 2c.27 To DMF/dichloromethane (v/v = 1/20, 40 mmol) at 0 °C was added POBr3 (3.17 mL, 11 mmol) in a dropwise manner in dichloromethane (15 mL) under argon. The reaction mixture was stirred at room temperature for 30 min before dropwise addition of 4-methoxy-3-pyrrolin-2-one 3c (0.5 g, 4.4 mmol) in dichloromethane (80 mL) at 0 °C. By increasing the temperature to 65 °C, the reaction mixture was stirred at this temperature for 4 h. Upon completion of the reaction, the reaction mixture was cooled down to 0 °C, and the reaction was quenched by dropwise addition of 2 M 3558


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δ 9.11 (s, 1H), 6.94 (s, 1H), 2.63 (t, J = 6.0 Hz, 2H), 2.46−2.35 (m, 7H), 2.28 (s, 3H), 2.19 (s, 3H), 2.03−2.00 (m, 6H), 1.20 (t, J = 6.0 Hz, 3H), 1.13−1.03 (m, 6H). 13C NMR (75 MHz, CDCl3): δ 153.6, 147.9, 144.6, 135.7, 133.1, 132.4, 131.6, 127.6, 125.7, 122.9, 121.6, 118.1, 116.8, 18.2, 17.8, 17.3, 15.8, 15.3, 14.6, 12.6, 12.0, 11.7, 11.0, 9.5. HRMS (ESI) calcd for C24H33N3BF2 [M + H]+: 412.2730; found, 412.2730. PyrrolylBODIPY 4f was obtained as greenish powder in 21% yield (42 mg) from 1c (102 mg, 0.5 mmol) and 2,4-dimethyl-3ethylpyrrole (1.3 mL, 10 mmol). 1H NMR (300 MHz, CDCl3): δ 10.12 (s, 1H), 6.99 (s, 1H), 6.07 (s, 1H), 3.95 (s, 3H), 2.48− 2.29 (m, 13H), 2.15 (s, 3H), 1.07 (t, J = 6.0 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 162.6, 148.2, 147.3, 132.6, 131.8, 131.6, 129.9, 129.8, 126.2, 126.0, 125.0, 119.0, 112.8, 96.8, 58.2, 17.4, 15.5, 14.9, 12.4, 12.2, 11.9, 9.3. HRMS (EI) calcd for C22H28N3BF2O [M]+: 399.2293; found, 399.2295.

