Accepted Manuscript Synthesis and properties of amphiphilic BODIPY derivatives bearing hydroxyl groups Matthew Vincent, Eva Beabout, Richard Bennett, Priya Hewavitharanage PII: S0040-4039(13)00206-2 DOI: http://dx.doi.org/10.1016/j.tetlet.2013.01.128 Reference: TETL 42504 To appear in: Tetrahedron Letters Received Date: 13 September 2012 Revised Date: 28 January 2013 Accepted Date: 29 January 2013 Please cite this article as: Vincent, M., Beabout, E., Bennett, R., Hewavitharanage, P., Synthesis and properties of amphiphilic BODIPY derivatives bearing hydroxyl groups, Tetrahedron Letters (2013), doi: http://dx.doi.org/ 10.1016/j.tetlet.2013.01.128 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Transcript
Accepted Manuscript
Synthesis and properties of amphiphilic BODIPY derivatives bearing hydroxyl
groups
Matthew Vincent, Eva Beabout, Richard Bennett, Priya Hewavitharanage
Synthesis and properties of amphiphilic BODIPY derivatives bearing hydroxyl groups
Matthew Vincenta Eva Beabouta; Richard Bennettb; Priya Hewavitharanage*a,
Leave this area blank for abstract info.
Tetrahedron Letters
1
Tetrahedron Letters j ourna l homepage: www.e lsev ier .com
Synthesis and properties of amphiphilic BODIPY derivatives bearing hydroxyl groups
Matthew Vincenta, Eva Beabouta, Richard Bennettb, Priya Hewavitharanage*,a aDepartment of Chemistry, University of Southern Indiana, Evansville, IN 47712, USA bDepartment of Biology, University of Southern Indiana, Evansville, IN 47712, USA Fax: +1-812-465-1052; Tel: +1-812-228-5009 E-mail: [email protected]
Fluorescence labeling of biomolecules is used extensively in various fields of biology and medicine to distinguish and identify the specific subcellular localization of molecules to various compartments in the cell and in vivo monitoring of biological processes.1 Fluorescent dyes can also be used to identify subcellular compartments such as the ER (Endoplasmic Reticulum), Golgi complex, and mitochondria.1 BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) derivatives are widely used in biological applications due to their superior properties, such as high fluorescence quantum yields, high molar extinction coefficients, narrow absorption and emission spectra, and high photochemical stability.2,3, 4 Unlike most other fluorescent dyes, BODIPY is electrically neutral, which prevents it from interacting with charged particles of the cell. The smaller size of BODIPY derivatives facilitates conjugation of them to biomolecules such as antibodies and peptides without altering their physicochemical properties.5 Insensitivity to pH and solvent polarity are additional advantages for biological applications.6,7 However, the photophysical properties of BODIPY derivatives can easily be tuned by changing substituents of the BODIPY core.3,8,5,9
Various types of BODIPY based fluorescence probes are commercially available for biological applications at high cost.1,2
BODIPY derivatives with basic amino groups have been shown to accumulate in acidic organelles.10 Those with fatty acids1, phospholipids1, or sphingolipids3,11 attached to the BODIPY core have been shown to accumulate in cell membranes. Thus, we reasoned that a BODIPY molecule with a neutral polar group would also be able to incorporate into cell membranes without further functionalization. To this end, a hydroxyl group was
selected as the initial neutral polar group while a nonyl group was introduced to the meso position to mimic the long hydrophobic tail of the cell membrane. Due to the amphiphilic nature, we expected that these molecules would accumulate in membrane structures such as ER in the configuration shown in Figure 1. In addition, the hydroxyl functional group can be used in further functionalization and bio-conjugation of BODIPY derivatives. In fact, BODIPY compounds with a hydroxyl group at the meso position have been synthesized before by hydrolyzing the corresponding ester and have been used to synthesize a BODIPY-tocopherol adduct which functions as an on/off fluorescent antioxidant indicator.12 To the best of our knowledge, amphiphilic BODIPY derivatives with free hydroxyl groups have not been used in cellular studies. Here we report the synthesis of new amphiphilic BODIPY derivatives with hydroxyl groups using Sonogashira coupling or organolithium chemistry (Scheme 1), and investigation of the localization of them in human osteosarcoma cells.
Figure 1. Schematic diagram of an amphiphilic BODIPY in a phospholipid bilayer.
