Nonbenzenoid BODIPY Analogues: Synthesis, Structural Organization, Photophysical Studies, and Cell Internalization of Biocompatible N-Alkyl-Aminotroponyl Difluoroboron (Alkyl-ATB) Complexes.

The alkyl-BODIPY derivatives are lipid types of fluorescent molecules that exhibit a unique structure and functions including sensing of hydrophobic microenvironments in living cells. Their synthesis involves multisteps from the core structure dipyrromethene scaffold. The alkyl-BODIPY analogues are sought to derivatize with minimal synthetic steps even by altering the core structures derived from benzenoid aromatic moiety. Recently, the nonbenzenoid scaffold (aminotropone) has been explored to synthesize troponyl-BODIPY analogues, which are fluorescent. In the repertoire of nonbenzenoid analogue, N-alkyl-aminotroponyl difluoroboron (alkyl-ATB) is rationally designed comprising long-chain hydrocarbons to explore the lipid type of fluorescent molecules. This report describes the synthesis, photophysical studies, structural organization, and biocompatibilities of ATB derivatives containing different lengths of alkyl chain at 2-aminotropone scaffold. The photophysical studies of ATB derivatives reveal their fluorescence behaviors in organic solvents (CH3OH/CH3CN) with a quantum yield of ∼10 to 15%. These ATB derivatives also exhibit fluorescence characters in the solid state though their quantum yield is relatively low. Cell permeability and cytotoxicity studies reveal that alkyl-ATB derivatives are permeable to HeLa/HEK293T cell lines and show negligible cytotoxicity. The biocompatibility of alkyl-ATB derivatives is studied and confirmed by cell viability (MTT) assay to the HeLa/HEK293T cell lines. Importantly, the cell internalization studies of the representative alkyl-ATB molecule by fluorescence microscopy show that octyl-ATB is efficiently detectable at the cytoplasmic membrane and cellular nucleus in HeLa cells. Hence, alkyl-ATB derivatives are potential fluorescent molecules for developing probes to visualize cellular components under a fluorescence microscope.

Introduction

Boron-dipyrromethene (BODIPY) derivatives are versatile synthetic fluorescent molecules for labeling biomolecules and synthesis of energy-related cassette entities (Figure A).[1−4] However, the unsubstituted BODIPY molecule is chemically less stable and nonfluorescent. Thus, various substituted dipyrromethene scaffolds are synthesized for tuning the structural and functional properties, including solubility, and dye properties.[5,6] For example, alkenyl-BODIPY exhibits absorption peaks in the near-infrared region (NIR);[7] dipyridylmethene (DIPYR) dyes[8] pyridine-based BODIPY analogues exhibit a wide range of photophysical properties, from nonemissive to green fluorescence with quantum yield 0.2–0.8;[9] BOIMPYs, benzimidazole-containing analogue, show red-emissive fluorophores, considered as NIR dyes.[10] Werz synthesized super-fluorophores comprising ethylene-bridged oligo-BODIPYS with quantum yield almost unity.[11] Recently, a new class of BODIPY dyes as N-BODIPYs (diaminoboron dipyrromethanes) are prepared for developing photonic materials.[12] In the literature, it is reported that BODIPY derivatives comprising longer linear alkyl chains can minimize their molecular aggregation problem owing to the π–π stacking and are capable of forming self-assembled supramolecular structures.[13] For example, long-alkyl-chain-containing fluorescence peptides have been applied as a probe for the fluidity changes in cell membranes or sol-to-gel transition.[9,14,15] Importantly, the lipophilic BODIPY analogues are used as fluorescence probes for the lipid environment.[16] However, the synthesis of lipid-BODIPY analogues involves multistep synthesis from nondipyrromethene scaffolds. Thus, nondipyrromethene scaffolds are sought to prepare BODIPY analogues from the benzenoid aromatic core structures that could be synthesized easily and elegantly. However, nonbenzenoid aromatic core structures are also known and are constituents of various natural products. These ligands are derived from tropolone. This nonbenzenoid aromatic molecule is a constituent of troponoid natural products, such as colchicine, thujaplicine, manicoline, theaflavin, and isoimerubrin.[17−19] Its related derivatives show unusual photophysical behavior owing to the π–π*, n−π*, and intramolecular charge transfer.[20−22] The fluorescence behavior of tropolone is sensitive to environments’ polarity and pH conditions, although its fluorescence quantum is low.[23,24] Tropolone and its synthetic derivatives are ligands of two various coordinating metal ions. Previously, we have explored nonbenzenoid aromatic scaffolds such as 2-aminotropones and 2-aminotropimines for the synthesis of BODIPY analogues from aminotropone derivatives via N,N-/N,O-chelation (Figure B,C).[25,26] These analogues exhibit fluorescence properties with a quantum yield of ca. ∼0 to 15%. However, N,O-chelated troponyl-BODIPY analogues comprise various amino acid carboxylate functionalities (Figure C).[26] In the literature, lipophilic BODIPY analogues are synthesized by conjugating the long-chain alkyl group at the BODIPY core structure for sensing cellular hydrophobic environments.[27,28] In the repertoire of lipophilic BODIPY analogues, we rationally designed alkyl-aminotroponyl difluoroboron complex containing different lengths of hydrocarbon chain (Figure D). This report describes their synthesis, structural analysis, and photophysical properties. This report also demonstrates their self-assembly structural morphologies and fluorescence properties in the solid-state surface. Herein, their biocompatibility is also explored in vivo with HeLa/HEK-292 cell lines.

Figure 1

Chemical structures previously reported and new compounds: (A) BODIPY; (B) aminotroponimine difluoroboron; (C) aminoacidyltropone difluoroboron; and (D) alkyl-aminotroponyl difluoroboron (this work).

