Synthesis and characterization of two novel red-shifted isothiocyanate BODIPYs and their application in protein conjugation

Article abstract of DOI:10.1016/j.dyepig.2020.108646

Fluorescent organic small molecules are very important tools for researchers in the fields of biochemistry and biotechnology, and boron-dipyrromethene (BODIPY) class of organic fluorophores gained much attention in recent years due to their remarkable pho

Full text of DOI:10.1016/j.dyepig.2020.108646

Journal Pre-proof
Synthesis and characterization of two novel red-shifted isothiocyanate BODIPYs and
their application in protein conjugation
Vanessa G. Amorim, Shaiani M.G. Melo, Ruan F. Leite, Paulo Arthur Coutinho, Sarah
M.P. da Silva, André R. Silva, Fernanda G. Amorim, Rita G.W. Pires, Juliana B.
Coitinho, Flavio S. Emery, Lucas C.D. Rezende
PII:
S0143-7208(20)31343-7
DOI:
https://doi.org/10.1016/j.dyepig.2020.108646
DYPI 108646
Reference:
To appear in: Dyes and Pigments
Received Date: 7 February 2020
Revised Date: 10 June 2020
Accepted Date: 10 June 2020
Please cite this article as: Amorim VG, Melo SMG, Leite RF, Coutinho PA, da Silva SMP, Silva AR,
Amorim FG, Pires RGW, Coitinho JB, Emery FS, Rezende LCD, Synthesis and characterization of
two novel red-shifted isothiocyanate BODIPYs and their application in protein conjugation, Dyes and
Pigments, https://doi.org/10.1016/j.dyepig.2020.108646.
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Author Statement
Vanessa G. Amorim: Methodology, Investigation, Writing - Original Draft Shaiani M. G. Melo:
Methodology, Investigation Ruan F. Leite: Investigation Paulo Arthur Coutinho: Methodology,
Investigation Sarah M. P. da Silva: Methodology, Investigation André R. Silva: Methodology, Writing -
Review & Editing Fernanda G. Amorim: Methodology, Investigation Rita G. W. Pires: Methodology,
Writing - Review & Editing Juliana B. Coitinho: Methodology, Writing - Review & Editing, Supervision
Flavio S. Emery: Conceptualization, Writing - Review & Editing, Funding acquisition, Supervision Lucas C.
D. Rezende: Conceptualization, Methodology, Writing - Original Draft, Writing - Review & Editing,
Supervision, Funding acquisition

Synthesis and characterization of two novel red-shifted isothiocyanate BODIPYs and their
application in protein conjugation
a
b
c
a
Vanessa G. Amorim , Shaiani M. G. Melo , Ruan F. Leite , Paulo Arthur Coutinho , Sarah M. P.
a,d
a,e
a,d
a
da Silva , André R. Silva , Fernanda G. Amorim, Rita G. W. Pires , Juliana B. Coitinho , Flavio
b
a,c*
S. Emery , Lucas C. D. Rezende
.
a
Graduate Program in Biochemistry, Federal University of Espírito Santo, Vitória, ES, Brazil.
Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto,
b
University of São Paulo (FCFRP-USP), Ribeirão Preto, SP, Brazil
c
Department of Natural Sciences, Federal University of Espírito Santo, São Mateus, ES, Brazil
d
Graduate Program in Biochemistry and Immunology, Federal University of Minas Gerais, Belo
Horizonte, ES, Brazil.
e
Federal Institute of Espírito Santo, Campus Aracruz, Aracruz, ES, Brazil
f
University of Vila Velha, Vila Velha, ES, Brazil.
*
Corresponding author: Lucas C D Rezende – rezendelcd@gmail.com
Abstract
Fluorescent organic small molecules are very important tools for researchers in the fields of
biochemistry and biotechnology, and boron-dipyrromethene (BODIPY) class of organic
fluorophores gained much attention in recent years due to their remarkable photophysical
properties and chemical versatility. Their application often involves conjugation with
biomolecules, which can be accomplished by inserting a reactive functionality to the
fluorescent dye. In this paper we present the preparation and characterization of two
unpublished BODIPYs bearing the electrophilic isothiocyanate group, widely used for
conjugation with proteins and biological amines. These dyes were prepared by inserting a
nitrobenzene group to the BODIPY core via direct arylation, which was later converted to the
isothiocyanate functionality. The arylated BODIPYs retained their fluorescence and showed
red-shifted absorption and fluorescence spectra, when compared to previously published
isothiocyanate-BODIPYs. Their application in bioconjugation was tested with bovine serum
albumin (BSA) and was confirmed from spectroscopic and electrophoretic analysis.
Graphical abstract
Keywords: BODIPY; Bioconjugation; Isothiocyanate; Protein; Fluorescence, Arylation
Highlights
-
-
-
-
Preparation of two unpublished amino-reactive BODIPYs
A red-shifted and highly fluorescent isothiocyanate-BODIPY is presented
Bioconjugation with a model protein was accomplished
BODIPY labelled proteins with fluorophore:protein (F:P) ratio between 0.5 and 5.5