ARTICLE INFO ABSTRACT
Article history: Received Received in revised form Accepted Available online
Functionalizable BODIPY compounds with a hydrophobic alkyl chain at the meso position and hydroxyl groups attached through alkynyl links at the 2, 6, and 4, 4' positions were conveniently synthesized using Sonogashira coupling or organolithium chemistry. The compounds are readily taken up and accumulated in human osteosarcoma cells. Some compounds showed excimer formation inside cells as well as in solution.
2009 Elsevier Ltd. All rights reserved.
Tetrahedron Letters 2 BODIPY 2 was synthesized in good yield as a one pot
reaction by condensation of 2,4-dimethyl-3-ethylpyrrole with decanoyl chloride and subsequent addition of boron trifluoride etherate (Scheme 1).13 The alcohol, 3-butyn-1-ol was protected using tert-butyldimetylsilyl chloride (TBDMSCl) in the presence of imidazole in DMF.14 Lithium derivative of 1 was prepared with n-BuLi at -78°C and reaction with 2 in THF at room temperature yielded 3 (63%) as a sticky orange solid. No monosubstituted compound was observed. Deprotection using TBAF in methanol yielded the β-γ unsaturated alcohol 4 as a bright orange solid in 90% yield. The hydroxyl group of 4 in 1H NMR appeared at 1.86 ppm as a broad peak (Fig. S6). Addition of D2O caused the complete disappearance of this peak. Encouraged by interesting properties of 4, we decided to synthesize BODIPY derivatives with hydroxyl groups (attached through alkynyl links) at the 2, 6 positions. BODIPY 5 was synthesized using a procedure similar to the synthesis of 2. Iodination of 5 using I2/HIO3 yielded the diiodo BODIPY 6 in 76% yield. Sonogashira coupling does not require protection of
the hydroxyl group hence coupling of 3-butyn-1-ol with 6 was attempted at room temperature using Pd(PPh3)4 as the catalyst. Complete consumption of 6 was observed after 36h to yield 8. However, it was extremely difficult to remove triphenylphosphine oxide, which is a decomposition product of the catalyst Pd(PPh3)4. Removal of triphenylphosphine oxide required multiple steps of column chromatography. Therefore, we decided to synthesize TBDMS protected alcohol 7. Because 7 is significantly less polar compared to 8, it was easily separated from Ph3PO and isolated as a dark red solid (96%). Unlike in 3, TBAF failed to deprotect 7 to yield 8. Addition of methanolic solution of TBAF caused immediate decomposition of 7. It is known that some BODIPY derivatives are sensitive to fluoride ions.15,16,17 Meng et al suggested that the decomposition is due to nucleophilic displacement which breaks a B-N bond to form a new B-F bond.16
Scheme 1. Reagents and conditions: (i) tert-butyldimethysilyl chloride, imidazole, DMF; (ii) 1, nBuLi, THF, -78 °C to rt; (iii) TBAF; THF (iv) HIO3, I2, Ethanol
Table 1. Photophysical properties of BODIPY compounds in CH2Cl2 at rt. compound λ absorption (nm) ε(M-1cm-1) λ emission (nm) Quantum yield (%)a Stokes Shift/nm
Tetrahedron Letters
3
2 520 77900 524 84 4
3 516 82800 527 92 11
4 516 82300 525 83 9
5
6
7
8
9
10
11
12
13
14
15
498 87000
528 93200
550 64300
548 59700
548 60000
541 63100
539 63600
552 76600
547 90600
550 74000
549 75500
504
542
564
562
562
560
554
566
561
565
566
80
2
88
78
81
10
5
85
83
76
80
6
14
14
14
14
19
15
14
14
15
17
aRhodamine B was used as reference.
Utilization of 0.3 mole equivalent of acetyl chloride in dry methanol successfully deprotected 7 to achieve the β-γ unsaturated alcohol 8 in 73% yield.18 No decomposition of 7 was observed. The hydroxyl group of 8 in 1H NMR appeared at 1.77 ppm as a triplet while the two hydrogens of the adjacent carbon was a quartet (Fig S14). Addition of D2O caused complete disappearance of the triplet at 1.77 ppm while the two hydrogens of the carbon adjacent to the hydroxyl group became a triplet. Partial deprotection of 7 was achieved with 0.15 mole equivalent of acetyl chloride. The reaction was quenched after 30 min. to avoid the formation of 8. Compound 9 was isolated as a dark red solid (8.8%) and the unreacted starting material 7 was recovered.
