A Synthetic Approach for the Rapid Preparation of BODIPY Conjugates and their use in Imaging of Cellular Drug Uptake and Distribution

Article abstract of DOI:10.1002/chem.201706093

A solid-phase synthetic (SPS) method was developed for the preparation of BODIPY-labeled bioactive compounds that allows for fast and simple synthesis of conjugates for use in fluorescent microscopy. The approach was used to visualize cellular uptake and

Full text of DOI:10.1002/chem.201706093

A Journal of
Accepted Article
Title: Synthetic approach for rapid preparation of BODIPY conjugates
and their use in imaging of cellular drug uptake and distribution
Authors: Sona Krajcovicova, Jarmila Stankova, Petr Dzubak, Marian
Hajduch, Miroslav Soural, and Milan Urban
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To be cited as: Chem. Eur. J. 10.1002/chem.201706093
Link to VoR: http://dx.doi.org/10.1002/chem.201706093
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Synthetic approach for rapid preparation of BODIPY conjugates
and their use in imaging of cellular drug uptake and distribution
Sona Krajcovicova,[b] Jarmila Stankova,[a] Petr Dzubak,[a] Marian Hajduch,[a] Miroslav Soural,* [b] and
Milan Urban*[a]
Abstract: Solid-phase synthetic (SPS) method was developed for the
preparation of BODIPY-labeled bioactive compounds that allows for
fast and simple synthesis of conjugates usable in fluorescent
microscopy. The approach was used to visualize cellular uptake and
distribution of cytotoxic triterpenes in cancer cells.
incompatible with the SPS concept due to the lack of stability
under the standard SPS reaction conditions.[3,6,7] Regardless of
this statement, we have been able to develop a versatile
procedure for the preparation of such conjugates using common
backbone amide linker and standard coupling reagents by careful
optimization of the reaction time and concentration of harsh
reagents such as piperidine and trifluoroacetic acid. BODIPY-FL
propanoic acid was selected as the most appropriate fluorescent
label in this project. To prove the concept, we have synthesized a
small set of BODIPY-labeled cytotoxic triterpenes in which we
expected different mechanisms of action and differences in
cellular uptake and distribution. Selected compounds (Figure 1)
have low micromolar cytotoxicity on various cancer cell lines,
whereas some were supposed to have a unique mechanism of
action: aldehyde 2,[8] monoketone 3,[9] diketone 4,[9] and pyrazine
5,[10] respectively (Figure 1). Betulinic acid 1 was used as a
standard since it is the most commonly studied cytotoxic
triterpene and its mechanism of action has been well-studied to
date.11,12
Introduction
Visualization of small molecules in cells became an essential tool
in drug discovery. The most commonly used method is
fluorescent microscopy in which the studied molecule is equipped
with a fluorescent label that allows direct visualization of the
cellular uptake and distribution of the drug within the cell. A
number of various conjugates of small molecules with a variety of
fluorescent tags were reported to date with the application as
probes,[1] photosensitizers[2] or luminescence switches and
sensors.[3] Among them, BODIPY dyes[2,4] are preferably used
fluorophores because of their superior physico-chemical
properties such as high photostability, high quantum yield of
fluorescence, total neutral charge and low polarity.[4] In contrast to
other dyes, wavelengths of absorption and emission can be tuned
easily by various substitutions on the BODIPY core.[4] The
simplest BODIPYs show fluorescein-like parameters, however,
unlike fluoresceins or rhodamines, they are prone to cellular
permeability and lack nonspecific binding to proteins or lipids.[5] In
this work, we designed a versatile solid-phase synthetic (SPS)
method for the synthesis of fluorescent conjugates of biologically
active molecules. The solid-phase synthesis allows for fast and
simple production of libraries of desired compounds with only
minimum effort and hands-on-time because it saves many
isolation and purification steps of the intermediates. Surprisingly,
there is only one example in the literature[6] that describes the use
of SPS for adding substituents to the BODIPY core. However,
there is no precedent of using the SPS in the synthesis of
conjugates of BODIPY and other molecules. In agreement with
this, there is a number of articles stating that BODIPY is
Figure 1. The selected triterpenoid structures.
Results and Discussion
Since the pharmacophores of the selected triterpenes are
suggested but yet unproven, chemical modification at a wrong
part of the molecule may negatively influence their biological
behavior. Therefore, we decided to attach the fluorescent dye
from three different sites and to compare the results. Triterpenes
1-5 were modified in positions C3, C28 or C30 (Figure 2).
[a]
[b]
MSc. J. Stankova, Dr. P. Dzubak, Assoc. Prof. M. Hajduch, Assoc.
Prof. M. Urban
Institute of Molecular and Translational Medicine
Palacky University in Olomouc, Faculty of Medicine and Dentistry
Hnevotinska 5, 77900, Olomouc, Czech Republic
E-mail: milan.urban@upol.cz
MSc. S. Krajcovicova, Assoc. Prof. M. Soural
Department of Organic Chemistry
Palacky University in Olomouc, Faculty of Science
17. Listopadu 12, 77100, Olomouc, Czech Republic
E-mail: miroslav.soural@upol.cz
Supporting information for this article is given via a link at the end of
the document.
Figure 2. Three different sites to attach the fluorescent dye.
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The reported syntheses[13,14] had to be slightly modified and
optimized to increase the overall yield. It started from the
commercially available pyrrole-2-carbaldehyde 15, which was
firstly converted to α,β-unsaturated ester 16 via the Horner–
Wadsworth–Emmons reaction with excellent selectivity yielding
only (E)-alkene. Such selectivity was obtained due to the
formation of stabilized ylide. The following reduction provided
intermediate 17, which was then subjected to POCl3 promoted
coupling with commercially available 3,5-dimethyl-1H-pyrrole-2-
carbaldehyde. Final treatment with BF3.OEt2 yielded BODIPY-FL
propanoate 18 in one-pot reaction sequence (Scheme 2 in main
manuscript). Subsequent acidic hydrolysis of ester yielded final
BODIPY-FL propanoic acid 19 in excellent overal yield of 40 %
(Scheme 2), indicating unusually long kinetic stability of BODIPY
dye in acidic media.[15] Importantly, the improved synthesis of
BODIPY-FL propanoic acid is scalable up to gram-scale
quantities, which is the solution to the most common problem in
the synthesis of BODIPY dyes.
Scheme 2. The synthetic route for the preparation of BODIPY-FL propanoic
acid 19. Reagents and conditions: i) methyl(triphenylphosphor-
anylidene)acetate, CH2Cl2, r.t., 84%; ii) Pd/C, H2, CH3OH, r.t., 90%; iii) a) 3,5-
dimethylpyrrole-2-carbaldehyde, POCl3, CH2Cl2, 0°C to r.t., b) BF3.OEt2, DIPEA,
CH2Cl2, 0°C to r.t., 63%; iv) THF/H2O/conc. HCl (3:2:1), 0°C to r.t., 85%.
Scheme 1. Synthesis of premodified triterpenes. Reagents and conditions: i)
SeO2, diglyme, 140°C; ii) KH2PO4, NaClO2, t-BuOH/2-methyl-2-butene (1:1), r.t.
(10; 90%); iii) succinic anhydride, DMAP, THF/DMF (2:1), 80°C (6; 80%, 9; 73%,
11; 73%); iv) benzylbromoacetate, K2CO3, THF, 50°C (7; 98%, 13; 91%); v)
Pd/C, H2, CH3OH, r.t. (8; 84%, 14; 49%).
In contrast to the synthesis of biotin-preloaded resins,[16,17] the
procedure for the preparation of BODIPY-preloaded resin 23 had
to be carefully optimized due to limited chemical stability of
BODIPY-FL. The aminomethyl resin was equipped with backbone
amide linker (BAL) and subjected to reductive amination with 2-
(2-aminoethoxy)ethanol to obtain immobilized secondary amine
20 (Scheme 3). The chemoselective protection of the secondary
amine with Fmoc was followed by acylation of the hydroxy group
with prepared BODIPY-FL propanoic acid 19 and afforded resin
21. Cleavage of the Fmoc-protective group and acylation with [2-
[2-(Fmoc-amino)ethoxy]ethoxy]acetic (FAEEAA) acid using the
standard DIC/HOBt technique yielded Fmoc-protected resin 22
which, upon deprotection with low concentration of piperidine in
DMF, yielded desired preloaded resin 23 in very good crude purity
(82%; calculated from UHPLC-MS traces). Subsequent acylation
with the premodified triterpenes 6, 8-12 and 14 afforded the final
conjugates 24-28, 30 and 31 (Scheme 3).
Because of the steric hindrances at some of the selected positions,
the original triterpenes had to be premodified by hemisuccinate at
C3-OH, glycolate at C28OOH, or oxidized to carboxylate at C30
(Scheme 1, see the experimental part [EP] and the SI file). Briefly,
the C3 modified hemisuccinates 6, 11 and 12 were obtained after
the reaction of betulinic acid 1, monoketone 3 or diketone 4, with
succinic anhydride in the presence of a base. C3 modified
hemisuccinic aldehyde 9 was obtained after two-stage oxidation
of betulinic acid
1 with selenium dioxide, followed by
abovementioned reaction of aldehyde 2 with succinic anhydride.
C28 modification of compounds 1 and 5 yielded protected
glyoxalates 7 and 13, which upon catalytic hydrogenation in the
presence of Pd/C afforded desired modified triterpenes 8 and 14,
respectively. Finally, the compound 10 was achieved after facile
and chemoselective Pinnick oxidation of the aldehyde 2, which
formed the carboxyl at the C30 position.
Synthesis of BODIPY-FL propanoic acid had to be optimized
(Scheme 2, see the EP and the SI file) to obtain better yields than
those previously reported in the literature.[13,14] Although BODIPY-
FL propanoic acid 19 (Scheme 2) is commercially available, due
to its extremely high price we synthesized the dye by ourselves.