1
. Introduction
In recent decades boron-dipyrromethene derivatives, more commonly known by the acronym
BODIPY”, found widespread application as fluorescent dyes in several fields of technology [1-
]. The BODIPY core is a pyrrolic dye characterized by a tricyclic structure in which the C-2 of
“
4
two pyrroles are connected by a methine bridge, and the pyrrolic nitrogen atoms are
complexed with a boron atom, usually as a part of a difluoroboryl group (Figure 1). The BODIPY
core is a highly emitting fluorophore, a consequence of their typical high fluorescence
quantum yields and molar absorptivity. The fluorescence spectra of the BODIPY core is centred
close to 500 nm and is weakly Stokes shifted, however their usual fluorescence properties can
be manipulated via chemical modifications [5,6].
Since their first report, more than 40 years ago [7], several methods for the preparation and
chemical modification of BODIPYs have been reported in the literature, rendering the BODIPY
core an excellent choice for the development of tailored fluorescent dyes [6,8]. Red-shifted
BODIPYs, for example, can be achieved from substitution reactions at positions 3 and 5, while
a very well-established method to prepare blue-shifted BODIPYs involves the insertion of
amines at position 8 [8-10].
The application of fluorescent dyes in biochemistry commonly involves bioconjugation with
biomolecules, such as antibodies and proteins in general. In this context, fluorescent dyes are
commonly functionalized with the isothiocyanate amino-reactive group, aiming to prepare
fluorophores for bioconjugation [11]. Fluorescein isothiocyanate (FITC) is the classic example
of an isothiocyanate-functionalized fluorophore and some BODIPY derivatives have also been
synthesized, however their peak fluorescence is usually around 500 nm [12,13]. For this paper
we designed two isothiocyanate-functionalized BODIPYs, with an aryl group at position 3 for a
slightly red-shifted emission (Figure 1).
Figure 1. Top left: General structure and numbering system of the BODIPY core. Bottom left:
Chemical structure of a previously published isothiocyanate-BODIPY. Right: Design of the red-
shifted BODIPYs presented in this paper.
2
. Results and discussion
2
.1 Synthesis

The synthesis of isothiocyanate BODIPYs (Scheme 1) started with the preparation of
dipyrromethanes 1 and 2, from the aqueous reaction of pyrrole with benzaldehyde (for
compound 1) or 2,6-dichlorobenzaldehyde (for compound 2). Based on our previous
experience the dipyrromethanes 1 and 2 were not isolated, instead, the crude products were
readily oxidized with DDQ to yield dipyrrins 3 and 4, respectively [14]. The complexation of
compounds 3 and 4 with BF ·Et O in organic basic medium yielded, respectively, BODIPYs 5
3
2
and 6 in 39% and 40%, for the three reaction steps. We used a direct arylation procedure
based on a radical reaction with an aryl diazonium salt to prepare compounds 7 and 8 (41%
and 45% yield) [15]. The objectives with the arylation reaction were to shift the absorption and
emission spectra and to introduce the nitro functionality as a precursor of the isothiocyanate
group. The formation of diarylated BODIPYs seemed as an unavoidable side reaction, however
the two products could be easily separated via silica gel column chromatography. The last two
steps were functional group conversions, starting with the reduction of nitro groups of 7 and 8
to the aromatic amines in 9 (84%) and 10 (95%) via catalytic hydrogenation [12]. Finally, the
conversion of amines 9 and 10 to the isothiocyanate derivatives 11 and 12 were accomplished
using different methods. Compound 9 was converted to compound 11 in 91% yield using 1,1'-
Thio-carbonyldi-2,2'-pyridone (TDP), while the preparation of 12 from 10 was done with
thiophosgene, in a reaction with 86% yield [12,13].
Scheme 1. Synthetic route applied for the preparation of isothiocyanato-BODIPYs 11 an 12.
The chemical structure of compounds 7 – 12 were confirmed by mass spectrometry and
nuclear magnetic resonance spectroscopy, and the characterization data for compounds 7 - 12
can be found as supplemental file. The two functional group conversions leading to the
isothiocyanate functionality (compounds 11 and 12) from the nitrophenyl substituted BODIPY
1
(
compounds 7 and 8) can be easily confirmed via H-NMR spectroscopy. In figure 2 a low field
1
section of the H-NMR containing the signals of the aromatic hydrogens of compounds 8, 10
and 12 are shown.
a
b
The hydrogens at the substituting aromatic ring, indicated as H and H , can be unambiguously
a
correlated with their respective signals from their multiplicity and integration. Hydrogens H
b
and H in compound 8 are strongly deshielded, due to the electron withdrawing effect of the