Results and Discussion

We began the synthesis of boron-aminotropone complexes from commercially available tropolone (Scheme ). Tropolone was converted to 2-tosyltropone (1) by following the previous method.[26] This tosylate derivative (1) was treated with various alkyl-amine under reflux conditions, producing N-substituted aminotropones derivatives (2). We used isobutyl-amine, butyl-amine, cyclohexyl-amine, propargyl-amine, hexyl-amine, octyl-amine, dodecyl-amine, and octadecyl-amine for the synthesis of respective N-alkyl-aminotropone ligands (2a–2h). These ligands (2a–2h) were purified by silica gel column chromatography and characterized by 1H/13C NMR and ESI-HRMS. Their characterization data are provided in the Supporting Information (Figures S1–S16). These alkyl-aminotropone ligands were treated with versatile difluoroboronating reagent BF3.OEt2 under the mild basic condition produced rationally designed alkyl-aminotropony-BODIPY (alkyl-ATB) analogues (3a–3h). We isolated isobutyl-ATB (3a), cyclohexyl-ATB (3b), propargyl-ATB (3c), butyl-ATB (3d), hexyl-ATB (3e), octyl-ATB (3f), dodecyl-ATB (3g), and octadecyl-ATB (3h). These complexes (3a–3h) were purified by silica gel column chromatography using ethylacetate/hexane (10:90) and isolated in good yields (80%), and were characterized by NMR (1H, 13C, 11B, 19F NMR) and ESI-HRMS. Their data are provided in the Supporting Information (Figures S17–S45).

Scheme 1

Synthesis of Alkyl-Aminotroponyl Difluoroboron Complexes

We attempted to crystallize alkyl-ATB derivatives in organic solvent systems for structural studies. Pleasantly, we obtained single crystals of four derivatives (3a/3b/3c/3g) in the DCM/MeOH solvent system and studied them using a single-crystal diffractometer. Their solved X-ray data were deposited at the Cambridge Crystallographic Data Centre (CCDC) with reference numbers 2158349 for 3a, 2158348 for 3b; 2158346 for 3c; and 2158347 for 3d. Their ORTEP and unit cell packing diagrams are provided in Figures and 3, while their other crystal structure parameters are tabulated in the Supporting Information (Figures S46–S49 and Tables S1–S4). The ORTEP diagram of isobutyl-ATB (3a) shows the chemical structure. In contrast, the unit cell packing diagram shows the presence of more than two molecules in the unit cell through the noncovalent interaction, mainly through intermolecular hydrogen bonding as F···H–C (Figure A,B). The selected bond length and bond angles are given in Table . Their lengths and bond angles are within the range. Generally, lipids form a bipolar self-assembly supramolecular structure by the interaction of head–head/tail–tail residue.[29,30] The packing arrangement of crystal 3a shows the orientation of troponyl difluoroboron residue (polar group, equivalent to the head of the lipid) and isobutyl residue (nonpolar group, comparable to the tail of lipid), which are nonplanar and do not exhibit lipid type of head–head/tail–tail residual interactions in the packing arrangement of crystal 3a.

Figure 2

X-ray analysis of propargyl-ATB (3a): ORTEP diagram (A); unit cell packing diagram (B).

Figure 3

X-ray analysis of cyclohexyl-ATB (3b): ORTEP diagram (A) and unit cell packing diagram (B); π–π interaction between troponyl ring (C).

Table 1

Selected Bond Lengths and Bond Angles of Boron Complexes 3a in the Solid State

selected bond lengths	selected bond angles	selected bond angles
C1–C7 = 1.446 Å	O1–B1–N1 = 99.31°	B1–O1–C1 = 99.31°
C1–O1 = 1.313 Å	F1–B1–O1 = 109.92°	B1–N1–C7 = 110.68°
C7–N1 = 1.329 Å	F2–B1–N1 = 112.34°	O1–C1–C7 = 110.55°
O1–B1 = 1.487 Å	F1–B1–N1 = 113.67°	N1–C7–C1 = 107.79°
N1–B1 = 1.548 Å	F2–B1–O1 = 111.02°	F1–B1–F2 = 110.13°

The OTEP diagram cyclohexane-ATB (3b) shows its chemical structure. In contrast, the unit cell packing diagram shows the presence of more than two molecules in the unit cell through a unique noncovalent π–π interaction between troponyl rings with a bond distance of 3.6 Å along with intermolecular hydrogen bonding as F···H–C (Figure A–C). In the packing arrangement of crystal 3b, the orientations of troponyl difluoroboron residue (polar group, equivalent to the head of the lipid) and cyclohexyl residue (nonpolar group, comparable to the tail of lipid) are almost in the same plane as a lipid type of head–head interaction.

The ORTEP diagram of propargyl-ATB (3c) shows its chemical structure. In contrast, the unit cell packing diagram exhibits the presence of more than one molecule through the noncovalent π-π interactions between troponyl rings with a bond distance of 3.7 Å (Figure A–C). Similarly, an intermolecular hydrogen bonding as F···H–C also occurs in packing arrangement. In the packing structure of crystal 3c, the orientations of troponyl difluoroboron residue (polar group, equivalent to the head of the lipid) and propargyl residue (nonpolar group, comparable to the tail of lipid) are almost in the opposite plane but exhibit a lipid type of head–head interaction.

Figure 4

X-ray analysis of propargyl-ATB (3c): ORTEP diagram (A); unit cell packing diagram propargyl-ATB derivative (B) and π–π interaction between troponyl rings (C).