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Scheme 3. Synthesis of BODIPY-preloaded resin and its use for conjugation with triterpenes. Reagents and conditions: i) BAL, HOBt, DIC, DMF/CH2Cl2 (1:1), r.t.;
ii) 2-(2-aminoethoxy)ethanol, NaBH(OAc)3, DMF/AcOH (10:1), r.t.; iii) FmocOsu, CH2Cl2, r.t.; iv) 19, HOBt, DIC, DMF/CH2Cl2 (1:1); v) DMF/piperidine (20:1), r.t.; vi)
FAEEAA, DIC, HOBt, DMF/CH2Cl2 (1:1), r.t.; vii) 6, 8-12, 14, DIC, DMF/CH2Cl2 (1:1), r.t.; viii) CH2Cl2/TFA (10:1), r.t.; ix) Ac2O, DMAP, CH2Cl2, r.t.
uptake or distribution and compound 33 was designed to show
the properties of the BODIPY-FL connected to linker without a
triterpene. In contrast to the construction of the intermediate 23,
omitting HOBt in the final acylation with triterpenes (Scheme 3,
step vii) increased the final crude purities of all conjugates
significantly. Additionally, TFA-mediated cleavage of the
conjugate 24 from the resin led to the formation of byproduct 32
in the equimolar ratio (calculated from UHPLC-MS traces) as a
result of Wagner-Meerwein rearrangement.[18] It is important to
mention, that the concentration of both piperidine and TFA had to
be considerably lowered, compared to standard cleavage
conditions (20-50% piperidine in DMF; 25-50% TFA in CH2Cl2), to
maintain good crude purities of all intermediates as well as final
conjugates and to prevent decomposition of BODIPY scaffold
(see SI for the details).
The excitation and emission spectra of BODIPY-FL propanoic
acid 19 were measured and compared to BODIPY conjugates 24-
33. All of the conjugates 24-33 had the same absorption/emission
wavelengths and Stokes shifts as the parent BODIPY-FL
propanoic acid (See SI for details). The conjugate's quantum
yields of fluorescence are lower in comparison to BODIPY-FL
propanoic acid (Φ = 98 for 19 vs. Φ = 0.14 - 0.30 for compounds
24-32). The diminished fluorescence is probably caused by the
static quenching between BODIPY dye and triterpenes, which is
consistent with recently published data.[19,20]
The cytotoxic activity of the parent compounds and fluorescent
conjugates was investigated in vitro against eight human cancer
cell lines and two non-tumor fibroblasts by using the standard
MTS test (Table 1). The cancer cell lines were derived from T-
lymphoblastic leukemia CCRF-CEM, leukemia K562 and their
multiresistant counterparts (CEM-DNR, K562-TAX), solid tumors
including lung (A549) and colon (HCT116, HCT116p53-/-)
carcinomas, osteosarcoma cell line (U2OS), and for comparison,
Figure 3. Structures of prepared conjugates 24 – 33. Compounds 32 formed as
on two human non-cancer fibroblast lines (BJ, MRC-5). In general,
the CCRF-CEM cell line was the most sensitive cancer cell line to
a byproduct and was not further studied since it lacks the cytotoxic activity.
the prepared compounds with only a few exceptions. Therefore,
SARs assumptions were mostly based on the activities in CCRF-
CEM cells.
Compound 29 containing an extended linker was prepared to
investigate the influence of the length of the linker on the cellular
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cytotoxic activity and selectivity. Conjugate of betulinic acid 24 at
the position C3 remained active, nevertheless, its conjugates at
the positions C28 and C30 (compounds 30 and 31,) were almost
inactive.
In fluorescent microscopy experiments (which were performed in
early intervals before the cytotoxic effect took place), we have
observed that all the tested fluorescent conjugates of triterpenes
are staining living cells and passing through the cellular
membrane into the cytoplasmic compartment (Figure 4, full
resolution image is in the SI). In addition, we used the BODIPY
conjugate 33 (which has the active triterpenic scaffold replaced
by acetate) as a negative control. According to the results, it is not
penetrating the cellular membrane indicating that it is the
triterpenoid part which is responsible for the cellular uptake. This
is likely because of the high lipophilicity of triterpenes. The
conjugate 25 containing Michael acceptor (acrolein moiety in this
case) resulted in different staining pattern - labeling cellular
cytoplasm homogenously, which is presumably caused by non-
specific covalent interaction with multiple intracellular proteins.
Staining is distinctly apparent when compared to another tested
compounds (24, 26, 27, 28, 29, 30, 31) which were labeling more
subtle cytoplasmic and membrane structures, likely mitochondria,
endoplasmic reticulum (ER), and the nuclear membrane. Co-
staining experiments are currently being performed to confirm this
unambiguously. Such results are in agreement with precedent
studies of another lupane triterpenes that were found to interact
with mitochondrion and ER.[21,22]
Conclusions
In this work we optimized the synthesis of BODIPY-FL propanoic
acid 19 to give better yields than procedures reported by other
authors and the synthesis is suitable for multi-gram scale
quantities. We prepared BODIPY-preloaded resin and applied it
to attach the fluorescent dye to cytotoxic triterpenic derivatives.
Despite previous reports on the limited applicability of BODIPYs
in solid-phase synthesis, due to their low stability under both basic
and acidic conditions,[3,6,7] we managed to develop and optimize
the synthetic protocols to overcome these problems. The reported
preloaded resin allows for routine and simple connecting of
various compounds to BODIPY label through a linker of choice
using simple laboratory equipment, common coupling reagents
and conditions, minimum hands-on-time and it can be even
commercialized similarly to biotin-preloaded resin (Biotin
NovaTag™, Novabiochem). Nine conjugates of BODIPY with
cytotoxic triterpenes were synthesized using the resin 23 and their
spectroscopic and biological properties were evaluated. In order
to prove that BODIPY with the linker do not interfere with the
biological study, we prepared a conjugate in which the triterpenic
part was replaced with acetate. Live cell studies focused on
fluorescence conjugates uptake have demonstrated non-specific
labeling in the aldehyde 25 and more specific labeling pattern in
the case of conjugates 24, and 26-31. Ongoing research is now
focused on more specific determination of which organelles,
proteins or protein complexes are targeted by our conjugates, and
Figure 4. Fluorescence imaging of U2OS cells stained by
BODIPY-triterpene conjugates. (For full resolution see SI).
Among the unmodified studied molecules 1-5, aldehyde 2 and
pyrazine 5 were cytotoxic against the CCRF-CEM line in a low
micromolar range of 1.53 and 0.53 μM, respectively. The
therapeutic index is rather low for the aldehyde 2 (4.7) but
surprisingly high in the case of pyrazine (more than 94). The
synthesized fluorescent conjugate of aldehyde 25 remained
highly but unselectively cytotoxic, probably due to the presence of
the reactive acrolein moiety. On the other hand, fluorescent
conjugates of pyrazine (28 and 29) had slightly decreased activity
and selectivity in comparison with the parent compound 5, which
indicates an important role of the free carboxyl group as a
pharmacophore. Interestingly, the length of the linker is affecting
the activity of the conjugates as well. This may be indicated by the
comparison of conujgates 28 and 29 from which the longer one
(29) was more active than the shorter one (28). Lastly, the
conjugate of diketone 27 was cytotoxic only on CCRF-CEM cell
line, whereas the monoketone conjugate 26 completely lost its
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it will be the aim of further proteomic and molecular biology
studies, e.g. co-localization experiments.
Table 1. Cytotoxic activity of all prepared compounds.
IC50 (μM/L)a
Comp. CCRF-
CEM
CEM-
DNR
HCT116 HCT116 K562
p53-/-
K562-TAX A549
U2OS
BJ
MRC-5
TIb
1c
2c
3c
4c
8.09
1.53
15.16
35.58
0.53
6.62
0.76
>50
14.04
7.66
20.16
35.98
0.63
>50
4.29
8.83
27.25
>50
14.09
12.43
34.67
43.11
11.6
>50
9.43
9.68
21.01
>50
15.78
7.73
24.25
>50
15.96
7.23
27.12
>50
20.75
7.51
40.06
>50
24.23
11.99
44.28
>50
28.18
2.39
42.67
>50
3.24
4.70
2.87
> 1.41
> 94.34
6.59
5c
11.54
>50
31.84
>50
34.41
>50
47.3
>50
32.43
33.75
1.46
>50
>50
>50
24
25
26
27
28
29
30
31
32
33
43.61
1.87
>50
43.6
1.62
>50
6.06
>50
1.65
>50
10.52
>50
1.96
>50
1.6
1.45
>50
2.30
>50
N.A.
3.4
>50
45.7
>50
>50
>50
>50
>50
>50
45.84
>50
44.26
>50
13.25
> 2.76
6.36
18.09
6.13
>50
20.28
9.27
>50
>50
>50
>50
>50
>50
18.92
>50
12.07
>50
29.02
>50
35.05
>50
29.14
>50
26.34
>50
39.74
>50
38.23
>50
N.A.
41.17
>50
40.68
>50
41.96
>50
>50
49.02
>50
40.6
>50
41.43
>50
45.02
>50
48.81
>50
40.75
>50
1.09
>50
N.A.
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
N.A.
aThe lowest concentration that kills 50 % of the cells. The standard deviation in cytotoxicity assays is typically up to 15 % of the average value.
bTherapeutic index is calculated for IC50 of CCRF-CEM line vs. an average of both fibroblasts (BJ and MRC-5).
cParent compounds used as a standard. Compounds with IC50 > 50 μM are considered inactive.