nitro group and the BODIPY core, which is reflected on their chemical shifts (8.33 ppm and
a
b
8
1
1
.14 ppm, respectively). As expected, an upfield shift was observed for H and H in compound
b
a
0 (7.97 ppm for H and 6.75 ppm for H ), and its conversion to the isothiocyanate derivative
a
b
2 resulted in an downfield shift of H (7.33 ppm) while the chemical shift of H was unaltered
(
8.01 ppm). A similar trend was observed for the chemical shifts of hydrogen atoms directly
linked to the BODIPY core. In general, the reduction of the nitro group of compound 8 to the
amino group in compound 10 resulted in an upfield shift, which was partially reverted when
the amino group was converted to isothiocyanate in compound 12. This observation confirms
the existence of a resonance interaction between the BODIPY core and the phenyl group at
c
position 3 and can be exemplified by the chemical shift of H , which was 7.95 ppm for
compound 8, 7.75 ppm for compound 10 and 7.88 ppm for compound 12. Moreover, the
absence of resonance interaction between the BODIPY core and the meso-aromatic
substituent is well reported, [16,17] and it explains the absence of any relevant upfield or
d
downfield shift for the hydrogens in the 2,6-dichlorophenyl substituent, indicated as H .
1
Figure 2. Low field section of the H-NMR of compounds 8, 10 and 12, confirming the
functional group conversions.
2
.2 Photophysical properties
The UV-Vis absorption and the fluorescence emission of compounds 11 and 12 were
characterized in selected solvents, to study their solvatochromic behaviour. Pentane (C H ),
5
12
dichloromethane (CH Cl ), chloroform (CHCl ), ethyl acetate (EtOAc), acetone (Me CO),
2
2
3
2
methanol (MeOH) and dimethylsulfoxide (DMSO) were selected for this characterization. A
solution in aqueous buffer (PBS pH 7.4) could not be tested, once both compounds were
completely insoluble in this solvent system. This was not surprising, as BODIPY dyes are often
water-insoluble and chemical modifications are commonly applied to enhance their solubility

[18]. For biochemical applications, DMF and DMSO are often applied as co-solvents to work
around the low solubility of organic fluorescent dyes.
The results of the photophysical characterization are presented in table 1 as the wavelength of
maximum absorption and emission (λmax), the full width at half maximum for absorption and
emission spectra (FHWM), the Stokes shift and the fluorescence quantum yield (φ ). As
fl
expected, a red-shifted λmax was observed for the absorption and emission of both compounds,
when compared to the isothiocyanate-BODIPYs previously described in the literature [12,13].
Absorption and fluorescence maxima of meso-(2,6-dichlorophenyl)-BODIPYs are known to be
slightly red-shifted when compared to meso phenyl-BODIPYs, [15,19] which was also observed
for compound 12 whose absorption and fluorescence spectra were consistently more red-
shifted (10 to 15 nm) when compared to compound 11. The FWHM parameter is shown as an
estimate for peak width, and a slightly narrower peaks were observed for compound 12 when
compared to 11. The effect of solvent polarity over absorption and emission spectra was very
weak and no clear trend was observed, as can be visualized in their spectra (supplemental file,
Figures S1 – S4).
Regarding fluorescence quantum yields, previous reports indicates a stronger fluorescence
emission for meso-(2,6-dichlorophenyl)-BODIPYs when compared to meso-phenyl BODIPYs
[15,19]. This observation is explained by nonradiative decay pathways related to
conformational changes of the S1 excited state in meso-phenyl BODIPYs, which is dramatically
diminished in sterically hindered substituents, such as 2,6-dichlorophenyl, that prevents aryl
rotation [16,17]. Our results are in accordance with these observations, as fluorescence
quantum yields of 11 ranged from 0.05 to 0.13, while for compound 12 the results were closer
to unity, ranging from 0.85 to 0.99.
Stokes shifts are presented in wavenumber and reflects the energy dissipation due to
vibrational relaxation and the reorganization of solvents around the excited state [20]. Our
results indicated slightly reduced Stokes shift for compound 12, which is probably due to their
red-shifted absorption and emission peaks, as the shifts are very similar if expressed in
wavelength (between 23 and 29 nm for BODIPY 11, and between 22 and 26 nm for BODIPY
1
2). A slightly shorter Stokes shift was observed in pentane, a hydrocarbon solvent. We have
previously reported a similar trend for other BODIPYs and it might reflect the absence of
solvent dipole reorientation due to its low polarity [20,21].
Table 1. Photophysical properties of compounds 11 and 12 in seven organic
solvents
Compound and
solvent
1
Absorption
Emission
Stokes
shift
a
b
a
b
b
φfl
λmax
FWHM
λmax
FWHM
1
C5H12
538
539
540
537
536
534
543
1562
1661
1665
1646
1666
1695
1717
561
566
567
562
561
561
572
1027
1094
1060
1062
1079
1075
1084
762
885
882
828
831
901
934
0.09
0.09
0.14
0.07
0.05
0.05
0.10
CH Cl
2
2
CHCl3
EtOAc
CH COCH
3
3
MeOH
DMSO
1
2
C5H12
551
552
554
1434
1530
1551
573
578
578
898
935
944
697
815
749
0.99
0.99
0.95
CH Cl
2
2
CHCl3