The ORTEP and packing diagrams of dodecyl-ATB (3g) are provided in Figure A–E. The crystal structure of dodecyl-ATB (3g) shows a unique bipolar arrangement in its unit cell (Figure B). Noncovalent π–π interactions are found between their troponyl ring with a distance of 3.7 Å, and hydrophobic interactions are found between lipophilic dodecyl residues (Figure C). The space-filled model shows the formation of sheet-type 3D structures (Figure D). Its long-chain and difluoroboron residues are oriented at ∼120°, unlike other ATB crystals 3a–3c (Figure E). This rearrangement strongly supports the formation of three-dimensional lipid-type structure through head–head and tail–tail interactions.

Figure 5

X-ray analysis of octadodecyl-ATB (3g): ORTEP diagram (A); unit cell packing diagram (B); π–π interaction between troponyl ring (C); space-filled model of unit cell (D); and π–π interaction in space-filled model (E).

Photophysical Studies

We recorded the absorption and emission spectra of newly synthesized N-alkyl-aminotroponyl-BODIPY lipid analogues (3a–3h) in organic solvent methanol (MeOH)/acetonitrile (ACN). Their full-range UV–vis/fluorescence spectra are provided in the Supporting Information (Figures S50 and S51). The normalized absorbance and fluorescence spectra of isobutyl-ATB complex (3a) in MeOH are depicted in Figure A. However, the normalized absorbance and emission spectra of alkyl-ATB (3a–3h) in both solvent MeOH/ACN are also provided the Supporting Information for finding the Strokes shift (Figures S53 and S54). We also extracted and calculated their photophysical parameters in both solvents (MeOH/ACN), which are summarized in the Supporting Information (Tables S5 and S6). Their absorption spectra exhibit three peaks at wavelengths (λ) ∼245, ∼340, and ∼390 nm in both solvents, mainly due to the π–π*, n−π*, and charge transfer transition in the aminotropone-difluoroboron residue. We also determined their extinction coefficients (ε) at λ390 nm in MeOH (Table S5, column 5). The octadecyl aminotroponyl difluoroboron complex (3h) has a lower ε value (∼5.8 × 103 M– cm–1) compared to other analogues (∼9.0 × 103 M–1 cm–1). Their emission spectra exhibit an intense peak at wavelength 455 nm (λem,455 nm) and excitation wavelength 340 nm (λex,340 nm) with Stock’s shift ca. ∼110 nm in MeOH. Thus these analogues are fluorescent, almost like our previously reported amino acid comprising BODIPY analogues.[26] We measured their (3a–3h) relative quantum yield (Φ) in MeOH/ACN at a temperature of 20 °C compared to reference standard 0.1 M quinine sulfate in H2SO4. Their values are provided in the Supporting Information (Tables S5–S8). We plotted a bar diagram of the quantum yield of 3a–3h vs alkyl-ATB in MeOH (Figure B). Their quantum yields are ca. 9–13% (Tables S5 and S6, column 8). However, the quantum yield of propargyl-ATB (3c) is slightly higher (by ∼3%) compared to other analogues (3a/3b/3d–3h) in MeOH, possibly owing to the π-electron-rich alkyne group, which may lower the HOMO–LUMO energy gap. The quantum yield of alkyl-ATB derivatives is less variable compared to the previously reported amino acidyl-troponyl difluoroboron complexes (Figure C).[26] The quantum yields of those amino acid fluorescent derivatives significantly varied with the bulkiness nature of substituents at α-C, such as glycine derivatives, which exhibit ∼15% while phenylalanine derivatives exhibit ∼6%. Their quantum yields have significantly dropped with branching α-C of amino acid residue and increasing carbon chain at the aminotropone ring. Herein, we also note that the quantum yield of cyclohexyl-substituted ATB derivative (6b) is decreased significantly with branching α-C compared to the alkynyl-substituted one (3c), while there are no significant changes with increasing carbon chain length. We also recorded the solvent-dependent quantum yield of long-chain octadecyl-ATB (3h) derivative (most lipophilic analogue) in nonpolar solvent systems such as DCM and cyclohexane, and chloroform. Their quantum yields and other photophysical parameters are provided in Table . Importantly, the quantum yield of 3h has slightly increased compared to protic solvent MeOH. We plotted a bar diagram of the quantum yield of 3h vs solvent (Figure B). We noted that the quantum yield of 3h is reasonably high in the nonpolar solvent cyclohexane, possibly due to the strong lipophilic interaction, which could provide more rigidity in its structure. Thus, alkyl-ATB derivatives have some sort of fluorescence selectivity in a nonpolar solvent. We also recorded the fluorescence spectra of long-chain alkyl-ATB (3f,3g/3h) derivatives in a solid state at two different excitation wavelengths (340/380 nm). Their emission spectra are provided in the Supporting Information (Figure S55). We noted that octyl-ATB (3f) exhibits better fluorescence with an emission peak at wavelength 430 nm compared to the dodecyl-ATB (3g) and octadecyl-ATB (3h) at excitation wavelengths 340 nm. However, octyl-ATB (3f) and dodecyl-ATB (3g) exhibit almost the same fluorescence with an emission peak at wavelength 430 nm and excitation wavelength 380 nm. To examine the photostability of alkyl-ATB derivatives, we irradiated three representative compounds (3d/3f/3g) with UV light (λ254 nm, Hand UV-Lamp) at different time intervals and sequentially recorded UV–vis spectra (Figure S56). Their UV absorbance significantly decreased wavelength (λ) 250 nm with time compared to λ350–400 nm with two isosbestic points λ266 nm and λ400 nm along with marginal redshift. These studies support the degradation of alkyl-ATB derivative with light. Hence, these alkyl-ATB derivatives are potential fluorescent analogues for applying in new fluorescence probe designs.

Figure 6

(A) Normalized absorption and emission spectra of isobutyl-ATB derivative (3a) in MeOH at the concentration (5 × 10–4 M); for other derivatives, see the Supporting Information. (B) Bar diagram of quantum yield of alkyl-ATB derivatives (3a–3h) insolvent MeOH.