NMR (500 MHz, CDCl3): δ 7.38 – 7.35 (m, 5H, Ph), 5.22 (d, J = 6.5 Hz, 1H,
OCH2Ph), 5.18 (d, J = 12.3 Hz, 1H, OCH2Ph), 4.73 (d, J = 2.0 Hz, 1H, H29
pro-Z), 4.65 (d, J = 2.3 Hz, 2H, OCH2CO), 4.61 – 4.60 (m, 1H, H29 pro-E), 3.21
Experimental Section
– 3.17 (dd, J = 11.0 Hz, 4.8 Hz, 1H, H3α), 2.97 (td, J = 11.3, 10.9 , 4.7 Hz,
1H, H19β), 2.30 (dt, J = 12.6, 2.7 Hz, 1H), 2.27 (td, J = 13.5, 13.4, 3.6 Hz
1H), 2.06 – 1.99 (m, 1H), 1.96 – 1.86 (m, 1H), 1.72 – 1.13 (m, 24H, overlap
with solvent), 0.97 (s, 6H, 2x CH3), 0.92 (s, 3H, CH3), 0.83 (s, 3H, CH3),
0.77 (s, 3H, CH3) ppm. 13C NMR (126 MHz, CDCl3): δ 175.6, 168.1, 150.7,
135.4, 128.8, 128.6, 109.8, 79.2, 67.2, 60.4, 56.7, 55.6, 50.8, 49.6, 47.0,
42.6, 40.9, 39.0, 38.9, 38.3, 37.4, 37.1, 34.5, 32.1, 30.6, 29.8, 28.2, 27.6,
25.8, 21.1, 19.6, 18.5, 16.3, 16.1, 15.5, 14.9 ppm. HRMS (ESI): m/z calcd
for C39H56O5 [M+H]+ = 605.4201, found [M+H]+ = 605.4207.
General technical information are available in the Supporting information
file and are analogous to our previous publications.[23,24] Note that the
yields of the final conjugates (24-33, usually between 10-30 %) are
calculated as overall yields of the entire synthetic procedure between
compound 20 and the final product in the scheme 3.
General procedure for the preparation of benzyl glyoxalates. To a
stirred solution of starting material in THF were added benzyl
bromoacetate (3 eq) and K2CO3 (3 eq). The reaction mixture was stirred
at 50 °C and monitored by TLC (hexane/EtOAc = 3:1, v/v) which indicated
its completion after overnight stirring. The reaction mixture was
concentrated, diluted with water (100 mL/ 1.32 mmol) and extracted with
EtOAc (5x 100 mL/ 1.32 mmol). Organic extracts were combined, dried
over MgSO4, filtered and evaporated under reduced pressure. The crude
products were purified by flash chromatography (hexane/EtOAc = 3:1 to
1:1, v/v) to yield desired compounds.
Benzyl glyoxalate of betulinic acid pyrazine (13). Compound 13 was
prepared by the General Procedure A using 5 (100 mg, 0.2 mmol), benzyl
bromoacetate (96 µL, 0.61 mmol) and K2CO3 (84 mg, 0.61 mmol) in THF
(8 mL); compound 13 was obtained as a yellowish oil (116 mg, 91% yield).
1H NMR (500 MHz, CDCl3): δ 8.30 (d, J = 2.1 Hz, 1H, pyrazine), 8.17 (d, J
= 2.4 Hz, 1H, pyrazine), 7.33 – 7.24 (m, 5H, Ph, overlap with solvent), 5.14
– 5.06 (m, 2H, OCH2Ph), 4.66 – 4.54 (m, 4H), 2.96 – 2.86 (m, 3H), 2.37 –
2.21 (m, 4H), 1.96 – 1.91 (m, 1H), 1.86 – 1.80 (m, 1H), 1.70 – 1.22 (m,
17H, overlap with solvent), 1.20 (s, 3H, CH3), 1.18 (s, 3H, CH3), 0.92 –
0.90 (m, 6H, 2x CH3), 0.70 (s, 3H, CH3) ppm. 13C NMR (126 MHz, CDCl3):
δ 175.5, 168.1, 159.8, 151.0, 150.5, 142.4, 141.6, 135.3, 128.8, 128.7,
128.6, 109.9, 67.2, 60.4, 56.7, 53.2, 49.5, 49.0, 48.9, 46.9, 42.6, 40.7, 39.6,
38.3, 37.1, 36.9, 33.4, 32.0, 31.7, 30.6, 29.7, 25.7, 24.2, 21.6, 20.2, 19.6,
2-(Benzyloxy)-2-oxoethyl betulinate (7). Compound 7 was prepared by
the General Procedure
A using 1 (600 mg, 1.32 mmol), benzyl
bromoacetate (633 µL, 4 mmol) and K2CO3 (552 mg, 4 mmol) in THF (20
mL); compound 7 was obtained as a white solid (783 mg, 98% yield). 1H
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16.3, 15.7, 14.8 ppm. HRMS (ESI): m/z calcd for C41H54N2O4 [M+H]+ =
(867 mg, 8.68 mmol) and DMAP (1.05 g, 8.68 mmol) in THF/DMF (2:1, 15
mL) for 40 h; compound 11 was obtained as a white solid (1.3 g, 73% yield).
1H NMR (500 MHz, CDCl3): δ 4.51 (dd, J = 11.0, 5.5 Hz, 1H, H3α), 3.69 (s,
3H, COOCH3), 3.22 – 3.16 (m, 1H), 2.70 – 2.60 (m, 4H), 2.49 – 2.43 (m,
2H), 2.13 (d, J = 18.6 Hz, 1H), 2.00 (dd, J = 12.5, 3.0 Hz, 1H), 1.92 – 1.23
(m, 9H, overlap with solvent), 1.21 (app s, 3H, CH3), 1.20 (app s, 3H, CH3),
1.02 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.84 (s, 3H, CH3),
0.83 (s, 3H, CH3) ppm. 13C NMR (126 MHz, CDCl3): δ 207.5, 177.7, 175.1,
172.1, 172.0, 145.9, 81.5, 55.6, 53.3, 52.7, 51.2, 47.8, 45.4, 41.5, 38.7,
38.0, 37.3, 35.0, 33.9, 29.5, 29.3, 29.2, 28.1, 27.8, 25.3, 23.7, 21.4, 20.3,
20.2, 18.3, 17.0, 16.8, 16.7, 16.1 ppm. HRMS (ESI): m/z calcd C35H52O7
for [M+H]+ = 585.3786, found [M+H]+ = 585.3796.
639.4156, found [M+H]+ = 639.4155.
3β-Hydroxylup-20(29)-ene-28,30-dioic acid (10). To a stirred solution of
aldehyde 2 (100 mg, 0.21 mmol) in t-BuOH/2-methyl-2-butene (1:1, 10 mL,
v/v) was added NaClO2 (96 mg, 1.06 mmol) and solution of KH2PO4 (550
mg, 4.04 mmol) in H2O (5 mL). The reaction mixture was stirred vigorously
at ambient temperature and monitored by TLC (CH2Cl2/CH3OH = 10:1, v/v),
which indicated its completion after 5 h. The reaction mixture was
concentrated, diluted with NH4Cl (50 mL) and extracted with EtOAc (5x 50
mL). Organic extracts were combined, dried over MgSO4, filtered and
evaporated under reduced pressure. The crude product was purified by
flash chromatography (CH2Cl2/CH3OH = 5:1, v/v) to afford compound 10
as a white crystalline solid (90 mg, 90% yield). 1H NMR (500 MHz, DMSO-
d6): δ 12.17 (br s, 2H, 2x COOH), 5.96 (s, 1H, H29 pro-Z), 5.60 (s, 1H, H29 pro-
E), 4.25 (d, J = 5.1 Hz, 1H), 4.08 – 4.07 (m, 1H), 2.98 – 2.94 (m, 1H), 2.22
– 2.07 (m, 3H), 1.99 – 1.91 (m, 1H), 1.82 – 1.75 (m, 2H), 1.62 – 1.52 (m,
2H), 1.43 – 1.24 (m, 16H), 0.90 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.86 (s,
3H, CH3), 0.76 (s, 3H, CH3), 0.65 (s, 3H, CH3) ppm. 13C NMR (126 MHz,
DMSO-d6): δ 177.2, 168.4, 147.7, 122.5, 76.7, 55.5, 54.8, 51.05, 49.8, 48.6,
41.9, 38.5, 38.2, 37.3, 36.7, 36.0, 33.9, 32.7, 31.6, 29.2, 28.1, 27.1, 26.8,
20.5, 17.9, 15.9, 15.8, 15.7, 14.3 ppm. HRMS (ESI): m/z calcd for C30H46O5
[M-H]- = 485.3262, found [M-H]- = 485.3252.
Hemisuccinate of 21,22-dioxoacid (12). Compound 12 was prepared by
a previously published procedure.7 1H NMR (500 MHz, DMSO-d6): δ 12.20
(br s, 1H, COOH), 4.40 (dd, J = 11.6, 4.7 Hz, 1H, H3α), 3.66 (s, 3H,
COOCH3), 3.37 – 3.30 (m, 1H, overlap with solvent), 2.72 – 2.69 (m, 1H),
2.52 – 2.44 (m, 4H, overlap with solvent), 2.25 – 2.22 (m, 1H), 1.97 – 1.87
(m, 3H), 1.74 – 1.31 (m, 14H), 1.19 (d, J = 3.5 Hz, 3H, CH3), 1.18 (d, J =
3.5 Hz, 3H, CH3), 1.00 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.86 (s, 3H, CH3),
0.80 (s, 3H, CH3), 0.79 (s, 3H, CH3) ppm. 13C NMR (126 MHz, DMSO-d6):
δ 194.4, 189.0, 173.4, 171.6, 170.5, 168.0, 149.2, 79.9, 58.1, 54.5, 53.5,
49.8, 45.6, 45.3, 41.0, 37.7, 37.4, 36.6, 33.9, 29.1, 28.7, 27.8, 27.5, 27.3,
26.4, 25.2, 23.3, 20.5, 19.9, 19.4, 17.7, 16.5, 16.4, 16.3, 15.6 ppm. HRMS
(ESI): m/z calcd for C35H50O8 [M-H]- = 597.3422, found [M-H]- = 597.3408.
