Novel sulphur-rich BODIPY systems that enable stepwise fluorescent O-atom turn-on and H2O2 neuronal system probing

Article abstract of DOI:10.1039/c2cc33076h

Bis-arylsulfide BODIPY systems were prepared and studied for multiple O-atom sensing (at 522 nm); 2- and 3-atom loading was optimal (50-fold, "turn on"). Neuronal studies showed greater H₂O₂ sensitivity than 2′,7′-dichlorofluorescein diacetate. The novel 1,3,6-trimethyl BODIPY formed as a biproduct under Lindsey conditions.

Full text of DOI:10.1039/c2cc33076h

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COMMUNICATION
Novel sulphur-rich BODIPY systems that enable stepwise fluorescent
O-atom turn-on and H2O2 neuronal system probingwz
Atul P. Singh,a Kang Mun Lee,a Dhiraj P. Murale,a Taehong Jun,a Hyunjeong Liew,b
Yoo-Hun Suhb and David G. Churchill*a
Received 29th February 2012, Accepted 22nd May 2012
DOI: 10.1039/c2cc33076h
Bis-arylsulfide BODIPY systems were prepared and studied for
multiple O-atom sensing (at 522 nm); 2- and 3-atom loading was
optimal (50-fold, ‘‘turn on’’). Neuronal studies showed greater
H2O2 sensitivity than 20,70-dichlorofluorescein diacetate. The
novel 1,3,6-trimethyl BODIPY formed as a biproduct under
Lindsey conditions.
The rational synthesis of well-defined systems paves the way
for novel synthetic probes with improved and varying functions.1–5
Oxidative transformations in synthetic systems have involved
boronate, quinone, olefin, reduced moieties, and lanthanide-
based systems, and the photomechanisms of PET, FRET,
phosphorescence etc.1
BODIPY (4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene) species
are resilient, easily derivatizable and have been prepared for a
host of optical applications.2 They are commonly synthesized
by the straight-forward Lindsey method.4 Sulphide- or thienyl-
functionalized BODIPY platforms for fluorescent ROS detection
are few.5 Direct chemical sulphide oxidation accesses valence
states 4 and 6. The oxidation of Met in proteins might access,
e.g., neurodegenerative disease pathways. Dyes with multiple
inputting are also important targets.3 Herein, we report a
full study of novel and fully characterized 4-tolyl sulphide-
containing thienyl BODIPY species that allow for ROS
sensing.
Reactive oxygen species (ROS) by their name and nature are
extremely unstable. High levels can cause biological damage
implicated in cancer, neurodegeneration, diabetes, etc. Molec-
ular level effects such as extensive lipid peroxidation, protein
oxidation, and mitochondrial DNA mutation arise from
excess biological hydrogen peroxide (H2O2), superoxide
ꢀ
(O2 ), hydroxyl (ꢁOH), singlet oxygen, and lipid hydroperoxide
concentrations. Selective and sensitive detection and quantifi-
cation of such species by luminescent molecules may allow for
a better understanding of the intricacies of biological processes.
Synthetically, 3-thiophene carboxaldehyde was selected as a
starting aldehyde to enable sequential asymmetric sulphide
incorporation. First, N-bromo succinimide (NBS) effected
dibromination of the thiophene carboxaldehyde (DMF).
Arylsulphide intermediates 2,5-bis(4-tolylsulfide)- (1) and
5-bromo-2-(4-tolylsulfide)-3-thiophene carboxaldehyde (1a)
were prepared efficiently by an adapted CuI/BtH-catalyzed
coupling of 2,5-dibromothiophene carboxaldehyde with
p-toluenethiol (see ESIw).6 The BODIPY derivatives 2 and
2a were then conveniently prepared via the Lindsey method
(Scheme 1);3 authentication was made by 1H, 13C, and
11B NMR spectroscopy, and high-resolution ES-mass spectro-
a Molecular Logic Gate Laboratory, Department of Chemistry,
Korea Advanced Institute of Science and Technology (KAIST),
373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701,
Republic of Korea. E-mail: dchurchill@kaist.ac.kr
b Department of Pharmacology, College of Medicine, National
Creative Research Initiative Center for Alzheimer’s Dementia and
Neuroscience Research Institute, MRC, Seoul National University,
Seoul 110-799, Republic of Korea
w Electronic supplementary information (ESI) available: Experimental
details, in-line spectral data, reproductions of NMR spectra, HR MS
spectra, Crystallographic data, structural bond lengths and angles, etc.