EtOAc
550
550
547
557
1559
1517
1606
1567
573
576
571
583
922
886
932
881
730
821
768
801
0.85
0.90
0.94
0.97
CH COCH
3
3
MeOH
DMSO
a
b
-1
data shown in nm data shown in cm
2
.3 Bioconjugation
BODIPYs 11 and 12 were designed as fluorescent probes for protein bioconjugation, and their
application was tested using BSA as a model protein. Each compound was used in independent
bioconjugation reactions with two different fluorophore:BSA molar ratio (10:1 and 25:1).
Aiming to eliminate non-conjugated fluorophore, the products were submitted to a centrifugal
dialysis against distilled water using a Vivaspin® Turbo 15 centrifugal concentrators (30 KDa
MWCO), which we will refer as protocol A. The purification via protocol A seemed inefficient as
the non-conjugated dye precipitated and was not visually observed in the dialysate, probably
due to the low water-solubility of BODIPY dyes. To test another purification protocol (which
we will refer to as protocol B), new batches of conjugation products were prepared using the
same procedure. For purification protocol B we added three cycles of centrifugal dialysis
against aqueous ethanol 70%, which better solubilizes the non-conjugated dye, followed by
three cycles of dialysis against water. In protocol B the elimination of non-conjugated dyes
could be visually observed by the colour and fluorescence appearing in ethanolic dialysate.
Additionally, no precipitation was observed during this protocol, and the conjugated protein
could be retrieved as a slightly coloured solid after lyophilization (supplemental file, Figures S5
and S6).
Figure 3 shows the absorption and emission spectra recorded for the BSA-BODIPY conjugates
prepared with 25 eq of each dye and purified using Protocol B. UV-Vis absorption spectroscopy
revealed the typical protein band at 280 nm, and the BODIPY band at 547 nm for BODIPY 11
and at 560 nm for BODIPY 12. Fluorescence spectroscopy indicated that the BODIPY dyes
retained their fluorescence after conjugation, with fluorescence emission peaks slightly shifted
to 575 nm and 590 nm for BODIPYs 11 and BODIPY 12, respectively. Normalized absorption
and fluorescence spectra of each BSA-BODIPY conjugate can be found in the supplemental file
(
Figiures S7 – S10).

Figure 3. Normalized absorption (full line) and fluorescence (dotted line) spectra of compounds
1 (black) and 12 (blue) after conjugation with BSA.
1
Even though fluorescence quantum yield was not calculated for the BSA-BODIPY, their
fluorescence intensity was seemingly less intense when compared to the non-conjugated dyes.
Larger slit opening was necessary to record the fluorescence spectra of BSA-BODIPY conjugates
when compared to solutions of the non-conjugated dyes with similar absorbance. This
reduction of fluorescence intensity does not prevent their application in bioconjugation
protocols and could be related to interactions with the protein/aqueous environment [24,25],
or result from the formation of thiourea functionality. To better understand this observation,
small-scale test reactions between the isothiocyanato-BODIPYs and an excess of diethylamine
were performed (Figure S11). TLC control indicated an instantaneous full conversion of
BODIPYs 11 and 12 to a more polar and less bright thiourea-BODIPY. Photophysical
characterization of these thiourea-BODIPYs in methanol (Figure S12) indicated a slightly red-
shifted absorption (λmax = 543 nm, for compound 11; λmax = 555 nm for compound 12) and
emission spectra (λmax = 572 nm, for compound 11; λmax = 581 nm for compound 12, and
reduced fluorescence quantum yields (φ = 0.03 for compound 11; φ = 0.11 com compound
fl
fl
1
2). This data suggests that the formation of thiourea functionality could explain the changes
in the photophysical properties observed after protein conjugation.
The ratio between the absorbance of protein and that of the dye was used to estimate the
degree of protein labelling (also known as F/P ratio), which estimates the number of
fluorophore molecules per molecule of protein [24]. As expected, a slightly higher degree of
protein labelling was observed when 25 equivalents of the fluorophore were used in the
bioconjugation reaction (Table 2). Additionally, a lower F:P ratio was observed when the
bioconjugation product was purified in the dialysis with ethanol, reinforcing the conclusion
that this a better purification method.
Table 2. Application of 11 and 12 in bioconjugation with BSA.
b
CF
a
ε
Comp.
Dye excess Purification F:P ratio
1
1
1
2
36000 0.36
10 eq
Protocol A
Protocol B
Protocol A
Protocol B
Protocol A
Protocol B
Protocol A
Protocol B
3.24
0.56
5.31
0.67
2.19
0.64
2.54
0.81
1
2
2
0 eq
5 eq
5 eq
28000 0.44
10 eq
1
2
2
0 eq
5 eq
5 eq
a
-1
-1
b
Molar extinction coefficient in DMSO (M cm ) Correction
factor calculated from the absorption spectra of the
unconjugated dyes in aqueous solution (DMSO as co-solvent).
To further confirm conjugation of the BODIPY dyes SDS-PAGE experiments were performed to
analyse the bioconjugation products. These experiments revealed strong fluorescent protein
bands, regardless of the molar excess used in the bioconjugation step or the purification
procedure. Figure 4 shows the SDS-PAGE of BSA conjugated with BODIPY 11 (10 eq in lane 1
and 25 eq. in lane 2) and BODIPY 12 (10 eq, in lane 3 and 25 eq. in lane 4), all of them purified
by purification protocol A. Non-conjugated BSA was loaded to the gel as a control and, as
expected, was only observed in the gel after comassie blue stain (lane 5). Similar results were