General procedure B for preparation of hemisuccinates. To a stirred
solution of starting material in THF/DMF (2:1) was added succinic
anhydride (6 eq) and DMAP (6 eq). The reaction mixture was stirred at
80 °C and monitored by TLC (CH2Cl2/CH3OH = 10:1, v/v) which indicated
its completion after 36 to 48 h. The reaction mixture was concentrated,
diluted with NH4Cl (150 mL/ 2.2 mmol) and extracted with EtOAc (5x 100
mL/ 2.2 mmol). Organic extracts were combined, dried over MgSO4,
filtered and evaporated under reduced pressure. The crude products were
purified by flash chromatography (CH2Cl2/CH3OH = 10:1, v/v) to afford
desired compounds.
General procedure C for preparation of glyoxalates. To a freshly
degassed solution of starting material in CH2Cl2/CH3OH (2:1) was added
Pd/C (10%, 3.5 mol%) and H2 was bubbled through the resulting reaction
mixture for 20 min. The reaction was monitored with TLC
(CH2Cl2/CH3OH/AcOH = 10:1:0.1, v/v) which indicated its completion after
1h. The reaction mixture was diluted with CH3OH (20 mL/ 0.83 mmol) and
filtered through bed of Celite. The residual solvent was evaporated under
reduced pressure and the crude product was purified by
chromatography (CH2Cl2/CH3OH/AcOH 10:1:0.1, v/v).
a flash
Betulinic acid 3-hemisuccinate (6). Compound 6 was prepared by the
General Procedure B using 1 (1 g, 2.2 mmol), succinic anhydride (1.32 g,
13.1 mmol) and DMAP (1.6 g, 13.1 mmol) in THF/DMF (2:1, 60 mL) for 36
h; yield compound 6 as a white solid (980 mg, 80% yield). 1H NMR (500
MHz, DMSO-d6): δ 12.14 (br s, 2H, 2x COOH), 4.69 (s, 1H, H29 pro-Z), 4.56
(s, 1H, H29 pro-E), 4.37 (dd, J = 11.7, 4.6 Hz, 1H, H3α), 2.97 – 2.92 (m, 1H,
H19β), 2.54 – 2.46 (m, 4H, overlap with solvent) 2.25 – 2.18 (m, 1H), 2.11
(m, 1H), 1.79 (m, 1H), 1.64 (s, 3H, CH3), 1.62 – 1.07 (m, 12H), 0.94 (s, 3H,
CH3), 0.87 (s, 3H, CH3), 0.80 (s, 3H, CH3), 0.78 (s, 6H, 2x CH3). All other
data were consistent with published results.6
Betulinic acid glyoxalate (8). Compound 8 was prepared by the General
Procedure C using 7 (500 mg, 0.83 mmol), Pd/C (30 mg) in CH2Cl2/CH3OH
(2:1, 7.5 mL); compound 8 was obtained as a white solid (357 mg, 84%
yield). 1H NMR (500 MHz, DMSO-d6): δ 12.91 (br s, 1H, COOH), 4.65 (d,
J = 2.0 Hz, 1H, H29 pro-Z), 4.53 (s, 1H, H29 pro-E), 4.50 (app s, 2H, OCH2CO),
4.21 (br s, 1H, H3α), 2.94 (m, 1H), 2.90 – 2.84 (m, 1H), 2.18 – 2.13 (m, 2H),
1.92 – 1.86 (m, 1H), 1.83 – 1.75 (m, 1H), 1.62 (s, 3H, CH3), 1.58 – 1.01
(m, 20H), 0.90 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.72 (s,
3H, CH3), 0.62 (s, 3H, CH3) ppm. 13C NMR (101 MHz, DMSO-d6): δ 174.6,
169.3, 150.1, 109.7, 76.7, 60.3, 55.7, 54.8, 49.9, 48.6, 46.5, 42.0, 38.5,
38.2, 37.4, 36.7, 36.2, 33.9, 31.3, 29.8, 28.9, 28.1, 27.1, 25.0, 20.4, 18.9,
17.9, 15.9, 15.8, 15.7, 14.3 ppm. HRMS (ESI): m/z calcd for C32H50O5 [M-
H]- = 513.3575, found [M-H]- = 513.3567.
30-Aldehyde 3-hemisuccinate of betulinic acid (9). Compound 9 was
prepared by the General Procedure B using 2 (1.5 g, 3.13 mmol), succinic
anhydride (1.88 g, 18.78 mmol) and DMAP (2.3 g, 18.78 mmol) in
THF/DMF (2:1, 30 mL) for 48 h; compound 9 was obtained as a white solid
(1.3 g, 73% yield). 1H NMR (500 MHz, DMSO-d6): δ 12.14 (br s, 2H, 2x
COOH), 9.49 (s, 1H, CHO), 6.47 (s, 1H, H29 pro-Z), 6.09 (s, 1H, H29 pro-E),
4.37 (dd, J = 11.3, 4.6 Hz, 1H, H3α), 3.25 (dd, J = 11.1, 4.8 Hz, 1H), 3.19
(d, J = 21.6 Hz, 2H), 2.52 – 2.46 (m, 4H), 2.20 – 2.12 (m, 2H), 1.97 – 1.89
(m, 1H), 1.81 – 1.77 (m, 2H), 1.58 – 1.11 (m, 17H), 0.91 (d, J = 7.2 Hz, 3H,
CH3), 0.86 (s, 3H, CH3), 0.78 (d, J = 3.6 Hz, 9H, 3x CH3) ppm. 13C NMR
(126 MHz, DMSO-d6): δ 195.6, 177.1, 175.0, 173.4, 171.6, 170.3, 156.3,
134.8, 79.9, 55.5, 54.6, 49.4, 41.9, 37.7, 37.4, 36.6, 36.1, 33.7, 31.6, 31.4,
29.2, 28.8, 27.6, 26.8, 23.3, 20.7, 20.5, 17.7, 16.4, 15.8, 15.6, 14.3, 14.1
ppm. HRMS (ESI): m/z calcd for C34H50O7 [M-H]- = 569.3473, found [M-H]-
= 569.3456.
Glyoxalate of pyrazine of betulinic acid (14). Compound 14 was
prepared by the General Procedure C using 13 (500 mg, 0.78 mmol), Pd/C
(27 mg) in CH2Cl2/CH3OH (2:1, 7.5 mL); compound 14 was obtained as a
white solid (210 mg, 49% yield). 1H NMR (500 MHz, DMSO-d6): δ 8.45 (d,
J = 2.3 Hz, 1H, pyrazine), 8.32 (d, J = 2.4 Hz, 1H, pyrazine), 4.71 (d, J =
1.9 Hz, 1H, H29 pro-Z), 4.59 (app s, 1H, H29 pro-E), 4.55 (app s, 2H, OCH2CO),
2.92 (td, J = 11. 2, 10.9, 4.7 Hz, 1H, H19β), 2.88 (d, J = 16.6 Hz, 1H, H1α),
2.46 (d, J = 16.6 Hz, 1H, H1β), 2.28 – 2.20 (m, 2H), 1.93 (dd, J = 11.9, 8.1
Hz, 1H), 1.87 – 1.79 (m, 1H), 1.67 (s, 3H, CH3), 1.67 – 1.28 (m, 15H), 1.24
(s, 3H, CH3), 1.20 (s, 3H, CH3), 1.17 – 1.05 (m, 2H), 1.00 (s, 3H, CH3),
0.93 (s, 3H, CH3), 0.72 (s, 3H, CH3) ppm. 13C NMR (126 MHz, DMSO-d6):
δ 174.7, 169.3, 158.5, 150.2, 150.1, 142.3, 141.7, 109.8, 60.2, 55.8, 52.0,
48.1, 47.8, 46.5, 42.1, 37.5, 36.2, 32.8, 31.3, 29.8, 29.0, 25.1, 24.0, 21.0,
Hemisuccinate of 21-oxoacid (11). Compound 11 was prepared by the
General Procedure B using 3 (700 mg, 1.45 mmol), succinic anhydride
This article is protected by copyright. All rights reserved.

10.1002/chem.201706093
Chemistry - A European Journal
FULL PAPER
19.5, 18.9, 15.8, 15.2, 14.4 ppm. HRMS (ESI): m/z calcd for C34H48N2O4
afford final compound 19 as a dark red crystalline solid (1.15 g, 85% yield).
1H NMR (500 MHz, DMSO-d6): δ 12.30 (s, 1H), 7.70 (s, 1H), 7.09 (d, J =
4.0 Hz, 1H), 6.38 (d, J = 4.0 Hz, 1H), 6.31 (s, 1H), 3.10 – 3.05 (t, J = 7.1
Hz, 2H), 2.64 (t, J = 8.5 Hz, 2H), 2.47 (s, 3H), 2.26 (s, 3H) ppm. 13C NMR
(126 MHz, DMSO-d6): δ 173.4, 159.5, 156.9, 144.3, 134.5, 133.0, 128.8,
125.4, 120.4, 116.5, 32.3, 23.5, 14.5, 11.0 ppm. 19F {1H} NMR (471 MHz,
CDCl3): δ -145.21 (d, J = 31.8 Hz), -145.36 (d, J = 31.8 Hz) ppm. HRMS
(ESI): m/z calcd for C14H15BF2N2O2 [M-H]- = 291.1111, found [M-H]- =
291.1105. Other spectral data were consistent with published results.3
[M-H]- = 547.3530, found [M-H]- = 547.3527.