CCDC 860722, 869054 and 869055. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c2cc33076h
z Crystallographic Data: Compound
7
(CCDC 860722):
C31H29BF2N2O3S3, F.W.: 622.55. Monoclinic, C2/c, Z = 8, a =
37.899(9) A, b = 7.743(2) A, c = 25.029(7) A, b = 122.015(11)1, V =
6227(3) A3, T = 296(2)K, No. reflections = 28247, No. independent
reflections = 7373 [R(int) = 0.1082], R1 = 0.0845, wR2 = 0.2158,
metry (ESIw). UV-PL data of 2 (FF = 0.01), and 2a (FF
=
0.06) were obtained (Fig. 1 and ESIw). Formal incorporation
of thiol groups greatly limits BODIPY fluorescence; com-
pound 2, because of its lower FF value (‘‘turned off’’), was
used further in potential ROS ‘‘turn on’’ assays.
m/mmꢀ1 = 0.285, Largest diff. peak and hole = 0.793 and 0.553 eꢀ/ꢀ3
.
Compound 8 (CCDC 869055): C31H29BF2N2O4S3, F.W. = 638.55,
monoclinic, C2/c, Z = 8, a = 38.241(9) A, b = 7.6428(18) A. c =
25.192(7) A, b = 121.40(2)1, V = 6285(3) A3, T = 296(2)K. No. of
reflections = 27 214, No. of independent reflections = 4422 [R(int) =
0.0713], R1 = 0.0508, wR2 = 0.1466, m/mmꢀ1 = 0.286, Largest diff.
peak and hole = 0.331 and ꢀ0.264 eꢀ/ꢀ3. Compound 9 (CCDC
869054): C12H13B1F2N2, F.W. = 234.05, Monoclinic, P21/c, Z = 4,
a = 6.350(3) A, b = 13.032(7) A. c = 14.253(7) A, b = 94.75(2)1, V =
1175.4(10) A3, T = 296(2)K. No of reflections = 16 333, No. of
independent reflections = 4133 [R(int) = 0.0857], R1 = 0.0992,
wR2 = 0.2003, m/mmꢀ1 = 0.100, Largest diff. peak and hole =
Compound 2 was assayed by ROS-type species to determine
the extent of sulphur oxidation (Fig. 2) as determined by
fluorescence changes (Fig. 3). m-CPBA (1.0 ꢂ 10ꢀ4 M) in
MeCN : H2O (70 : 30 by vol) with 2 (1.0 ꢂ 10ꢀ6 M) gave a
‘‘turn-on’’ response (2 h: 11-fold; 15 h: 50-fold) at 522 nm
(Fig. 1 and ESIw). Interestingly, 2 was unresponsive with
H2O2, NaOCl, KO2, ꢁOH, ꢁOtBu, and tBuOOH under analogous
conditions. Interestingly, this reactivity pattern is different
0.287 and ꢀ0.178 eꢀ/ꢀ3
.
c
7298 Chem. Commun., 2012, 48, 7298–7300
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from that reported for the related 2-SMe-substitution, selective
for NaOCl.5
Next, cellular fluorescence microscopy studies with SH-SY5Y
neuroblastoma were carried out on 2 and the commercial
20,70-dichlorofluorescein diacetate (DCFDA) for comparison
(Fig. 1 and ESIw); 2 compartmentalized in the cytosol and was
found to be sensitive to endogenous H2O2, in fact moreso than
the fluorescein species was (30 min).7 Various conditions were
measured (ESIw). Furthermore, compound 2 was found non-
neurotoxic at 100 mM, as determined by LDH assaying (ESIw).