Monosubstituted compound 10 was synthesized using a similar protocol that was used in the synthesis of 7 except a 1:1 molar ratio of BODIPY: protected alcohol was used with a shorter reaction time (12h, 44%). Small amount of disubstituted product 7 was also formed (~ 10%). Deprotection of 10, similar to 7, yielded 11 as a dark purple solid with a 90% yield. In compounds 1-7, methyl groups at 3, 5, and 1, 7 positions appear as two separate singlets. However, the presence of iodine at the 6 position in 10 and 11 caused the methyl groups to produce four separate singlets (appeared as two doublets, Fig S17, S19). The compound 12 was synthesized by direct coupling of hex-5-yn-1-ol with 6. Decreased polarity of 12 compared to 8 due to the extended alkyl chain of the alcohol facilitated purification of 12 from triphenylphosphine oxide impurity.
In order to demonstrate the synthetic utilities of the hydroxyl group in further functionalization of the BODIPY chromophore, two transformations were performed as outline in Scheme 2. The two hydroxyl groups of 8 were easily esterified with butanoic acid using EDC/DMAP to yield the ester 13 (38%).12 Esterification of the hydroxyl group can be used in bioconjugation of BODIPY and synthesis of BODIPY based energy transfer cassettes.12,19 The hydroxyl groups of 12 were oxidized using Dess-Martin periodinane to yield 14 and 15. The β-γ unsaturated aldehyde yielded by oxidation of 8 could not be isolated probably due to decomposition during purification. Aldehyde is a versatile functional group that can be used in
bioconjugation20,21,22,23 or further functionalization of the BODIPY chromophore. Hydroxyl group-containing compounds 4, 8, 9, 11 and 12 are highly stable at room temperature and quite
soluble in polar solvents such as DMSO but less soluble in nonpolar solvents such as hexane.
The UV-vis absorption experiments of BODIPY compounds were carried out in CH2Cl2 (1.0 × 10-5 mol/L). S0-S1 (π-π*) transition of the BODIPY derivatives 2, 3 and 4 appeared ~ 524 nm (Fig 2, Table 1). The weak band around 370 nm of BODIPY derivatives (Fig 2) is attributed to the S0-S2 (π-π*) transition.24 No significant change in the absorption maxima were observed in 3 and 4 compared to the absorption of 2 (Table 1). This is because ethynyl groups on boron are not in conjugation with the BODIPY core due to unavailability of the empty p orbital in tetrahedral boron. However, 2,6 ethynyl substituted compound 7 shows a 52 nm red shift of absorption and a 60 nm red shift of emission compared to the unsubstituted compound 5 due to extended conjugation (Fig. 2, Table 1).
Figure 2. A: Normalized absorption; B: normalized emission spectra of BODIPY derivatives in CH2Cl2.
Tetrahedron 4
Figure 3. Fluorescence images of Saos-2 cells incubated with A: 4 (0.5 µmol), B: Nuclear stain DAPI, C: Localization of Red ER Tracker (ER specific dye). D: Overlay of A, B and C.
No significant change in absorption maxima was observed in deprotected alcohol 4, 8, 9 and 11 in comparison to protected alcohols 3, 7 and 10. However, emission maxima were slightly blue shifted upon deprotection.
Quantum yields of BODIPY compounds were measured in CH2Cl2 using Rhodamine B (ф = 65% in ethanol) as the reference. All the BODIPY compounds fluoresce in high quantum yields except 6, 10 and 11. Presence of iodine in these compounds quenches fluorescence through heavy atom effect.25,26 Interestingly, quantum yields of protected alcohols 3,7 and 10 were 5-10% higher than those with free hydroxyl groups.
Figure 4. Fluorescence image of Saos-2 cells incubate with 3 (5 µmol) A: under 470/40 excitation and 525/50 emission filters, B: under 565/30 excitation and 620/60 emission filters, C: Overlay of A and B.