Methyl (E)-3-(1H-pyrrol-2-yl)acrylate (16). To a stirred solution of
pyrrole-2-carbaldehyde 15 (1.5 g, 15.78 mmol) in CH2Cl2 (30 mL) was
added methyl (triphenylphosphoranylidene)acetate (10.5 g, 31.57 mmol)
in CH2Cl2 (50 mL). The resulting mixture was stirred at room temperature
overnight. The residual solvent was evaporated and the crude oily product
was purified by a flash chromatography (hexane/EtOAc = 2:1, v/v) to afford
compound 16 as a pale pink solid (2 g, 84% yield). 1H NMR (500 MHz,
CDCl3): δ 8.65 (br s, 1H), 7.56 (d, J = 16.0 Hz, 1H), 6.94 – 6.93 (m, 1H),
6.58 – 6.57 (m, 1H), 6.31 – 6.28 (m, 1H), 6.00 (d, J = 16.0 Hz, 1H), 3.78
(s, 3H) ppm. 13C NMR (126 MHz, CDCl3): δ 168.2, 134.5, 128.5, 122.6,
114.7, 111.2, 111.0, 51.8 ppm. HRMS (ESI): m/z calcd for C8H9NO2
[M+H]+ = 152.0706, found [M+H]+ = 152.0705.
Preparation of BAL resin. Aminomethyl polystyrene resin (1 g, loading
0.98 mmol/g) was swollen in CH2Cl2 (10 mL) for 30 min, washed with DMF
(3x 10 mL), neutralized in DMF/piperidine (5:1, 10 mL) for additional 30
min and then again washed with DMF (5x 10 mL). Backbone amide linker
(700 mg, 2.94 mmol) and HOBt (450 mg, 2.94 mmol) were dissolved in
DMF/CH2Cl2 (1:1, 10 mL, v/v) and DIC (460 µL, 2.94 mmol) was added.
The resulting solution was added to polypropylene fritted syringe with
aminomethyl resin. The reaction slurry was shaken at ambient
temperature overnight, followed by wash with DMF (3x 10 mL) and CH2Cl2
(3x 10 mL). Bromphenol blue test confirmed quantitative acylation of amino
groups.
Methyl 3-(1H-pyrrol-2-yl)propanoate (17). To
a freshly degassed
solution of 16 (2 g, 13.25 mmol) in CH3OH (20 mL) was added Pd/C (10%
loading, 3.5 mol%, 500 mg) and H2 was bubbled through the resulting
reaction mixture for 20 min. The reaction was monitored with UHPLC-MS,
which indicated its completion after 1h. The reaction mixture was diluted
with CH3OH (20 mL) and filtered through bed of Celite. The residual
solvent was evaporated and the crude product was purified by a flash
chromatography (hexane/EtOAc = 2:1, v/v) to afford compound 17 as a
pale yellow oil (1.67 g, 90% yield). 1H NMR (500 MHz, CDCl3): δ 8.51 (br
s, 1H), 6.69 – 6.67 (m, 1H), 6.11 (dd, J = 5.7, 2.8 Hz, 1H), 5.92 – 5.91 (m,
Procedure for reductive amination. BAL resin (1 g, loading 0.98 mmol/g)
was swollen in CH2Cl2 (10 mL) for 30 min, then washed with dry THF (3x
10 mL) and dry DMF (3x 10 mL). The solution of 2-(2-aminoethoxy)ethanol
(490 µL, 4.9 mmol) in DMF/AcOH (10:1, 10 mL, v/v) was added to
polypropylene fritted syringe with BAL resin and it was shaken overnight
at ambient temperature. Then, NaBH(OAc)3 (210 mg, 2.94 mmol) in
DMF/AcOH (20:1, 5 mL, v/v) was added portionwise to the reaction mixture
during period of 4 h, followed by washing with DMF (5x 10 mL) and CH2Cl2
(3x 10 mL) and neutralization with DMF/TEA (10:1, 10 mL, v/v) for
additional 30 min to obtain resin 20. The loading was determined
according to published procedure8 (0.4 – 0.6 mmol/g).
1H), 3.71 (s, 3H), 2.92 (t, J = 6.8 Hz, 2H), 2.65 (t, J = 6.8 Hz, 2H) ppm. 13
C
NMR (101 MHz, CDCl3): δ 174.7, 131.1, 117.0, 108.2, 105.7, 52.0, 34.5,
22.7 ppm. HRMS (ESI): m/z calcd for C8H11NO2 [M+H]+ = 154.0863, found
[M+H]+ = 154.0863.
BODIPY-FL-methyl propanoate (18). To a stirred solution of 17 (1.67 g,
10.92 mmol) and 3,5-dimethylpyrrole-2-carboxaldehyde (1.54 g, 12.01
mmol) in CH2Cl2 (80 mL) was added dropwise POCl3 (1.12 mL, 12.01
mmol) at 0°C and the reaction mixture was allowed to warm to ambient
temperature. The reaction was monitored with UHPLC-MS, which
indicated formation of dipyrromethane intermediate after 3 h. Then the
reaction mixture was cooled to 0°C and DIPEA (8.1 mL, 49.14 mmol) was
added dropwise, followed by stirring for 20 min at 0°C. BF3.OEt2 (5.4 mL,
43.68 mmol) was added subsequently and the reaction was stirred
overnight at ambient temperature. The mixture was diluted with CH2Cl2 (50
mL) and brine (100 mL), filtered through bed of Celite, again diluted with
brine (100 mL) and extracted with CH2Cl2 (5x 200 mL). Organic extracts
were combined, dried over MgSO4, filtered and evaporated under reduced
pressure. The crude product was purified by a flash chromatography
(100% CH2Cl2) to obtain compound 18 as a dark green crystalline solid
(2.13 g, 63% yield). 1H NMR (500 MHz, CDCl3): δ 7.09 (s, 1H), 6.89 (d, J
= 3.9 Hz, 1H), 6.27 (d, J = 4.0 Hz, 1H), 6.12 (s, 1H), 3.70 (s, 3H), 3.31 (t,
J = 7.6 Hz, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.58 (s, 3H), 2.26 (s, 3H) ppm.
13C NMR (126 MHz, CDCl3): δ 173.2, 160.8, 144.1, 135.5, 133.5, 128.3,
124.1, 120.7, 116.9, 52.0, 33.5, 30.0, 24.3, 15.2, 11.5 ppm. 19F {1H} NMR
(471 MHz, CDCl3): δ -145.23 (d, J = 31.4 Hz), -145.37 (d, J = 31.4 Hz) ppm.
HRMS (ESI): m/z calcd for C15H17BF2N2O2 [M-H]- = 305.1267, found [M-
H]- = 305.1266. Other spectral data were consistent with literature
precedencies.3
Procedure for protection with Fmoc. Resin 20 (250 mg) was swollen in
CH2Cl2 (3 mL) for 30 min and then washed with CH2Cl2 (3x 5 mL). Fmoc-
OSu (505 mg, 1.5 mmol) was dissolved in CH2Cl2 (3 mL) and added to
polypropylene fritted syringe with the resin. The reaction slurry was shaken
at ambient temperature overnight, followed by washing with CH2Cl2 (5x 5
mL). Analytical sample was cleaved from the resin and UHPLC-MS
analysis confirmed the presence of desired product. MS (ESI): m/z [M+H]+
= 328.
Procedure for acylation with BODIPY-FL propanoic acid. Resin 20
equipped with Fmoc (250 mg) was swollen in CH2Cl2 (3 mL) for 30 min
and then washed with DMF (3x 3 mL) and CH2Cl2 (3x 3 mL). BODIPY-FL
propanoic acid 19 (220 mg, 0.75 mmol), HOBt (115 mg, 0.75 mmol) and
DMAP (92 mg, 0.75 mmol) were dissolved in DMF/CH2Cl2 (1:1, 2.5 mL,
v/v) and DIC (117 µL, 0.75 mmol) was added. The resulting solution was
added to polypropylene fritted syringe the resin. The reaction slurry was
shaken at ambient temperature overnight, followed by washing with DMF
(10x 3 mL) and CH2Cl2 (10x 3 mL) to give resin 21. Analytical sample was
cleaved from the resin and UHPLC-MS analysis confirmed the presence
of desired product. MS (ESI): m/z [M-H]- = 600.
General procedure D for deprotection of Fmoc. Resin 21 (250 mg) was
swollen in CH2Cl2 (3 mL) for 30 min and then washed with DMF (3x 3 mL).
The freshly prepared solution of DMF/piperidine (20:1, 2.5 mL, v/v) was
added to polypropylene fritted syringe with the resin. The reaction slurry
was shaken at ambient temperature for 30 min, followed by wash with
CH2Cl2 (3x 3 mL), THF (3x 3 mL), DMF (3x 3 mL), THF (3x 3mL) and
CH2Cl2 (3x 3 mL).
BODIPY-FL-propanoic acid (19). To a stirred solution of 18 (1.43 g, 4.67
mmol) in THF (30 mL) was added water (20 mL) and conc. HCl (10 mL) at
0°C. The reaction mixture was stirred at ambient temperature and
monitored by UHPLC-MS, which indicated its completion after 52h. Then
was the reaction mixture diluted with water (100 mL) and extracted with
CH2Cl2 (3x 150 mL). Organic extracts were combined, dried over MgSO4,
filtered and evaporated under reduced pressure. Following purification of
the crude product by a flash chromatography (CH2Cl2/AcOH 100:1, v/v) to
This article is protected by copyright. All rights reserved.

10.1002/chem.201706093
Chemistry - A European Journal
FULL PAPER
Procedure for acylation with FAEEAA. Fmoc deprotected resin 21 (250
mg) was swollen in CH2Cl2 (3 mL) for 30 min and then washed with DMF
(3x 3 mL) and CH2Cl2 (3x 3 mL). [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic
acid (334 mg, 0.9 mmol) and HOBt (137 mg, 0.9 mmol) were dissolved in
DMF/CH2Cl2 (1:1, 3 mL, v/v) and DIC (140 µL, 0.9 mmol) was added. The
resulting solution was added to polypropylene fritted syringe with the resin.