The calculated c log P value for 2 (2.75) indicates promise for
possible blood-brain barrier penetrability.
Next, to consider how the emission signal and molecular
geometry are attenuated upon stepwise [O]-atom incorporation,
we synthesised, isolated and characterized compounds 6–8, the
discrete proposed BODIPY products of oxidation (Scheme 2,
Fig. 2, ESIw), Aldehyde 1 was oxidized (m-CPBA) and its
products were isolated and characterized for convenient
BODIPY (6–8) preparation (ESIw). 1H NMR spectra show
inequivalent 1,3,5,7-position methyls and 2,6-position
methines for 6 and 7 (ESIw). These compounds are also highly
fluorescent as predicted (FF = B1.00). To confirm that 6–8
are genuine products of m-CPBA oxidation, 2 was treated
separately with excess m-CPBA (CH2Cl2, 0 1C); 1H NMR
spectral analysis revealed three major products assigned
to 6–8.
Scheme 1 Synthesis of the mono- and dithiol-derivatized aldehydes
(1 and 1a) from 3-thiophene carboxaldehyde, and BODIPY compounds
2 and 2a (ESIw).
Fig. 1 (left) Cuvette emission spectra of compound 2 (1 ꢂ 10ꢀ6 M)
and ‘‘turn-on’’ response (50-fold, 522 nm) with m-CPBA (100 eq) in
acetonitrile/water (70 : 30) after 2 and 15 h (lexc = labs = 506 nm)
(ESIw). (right) Fluorescence microscopy images of cultured neuro-
blastoma for 2 at 100 mmol with H2O2 after incubation (2 h, 37 1C;
lexc = 543 nm, lem = 592 nm) (see ESIw).
Structural data was also obtained and can be brought into
line with spectroscopic data. Crystal structures of 7 and 8
confirm the expected BODIPY species with the trioxidized
arylsulfoxide–arylsulfone and tetraoxidized bis-arylsulfone
moieties, respectively (Fig. 2). The crystal structure of 7 reveals
one sulfoxide and one sulfone (Fig. 2). In contrast, that for
compound 8 reveals two sulfones and is Cs-symmetric by
1H NMR spectroscopy, suggesting rapid arylsulfone motion
on the NMR time-scale (RT, CDCl3). The final oxidation site
is at the proximal sulfide. The FF value of 8 (B0.59) is 40%
lower than that of 7. From the emission data, importantly, the
Fig. 2 BODIPY crystal structures of (left) the trioxidized 7, an
arylsulfoxide–arylsulfone and (right) the tetraoxidized 8, a bis-arylsulfone
(CCDC #’s: 860722 & 869055, respectively). Hydrogen atoms are
omitted for clarity, percentage of thermal ellipsoids = 30%.
Scheme 2 Preparation and characterization of BODIPY oxidation
products of 2 with p-chloranil, and the simple BODIPY by-product
species 9.
Fig. 3 (left) Selectivity of 2 for m-CPBA, and (right) comparison of
FF values for compounds 2, 6, 7, and 8 (522 nm).
c
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Chem. Commun., 2012, 48, 7298–7300 7299

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proximal to the chromophore is oxidized; cuvette optical data
show selective ‘‘turn-on’’ behavior (50-fold) with m-CPBA
t
(over H2O2, NaOCl, KO2, ꢁOH, ꢁOtBu, and BuOOH) in
30+% water. Neuroblastoma cellular trials with 2 show
responses for H2O2. Structural data support the asymmetric
findings gleaned from NMR spectral data. Geometric imposi-
tions give rise to increased molecular fluorescence to a point.
Lastly, bis-arylsulfone aldehyde 5 facilitates the unexpected
formation of a simple, asymmetric trimethyl BODIPY species
(1,3,6-trimethyl-4,4-difluoroboradiazaindacene) (FF = B1.00).