Table 2

Quantum Yields of Octadecyl-ATB (3h)a

solvents	λabs (nm)	abs	λem (nm)	Stoke’s shift (nm)	OD/abs (nm)	Φf
methanol	334, 380	0.06085	417, 438	104	334	0.10
acetonitrile	335, 380	0.07202	415, 441	106	340	0.10
DCM	335, 382	0.08749	416, 443	108	346	0.12
cyclohexane	337, 385	0.08650	418, 442	105	345	0.13
chloroform	343, 394	0.06974	431, 556	113	346	0.13

All measurements and quantum yields were determined for boron complex (3h) by considering quinine sulfate in 0.1 M H2SO4 as the standard reference.

The lipid molecules are prone to form liposome and micelles types of self-assembly supramolecular structures.[31] The packing diagram of the alkyl-ATB derivative’s crystal reveals the formation of a self-assembly structure in the solid state through noncovalent interactions such as C–H···F hydrogen bonding, π–π interactions, and hydrophobic interactions. In ethanol, we dissolved alkyl-ATB derivatives (3b/3e/3f/3g/3h). We prepared their respective thin-layer surfaces at silicon wafers for studying surface morphologies with field emission scanning electron microscopy (FE-SEM). The SEM images of alkyl-ATB (3e–3h) at the 10 uM scale are provided in Figure , while that of cyclohexyl-ATB (3b) is provided in the Supporting Information (Figure S56); their SEM images at other scales are provided in the Supporting Information (Figures S56–S60). The SEM images of cyclohexyl-ATB (3b) show the formation of crystalline structure, possibly owing to the π–π interactions between head groups as troponyl ring (Figure S57). However, the SEM images of hexyl-ATB (3e), octyl-ATB (3f), dodecyl-ATB (3g), and octadecyl-ATB (3h) are significantly different from cyclohexyl-ATB (3b). The hexyl-ATB derivative (3e) forms a ring-type structure at the surface (Figure A). The octyl-ATB with lipophilic interaction and head–tail-type orientation. The octanyl-ATB (3e) forms a honey-comb type of surface morphology (Figure B). The dodecyl-ATB (3g) shows the flower-type surface structure, possibly due to the head–tail type of lipid structure (Figure C). The octadecyl-ATB (3h) also shows complex flower-type structural morphology that is more compact and dense, possibly due to the head–head and tail–tail orientation (Figure D). The unique surface morphologies of alkyl-ATB derivatives are possibly formed owing to the bipolar self-assembly orientation and strong lipophilic interactions between their long-chain hydrocarbons (tail–tail) along with π–π interactions between troponyl ring (head–head). Nevertheless, we performed elemental mapping at the unique morphological surface of long-alkyl-chain-containing ATB derivatives (3g/3h) by SEM-associated energy-dispersive X-ray (EDX) spectrometer. Energy-dispersive X-ray analysis (EDXA) is a technique used for the elemental analysis and concentration determination of nanoparticles by SEM.[32] However, this method has some limitations with regard to accurate dimension and elemental analyses. The EDX data of representative alkyl-ATB derivatives (3g)/(3h) are provided in the Supporting Information (Figures S60A–S63B). Herein, elemental composition (atom %) of 3g/3h are extracted from their restive EDX spectrum as C (72.53%), N (5.12%), O (5.48%), B (8.41%), and F (8.46%) for 3g and C (72.46%), N (5.20%), O (5.74%), B (8.15%), and F (8.46%) for 3h. These analyses strongly support the presence of 3g/3h compounds at their respective surfaces. Hence, ATB derivatives are capable to form self-assembled supramolecular structures owing to the lipid type of noncovalent interactions.

Figure 7

FE-SEM images of hexyl-ATB derivative 3b (A), octyl-ATB derivative 3e (B), dodecyl-ATB derivative 3f (C), and octadecyl-ATB derivative 3g (D) at scale size of 10 μm.

The lipid biomolecules are prone to form liposome and micelle types of self-assembly supramolecular structures. Since alkyl-ATB exhibits fluorescence characters in the polar and nonpolar solvent with almost equal quantum efficiency, we attempted to record the fluorescence image of alkyl-ATB in the solid state using the confocal microscopic technique, unique for single-molecule sensitivity. Fluorescence lifetime imaging (FLIM) is a technique that resolves and displays the lifetimes of individual fluorophores rather than their emission spectra.[33,34] Fluorophores’ lifetime can be influenced by environmental parameters such as pH, ion, oxygen concentration, or molecular bonding. It can be used to distinguish fluorophores. We dissolved fluorescent alkyl-ATB derivatives (3d/3f/3g/3h) in ethanol and prepared the thin layer of the respective derivative at the glass surface. We recorded their separate fluorescence lifetime images at a scale of 10 μm (Figure ) while at different scales; their pictures are provided in the Supporting Information (Figures S64–S67). We also extracted lifetime fluorescence decay from respective fluorescence images (Figure ). The butyl-ATB (4d) exhibits a solid fluorescence surface with irregular morphology with an average lifetime of 4.6 ns, possibly due to the lipid type of structure (Figure A). The octyl-ATB (3f) shows the formation of a ring type of unique fluorescence surface morphology with an average lifetime of 3.2 ns, possibly due to the lipid type of structural organization (Figure B). The dodecyl-ATB (3g) exhibits a fluorescence surface with irregular morphologies with an average lifetime of 3.3 ns (Figure C). The octadecyl-ATB (3h) exhibits a fluorescence surface, which is compact with a dense average lifetime of 3.5 ns, possibly due to the strong lipophilic interactions of the long hydrocarbon chain (Figure D). We also noted that more minor lipophilic derivatives have a little higher fluorescence lifetime. Their images also support the formation of a self-assembly fluorescent structure with a unique arrangement, possibly owing to the lipophilic noncovalent interactions. For comparison, we recorded FLIM of amino acid derivative of troponyl-BODIPY analogue (alanyl-troponyl difluoroboron). Its FLIM images are provided in the Supporting Information (Figure S68), which exhibit its fluorescence behavior scattered over the surface, unlike the long-chain-hydrocarbon-containing ATB derivatives. The fluorescence lifetime of amino acid derivatives is relatively lower than that of ATB derivatives possibly owing to weak fluorescence behavior and poorly aggregated structure in the solid state. These results strongly support that alkyl-ATB derivatives (3d/3f/3g/3h) are fluorescent in the solid state with an average lifetime of 3–4 ns, and they have the ability to form a unique type of self-assembly structure.