The reaction slurry was shaken at ambient temperature overnight, followed
by washing with DMF (10x 3 mL) and CH2Cl2 (10x 3 mL) which gave resin
22. Analytical sample was cleaved from the resin and UHPLC-MS analysis
confirmed the presence of desired product. MS (ESI): m/z [M-H]- = 745.
The Fmoc was deprotected from the resin 22 by the general procedure to
give resin 23.
= 31.5 Hz) ppm. HRMS (ESI): m/z calcd for C58H85BF2N4O11 [M-H]- =
1061.6276, found [M-H]- = 1061.6275. UV/Vis (CH2Cl2): λmax = 507 nm; λem
= 513 nm; Φ = 0.20.
BODIPY-FL-triterpene conjugate 25. Dark red oil (10.0 mg, 10% overall
yield). 1H NMR (500 MHz, CDCl3): δ 9.52 (s, 1H, CHO), 7.17 (t, J = 5.2 Hz,
1H), 7.09 (s, 1H), 6.88 (d, J = 3.9 Hz, 1H), 6.36 (t, J = 5.4 Hz, 1H), 6.29 –
6.26 (m, 2H), 6.12 (s, 1H, H29 pro-Z), 5.91 (s, 1H, H29 pro-E), 4.47 – 4.43 (m,
1H), 4.27 – 4.24 (m, 2H), 4.01 (s, 2H), 3.69 – 3.64 (m, 5H), 3.61 – 3.55 (m,
5H), 3.52 – 3.47 (m, 5H), 3.45 – 3.43 (m, 2H), 3.29 (t, J = 7.6 Hz, 2H), 2.79
(t, J = 7.6 Hz, 2H), 2.64 (t, J = 6.9 Hz, 2H), 2.55 (s, 3H), 2.48 (t, J = 7.0 Hz,
2H), 2.25 (s, 3H), 2.21 – 2.09 (m, 2H), 2.00 – 1.95 (m, 2H), 1.73 – 1.16 (m,
19H, overlap with solvent), 0.93 (s, 3H, CH3) 0.91 (s, 3H, CH3), 0.82 (s,
6H, 2x CH3), 0.81 (s, 3H, CH3) ppm. 13C NMR (101 MHz, CDCl3): δ 195.2,
180.7, 172.9, 172.7, 171.9, 170.5, 160.8, 156.9, 144.3, 135.5, 133.4, 128.2,
124.1, 120.8, 116.8, 81.4, 71.1, 70.8, 70.4, 70.2, 70.1, 69.1, 63.7, 56.6,
55.6, 50.4, 42.5, 40.8, 39.5, 38.9, 38.4, 38.0, 37.3, 37.1, 34.4, 33.5, 32.1,
31.1, 30.1, 29.8, 28.2, 27.4, 24.1, 23.8, 21.0, 18.3, 16.7, 16.3, 16.2, 15.2,
14.8, 11.5 ppm. 19F {1H} NMR (471 MHz, CDCl3): δ -145.85 (d, J = 31.8
Procedure for acylation with acetic anhydride. Resin 23 (200 mg) was
swollen in CH2Cl2 (2 mL) for 30 min and then washed with DMF (3x 2 mL)
and CH2Cl2 (3x 2 mL). DMAP (92 mg, 0.75 mmol) was dissolved in CH2Cl2
(2.5 mL) and acetic anhydride (71 µL, 0.75 mmol) was added. The
resulting solution was added to polypropylene fritted syringe with the resin.
The reaction slurry was shaken at ambient temperature overnight, followed
by wash with CH2Cl2 (5x 3 mL). Subsequent cleavage from the resin and
UHPLC-MS analysis confirmed the presence of desired product 33.
Hz), -145.99 (d,
J = 31.8 Hz) ppm. HRMS (ESI): m/z calcd for
C58H83BF2N4O12 [M-H]- = 1075.6069, found [M-H]- = 1075.5985. UV/Vis
(CH2Cl2): λmax = 507 nm; λem = 513 nm; Φ = 0.14.
General procedure E for acylation with triterpene derivatives. Resin
23 (250 mg) was swollen in CH2Cl2 (3 mL) for 30 min and then washed
with DMF (3x 3 mL) and CH2Cl2 (3x 3 mL). To each solution of premodified
triterpenes 6, 8-12, 14 (0.9 mmol) in DMF/CH2Cl2 (1:1, 3 mL, v/v) was
added DIC (70 µL, 0.45 mmol) and the resulting mixture was added to
polypropylene fritted syringe with starting material. The reaction slurry was
shaken at ambient temperature overnight, followed by wash with CH2Cl2
(10x 3 mL). The final compounds were cleaved according to General
Procedure F.
BODIPY-FL-triterpene conjugate 26. Dark red solid (10.7 mg, 15%
overall yield). 1H NMR (500 MHz, CDCl3): δ 7.15 (t, J = 4.7 Hz, 1H), 7.09
(s, 1H), 6.88 (d, J = 3.9 Hz, 1H), 6.32 (t, J = 5.2 Hz, 1H), 6.27 (d, J = 4.0
Hz, 1H), 6.12 (s, 1H), 4.50 – 4.47 (m, 1H), 4.27 – 4.24 (m, 2H), 4.00 (s,
2H), 3.70 – 3.65 (m, 5H), 3.69 (s, 3H, COOCH3) 3.61 – 3.56 (m, 5H), 3.52
– 3.48 (m, 5H), 3.45 – 3.42 (m, 2H), 3.29 (t, J = 7.6 Hz, 2H), 3.21 – 3.17
(m, 1H), 2.79 (t, J = 7.6 Hz, 2H), 2.67 – 2.62 (m, 3H), 2.55 (s, 3H), 2.51 –
2.46 (m, 3H), 2.25 (s, 3H), 2.14 – 1.23 (m, 15H, overlap with solvent), 1.21
(d, J = 1.2 Hz, 3H, CH3), 1.20 (d, J = 1.3 Hz, 3H, CH3), 1.02 (s, 3H, CH3),
0.93 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.82 (s, 3H, CH3)
ppm. 13C NMR (101 MHz, CDCl3): δ 207.4, 175.1, 172.9, 172.7, 171.9,
171.8, 170.4, 160.8, 156.9, 145.9, 144.2, 135.5, 133.4, 128.2, 124.1, 120.8,
116.8, 81.2, 71.1, 70.8, 70.4, 70.2, 70.1, 69.1, 63.7, 55.6, 53.2, 52.7, 51.2,
47.8, 45.4, 41.5, 39.5, 38.8, 38.7, 38.0, 37.3, 35.0, 33.9, 33.5, 31.0, 29.9,
29.6, 28.1, 27.8, 25.3, 24.1, 23.8, 21.4, 20.3, 20.2, 18.3, 17.0, 16.8, 16.7,
16.1, 15.2, 14.3, 11.5 ppm. 19F {1H} NMR (471 MHz, CDCl3): δ -145.71 (d,
J = 34.5 Hz), -145.85 (d, J = 34.5 Hz) ppm. HRMS (ESI): m/z calcd for
C59H85BF2N4O12 [M-H]- = 1089.6225, found [M-H]- = 1089.6141. UV/Vis
(CH2Cl2): λmax = 507 nm; λem = 513 nm; Φ = 0.25.
General procedure F for cleavage from the resin. Cleavage of
intermediates 24-31 and 33 in analytical scale (~5 mg) prior to analysis
was carried out in CH2Cl2/TFA (10:1, 1 mL, v/v) for 30 min according to the
General Information.
Cleavage of intermediates 24-31 and 33 in preparative scale (~250 mg):
The corresponding resin was swollen in CH2Cl2 (3 mL) for 30 min and then
washed with CH2Cl2 (5x 3 mL). Solution of CH2Cl2/TFA (10:1, 3 mL, v/v)
was added to each polypropylene fritted syringe with resin. The reaction
slurry was shaken at ambient temperature for 90 min (28-30) or 2 h (24-
27, 31, 33) and then washed with CH2Cl2/TFA (10:1, 3x 3 mL, v/v) and
CH2Cl2 (3x 3 mL). The cleavage cocktail with combined washes was
evaporated under a stream of nitrogen, the crude products were dissolved
in CH3CN (3 mL) and purified by RP-HPLC to afford final compounds 24-
33.