The ability of DMSO solutions of BODIPY 3, 4, 7, 8, 9, 10, 11, 12 and 13 to stain live cells were tested using human osteosarcoma (Saos-2). All compounds were taken up within 1 hour of exposure. Cells were analyzed on a Zeiss AxioImager A.2 fluorescent microscope (Carl Zeiss) and images were captured with an AxioCam MRm digital camera (Carl Zeiss). BODIPY with free hydroxyl groups showed more or less uniform distribution in the cytoplasm (Fig 3A) while BODIPY with TDBMS exhibited punctate distribution (Fig 5). Cells fluoresced in bright green when analyzed using a 470/40 excitation filter and a 525/50 emission filter (Fig. 3). Fluorescence of 10 and 11 was weak thereby confirming the fluorescence quenching due to iodine. Cells also fluoresced in the red region when incubated with 8, 9, 11, 12 and 13 and analyzed using a 565/30 excitation and a 620/60 emission filter set. Because the emission spectra of these compounds extend beyond 600 nm (Fig. 2), red emission was expected. Interestingly, at high concentrations (> 0.5µmol),
3 and 4 which emit only in the green region also showed dual color fluorescence (green under 470/40 excitation and a 525/50 emission filter, red under 565/30 excitation and 620/60 emission filters). Red emission was more pronounced in cells stained with TBDMS protected BODIPY 3 (Fig. 4B), and 7 due to punctate distribution. It is known that at high concentrations some BODIPY derivatives emit at a longer wavelength due to excimer formation, which results in efficient energy transfer between nearby BODIPY molecules.1,12,27,28,29 To confirm the excimer emission, the fluorescence spectra of 3 and 4 were measured at high concentrations in 0.5mm glass capillary tubes (to avoid inner filter effect). As the concentration of 4 increased, a new peak at 571 nm appeared (Fig. 5) which is a 46 nm red shift of the emission compared to a dilute solution. Further increase in concentration caused a concomitant decrease of emission at 525 nm and an increase in the 571 nm peak (excimer emission). Excimer of the TBDMS protected BODIPY 3 emits at 564 nm (λmax). BODIPY excimer emission has been used in detection of lipid peroxidation on erythrocytes30 and has potential applications in study of protein structure and folding and monitoring lipid traffic between cell membrane processes by fluorescence imaging microscopy.31
Subcellular distribution of BODIPY dyes 3, and 4, was investigated by incubating cells with BODIPY for 1h followed by a wash and incubation with red ER Tracker® (Endoplasmic reticulum specific dye, Invitrogen, Fig. 3). Concentrations of 3 and 4 were kept low (0.5 µmol) to avoid any excimer formation which might complicate interpretation of results. Overlay image (Fig. 3D) confirmed the main localization site for 4 as the ER while the TBDMS protected 3 localized in the ER and also exhibited punctate distribution (Fig 4). Subcellular distribution studies of 2, 6 ethynyl substituted dyes 7 and 10 showed distribution patterns similar to 3 while 8, 9 and 11 were similar to 4. However, because the emission spectrum of red ER tracker slightly overlaps with the emission spectra of 2,6 ethynyl substituted BODIPY derivatives, the experiment should be repeated with a different ER tracker. Photophysical properties and cellular distribution of 3 is similar to commercially available BODIPY-labeled sphingolipid and cholesterol analogs. Such lipophilic fluorescence probes are known to accumulate in lipid droplets32 and apolar region of the lipid bilayer.11,31 Comparison of the BODIPY probes with free hydroxyl groups to other BODIPY probes revealed that their photophysical properties and cellular uptake properties are similar to those of commercially available BODIPY-fatty acid series.33 However, BODIPY-fatty acids are known to be metabolized by living cells indicated by accumulation of them in cytoplasmic droplets and confirmed by analysis of lipid extracts of cells.31 Free hydroxyl group containing BODIPY (4, 8, 9, 11) showed no indication of such accumulation.
Figure 5. Normalized emission of 4 in CH2Cl2 A: 8 x 10-2 mol/L, B: 1 x 10-5 mol/L, C: 6 x 10-3 mol/L
Tetrahedron Letters
5
In conclusion, we have synthesized functionalizable and photochemically tunable BODIPY dyes with terminal hydroxyl groups which are permeable to live cell membrane. Detailed studies on excimer formation, and cellular localization of these dyes are underway.
Acknowledgments
Financial support by the Faculty Research and Creative Work Award and the Science and Engineering Research Grant Award of the University of Southern Indiana is gratefully acknowledged. Authors greatly acknowledge Dr. Alexandre Hafale of Dr. Felix N. Castellano research group at the Center for Photochemical Sciences, Bowling Green State University for photophysical characterization of compounds 2 and 5. Authors also acknowledge Alexander Clayton and Brooke Spitler for assistance given in some synthetic steps and Professor Jeff Seyler for valuable discussion.
Supplementary data associated with this article can be found, in
the online version, at…….
References and notes
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