We hope to fine-tune the concept of stepwise oxidation for
neurodegenerative disease research through future studies.
The Molecular Logic Gate Laboratory operated by D.G.C.
acknowledges research support from the National Research
Foundation (NRF) (Grant # 2009–0070330 & 2010–0013660,
2011-0017280). Mr Hack Soo Shin and Prof. Youngkyu Do
are acknowledged, respectively, for facilitating the acquisition
of NMR spectroscopic and crystallographic data. The
research support staff at KAIST facilitated the acquisition of
our MS data. We thank Deposition Coordinator Kamila
Nowakowska (CCDC).
Fig. 4 Crystal structure of 9 (CCDC #: 869054). Hydrogen atoms are
omitted for clarity, percentage of thermal ellipsoids = 30%.
first-oxygenation of the proximal sulphur allows for a B100-fold
increase in FF value; upon oxidation, the entire 8-aryl moiety
gains steric hindrance, rotational inhibition allows the fluoro-
phore to possess higher fluorescence.3,8 The sulfone in 8,
involves a combined electronic and steric attenuation of
fluorescence (ESIw); structurally, the fourth [O] must direct
itself toward the BODIPY fluorophore (Fig. 2). Red shifting
for the series (6 o 7 o 8: lABS: 509 o 512 o 515; lEM: 523 o
524 o 527) suggests a growing heteroatomic influence of the
thienyl donor moiety to the BODIPY acceptor. Oxidation at
the distal sulfide imparts a subtle photophysical effect not
underscored here. Compound 2 was also assayed with poten-
tially interfering metal cations (50 equiv); no fluorescence
perturbations were found, except for minor quenching effect
for Cu2+ (ꢀ20%) and Hg2+ (ꢀ15%). For Hg2+, very slight
bathochromic shifting for 6 (3 nm) and 7 (4 nm), and
hypsochromic shifting for 8 (2 nm) was found in MeCN
(ESIw). c log P values were calculated and found to decrease:
6: 1.62 4 7: 1.06 4 8: 0.498, suggesting worsening BBB
penetrability with more O-loading.
References
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Surprisingly, the reaction of 5 with 2,4-dimethyl pyrrole
gave the novel and highly fluorescent 1,3,6-trimethyl-4,4-
difluoroboradiazaindacene (FF = 0.98) as a side product in
9.9% yield (Rf 0.70, CH2Cl2). High resolution-MS confirmed a
low MW molecule (257.1038 (cal.), 257.1033 (exp.)). The
structure by solution 1H NMR spectroscopy reveals four
aromatic singlets (d 7.46, 7.06, 6.68, 6.09) and three aliphatic
singlets (d 2.54, 2.23, 2.10). The asymmetric nature of 9, where
two methyls exist on one side and a single methyl is present on
the opposite side of the molecule, is supported structurally as
well (Fig. 4). Interestingly, in a deviation from the known
Lindsey reaction, the formation of 9 requires 5, but not TFA.
Remarkably, no product formation was detected in the
presence of 4, suggesting the subtlety of proximal sulfone/
sulfoxide sterics in product formation in which the 4-methyl
carbon of the first 2,4-dimethylpyrrole bonds to the ring
5-carbon of the second 2,4-dimethylpyrrole via oxidation/
condensation.
Herein, we report the preparation and characterization of
various closely-related sulphur-containing BODIPY species
for ROS sensing. Stepwise chemical S-oxidations effected by
m-CPBA or H2O2 can greatly enhance molecular fluorescence.
Specifically, oxygen atom loading gives respective sulfoxide/
sulfone groups: enhancement is found with 3[O]-atom loading
(7); a diminished capacity is found for 4[O]-atom loading (8).
The greatest emission changes are seen when the sulfide that is
c
7300 Chem. Commun., 2012, 48, 7298–7300
This journal is The Royal Society of Chemistry 2012