BODIPY-FL-triterpene conjugate 27. Dark red solid (10.0 mg, 15%
overall yield). 1H NMR (500 MHz, CDCl3): δ 7.17 (t, J = 5.3 Hz, 1H), 7.09
(s, 1H), 6.88 (d, J = 3.9 Hz, 1H), 6.34 (t, J = 5.3 Hz, 1H), 6.27 (d, J = 4.0
Hz, 1H), 6.12 (s, 1H), 4.50 – 4.47 (m, 1H), 4.27 – 4.24 (m, 2H), 4.01 (s,
2H), 3.72 (s, 3H, COOCH3), 3.69 – 3.65 (m, 4H), 3.61 – 3.56 (m, 4H), 3.53
– 3.48 (m, 4H), 3.46 – 3.42 (m, 2H), 3.39 – 3.33 (m, 1H), 3.31 – 3.28 (m,
2H), 2.81 – 2.76 (m, 2H), 2.67 – 2.64 (m, 2H), 2.56 (s, 3H), 2.51 – 2.48 (m,
2H), 2.25 (s, 3H), 2.11 – 1.91 (m, 6H), 1.76 – 1.32 (m, 14H, overlap with
solvent), 1.28 (d, J = 6.9 Hz, 3H, CH3), 1.25 (d, J = 6.9 Hz, 3H, CH3), 1.06
(s, 3H, CH3), 0.97 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.83
(s, 3H, CH3) ppm. 13C NMR (101 MHz, CDCl3): δ 194.5, 189.5, 176.3,
172.9, 172.1, 171.9, 171.2, 170.6, 168.3, 160.8, 156.8, 150.8, 144.3, 135.5,
133.4, 128.1, 124.1, 120.7, 116.7, 81.3, 81.1, 71.1, 70.8, 70.4, 70.2, 70.0,
69.1, 63.7, 58.6, 55.6, 53.6, 51.0, 46.3, 45.7, 41.7, 39.5, 38.7, 38.0, 37.3,
34.7, 33.4, 31.0, 29.9, 29.5, 29.0, 28.6, 28.1, 27.6, 26.1, 24.0, 23.7, 21.2,
20.0, 18.2, 17.0, 16.9, 16.7, 16.3, 15.1, 11.5 ppm. 19F {1H} NMR (471 MHz,
CDCl3): δ -145.04 (d, J = 34.4 Hz), -145.18 (d, J = 34.4 Hz) ppm. HRMS
(ESI): m/z calcd for C59H83BF2N4O13 [M-H]- = 1103.6018, found [M-H]- =
1103.5925. UV/Vis (CH2Cl2): λmax = 507 nm; λem = 513 nm; Φ = 0.26.
BODIPY-FL-triterpene conjugate 24. Dark red crystalline solid (11.4 mg,
14% overall yield). 1H NMR (500 MHz, CDCl3): δ 7.19 (t, J = 5.6 Hz, 1H),
7.09 (s, 1H), 6.87 (d, J = 4.0 Hz, 1H), 6.37 (t, J = 5.5 Hz, 1H), 6.27 (d, J =
4.0 Hz, 1H), 6.11 (s, 1H), 4.73 (d, J = 1.5 Hz, 1H, H29 pro-Z), 4.60 (s, 1H, H29
pro-E), 4.48 – 4.44 (m, 1H), 4.26 – 4.24 (m, 2H), 4.01 (s, 2H), 3.69 – 3.65
(m, 5H), 3.60 – 3.56 (m, 5H), 3.52 – 3.47 (m, 5H), 3.43 (t, J = 5.6 Hz, 2H),
3.28 (t, J = 7.6 Hz, 2H), 3.02 – 2.97 (m, 1H, H19β), 2.79 (t, J = 7.6 Hz, 2H),
2.55 (s, 3H), 2.48 (t, J = 6.9 Hz, 2H), 2.25 (s, 3H), 1.68 (s, 3H, CH3), 1.64
– 1.15 (m, 23H, overlap with solvent), 0.96 (s, 3H, CH3), 0.92 (s, 3H, CH3),
0.83 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.81 (s, 3H, CH3) ppm. 13C NMR (101
MHz, CDCl3): δ 181.0, 172.9, 172.7, 171.9, 170.5, 160.8, 156.9, 150.6,
144.2, 135.5, 133.4, 128.2, 124.1, 120.8, 116.8, 109.9, 81.4, 71.1, 70.8,
70.4, 70.2, 70.1, 69.1, 63.7, 56.5, 55.6, 50.6, 49.4, 47.1, 42.6, 41.0, 40.9,
39.5, 38.8, 38.5, 38.0, 37.3, 34.4, 33.4, 32.3, 31.1, 30.7, 30.0, 29.9, 28.2,
25.6, 24.1, 23.9, 21.1, 19.6, 18.4, 16.7, 16.4, 16.2, 15.2, 14.9, 11.5 ppm.
19F {1H} NMR (471 MHz, CDCl3): δ -145.82 (d, J = 31.5 Hz), -145.96 (d, J
This article is protected by copyright. All rights reserved.

10.1002/chem.201706093
Chemistry - A European Journal
FULL PAPER
BODIPY-FL-triterpene conjugate 28. Dark red solid (21.7 mg, 30%
overall yield). 1H NMR (500 MHz, CDCl3): δ 8.41 (d, J = 2.3 Hz, 1H,
pyrazine), 8.27 (d, J = 2.4 Hz, 1H, pyrazine), 7.08 (s, 1H), 7.07 – 7.04 (m,
1H), 6.87 (d, J = 4.0 Hz, 1H), 6.52 – 6.51 (m, 1H), 6.27 (d, J = 4.0 Hz, 1H),
6.11 (s, 1H), 4.75 (d, J = 1.6 Hz, 1H, H29 pro-Z), 4.64 (d, J = 1.3 Hz, 1H, H29
pro-E), 4.59 (d, J = 15.1 Hz, 1H, OCH2CO), 4.53 (d, J = 15.1 Hz, 1H,
OCH2CO), 4.26 – 4.24 (m, 2H), 3.99 (s, 3H), 3.68 – 3.64 (m, 4H), 3.62 –
3.61 (m, 2H), 3.58 – 3.54 (m, 4H), 3.52 – 3.47 (m, 4H), 3.29 (t, J = 7.6 Hz,
2H), 3.09 (d, J = 16.6 Hz, 1H, H1), 2.97 (dd, J = 11.0, 4.7 Hz, 1H), 2.79 (t,
J = 7.4 Hz, 2H), 2.55 (s, 3H), 2.44 (d, J = 16.6 Hz, 1H, H1), 2.31 – 2.26
(m, 2H), 2.25 (s, 3H), 2.00 – 1.96 (m, 1H), 1.93 – 1.89 (m, 1H), 1.79 – 1.74
(m, 1H), 1.70 (s, 3H, CH3), 1.68 – 1.32 (m, 13H, overlap with solvent), 1.29
(s, 3H, CH3), 1.27 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.79
(s, 3H, CH3) ppm. 13C NMR (101 MHz, CDCl3): δ 174.8, 172.6, 170.1,
167.7, 160.8, 159.9, 156.9, 150.9, 150.7, 150.2, 144.2, 142.5, 141.5, 135.5,
133.4, 128.1, 124.0, 120.7, 116.7, 110.2, 71.1, 70.9, 70.4, 70.0, 69.9, 69.1,
63.7, 62.6, 56.8, 53.2, 49.5, 49.0, 48.8, 46.9, 42.7, 40.7, 39.7, 39.0, 38.8,
38.3, 37.1, 37.0, 33.5, 32.1, 31.7, 30.6, 29.9, 25.6, 24.2, 24.1, 21.6, 20.2,
19.6, 16.3, 15.8, 15.1, 14.8, 11.5 ppm. 19F {1H} NMR (471 MHz, CDCl3): δ
-145.04 (d, J = 31.8 Hz), -145.18 (d, J = 31.8 Hz) ppm. HRMS (ESI): m/z
calcd for C58H81BF2N6O9 [M-H]- = 1053.6126, found [M-H]- = 1053.6134.
UV/Vis (CH2Cl2): λmax = 507 nm; λem = 513 nm; Φ = 0.21.
BODIPY-FL-triterpene conjugate 31. Dark red solid (15.2 mg, 21%
overall yield). 1H NMR (500 MHz, CDCl3): δ 7.19 (t, J = 5.5 Hz, 1H), 7.09
(s, 1H), 6.88 (d, J = 4.0 Hz, 1H), 6.40 (t, J = 5.2 Hz, 1H), 6.27 (d, J = 4.0
Hz, 1H), 6.12 (s, 1H), 5.67 (app s, 1H, H29 pro-Z), 5.29 (app s, 1H, H29 pro-E),
4.27 – 4.24 (m, 2H), 4.03 (s, 2H), 3.71 – 3.66 (m, 4H), 3.64 – 3.50 (m, 10H),
3.29 (t, J = 7.6 Hz, 2H), 3.17 (dd, J = 11.0, 5.1 Hz, 2H), 2.79 (t, J = 7.6 Hz,
2H), 2.55 (s, 3H), 2.25 (s, 3H), 2.22 – 2.13 (m, 2H), 1.96 – 1.84 (m, 3H),
1.67 – 1.34 (m, 15H), 1.25 – 1.16 (m, 4H), 0.95 (s, 6H, 2x CH3), 0.91 (s,
3H, CH3), 0.80 (s, 3H, CH3), 0.74 (s, 3H, CH3) ppm. 13C NMR (101 MHz,
CDCl3): δ 179.8, 172.6, 170.7, 169.1, 160.8, 156.9, 150.7, 144.3, 135.5,
133.4, 128.2, 127.9, 127.2, 125.1, 124.1, 120.8, 116.8, 79.1, 71.3, 70.8,
70.7, 70.3, 70.0, 69.1, 63.6, 56.5, 55.5, 50.9, 50.6, 42.6, 40.9, 39.4, 39.0,
38.9, 38.4, 37.4, 36.8, 34.5, 33.5, 32.9, 32.0, 29.8, 28.2, 27.5, 24.1, 21.1,
18.4, 16.3, 16.2, 15.6, 15.2, 14.9, 11.5 ppm. 19F {1H} NMR (471 MHz,
CDCl3): δ -145.75 (d, J = 35.6 Hz), -145.97 (d, J = 35.6 Hz) ppm. HRMS
(ESI): m/z calcd for C54H79BF2N4O10 [M-H]- = 991.5857, found [M-H]- =
991.5852. UV/Vis (CH2Cl2): λmax = 507 nm; λem = 513 nm; Φ = 0.17.
BODIPY-FL-triterpene conjugate 32. Dark red oil (4.5 mg, 7% overall
yield). 1H NMR (500 MHz, CDCl3): δ 7.17 (t, J = 5.3 Hz, 1H), 7.09 (s, 1H),
6.88 (d, J = 3.8 Hz, 1H), 6.33 (t, J = 5.2 Hz, 1H), 6.27 (d, J = 3.8 Hz, 1H),
6.12 (s, 1H), 4.49 – 4.45 (m, 1H), 4.27 – 4.24 (m, 2H), 4.01 (s, 2H), 3.93
(s, 1H), 3.67 (dd, J = 9.2, 5.3 Hz, 4H), 3.61 – 3.56 (m, 4H), 3.53 – 3.48 (m,
4H), 3.46 – 3.42 (m, 2H), 3.29 (t, J = 7.5 Hz, 2H), 2.79 (t, J = 7.6 Hz, 2H),
2.66 – 2.62 (m, 2H), 2.55 (s, 3H), 2.50 – 2.47 (m, 2H), 2.25 (s, 3H), 1.85
(d, J = 13.4 Hz, 1H), 1.79 (d, J = 11.1 Hz, 1H), 1.72 – 1.18 (m, 24H, overlap
with solvent), 1.02 (s, 3H, CH3), 0.95 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.86
(s, 3H, CH3), 0.85 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.82 (s, 3H, CH3) ppm.