Figure 8

Confocal fluorescence lifetime images and lifetime decay profile of butyl-ATB derivative 3d (A); octyl-ATB 3f (B); dodecyl-ATB 3g (C); and octadecyl-ATB 3h (D).

The MTT assay (in vitro toxicology assay) is a colorimetric assay for assessing cell metabolic activity and cell viability.[35,36] We examined the cell viability of alkyl-aminotropones (2d–2h) and alkyl-ATB derivatives (3a–3h) by dose-dependent MTT assay to the cancerous HeLa cell line and normal human HEK293T cell line (see Figures S69 and S70). Our MTT assay results strongly support that alkyl-ATB derivatives (3a–3h) are nontoxic to both types of cells. Then, we examined the transfection of alkyl-ATB derivatives into HeLa cells in vitro conditions. These derivatives were incubated for 18 h before fixing the cells for imaging under a fluorescence microscope. Their fluorescence images are provided in the Supporting Information (Figures S71–S73). We noted that the octyl-ATB derivative (3f) has efficiently transfected into HeLa cells and localized into the cellular cytoplasm/nucleus. For co-localization studies, we also recorded confocal images of the fixed cells and studied their co-localization with nucleus staining dye (DAPI) and lipid staining dye (BODIPY 493/503). Their confocal images are provided in Figure . These images clearly indicate that compound 3f is localized at the nucleus of cells (Figure A–C) and lipid environment (Figure D,E). In the literature, Pearson’s coefficient (r) is used to quantify the co-localization of two probes by fluorescence microscope.[37] We also extracted Pearson’s coefficient (r) of compound 3f with standard probes DAPI and BODIPY (493/503) by superimposing their respective images using co-localization quantifying software (JACoP plugin in Fiji: ImageJ).[38] The Pearson’s coefficient value of 3f with DAPI is ∼0.88, while with BODIPY staining agents, it is ∼0.77. These results strongly support that compound 3f is localized in the cellular lipid environment and nucleus. Thus long-chain-containing alkyl-ATB derivatives are localized at the nucleus of cells, which could be applied to develop fluorescent probes of the cellular nucleus environment.

Figure 9

Confocal Image of HeLa cells treated with octyl-ATB derivative (3f): (A) nuclei were stained with DAPI (blue); (B) cells stained with compound 3f; (C) merged image of (A) and (B) sections; (D) visualization of compound stained with 3f (100 μM) in TRITC channel (red); (E) cells stained with commercial BODIPY (BODIPY 493/503; 2 μM) stain (green); and (F) merged image of (A) and (B) sections. All images were taken at 63×, and a scale bar is provided for reference.

Conclusions

We have successfully synthesized lipophilic aminotroponyl difluoroboron complexes and nonbenzenoid BODIPY analogues from Tropolone. We have also shown the structural organization of four crystal complexes in solid states. Their crystal packing diagram shows the formation of supramolecular self-assembly structure with an intermolecular hydrogen bonding between C–H···F. Importantly, these complexes exhibit fluorescence character in the organic solvents (MeOH/ACN) with quantum yield ∼10 to 13% and Stoke’shift 110 nm. Their surface morphologies are demonstrated by the SEM imaging technique, which reveals the role of lipophilic alkyl substituents in the formation of unique self-assembly structures. These complexes also exhibit fluorescence characters in the solid state with a relatively low quantum yield compared to solution. Their self-assembly surface morphologies are also demonstrated by confocal imaging techniques that strongly support supramolecular structure formation with lipophilic alkyl substituents. We have shown their biological relevance by examining cell cytotoxicity and cell transfection in human cells (HEK292). In vitro studies reveal that these analogies are nontoxic to normal human cells. We successfully showed the delivery of octyl-aminotroponyl-BODIPY analogue into the HeLa cell line, which becomes fluorescent. Hence, these BODIPY analogues are the potential candidate to design the fluorescence probes of cellular components.

Experimental Procedure

General Information

Unless noted, all required materials and solvents were purchased from commercial suppliers and used without any further purification unless noted. Anhydrous dichloromethane was freshly prepared by distilling over calcium hydride. Reactions were monitored by thin-layer chromatography and visualized by UV and Ninhydrin. Column chromatography was performed in 100–200 mesh silica. Mass spectra were obtained from a Bruker micrOTOF-Q II spectrometer, and the samples were prepared in methanol and injected into a methanol and water mixture. NMR spectra were recorded on a Bruker AV-400 at room temperature (1H: 400 MHz, 13C: 100.6 MHz, 11B: 128 MHz, 19F: 377 MHz). 1H, 13C, 11B, and 19F NMR chemical shifts were recorded in ppm, downfield from tetramethylsilane. Splitting patterns are abbreviated as s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; m, multiplet. All boron complex crystal data were collected on a Rigaku Oxford diffractometer at 293 K, respectively. Absorption spectra were obtained using a Jasco V-730 spectrometer. Fluorescence spectra were obtained from a PerkinElmer LS-55 using a xenon lamp. All spectroscopic measurements were carried out with spectroscopic-grade nondegassed solvents and at 20 °C. Relative fluorescence quantum yields were compared with quinine sulfate quantum yield in 0.1 M H2SO4 (0.54). The obtained values were substituted in the following equation.