observed for the BSA-BODIPY conjugates purified via protocol B, despite their lower F:P ratio
supplemental file, Figure S11).
(
Figure 4. SDS-PAGE gel of BSA-BODIPY conjugates after purification via protocol A. Top:
Visualised for fluorescence emission before staining. Bottom: Visualised after comassie blue
staining. Lane 1: Product of the conjugation of BSA with 10 eq of BODIPY 11. Lane 2: Product of
the conjugation of BSA with 25 eq of BODIPY 11. Lane 3: Product of the conjugation of BSA
with 10 eq of BODIPY 12. Lane 4: Product of the conjugation of BSA with 25 eq of BODIPY 12.
3
. Concluding Remarks
Herein we showed the synthesis and photophysical characterization of two isothiocyanate-
functionalized BODIPY dyes, designed for application in bioconjugation reactions. As planned,
the fluorescence of both compounds was red-shifted when compared to previously published
isothiocyanate-BODIPYs, and the fluorescence quantum yield strongly depends on the
aromatic substituent at position 8. Both compounds were completely non-water soluble,
which makes the use of co-solvents necessary. Nevertheless, both were successfully applied in
bioconjugation reactions with a model protein and their application in the conjugation with
antibodies will be further studied by our research group. Additionally, enhancing their
solubility in water could result in simpler bioconjugation and purification protocols, and is a
current goal our research group working on.
4
. Experimental
4
.1 Syntheses
The syntheses were performed in appropriate round-bottom glass flasks with magnetic
stirring. The reactions were monitored using silica gel thin layer chromatography (TLC) in
aluminium foils with fluorescent indicator. Mixtures of hexane, ethyl acetate and

dichloromethane were used as mobile phase for TLC analyses, which were visualized under UV
light. Purification was performed via column chromatography on silica gel (0.060–0.200 mm,
6
0 Å), and organic solvents were evaporated using a rotary evaporator at 40–50 °C. For novel
1
3
1
compounds the chemical structures were determined by C NMR (75 or 101 MHz), H NMR
300, 400 or 600 MHz), low-resolution mass spectrometry (electrospray ionization) and high-
(
resolution mass spectrometry (electrospray ionization with time-of-flight). The preparation of
compounds 5 and 6 is well stablished in our group and were performed according to previously
published procedures [25-27].
Preparation of BODIPYs 7 and 8 by arylation: Approximately 0,9 mmol of the starting BODIPYs
5
(300 mg) and 6 (241 mg) were dissolved in acetone (5 mL) in different round-bottom flasks at
room temperature under magnetic stirring. Approximately 1 equivalent (0,9 mmol) of 4-
nitrobenzenediazonium tetrafluorborate (212 mg) was added to the stirring solution. A strict
stoichiometric relation should be followed, as the formation of diarylated BODIPY is an
important side reaction. Following, 20 mol% of Ferrocene (33 mg, 0,18 mmol) dissolved in
acetone (5 mL) was added dropwise for 10 minutes. After the ferrocene addition was
completed the reaction was left stirring at room temperature for 15 minutes, after which TLC
control indicated partial conversion to the desired product and the formation of a small
amount of the diarylated BODIPY. Each reaction was separately poured into diethyl ether (100
mL), extracted three times with distilled water and dried over magnesium sulphate. Each
solution was filtered, evaporated in a rotary evaporator, and the residue was purified
chromatographically (hexane/ethyl acetate) to yield the desired products 7 (144 mg, 0,368
mmol, 41% yield) and 8 (185 mg, 0,405 mmol, 45% yield).
Preparation of BODIPYs 9 and 10 by catalytic hydrogenation: Approximately 0,36 mmol of the
starting BODIPYs 7 (140 mg) and 8 (165 mg) were dissolved in a mixture of dichloromethane (9
mL) and ethanol (1 mL) in different round-bottom flasks at room temperature under magnetic
stirring. Palladium on activated charcoal (20 mol%) was added to the solution as a
heterogenous catalyst. The reaction flask was purged with hydrogen gas, sealed with
appropriated silicon septum and left under magnetic stirring for 15 min at room temperature
and H atmosphere (1 atm). TLC control indicated full conversion of the starting material and
2
the formation of new products, which were purified chromatographically (hexane/ethyl
acetate) to yield the desired products 9 (109 mg, 0,304 mmol, 84% yield) and 10 (148 mg,
0
,345 mmol, 95% yield).
Preparation of BODIPY 11: To a stirring solution of BODIPY 9 (42 mg, 0,117 mmol) in
chloroform (5 mL) at room temperature 1,1'-Thio-carbonyldi-2,2'-pyridone (46 mg, 0,194
mmol) was added. After 1.5 hour under magnetic stirring at room temperature TLC control
indicated the full conversion of the starting material to a new compound, which was purified
chromatographically (hexane/ethyl dichloromethane) to yield the desired product 11 (43 mg,
0
,106 mmol, 91% yield).
Preparation of BODIPY 12: To a stirring solution of BODIPY 10 (80 mg, 0,187 mmol) in
o
dimethylformamide (5 mL) under cooling a bath (0 C) and an inert atmosphere, Na CO was
2
3
added (95 mg, 0,9 mmol). After 15 minutes under magnetic stirring, phosgene (67 uL, 101 mg,
,9 mmol) was added to the solution, which was kept stirring at cooling bath and inert
0
atmosphere. After 1 hour, TLC control showed full conversion of the starting material to a new
compound. The reaction was poured into ethyl acetate, extracted three times with distilled
water and dried over magnesium sulphate. The solution was filtered, solvent was evaporated