13C NMR (126 MHz, CDCl3): δ 180.1, 172.9, 172.6, 171.8, 170.4, 160.8,
156.9, 144.2, 135.5, 133.5, 128.2, 124.1, 120.8, 116.8, 109.9, 86.2, 81.3,
71.1, 70.8, 70.4, 70.2, 70.1, 69.1, 63.7, 55.8, 51.4, 46.9, 46.3, 40.8, 40.1,
39.5, 38.9, 38.8, 38.1, 37.4, 36.2, 33.9, 33.8, 33.5, 32.5, 32.1, 31.1, 30.1,
28.9, 28.1, 28.1, 26.7, 24.2, 24.1, 23.8, 21.1, 18.2, 16.8, 16.7, 15.7, 15.2,
13.8, 11.5 ppm. 19F {1H} NMR (471 MHz, CDCl3): δ -145.31 (d, J = 31.8
BODIPY-FL-triterpene conjugate 29. Dark red solid (16.7 mg, 20%
overall yield). 1H NMR (500 MHz, CDCl3): δ 8.46 (app s, 1H, pyrazine),
8.28 (d, J = 2.4 Hz, 1H, pyrazine), 7.08 (s, 1H), 7.07 – 7.06 (m, 1H), 6.87
(d, J = 3.9 Hz, 1H), 6.57 (t, J = 5.1 Hz, 1H), 6.27 (d, J = 4.0 Hz, 1H), 6.11
(s, 1H), 4.75 (app s, 1H, H29 pro-Z), 4.64 (app s, 1H, H29 pro-E), 4.60 (d, J =
15.1 Hz, 1H, OCH2CO), 4.53 (d, J = 15.1 Hz, 1H, OCH2CO), 4.26 – 4.23
(m, 2H), 4.00 (s, 4H), 3.68 – 3.46 (m, 24H), 3.28 (t, J = 7.6 Hz, 2H), 3.09
(d, J = 16.7 Hz, 1H), 3.01 – 2.96 (m, 1H), 2.79 (t, J = 7.6 Hz, 2H), 2.55 (s,
3H), 2.46 (d, J = 16.7 Hz, 1H), 2.30 – 2.27 (m, 2H), 2.25 (s, 3H), 2.00 –
1.93 (m, 2H), 1.77 (d, J = 11.5 Hz, 1H), 1.70 (s, 3H, CH3), 1.68 – 1.34 (m,
14H, overlap with solvent), 1.30 (s, 3H, CH3), 1.28 (s, 3H, CH3), 1.01 (s,
3H, CH3), 0.98 (s, 3H, CH3), 0.80 (s, 3H, CH3) ppm. 13C NMR (101 MHz,
CDCl3): δ 174.8, 172.6, 170.1, 167.7, 160.8, 160.4, 157.1, 156.9, 151.0,
150.2, 144.2, 142.7, 135.5, 133.4, 128.2, 124.1, 120.7, 116.8, 110.2, 71.0,
70.9, 70.4, 70.1, 70.0, 69.0, 63.7, 62.6, 56.8, 53.1, 49.5, 48.9, 48.4, 46.9,
42.7, 40.7, 39.7, 39.0, 38.8, 38.3, 37.1, 37.0, 33.5, 32.2, 31.7, 30.6, 29.9,
25.6, 24.2, 24.1, 22.9, 21.6, 20.2, 19.6, 16.3, 15.8, 15.2, 14.8, 14.3, 11.5
ppm. 19F {1H} NMR (471 MHz, CDCl3): δ -145.85 (d, J = 34.2 Hz), -145.99
(d, J = 34.2 Hz) ppm. HRMS (ESI): m/z calcd for C64H92BF2N7O12 [M+H]+
= 1200.6865, found [M+H]+ = 1200.6848. UV/Vis (CH2Cl2): λmax = 507 nm;
λem = 513 nm; Φ = 0.30.
Hz), -145.45 (d,
J = 31.8 Hz) ppm. HRMS (ESI): m/z calcd for
C58H85BF2N4O11 [M-H]- = 1061.6276, found [M-H]- = 1061.6278. UV/Vis
(CH2Cl2): λmax = 507 nm; λem = 513 nm; Φ = 0.20.
BODIPY-FL-2,11-dioxo-6,9,15-trioxa-3,12-diazaheptadecan-17-yl
propionate 33. Dark red crystalline solid (2.49 mg, 11% overall yield). 1H
NMR (500 MHz, CDCl3): δ 7.18 (t, J = 5.5 Hz, 1H), 7.09 (s, 1H), 6.88 (d,
J = 4.0 Hz, 1H), 6.29 – 6.27 (m, 2H), 6.12 (s, 1H), 4.27 – 4.24 (m, 2H),
4.01 (s, 2H), 3.69 – 3.66 (m, 4H), 3.61 – 3.57 (m, 4H), 3.53 – 3.47 (m,
4H), 3.45 – 3.41 (m, 2H), 3.29 (t, J = 7.6 Hz, 2H), 2.79 (t, J = 7.4 Hz, 2H),
2.56 (s, 3H), 2.25 (s, 3H), 1.98 (s, 3H) ppm. 13C NMR (126 MHz, CDCl3):
δ 172.7, 170.6, 170.5, 160.9, 156.9, 144.3, 135.5, 133.5, 128.2, 124.1,
120.1, 116.8, 71.2, 70.9, 70.5, 70.3, 70.1, 69.1, 63.7, 39.5, 38.8, 33.5,
24.1, 23.3, 15.2, 11.5 ppm. 19F {1H} NMR (471 MHz, CDCl3): -145.64 (d,
J = 35.6 Hz), -145.79 (d, J = 35.6 Hz) ppm. HRMS (ESI): m/z calcd for
C26H37BF2N4O7 [M-H]- = 565.2723,
BODIPY-FL-triterpene conjugate 30. Dark red solid (8.5 mg, 12% overall
yield). 1H NMR (500 MHz, CDCl3): δ 7.09 (s, 1H), 7.07 – 7.06 (m, 1H), 6.88
(d, J = 3.9 Hz, 1H), 6.50 – 6.48 (m, 1H), 6.27 (d, J = 4.0 Hz, 1H), 6.12 (s,
1H), 4.73 (app s, 1H, H29 pro-Z), 4.60 (d, J = 2.0 Hz, 1H, H29 pro-E), 4.59 (d, J
= 15.5 Hz, OCH2CO), 4.52 (d, J = 15.1 Hz, 1H, OCH2CO), 4.27 – 4.24 (m,
2H), 3.99 (s, 2H), 3.68 – 3.63 (m, 4H), 3.62 – 3.60 (m, 2H), 3.58 – 3.53 (m,
4H), 3.52 – 3.47 (m, 4H), 3.29 (t, J = 7.6 Hz, 2H), 3.18 (dd, J = 11.4, 4.8
Hz, 1H), 2.99 – 2.94 (m, 1H), 2.79 (t, J = 7.6 Hz, 2H), 2.56 (s, 3H), 2.25 (s,
3H), 2.25 – 2.20 (m, 2H), 1.98 – 1.86 (m, 3H), 1.68 (s, 3H, CH3), 1.65 –
1.15 (m, 19H, overlap with solvent), 0.96 (s, 3H, CH3), 0.96 (s, 3H, CH3),
0.90 (s, 3H, CH3), 0.81 (s, 3H, CH3), 0.75 (s, 3H, CH3) ppm. 13C NMR (101
MHz, CDCl3): δ 174.8, 172.6, 170.1, 167.7, 156.9, 150.3, 144.3, 128.1,
124.1, 120.8, 116.8, 110.1, 79.2, 71.1, 70.9, 70.4, 69.9, 69.1, 63.7, 62.6,
56.8, 55.5, 50.7, 49.6, 47.0, 42.6, 40.9, 39.0, 38.9, 38.8, 38.3, 37.4, 37.1,
34.5, 33.5, 32.2, 30.6, 29.9, 28.2, 27.6, 25.7, 24.1, 21.0, 19.5, 18.5, 16.3,
16.2, 15.5, 15.2, 14.9, 11.5 ppm. 19F {1H} NMR δ -145.76 (d, J = 30.5 Hz),
-145.90 (d, J = 30.5 Hz) ppm. HRMS (ESI): m/z calcd for C56H83BF2N4O10
[M-H]- = 1019.6170, found [M-H]- = 1019.6171. UV/Vis (CH2Cl2): λmax = 507
nm; λem = 513 nm; Φ = 0.17.
found [M-H]- = 565.2736. UV/Vis (CH2Cl2): λmax = 507 nm; λem = 513 nm;
Φ = 0.71.
Acknowledgements
This work was mainly supported by the Czech Science
Foundation (15-05620S), microscopy and part of the salaries
were paid from the Technology Agency of the Czech Republic
(TE01020028), the Czech Ministry of Education, Youth and
Sports (Grant Numbers: LO1304, LM2015063) and internal grant
This article is protected by copyright. All rights reserved.

10.1002/chem.201706093
Chemistry - A European Journal
FULL PAPER
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[1]
[2]
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10.1002/chem.201706093
Chemistry - A European Journal
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Sona Krajcovicova, Jarmila Stankova,
Petr Dzubak, Marian Hajduch, Miroslav
Soural,* and Milan Urban*
Page No. – Page No.
Synthetic approach for rapid
((Insert TOC Graphic here: max.
preparation of BODIPY conjugates
and their use in imaging of cellular
drug uptake and distribution
width: 5.5 cm; max. height: 5.0 cm))
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