Solid emission of boron complex 3f, 3g, and 3h (excited at 340 and 380 nm) is measured by Edinburgh Instruments FLS 920. HRMS was analyzed with an Agilent Q-TOF 6500.

General Procedure for the Synthesis of Alkyl-Aminotropone (2a–2h)

All aminotropones were synthesized by following the reported procedure. 2-Tosyloxytropone and amines (1.2 equiv) were dissolved in ethanol; to this, Et3N (3.0 equiv) was added. The reaction mixture was allowed to reflux for 24–36 h. TLC monitored the completion of the reaction. All volatiles were evaporated under reduced pressure after reaction completion. To the crude product, 1.0 N HCl was added and extracted with dichloromethane (thrice), and the combined organic layers were dried over Na2SO4 and evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography using ethylacetate and hexane mixture as the mobile phase.

Isobutyl-Aminotropone or 2-(Sec-butylamino)cyclohepta-2,4,6-trienone (2a)

The pure product was obtained as a yellowish solid (552 mg, 86%). 1H NMR (400 MHz, CDCl3) δ 7.27–7.12 (m, J = 28.0, 11.7 Hz, 3H), 6.65 (t, J = 9.4 Hz, 1H), 6.56 (d, J = 10.5 Hz, 1H), 3.66–3.60 (m, 1H), 2.00 (s, 1H), 1.75–1.60 (m, 2H), 1.28 (d, J = 6.4 Hz, 3H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 176.51, 155.03, 137.16, 136.29, 128.03, 121.77, 108.82, 49.45, 29.10, 19.42, 10.40. HRMS (ESI-TOF) m/z: [M + Na] calc. for C11H15NO 200.1046, found 200.1046.

Cyclohexyl-Aminotropone or 2-(Cyclohexylamino)cyclohepta-2,4,6-trienone (2b)

The pure product was obtained as a yellowish solid (352 mg, 95%). 1H NMR (400 MHz, CDCl3) δ 7.27–7.12 (m, 3H), 6.67–6.58 (m, 2H), 3.50 (t, J = 4 Hz, 1H), 2.05 (d, J = 12 Hz, 2H), 1.84–1.67 (m, 4H), 1.44–1.31 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 176.53, 163.43, 154.63, 137.15, 136.28, 128.06, 121.78, 108.93, 51.06, 32.07, 25.57, 24.66. HRMS (ESI-TOF) m/z: [M + nH] calc. for C13H17NO 204.1383, found 204.1393.

Propargyl-Aminotropone or 2-(Prop-2-yn-1-ylamino)cyclohepta-2,4,6-trienone (2c)

The pure product was obtained as a yellowish solid (850 mg, 74%). 1H NMR (400 MHz, CDCl3) δ 7.33–7.27 (m, 2H), 7.21 (t, J = 12 Hz, 1H), 6.75 (t, J = 8 Hz, 1H), 6.62 (d, J = 8 Hz, 1H), 4.14 (dd, J = 5.9, 2.4 Hz, 2H), 2.30 (t, J = 2.4 Hz, 1H), 1.67 (s, 1H). 13C{H} NMR (101 MHz, CDCl3) δ 177.19, 154.49, 137.49, 136.05, 130.07, 123.40, 109.22, 77.87, 72.51, 32.50. HRMS (ESI-TOF) m/z: [M + nH] calc. for C10H9NO 160.0757, found 160.0759.

Butyl-Aminotropone or 2-(Butylamino)cyclohepta-2,4,6-trienone (2d)

The pure product was obtained as a yellowish solid (327 mg, 94%). 1H NMR (400 MHz, CDCl3) δ 7.28–7.21 (m, 3H), 7.14 (d, J = 12 Hz, 1H), 6.66 (t, J = 12 Hz, 1H), 6.54 (d, J = 12 Hz, 1H), 3.33–3.28 (m, 2H), 1.75–1.70 (m, 2H), 1.47 (dd, J = 16, 8 Hz, 2H), 0.98 (t, J = 8 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 176.58, 155.71, 137.27, 136.34, 128.28, 121.97, 108.65, 42.59, 30.47, 20.31, 13.75. HRMS (ESI-TOF) m/z: [M + nH] calc. for C11H15NO 178.1226, found 178.1237.

Hexyl-Aminotropone or 2-(Hexylamino)cyclohepta-2,4,6-trienone (2e)

The pure product was obtained as a yellowish solid (337 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 7.24 (dd, J = 20.3, 10.4 Hz, 3H), 7.14 (d, J = 8 Hz, 1H), 6.66 (t, J = 8 Hz, 1H), 6.52 (d, J = 12 Hz, 1H), 3.29 (dd, J = 12, 6.5 Hz, 2H), 1.77–1.70 (m, 2H), 1.45–1.42 (m, 2H), 1.33 (d, J = 2.9 Hz, 4H), 0.90 (t, J = 5.9 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 176.57, 155.68, 137.22, 136.31, 128.27, 121.93, 108.61, 42.89, 31.46, 28.41, 26.81, 22.53, 14.00. HRMS (ESI-TOF) m/z: [M + nH] calc. for C13H19NO 206.1539, found 206.1548.

Octyl-Aminotropone or 2-(Octylamino)cyclohepta-2,4,6-trienone (2f)

The pure product was obtained as a yellowish solid (834 mg, 98%). 1H NMR (400 MHz, CDCl3) δ 7.09 (s, 1H), 6.97–6.84 (m, 3H), 6.37–6.29 (m, 1H), 6.23–6.18 (m, 1H), 2.98–2.91 (m, 2H), 1.43–1.36 (m, 2H), 1.11–1.00 (m, 10H), 0.63–0.59 (m, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 176.19, 155.37, 136.77, 136.04, 127.88, 121.52, 108.24, 42.61, 31.57, 29.07, 28.98, 28.21, 26.94, 22.44, 13.89. HRMS (ESI-TOF) m/z: [M + nH] calc. for C16H23NO 234.1852, found 234.1843.