in a rotary evaporator and the residue was purified chromatographically (hexane/ethyl
acetate) to yield the desired product 12 (76 mg, 0,161 mmol, 86% yield).
4
.2 Photophysical characterization
UV-Vis absorption spectra of the fluorescent dyes in seven organic solvents were recorded in a
TM
Genesys 10S spectrophotometer (Thermo Scientific). Fluorescence spectra of the fluorescent
dyes in the same seven solvents were recorded in a Cary Eclipse fluorescence
spectrophotometer (Agilent). UV-Vis absorption spectra of the conjugated protein were
recorded in Evolution® 300 spectrophotometer (Thermo-Scientific). Fluorescence quantum
yields were calculated by a comparative method using Rhodamine-101 in acidified methanol as
a reference (ϕ = 0.96, excitation at 535 nm for compound 11 and at 550 nm para for
compound 12). In this experiment, the absorbance of five diluted solutions of the fluorescent
compound was recorded at 535 nm for compound 11 and at 550 nm for compound 12, so that
the absorbance of each solution was no higher than 0.1 and no lower than 0.01. These same
solutions were analysed in a fluorescence spectrophotometer, and the area under the
emission band was calculated using the equipment’s software. Following, absorbance and
integrated fluorescence are plotted and the angular coefficient of the linear regression is used
in the following equation to calculate the fluorescence quantum yield. Where ꢀ and ꢀ are
ꢁ
ꢂꢃ
the fluorescence quantum yield of the studied dye and the reference, ꢄ and ꢄꢂꢃ are the
ꢁ
angular coefficient calculated for the studied dye and the reference, and ꢅ and ꢅꢂꢃ are the
ꢁ
refractive index of the solvents used to solubilize the studied dye and the reference.
ꢈ
ꢄꢁ ꢅꢁ
ꢀꢁ
= ꢀꢂꢃ ꢆ
ꢇ ꢆ
ꢇ
ꢄꢂꢃ ꢅꢂꢃ
4
.3 Bioconjugation
The bioconjugation reactions were performed in 2 mL of aqueous bicarbonate buffer (0,5 M;
pH = 9,2) containing DMSO (5 % v/v) as co-solvent. The final concentration of BSA was 1
mg/mL (approximately 15 μM) and the fluorescent dye was added in a final concentration of
1
3
1
50 μM (for 10 eq experiments) or 375 μM (for 25 eq experiments). After 1 hour reacting at
o
8 C in a heating bath, the solution was submitted to a centrifugal dialysis using Vivaspin turbo
5 (Sartorius), with a 30 kDa molecular weight cut-off. Two distinct dialysis protocols were
tested in different bioconjugation reactions: a single dialysis against distilled water (4000 RPM,
0 minutes – Protocol A); a triple dialysis against aqueous ethanol 70% v/v (4000 RPM, 20
1
minutes – Protocol B). Purification procedures using ethanol 70% were followed by a solvent
exchange step in which the solution was dialysed against distilled water for three times (4000
RPM, 10 minutes). After the dialysis the aqueous solutions were evaporated in a centrifugal
evaporator CentriVap (LABCONCO).
Degree of protein labelling: UV-Vis absorption spectra of the conjugated protein were
recorded in Evolution® 300 spectrophotometer (Thermo-Scientific) and used to estimate the
dye:protein molar ratio (F/P ratio). The molar extinction coefficient of each fluorescent dye (ε)
was calculated from the absorbance at maximum wavelength of each compound in DMSO
solutions of known concentration. Absorbance was plotted against molar concentration and a
linear regression curve was plotted for the dataset with a linear relation. ε was calculated from
the angular coefficient of the linear regression. The correction factor (CF) equals the
absorbance at 280 nm (A ) of the dye divided by the absorbance at peak maxima (Amax) of the
280

