Some observations relating to the stability of the BODIPY fluorophore under acidic and basic conditions

Article abstract of DOI:10.1016/j.dyepig.2011.03.027

4,4-Difluoro-4-bora-1,3,5,7-tetramethyl-2,6-diethyl-8-methyl-3a, 4a-diaza-s-indacene (BODIPY) fluorophore was transformed into its corresponding 4,4-dimethyl, 4,4-dimethoxy and 4,4-diphenyl analogues. The stabilities of these BODIPY fluorophores in acidic

Full text of DOI:10.1016/j.dyepig.2011.03.027

Dyes and Pigments 91 (2011) 264e267
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Short communication
Some observations relating to the stability of the BODIPY fluorophore
under acidic and basic conditions
,
Lijing Yang a, Razvan Simionescu a, Alan Lough b, Hongbin Yan a
*
a Department of Chemistry, Brock University, 500 Glenridge Ave., St. Catharines, ON L2S 3A1, Canada
b Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
4,4-Difluoro-4-bora-1,3,5,7-tetramethyl-2,6-diethyl-8-methyl-3a,4a-diaza-s-indacene (BODIPY) fluo-
rophore was transformed into its corresponding 4,4-dimethyl, 4,4-dimethoxy and 4,4-diphenyl
analogues. The stabilities of these BODIPY fluorophores in acidic (di- and trichloroacetic acid) and basic
conditions (aqueous ammonium hydroxide) were investigated using 11B NMR spectroscopy.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 9 March 2011
Received in revised form
25 March 2011
Accepted 26 March 2011
Available online 2 April 2011
Keywords:
BODIPY
11B NMR
Stability
Fluorophore
Acids and bases
Crystal structure
1. Introduction
attempts to incorporate BODIPY analogues into oligonucleotides by
the phosphoramidite chemistry-based solid-phase synthesis,
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY 1, Fig. 1)
fluorophores were first synthesized in 1968 by Treibs and Kreuzer
[1], and have attracted much attention in recent years in various
applications. Many BODIPY fluorophores possess high molar
extinction coefficients and are highly fluorescent [2e4]. In addition,
many BODIPY fluorophores are relatively insensitive to changes in
pH and polarity, and are more photochemically stable than many
other commercially available fluorophores.
While some reactivities of BODIPY have been documented in the
literature [2,3], detailed studies on the stability of BODIPY and
analogues under acidic and basic conditions, however, have been
very rare [5]. One recent publication relates to the replacement of
the fluorine atoms in BODIPY by hydroxyl in the presence of
a strong Lewis acid such as aluminium chloride [6]. In another
report, it was shown that BODIPY analogues can be converted to
their corresponding dipyrrins when treated with potassium but-
oxide under microwave conditions [7]. It is noted that BODIPY has
been given credit in numerous articles to be chemically stable. This
contrasts the observations made in our laboratory during our
particularly during acid and base treatments [8]. The work pre-
sented herein aims at comparing the stability of a series of
4,4-disubstituted BODIPY analogues under acidic and basic condi-
tions that are commonly used in solid-phase synthesis of oligo-
nucleotides. In this respect, building blocks are exposed to repeated
acid treatment, e.g. di- or trichloroacetic acid. The BODIPY
analogues are also tested for their stability under the deprotection
conditions that are used in solid-phase oligonucleotide synthesis,
e.g. treatment with aqueous ammonium hydroxide at 55 ꢀC for 15 h.
2. Materials and methods
2.1. Instrumentation
1H NMR spectra were measured at 300 and 600 MHz with
Bruker Avance spectrometers; spectra were calibrated to residual
undeuterated NMR solvents; J values are given in Hz. 13C and 19F
NMR spectra were measured at 75.5 and 282.4 MHz, respectively,
and 11B NMR spectra were measured at 96.3 and 192.6 MHz with
the same spectrometers. Chemical shifts are given in ppm. Low and
high resolution mass spectra were obtained with Kratos Concept 1S
high resolution mass spectrometer using electron impact or fast
* Corresponding author. Tel.: þ1 905 688 5550; fax: þ1 905 682 9020.
E-mail address: tyan@brocku.ca (H. Yan).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.03.027

L. Yang et al. / Dyes and Pigments 91 (2011) 264e267
265
hydroxide solution (600
4, 6, 8, 11, 13, 24 h, two vials were removed from heating, cooled,
and combined. After solvents were removed under reduced pres-
m
l, 28%), sealed and heated at 55 ꢀC. After 2,
8 (meso)
1
3
7
6
2
sure, the residue was dissolved in dichloromethane (600 ml) and
N
N
B
transferred to an NMR tube. 11B NMR spectra were recorded with
a sealed capillary tube insert containing a solution of sodium tet-
raphenylborate in D2O.
5
4
F
1
F
2.5. X-ray crystallography
Fig. 1. BODIPY core structure.
Single crystals of compound 5 were obtained by slow evaporation
of solvents from hexaneedichloromethane. CCDC 812198 contains
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.
atom bombardment sources interfaced with DART 32 bit acquisi-
tion system through a Sun Sparcstation 10 and Mach 3 software.
2.2. Synthesis of 4,4-difluoro, 4,4-dimethyl, 4,4,-dimethoxy,
4,4,-diphenyl, and 4,4-bis(dichloroacetyl) BODIPY analogues
Datawere collected on a Bruker-Nonius Kappa-CCDdiffractometer
using monochromated Mo-K
a
u
radiation and were measured using
scans with offsets, to fill the Ewald
Detailed synthetic procedures, NMR and mass spectroscopic
data of these compounds are provided in the Supplementary
materials.
a combination of scans and
4
k
sphere. The data were processed using the Denzo-SMN package [9].
Absorption corrections were carried out using SORTAV [10]. The
structure was solved and refined using SHELXTL V6.1 [11] for full-
matrix least-squares refinement that was based on F2. All H atoms
were included in calculated positions and allowed to refine in
riding-motion approximationwith Uwisow tied tothe carrier atom.
2.3. Procedures for the stability of BODIPY analogues in the
presence of dichloroacetic acid
2.3.1. Example: stability of 4,4-diphenyl-1,3,5,7,8-pentamethyl-2,
6-diethyl-4-bora-3a,4a-diaza-s-indacene 4d in dichloroacetic
acidedichloromethane
3. Results and discussion
4,4-Diphenyl-1,3,5,7,8-pentamethyl-2,6-diethyl-4-bora-3a,4a-
diaza-s-indacene 4d (10 mg, 0.023 mmol) was dissolved in dry
dichloromethane (0.4 ml) in an NMR tube, followed by addition of
As a model for the stability experiments, 4,4-difluoro-4-bora-
1,3,5,7-tetramethyl-2,6-diethyl-8-methyl-3a,4a-diaza-s-indacene
4a was chosen since the dipyrromethene ring is fully substituted to
avoid potential reactions at these sites. This compound was
readily prepared using literature procedures by the treatment of
2,4-dimethyl-3-ethylpyrrole 2 with acetyl chloride, followed by
complexation with boron trifluoride diethyl etherate in the pres-
ence of triethylamine [7,12]. In addition, three other analogues, i.e.
4,4-dimethyl 4b [13], 4,4-dimethoxy 4c [14] and 4,4-diphenyl 4d
[15] BODIPY were prepared according to methods documented in
the literature (Scheme 1).
The stability of the BODIPY fluorophores 4aed in acidic solu-
tions at room temperature was followed by recording 11B NMR
spectra at appropriate intervals. A sealed capillary insert of a solu-
tion of sodium tetraphenylborate in D2O was used as an internal
standard. In the case of an unstable substrate, the disappearance of
substrate was monitored. In order to eliminate the broad 11B
resonance signal from the NMR tube, which overlaps with both
substrate and internal standard signals, a linear prediction method
[16] was used to process the 11B NMR data. As shown in Fig. 2, this
linear prediction method allows for the suppression of background
signals from the NMR tube.
dichloroacetic acid (95
ml, 1.15 mmol, 50 mol equiv). A sealed
capillary tube containing a solution of sodium tetraphenylborate in
D2O was inserted. 11B NMR spectra were acquired at appropriate
interval.
When processing the 11B spectra, the following processing
parameters were used:
Lb ¼ 10 Hz
ME_mod ¼ LPbr
NCOER ¼ 32
LPBIN ¼ 1024
Tdoff ¼ 32
FCOR ¼ 1
PKNL ¼ TRUE
FT_mod ¼ FSC
2.4. The stability of BODIPY analogues in base condition
2.4.1. Example: 1,3,5,7,8-pentamethyl-2,6-diethyl-4,
4-dimethoxy-4-bora-3a,4a-diaza-s-indacene 4c
It was found that 4,4-diphenyl BODIPY 4d is stable in a solution of
dichloroacetic acid (50 mol equiv) in dichloromethane for 3 days (No
new signals appeared after 3 days when the experiment was
terminated). 4,4-Difluorine 4a is less stable under the same condi-
tions, with 8% decomposition after 24 h. Treatment of 4,4-dimethyl
1,3,5,7,8-Pentamethyl-2,6-diethyl-4,4-dimethoxy-4-bora-3a,4a-
diaza-s-indacene 4c (42 mg, 0.123 mmol) was dissolved in THF
(4 ml). This solution was divided equally into 16 reacti-vials (i.e.
250
m
l each). To each of the vial was added aqueous ammonium
iii
4b; Y = Me
O
i, ii
iv
4c; Y = OMe
4d; Y = Ph
N
Y
N
Y
Cl
N
B
3
v
H
2
4a; Y = F
Scheme 1. Reagents and conditions: i). CH2Cl2, reflux, 3 h; ii). CH2Cl2, NEt3, BF3$Et2O, reflux; iii). MeMgBr, CH2Cl2, 0 ꢀC; iv). PhMgBr, CH2Cl2, 0 ꢀC; v). NaOMe, MeOH, CH2Cl2, reflux.

266
L. Yang et al. / Dyes and Pigments 91 (2011) 264e267
Fig. 2. 11B NMR spectra of 4b recorded in the presence of an internal stand (NaBPh4). a). Before applying the linear backward prediction algorithm, and b). after applying the linear
backward prediction algorithm.
BODIPY 4b under the same conditions led to complete decomposi-
tion over 6 h to give a single boron-containing product, which was
not identified (see Supplementary material for 11B NMR spectra).
When 4,4-dimethoxy BODIPY 4c was treated with 10 mol equiv of
dichloroacetic acid in dichloromethane, full conversion to a single
product was observed in 12 h (see Supplementary material for 11B
NMR spectra). When this reaction was carried out on a preparative
scale (Scheme 2), the product was identified as the corresponding
anhydride 5 (see Supplementary material for details). The identity of
this product 5 was confirmed by X-ray crystallography (Fig. 3).
When a stronger acid, i.e. trichloroacetic acid (50 mol equiv), was
used, both 4,4-difluorine 4a and 4,4-diphenyl 4d BODIPY became
unstable. Thus, 50% of 4,4-difluorine BODIPY 4a undergoes decom-
position after incubation in dichloromethane in the presence of
50 mol equiv of trichloroacetic acid for 3 h. Under the same conditions,
50% of 4,4-diphenyl BODIPY 4d undergoes decomposition in 4 h.
Stability of the BODIPY analogues under basic conditions was
assessed as follows. The substrate was dissolved in tetrahydrofuran
followed by addition of aqueous ammonia and methanol (Methanol
was not added in the case of 4,4-dimethoxy BODIPY 4c). THF and
methanol were added in order to generate a homogeneous solu-
tion. A series of this identical solution were prepared and heated to
55 ꢀC in sealed vials. At appropriate time intervals, samples were
removed and 11B NMR spectra were recorded. The results showed
that 4,4-dimethoxy BODIPY 4c is the least stable under this
condition. Within about 8 h, half of the starting materials degraded,
forming a product with a 11B shift of 1.2 ppm (see Supplementary
material for 11B NMR spectra). Under this condition 4,4-dimethyl
4b and 4,4-difluoro BODIPYs 4a are stable for up to 8 h,
4,4-diphenyl BODIPY 4d is stable for 7 days.
i
N
N
N
N
B
B
Cl2HCOCO
MeO
OCOCHCl2
OMe
4c
5
Scheme 2. Reagents and conditions: i). CH2Cl2, Cl2CHCOOH, r.t.
4. Conclusions
From the results obtained in this study, 4,4-diphenyl substituted
BODIPY 4d appears to be the most stable under both acidic and
basic conditions. It is also noted that substitution of the fluorine by
phenyl does not compromise the fluorescent intensity of the fluo-
rophore (
F
¼ 0.91 in dichloromethane for 4d v 0.83 for 4a) [3]. The
phenyl analogue 4d shows a larger Stokes shift (35 nm) than the
fluorine BODIPY 4a (21 nm) [3]. It is possible that the 4,4-diphenyl
substituted BODIPY analogues could be more suitable for oligonu-
cleotide labelling via the phosphoramidite chemistry-based solid-
phase synthesis. Work in this area is currently underway.
Acknowledgments
The authors thank Natural Sciences and Engineering Research
Council of Canada and Ontario Partnership for Innovation and
Commercialization for financial support of this work.
Fig. 3. The molecular structure of 5 with 30% probability displacement ellipsoids for
non-H atoms (prepared with PLATON) [17].

L. Yang et al. / Dyes and Pigments 91 (2011) 264e267
267
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oscillation mode. In: Carter CW, Sweet RM, editors. Methods in enzymology,
macromolecular crystallography, part A, vol. 276. London: Academic press;
1997. p. 307e26.
Supplementary material related to this article can be found
online at doi:10.1016/j.dyepig.2011.03.027.
[10] Blessing RH. An empirical correction for absorption anisotropy. Acta Crys-
tallogr A 1995;51:33e8.
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