Dodecyl-Aminotropone or 2-(Dodecylamino)cyclohepta-2,4,6-trienone (2g)

The pure product was obtained as a yellowish solid (448 mg, 85%). 1H NMR (400 MHz, CDCl3) δ 7.28–7.12 (m, 4H), 6.64 (t, J = 8 Hz, 1H), 6.51 (d, J = 12 Hz, 1H), 3.28 (d, J = 8 Hz, 2H), 1.76–1.69 (m, 2H), 1.42–1.26 (m, 20H), 0.87 (t, J = 8 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 176.56, 155.67, 137.20, 136.29, 128.26, 121.90, 108.58, 42.89, 31.90, 29.62, 29.61, 29.56, 29.50, 29.33, 29.29, 28.45, 27.14, 22.68, 14.11. HRMS (ESI-TOF) m/z: [M + nH] calc. for C19H31NO 290.2478, found 290.2493.

Octadecyl-Aminotropone or 2-(Octadecylamino)cyclohepta-2,4,6-trienone (2h)

The pure product was obtained as a yellowish solid (649 mg, 96%). 1H NMR (400 MHz, CDCl3) δ 7.28–7.20 (m, 3H), 7.14 (d, J = 12 Hz, 1H), 6.66 (t, J = 8 Hz, 1H), 6.53 (d, J = 12 Hz, 1H), 3.32–3.27 (m, 2H), 1.76–1.68 (m, 3H), 1.45–1.42 (m, 2H), 1.40–1.28 (m, 29H), 0.88 (t, J = 6.7 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 176.61, 155.70, 137.25, 136.30, 128.31, 121.93, 108.59, 42.92, 31.93, 29.71, 29.67, 29.58, 29.51, 29.37, 29.30, 28.47, 27.16, 22.70, 14.12. HRMS (ESI-TOF) m/z: [M + nH] calc. for C25H43NO 374.3417, found 374.3436.

General Procedure for the Synthesis of Alkyl-Amintroponyl Difluoroboron (Alkyl-ATB) Complex Boron-Aminotropone (3)

Aminotropone (2a) (1 equiv) was dissolved in anhydrous dichloromethane, and Et3N (15 equiv) was added. For further reaction, BF3·OEt2 (15 equiv) was added to the resultant reaction mixture and stirred at room temperature. TLC monitored the completion of the reaction, and water was added to quench unreacted BF3·OEt2 that is extracted with dichloromethane (thrice). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified through silica gel column chromatography using 100% DCM as the mobile phase. Boron complex 3a was obtained in 90% yield. The remaining boron complexes were synthesized by following this general procedure.

NMR Data of Boron-Aminotropones Isobutyl-ATB or 3-(Sec-butyl)-2,2-difluoro-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3a)

The pure product was obtained as a reddish-brown solid (290 mg, 76%).1H NMR (400 MHz, CDCl3) δ 7.63–7.58 (m, 1H), 7.46 (t, J = 12 Hz, 1H), 7.25 (t, J = 8 Hz, 1H), 7.15 (d, J = 12 Hz, 1H), 7.06 (t, J = 8 Hz, 1H), 3.83 (s, 1H), 1.98–1.91 (m, 1H), 1.81–1.74 (m, 1H), 1.44 (d, J = 4 Hz, 3H), 0.98 (t, J = 8 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 167.33, 159.59, 141.10, 139.42, 127.23, 118.54, 117.92, 53.08, 28.02, 18.16, 11.33. 11B NMR (128 MHz, CDCl3) δ 6.08 (t, J = 20.0 Hz). 19F NMR (377 MHz, CDCl3) δ −130.88 (s), −130.93 (s), −130.99 (s), −131.04 (s), −131.11 (s), −131.16 (s), −131.22 (s), −131.27 (s), −132.59 (dd, J = 39.1, 19.1 Hz), −132.82 (dd, J = 39.1, 19.2 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C11H14BF2NO 248.1031, found 248.1036.

Cyclohexyl-ATB or 3-Cyclohexyl-2,2-difluoro-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3b)

The pure product was obtained as a reddish-brown solid (180 mg, 77%). 1H NMR (400 MHz, CDCl3) δ 7.65–7.60 (m, 1H), 7.46 (t, J = 10 Hz, 1H), 7.21 (d, J = 12 Hz, 1H), 7.14 (d, J = 12 Hz, 1H), 7.06 (t, J = 10 Hz, 1H), 3.64 (s, 1H), 2.05–2.02 (m, 2H), 1.91–1.71 (m, 4H), 1.41–1.26 (m, 4H). 13C{H} NMR (101 MHz, CDCl3) δ 141.20, 139.38, 127.33, 118.51, 117.94, 55.17, 30.58, 25.56, 25.26. 11B NMR (128 MHz, CDCl3) δ 6.05 (t, J = 19.2 Hz). 19F NMR (377 MHz, CDCl3) δ −131.55 (dd, J = 41.4 Hz, 19.2 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C13H16BF2NO 274.1188, found 274.1193.