dye in aqueous solution containing 30% of DMSO as co-solvent. Finally, F/P ratio could be
calculated from the following equation [22].
ꢉꢊꢋꢁ × ꢌꢍꢎꢏꢃ
(
ꢉꢈꢐꢑ − ꢉꢊꢋꢁꢒꢓ) × ꢌꢔꢕꢖꢏꢎ
Polyacrylamide gel electrophoresis: SDS-PAGE was carried out on the Mini-Protean Tetra Cell
apparatus (BioRad, Hercules, USA). Polyacrylamide resolving gel mixture (5 mL) was prepared
using polyacrylamide 12%, bisacrylamide 0.3%, SDS 0.1% in Tris-HCl buffer (0.375 mM, pH 8.8)
and polymerized with ammonium persulfate 0.1% and tetramethylethylenediamine 20 µL. This
gel mixture was poured into a cassette and after complete polymerization the polyacrylamide
stacking gel was prepared and poured over. Polyacrylamide stacking gel mixture (3 mL) was
prepared using polyacrylamide 3%, bisacrylamide 0.3%, SDS 0.1%, in Tris-HCl buffer (0.125
mM, pH 6.8) and polymerized with ammonium persulfate 0.1% and
tetramethylethylenediamine 20 µL. After polymerization, the gel was placed into the
electrophoresis tank and covered in running buffer (0.025 mM Tris, 0.192 mM glycine, 0.1%
SDS). The samples were prepared by diluting the protein samples in distilled water in a final
concentration of 4 mg/mL. This solution was mixed with loading buffer (0.31 M Tris- HCl pH
6
.8, 0,2% SDS, 50% glycerol, 0.01% bromophenol blue), and a 20 µL sample was loaded onto
the gel. The gel was developed at a voltage of 120 V for two hours. The gel was illuminated
with UV light and registered photographically, before staining overnight in staining solution
(
0.025% “Coomassie Brilliant Blue” R250, 15% methanol, 7% acetic acid, in distilled water),
followed by several changes of destain solution (50% methanol, 10% acetic acid, in distilled
water).
Acknowledgements
This work was supported by Fundação de Amparo à Pesquisa e Inovação do Espírito Santo
(
FAPES, grant #038/2019), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP,
grants #2011/23342-9, 2013/24408-9, 2014/18973-8), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES).
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Supplementary Data
Synthesis and characterization of two novel red-shifted isothiocyanate BODIPYs and their
application in protein conjugation.
Vanessa G. Amorim, Shaiani M. G. Melo, Ruan F. Leite, Paulo Arthur Coutinho, Sarah M. P. da
Silva, André R. Silva, Fernanda G. Amorim, Rita G. W. Pires, Juliana B. Coitinho, Flavio S. Emery,
Lucas C. D. Rezende.
Spectroscopical characterization data
1
BODIPY 7: H NMR (300 MHz, CDCl ) δ 7.99 (d, J = 9.0Hz, 2H), 7,52 (s, 1H), 7,52 – 7.40 (m, 5H),
3
7
6
1
5
,02 (d, J = 9.0 Hz, 2H), 6,97 (d, J = 4.5 Hz, 1H), 6.80 (d, J = 4.3 Hz, 1H), 6.70 (d, J = 4.5 Hz, 1H),
13
.49 (dd, J = 4.3, 2.0 Hz, 1H), 3.81 (s, 3H). C NMR (101 MHz, CDCl ) δ 161.5, 160.9, 1445,
3
41.2, 137.6, 134.4, 134.0, 133.2, 131.5, 130.6, 130.4, 128.7, 128.5, 124.6, 121.2, 117.7, 114.1,
+
+
+
5.5. LRMS (ESI) 375 (M + H) , 397 (M + Na) , 413 (M + K) .
1
BODIPY 8: H NMR (300 MHz, CDCl ) δ 8.33 (d, J = 8.7 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 7.95 (s,
3
13
1
H), 7.56 – 7.40 (m, 3H), 6.75 (s, 1H), 6.69 (d, J = 4.3 Hz, 1H), 6.57 (d, J = 4.0 Hz, 1H). C NMR
(
75 MHz, CDCl ) δ 156.6, 148.2, 146.3, 140.3, 138.1, 136.9, 135.2, 134.9, 131.5, 131.2, 130.4,
3
1
30.3, 130.3, 128.3, 123.5, 120.9, 120.1. LRMS (EI): 457, 438 (M - F), 422 (M - Cl).
1
BODIPY 9: H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 8.9 Hz, 2H), 7.73 (s, 1H), 7.65 – 7.54 (m,
5
1
1
1
+
H), 7.12 (d, J = 4.8 Hz, 1H), 6.99 (d, J = 4.8 Hz, 1H), 6.67 (d, J = 8.9 Hz, 2H), 6.64 (d, J = 3.9 Hz,
13
H), 6.51 (dd, J = 3.9, 2.1 Hz, 1H), 6.24 (br, 2H). C NMR (101 MHz, DMSO-d6) δ 162.0, 152.4,
39.8, 137.5, 137.4, 133.8, 133.5, 132.3, 132.2, 130.3, 130.1, 128.5, 124.9, 122.4, 117.1,
+
16.4, 113.3. HRMS (ESI): calculated for C H BF N , [M + H] : 360.1484, found: 360.1459, [M
21
16
2
3
+
Na] : 382.1303, found: 382.1285.
1
BODIPY 10: H NMR (300 MHz, CDCl ) δ 7.97 (d, J = 8.8 Hz, 2H), 7.75 (s, 1H), 7.54 – 7.34 (m,
3
13
3
H), 6.82 – 6.66 (m, 4H), 6.49 (d, J = 3.8 Hz, 1H), 6.43 (dd, J = 3.8, 1.9 Hz, 1H), 4.11 (br, 2H). C
NMR (75 MHz, CDCl ) δ 163.1, 149.5, 140.1, 137.9, 135.7, 132.9, 132.2, 132.1, 132.0, 131.6,
3
1
31.0, 128.3, 125.3, 122.2, 121.2, 117.2, 114.6. HRMS (ESI): calculated for C H BCl F N , [M +
21 14 2 2 3
+
+
+
H] : 428.0699, found: 428.0686, [M + Na] : 450.0524, found: 450.0505, [M + K] : 466.0263,
found: 466.0290.
1
BODIPY 11: H NMR (400 MHz, CDCl ) δ 7.90 (d, J = 8.8 Hz, 2H), 7.81 (s, 1H), 7.44 – 7.54 (m,
3
5
1
1
H), 7.26 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 4.4 Hz, 1H), 6.83 (d, J = 4.1 Hz, 1H), 6.60 (d, J = 4.3 Hz,
13
H), 6.48 (dd, J = 4,1, 1,6 Hz, 1H). C NMR (101 MHz, CDCl ) δ 157.9, 146.0, 143.3, 137.3,
3
36.7, 134.5, 134.0, 132.6, 131.1, 130.8, 130.7, 130.6, 130.5, 128.5, 125.7, 120.5, 118.6.
1
BODIPY 12: H NMR (300 MHz, CDCl ) δ 8.01 (d, J = 8.8 Hz, 2H), 7.88 (s, 1H), 7.40 – 7.52 (m,
3
3
1
1
H), 7.33 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 4.4 Hz, 1H), 6.66 (d, J = 4.4 Hz, 2H), 6.51 (d, J = 2.8 Hz,
13
13
H). C NMR (75 MHz, CDCl ) δ C NMR (75 MHz, CDCl ) δ 159.1, 144.4, 139.2, 137.2, 135.5,
3
3
34.2, 132.9, 131.6, 131.4, 131.0, 130.9, 130.8, 128.9, 128.4, 125.9, 121.3, 119.2. HRMS (ESI):
+
calculated for C H BCl F N S, [M + H] : 470.0268, found: 470.0268.
22
12
2
2
3