vacuum. The residue was recrystallized from MeOH to give 1c28 as a light yellow solid in 69% yield (0.57 g). 1H NMR (300 MHz, CDCl3): δ 10.47 (s, 1H), 9.37 (s, 1H), 5.92 (s, 1H), 3.79 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 174.0, 158.4, 120.3, 112.0, 98.7, 58.2, 31.0. General Procedure for the Preparation of PyrrolylBODIPYs 4a−f. To the mixture of 5-halo-2-formylpyrrole 1 (0.5 mmol) and pyrrole derivatives (10 mmol) were added POCl3 (0.47 mL, 5 mmol) in 80 mL 1,2-dichloroethane at ice-cold condition under argon. The reaction mixture was stirred at 80 °C for 6−8 h. TLC was used to follow up this reaction. Upon the disappearance of 1, Et3N (1.5 mL) was added to the reaction mixture under ice-cold condition. The reaction mixture was stirred for 10 min before adding BF3·OEt2 (2.5 mL) under ice-cold condition through syringe. The reaction mixture was stirred overnight, poured into water, and extracted with CH2Cl2 (50 mL × 3). The organic layers were combined, and the solvent was removed under vacuum. The crude product was purified from chromatograph (silica gel, petroleum/CH2Cl2 = 2/1, v/v) to afford pyrrolylBODIPYs 4 as reddish or greenish powder. PyrrolylBODIPY 4a was prepared as greenish powder in 23% yield (36 mg) from 5-chloro-2-formylpyrrole 1a (115 mg, 0.5 mmol) and 2,4-dimethylpyrrole (1 mL, 10 mmol). 1H NMR (300 MHz, CDCl3): δ 10.08 (s, 1H), 6.99 (d, J = 1.5 Hz, 1H), 6.93 (s, 1H), 6.80 (d, J = 1.5 Hz, 1H), 6.04 (s, 1H), 5.90 (s, 1H), 2.56 (s, 3H), 2.36−2.34 (m, 6H), 2.24 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 153.3, 148.7, 138.1, 135.6, 134.2, 133.1, 130.1, 128.3, 119.8, 119.3, 119.2, 118.3, 112.9, 15.0, 14.7, 13.6, 11.2. HRMS (EI) calcd for C17H18N3BF2 [M]+: 313.1562; found, 313.1566. PyrrolylBODIPY 4b was prepared as greenish powder in 29% yield (51 mg) from 1b (132 mg, 0.5 mmol) and 2,4dimethylpyrrole (1 mL, 10 mmol). 1H NMR (500 MHz, CDCl3): δ 9.20 (s, 1H), 6.99 (s, 1H), 6.01 (s, 1H), 5.88 (s, 1H), 2.63 (q, J = 5.0 Hz, 2H), 2.48 (s, 3H), 2.33 (s, 3H), 2.25 (s, 3H), 2.09 (s, 3H), 2.01 (s, 3H), 1.21 (t, J = 5.0 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 154.8, 148.9, 145.4, 139.4, 133.5, 133.1, 131.5, 126.4, 123.6, 118.9, 118.5, 117.8, 110.6, 18.2, 15.7, 14.6, 13.5, 13.4, 11.3, 10.8. HRMS (ESI) calcd for C20H25N3BF2 [M]+: 356.2104; found, 356.2103. PyrrolylBODIPY 4c was prepared as greenish powder in 26% yield (44 mg) from 1c (102 mg, 0.5 mmol) and 2,4dimethylpyrrole (1 mL, 10 mmol). 1H NMR (300 MHz, CDCl3): δ 10.21 (s, 1H), 7.02 (s, 1H), 6.06 (s, 1H), 5.99 (s, 1H), 5.90 (s, 1H), 3.95 (s, 3H), 2.51 (s, 3H), 2.36 (s, 6H), 2.21 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 163.2, 149.0, 134.7, 134.6, 130.7, 129.0, 126.7, 119.6, 116.7, 113.4, 113.2, 96.8, 58.2, 15.0, 14.4, 13.4, 11.1. HRMS (EI) calcd for C18H20N3BF2O [M]+: 343.1667; found, 343.1676. PyrrolylBODIPY 4d was prepared as greenish powder in 24% yield (44 mg) from 1a (115 mg, 0.5 mmol) and 2,4dimethyl-3-ethylpyrrole (1.3 mL, 10 mmol). 1H NMR (300 MHz, CDCl3): δ 9.97 (s, 1H), 6.94 (d, J = 1.5 Hz, 1H), 6.88 (s, 1H), 6.76 (d, J = 3.0 Hz, 1H), 2.53 (s, 3H), 2.47−2.37 (m, 4H), 2.32 (s, 3H), 2.28 (s, 3H), 2.16 (s, 3H), 1.08 (t, J = 6.0 Hz, 6H). 13C NMR (75 MHz, CDCl3): δ 152.3, 148.0, 135.3, 134.9, 132.5, 131.5, 130.8, 129.2, 125.2, 124.8, 119.2, 118.7, 118.5, 17.4, 15.5, 14.7, 12.6, 12.3, 11.9, 9.4. HRMS (EI) calcd for C21H26N3BF2 [M]+: 369.2188; found, 369.2189. PyrrolylBODIPY 4e was obtained as greenish powder in 18% yield (37 mg) from 1b (132 mg, 0.5 mmol) and 2,4-dimethyl-3ethylpyrrole (1.3 mL, 10 mmol). 1H NMR (300 MHz, CDCl3):

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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/acsomega.7b00444. Crystal structure data and CIF files, additional photophysical data and spectra, copies of NMR spectra, highresolution mass spectra, and additional computational data for all new compounds (PDF) Crystallographic data of crystals of 4b (CCDC 1055369) (CIF) Crystallographic data of crystals of 4c (CCDC 978649) (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], [email protected] (C.Y.). *E-mail: [email protected] (L.J.). ORCID

Changjiang Yu: 0000-0002-9509-7778 Lijuan Jiao: 0000-0002-3895-9642 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by the National Nature Science Foundation of China (grants nos. 21372011, 21402001 and 21472002), and Nature Science Foundation of Anhui Province (grants no. 1508085J07). The numerical calculations in this paper have been done on the supercomputing system in the Supercomputing Center of The University of Science and Technology of China.

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REFERENCES

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