Propargyl-ATB or 2,2-Difluoro-3-(prop-2-yn-1-yl)-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3c)

The pure product was obtained as a yellowish solid (800 mg, 72%). 1H NMR (400 MHz, CDCl3) δ 7.78 (t, J = 10 Hz, 1H), 7.63 (t, J = 12 Hz, 1H), 7.38 (dd, J = 25.0, 10.6 Hz, 2H), 7.27–7.21 (m, 1H), 4.32 (s, 2H), 2.31 (s, 1H). 13C{H} NMR (101 MHz, CDCl3) δ 168.25, 159.89, 142.05, 140.54, 128.45, 120.59, 118.42, 73.00, 32.14, 29.70. 11B NMR (128 MHz, CDCl3) δ 5.76 (t, J = 17.8 Hz). 19F NMR (377 MHz, CDCl3) δ −139.06 (dd, J = 35.8, 17.6 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C10H8BF2NO 230.0561, found 230.0563.

Butyl-ATB 3-butyl-2,2-difluoro-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3d)

The pure product was obtained as a reddish-brown solid (250 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 7.65–7.59 (m, 1H), 7.47 (t, J = 10 Hz, 1H), 7.27–7.23 (m, 1H), 7.11–7.04 (m, 2H), 3.56 (t, J = 6 Hz, 2H), 1.79–1.75 (m, 2H), 1.47–1.41 (m, 2H), 0.98 (t, J = 8 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 167.43, 159.89, 141.30, 139.48, 127.27, 118.65, 117.49, 43.44, 29.86, 20.57, 13.76. 11B NMR (128 MHz, CDCl3) δ 5.99 (t, J = 18.8 Hz). 19F NMR (377 MHz, CDCl3) δ −138.28 (dd, J = 37.5, 18.2 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C11H14BF2NO 248.1031, found 248.1044.

Hexyl-ATB or 2,2-Difluoro-3-hexyl-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3e)

The pure product was obtained as a reddish-brown solid (310 mg, 83%). 1H NMR (400 MHz, CDCl3) δ 7.66–7.61(m, 1H), 7.47 (t, J = 10 Hz, 1H), 7.23 (d, J = 12 Hz, 1H), 7.12–7.05 (m, 2H), 3.54 (t, J = 6 Hz, 2H), 1.81–1.73 (m, 2H), 1.42–1.31(m, 6H), 0.89 (t, J = 8 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 167.31, 159.85, 141.40, 139.46, 127.40, 118.65, 117.56, 47.08, 43.66, 31.45, 27.79, 26.98, 22.53, 13.99, 8.71. 11B NMR (128 MHz, CDCl3) δ 5.97 (t, J = 18.7 Hz). 19F NMR (377 MHz, CDCl3) δ −138.17 (dd, J = 37.6, 18.0 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C13H18BF2NO 276.1344, found 276.1354.

Octyl-ATB 2,2-Difluoro-3-octyl-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3f)

The pure product was obtained as a reddish-brown solid (216 mg, 81%). 1H NMR (400 MHz, CDCl3) δ 7.65–7.60 (m, 1H), 7.47 (t, J = 10.2 Hz, 1H), 7.24 (t, J = 11.0 Hz, 1H), 7.11–7.05 (m, 2H), 3.54 (t, J = 7.6 Hz, 2H), 1.81–1.74 (m, 2H), 1.40–1.27 (m, 10H), 0.88 (t, J = 8 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 167.35, 159.86, 141.35, 139.45, 127.34, 118.62, 117.55, 43.68, 31.77, 29.25, 29.18, 27.84, 27.33, 22.61, 14.08. 11B NMR (128 MHz, CDCl3) δ 5.98 (t, J = 18.5 Hz). 19F NMR (377 MHz, CDCl3) δ −138.20 (dd, J = 36.7, 15.2 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C15H22BF2NO 304.1657, found 304.1659.

Dodecyl-ATB or 3-Dodeyl-2,2-difluoro-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3g)

The pure product was obtained as a reddish-brown solid (326 mg, 87%). 1H NMR (400 MHz, CDCl3) δ 7.62 (t, J = 10 Hz 1H), 7.48 (t, J = 10 Hz, 1H), 7.28–7.25 (m, 1H), 7.12–7.06 (m, 2H), 3.56 (t, J = 8 Hz, 2H), 1.81–1.77 (m, 2H), 1.42–1.28 (m, 18H), 0.90 (t, J = 6 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 167.49, 159.86, 141.27, 139.46, 127.24, 118.60, 117.48, 43.70, 31.91, 29.62, 29.57, 29.52, 29.33, 29.30, 27.84, 27.35, 22.68, 14.11. 11B NMR (128 MHz, CDCl3) δ 5.98 (t, J = 18.3 Hz). 19F NMR (377 MHz, CDCl3) δ −138.27 (dd, J = 37.1, 17.0 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C19H30BF2NO 360.2284, found 360.2296.

Octadecyl-ATB or 2,2-Difluoro-3-octadecyl-2,3-dihydrocyclohepta[d][1,3,2]oxazaborol-1-ium-2-uide (3h)

The pure product was obtained as a white solid (270 mg, 80%).1H NMR (400 MHz, CDCl3) δ 7.61 (t, J = 10 Hz, 1H), 7.47 (t, J = 10 Hz, 1H), 7.26–7.23 (m, 1H), 7.09–7.04 (m, 2H), 3.54 (t, J = 8 Hz, 2H), 1.81–1.74 (m, 2H), 1.61 (s, 1H), 1.40–1.26(m, 30H), 0.88 (t, J = 6.4 Hz, 3H). 13C{H} NMR (101 MHz, CDCl3) δ 167.46, 159.84, 141.24, 139.47, 127.21, 118.62, 117.48, 43.71, 31.93, 29.71, 29.67, 29.64, 29.58, 29.54, 29.37, 29.31, 27.84, 27.36, 22.70, 14.13. 11B NMR (128 MHz, CDCl3) δ 5.98 (t, J = 18.1 Hz). 19F NMR (377 MHz, CDCl3) δ −138.29 (dd, J = 36.8, 16.6 Hz). HRMS (ESI-TOF) m/z: [M + Na] calc. for C25H42BF2NO 444.3224, found 444.3232.