UV-Vis absorption spectra
BODIPY 11
Figure S1. Normalized UV-Vis absorption spectra of BODIPY 11 in seven organic solvents.
BODIPY 12
Figure S2. Normalized UV-Vis absorption spectra of BODIPY 12 in seven organic solvents.

Fluorescence emission spectra
BODIPY 11
Figure S3. Normalized fluorescence spectra of BODIPY 11 in seven organic solvents.
BODIPY 12
Figure S4. Normalized fluorescence spectra of BODIPY 12 in seven organic solvents.

Bioconjugation Purification Protocol
Figure S5. Appearance of dialysis solution of two BSA-BODIPY conjugates purified with Protocol
B after a dialysis cycle. A: Compound 11, 25 eq. B: Compound 12, 25 eq. No colour was
observed when dialysis was performed in water (Protocol A).
Figure S6. Appearance of aqueous solutions of BSA-BODIPY conjugates prepared with
compounds 11 or 12 and purified with Protocol A or Protocol B. Solubilization of BSA-BODIPY
conjugates purified via Protocol A was less efficient, probably due to the lower solubility of
non-conjugated dyes in water, hindering the dialysis in this purification protocol. No
solubilization issues were observed for the BSA-BODIPY conjugates purified via Protocol B.

Spectra of BSA-BODIPY conjugates
Figure S7. UV-Vis absorption spectra of BSA-BODIPY conjugates prepared using compound 11
in different molar ratios (10 eq or 25 eq) and purified with different protocols (Protocol A or
Protocol B). Spectra were normalized for the maximum wavelength of BODIPY absorption. Full
black line: 10 eq, Protocol A. Dotted black line: 10 eq, Protocol B. Full grey line: 25 eq,
Protocol A. Dotted grey line: 25 eq, Protocol B.
Figure S8. UV-Vis absorption spectra of BSA-BODIPY conjugates prepared using compound 12
in different molar ratios (10 eq or 25 eq) and purified with different protocols (Protocol A or
Protocol B). Spectra were normalized for the maximum wavelength of BODIPY absorption. Full
black line: 10 eq, Protocol A. Dotted black line: 10 eq, Protocol B. Full grey line: 25 eq,
Protocol A. Dotted grey line: 25 eq, Protocol B.

Figure S9. Normalized fluorescence emission spectra of BSA-BODIPY conjugates prepared using
compound 11 in different molar ratios (10 eq or 25 eq) and purified with different protocols
(
Protocol A or Protocol B). Full black line: 10 eq, Protocol A. Dotted black line: 10 eq, Protocol
B. Full grey line: 25 eq, Protocol A. Dotted grey line: 25 eq, Protocol B.
Figure S10. Normalized fluorescence emission spectra of BSA-BODIPY conjugates prepared
using compound 12 in different molar ratios (10 eq or 25 eq) and purified with different
protocols (Protocol A or Protocol B). Full black line: 10 eq, Protocol A. Dotted black line: 10 eq,
Protocol B. Full grey line: 25 eq, Protocol A. Dotted grey line: 25 eq, Protocol B.

Reaction of isothiocyanato-BODIPYs with diethylamine
Figure S11. Left panel: Test reactions between compounds 11 and 12 with diethylamine for the
formation of thiourea-BODIPYs ThU-1 and ThU-2. Right panel: Solutions of thiourea BODIPYs
ThU-1 and ThU-2 in acetone visualized under white light (WL) and black light (BL).
Figure S12. Absorption and emission spectra of thiourea-BODIPYs ThU-1 and ThU-2 in
Methanol. Full black line: Absorption spectra of ThU-1. Full grey line: Absorption spectra of
ThU-2. Dotted black line: Emission spectra of ThU-1. Dotted grey line: Emission spectra of
ThU-2.

SDS-PAGE
Figure S13. SDS page of BSA-BODIPY conjugates purified via protocol B. Gel 1, lane 1:
compound 11, 10 eq; Gel 1, lane 2: L compound 12, 10 eq; Gel 1, lane 3: non-conjugated BSA;
Gel 2, lane 1: compound 11, 25 eq; Gel 2, lane 2: compound 12, 25 eq; Gel 2, lane 3: non-
conjugated BSA.

Highlights
•
•
•
•
Synthesis of two amino-reactive red-shifted BODIPYs with isothiocyanate group
NMR and photophysical characterization
Amino-reactive BODIPYs successfully conjugated with BSA as a model protein
BODIPY-BSA conjugates retained fluorescence and were visualized via SDS-PAGE

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: