Acetylene functionalized BODIPY dyes and their application in the synthesis of activity based proteasome probes

Article abstract of DOI:10.1016/j.bmcl.2007.09.025

The synthesis of three acetylene functionalized BODIPY dyes is described. These dyes are used to fluorescently modify an azido functionalized epoxomicin analogue employing the Huisgen 1,3-dipolar cycloaddition, resulting in a panel of fluorescent epoxomic

Full text of DOI:10.1016/j.bmcl.2007.09.025

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6169–6171
Acetylene functionalized BODIPY dyes and their application
in the synthesis of activity based proteasome probes
Martijn Verdoes,ꢀ Ulrik Hillaert,ꢀ Bogdan I. Florea,
Myra Sae-Heng, Martijn D. P. Risseeuw, Dmitri V. Filippov,
Gijsbert A. van der Marel and Herman S. Overkleeft*
Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
Received 20 August 2007; revised 3 September 2007; accepted 5 September 2007
Available online 8 September 2007
Abstract—The synthesis of three acetylene functionalized BODIPY dyes is described. These dyes are used to fluorescently modify an
azido functionalized epoxomicin analogue employing the Huisgen 1,3-dipolar cycloaddition, resulting in a panel of fluorescent epox-
omicin derived proteasome probes.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The ease of detection by direct in-gel readout and visu-
alization in living cells make fluorophores powerful
tools in activity based proteomics. Tagging a probe with
a fluorescent dye enables rapid assessment of the label-
ing profile, and in certain examples allows monitoring
of intracellular localization of the target proteins. Tag-
ging can be achieved by connecting the dye to the activ-
ity based probe directly1,2 or in a two-step labeling
fashion.3 Boradiazaindacenes (BODIPY dyes)4 are fluo-
rescent dyes with attractive properties, such as their
photochemical stability, high molar absorptivity, high
fluorescence quantum yield, and the fact that their fluo-
rescence properties can be altered by varying the substi-
tution pattern on the core and the flanking pyrroles.
Symmetrical BODIPY dyes are relatively easy to access
by condensation of an acid chloride with two equiva-
lents of any given 1H-pyrrole.5 We envisaged that con-
densing a variety of pyrroles with 6-heptynoic acid
chloride would lead to a panel of optical diverse fluores-
cent dyes bearing a terminal alkyne. These dyes can be
used to fluorescently label an azido functionalized inhib-
itor in a chemo selective manner employing the copper
(I) catalyzed Huisgen 1,3-dipolar cycloaddition (‘click’
reaction),6 gaining potential activity based profiling
probes.
The natural product epoxomicin is a potent and selec-
tive proteasome inhibitor.7 The selectivity of epoxomicin
for the proteasome is governed by the a0,b0-epoxyketone
warhead, which, upon reaction with the N-terminal
threonine of one of the three catalytically active b-sub-
units of the proteasome, forms a morpholine ring.8
Because of the potency and selectivity of epoxomicin,
fluorescently labeled analogues thereof could be very
useful proteasome probes. Herein we describe the syn-
thesis of three alkyne functionalized BODIPY dyes
and the application of these in the synthesis of three
fluorescently labeled epoxomicin analogues.
The synthesis of the alkyne functionalized BODIPY
dyes commenced with the treatment of 6-heptynoic acid
(1) with oxalyl chloride (Scheme 1). Crude acid chloride
2 was condensed with either 2-(4-methoxyphenyl)-1H-
pyrrole (3a), 3-ethyl-2,4-dimethyl-1H-pyrrole (3b) or
2,4-dimethyl-1H-pyrrole (3c), followed by treatment
with DiPEA and BF3 etherate, giving BODIPY dyes
4a (pink), 4b (orange), and 4c (green). The extinction
coefficients e of the three new dyes were determined to
be in the range of that reported for commercial BODI-
PY-TMR (around 60,000, see Scheme 1).
Next, we synthesized azido functionalized epoxomicin
analogue 10 (Scheme 2). Tripeptide 5 bearing an N-ter-
minal azido acetyl cap was prepared by standard Fmoc-
based solid phase peptide synthesis. Treatment of 5 with
TMS-diazomethane gave methyl ester 6, which in turn
was transformed into hydrazide 7 upon treatment with
Keywords: Fluorophore; Proteasome; Inhibitor; Bioconjugate.
*
Corresponding author. Tel.: +31 71 5274342; fax: +31 71 5274307;
e-mail: h.s.overkleeft@chem.leidenuniv.nl
These authors contributed equally to this work.
ꢀ
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.025

6170
M. Verdoes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6169–6171
excess hydrazine. In situ generation of the acyl azide
using t-BuONO under acidic conditions and subsequent
neutralization with DiPEA and treatment with leucine
epoxyketone 8 gave protected epoxomicin analogue 9
as a single diastereomer (block-coupling of the leucine
epoxyketone 8 with peptide 5 using BOP as a condensat-
ing agent resulted in a mixture of diastereomers due to
epimerization at the threonine a-position). After depro-
tection, azidomicin 10 was reacted with the alkyne func-
tionalized BODIPY dyes under the influence of CuSO4
and sodium ascorbate resulting in the three fluorescent
peptide epoxyketones 11a, 11b, and 11c in good to excel-
lent yields.
Having three fluorescently labeled epoxomicin ana-
logues in hand, we set out to establish their labeling pat-
tern. Lysates of the murine EL4 cell line (expressing
both the constitutive proteasome and the immunopro-
teasome) were incubated with increasing concentrations
of 11a–c at 37 ꢁC. After one hour the proteins were
denatured and separated on SDS–PAGE, and the wet
gel slabs were scanned on a fluorescence scanner. All
three fluorescent epoxomicin analogues revealed bands
of labeled proteins, the molecular weight of which corre-
spond to the proteolytically active proteasomal b-sub-
units (Fig. 1A–C).2 Although there are no big
differences in potency, 11a seems to be the least potent
probe of the three. It should be noted that the efficiency
of proteasome labeling is the combined result of binding
affinity and the extinction coefficient of the probes, and
proteasome probe 11a contains the BODIPY dye that
has the lowest e value.
Scheme 1. Synthesis and spectroscopical data of acetylene function-
alized BODIPY dyes. Reagents and conditions: (a) oxalylchloride
(1.5 equiv), DMF (cat.), Tol., 3 h; (b) i—1 M 2 in DCE, 3a–c
(2.1 equiv), 2 h 65 ꢁC; ii—BF3ÆOEt2 (5 equiv), DiPEA (4 equiv), 4a
21%, 4b 14%, 4c 26%.
The cell permeability of the three probes was scrutinized
by exposing living EL4 cells to increasing concentrations
of 11a–c for 2 h. After washing, the cells were lysed, the
proteins denatured, and separated on SDS–PAGE. The
scans revealed a marked difference in cell permeability
between the three probes (Fig. 1D–F). Probe 11b seems
to be better cell permeable than 11a, bearing the biggest
and most hydrophobic BODIPY dye of the three. The
smallest and the least hydrophobic epoxomicin analogue
(11c) appears to be best capable of crossing the cell
membrane and labeling the proteasome.
In conclusion, we have synthesized three acetylene func-
tionalized BODIPY dyes 4a (pink), 4b (orange), and 4c
(green), and used these in the synthesis of fluorescently
labeled epoxomicin analogues 11a–c. Our work results
in the addition of three more fluorescently labeled pro-
teasome probes to the proteasome profiling toolkit.2,7–
10
Apart from this, the alkyne functionalized BODIPY
dyes can be used to conjugate to any azide containing
activity based profiling probe and azido modified
metabolite, potentially leading to valuable fluorescent
biochemical tools.11 Varying the substitution pattern
on the core and the flanking pyrroles of the BODIPY
not only changes the fluorescence properties of the
dye, but also has a dramatic effect on the bioavailability
of the fluorophore. Currently, our lab is focussing on the
two-step labeling of azido modified target proteins in the
proteome using the acetylene functionalized BODIPY
dyes.
Scheme 2. Synthesis of fluorescent epoxomicin analogues 11a–c.
Reagents and conditions: (a) TMS-diazomethane (2 equiv), MeOH/
Tol. (1:1), 15 min, 97%; (b) hydrazine monohydrate (60 equiv), MeOH,
reflux, 37%; (c) i—t-BuONO, HCl, dioxane/DMF; ii—DiPEA, 8, 11%;
(d) TFA, 30 min; (e) 4a–c, CuSO4 (10 mol %), sodium ascorbate
(15 mol %), t-BuOH/Tol./H2O (1:1:1), 80 ꢁC, 12 h, 11a 91%, 11b 82%,
11c 65%, two steps from 9.

M. Verdoes et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6169–6171
6171
2. Verdoes, M.; Florea, B. I.; Menendez-Benito, V.; May-
nard, C. J.; Witte, M. D.; van der Linden, W. A.; van den
Nieuwendijk, A. M. C. H.; Hofmann, T.; Berkers, C. R.;
van Leeuwen, F. W.; Groothuis, T. A.; Leeuwenburgh, M.
A.; Ovaa, H.; Neefjes, J. J.; Filippov, D. V.; van der
Marel, G. A.; Dantuma, N. P.; Overkleeft, H. S. Chem.
Biol. 2006, 13, 1217.
3. (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007; (b)
Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem.
Soc. 2003, 125, 4686.
4. (a) Treibs, A.; Kreuzer, F.-H. Liebigs Ann. Chem. 1968,
718, 208; (b) Vos de Wael, E.; Pardoen, J. A.; van
Koeveringe, J. A.; Lugtenburg, J. Recl. Trav. Chim. Pays-
Bas 1977, 96, 306; (c) Karolin, J.; Johansson, L. B.-A.;
Strandberg, L.; Ny, T. J. Am. Chem. Soc. 1994, 116, 7801.
5. Li, Z.; Mintzer, E.; Bittman, R. J. Org. Chem. 2006, 71,
1718.
6. (a) Huisgen, R.; Szeimies, G.; Moebius, L. Chem. Ber.
1967, 100, 2494; (b) Tornøe, C. W.; Christensen, C.;
Meldal, M. J. Org. Chem. 2002, 67, 3057; (c) Rostovtsev,
V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. Engl. 2002, 41, 2596.
7. (a) Hanada, M.; Sugawara, K.; Kaneta, K.; Toda, S.;
Nishiyama, Y.; Tomita, K.; Yamamoto, H.; Konishi, M.;
Oki, T. J. Antibiot. 1992, 45, 1746; (b) Sin, N.; Kim, K. B.;
Elofsson, M.; Meng, L.; Auth, H.; Kwok, B. H.; Crews, C.
M. Bioorg. Med. Chem. Lett. 1999, 9, 2283; (c) Meng, L.;
Mohan, R.; Kwok, B. H.; Elofsson, M.; Sin, N.; Crews, C.
M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10403; (d) Kim,
K. B.; Myung, J.; Sin, N.; Crews, C. M. Bioorg. Med.
Chem. Lett. 1999, 9, 3335.
8. Borissenko, L.; Groll, M. Chem. Rev. 2007, 107, 687.
9. (a) van Swieten, P. F.; Samuel, E.; Hernandez, R. O.;
van den Nieuwendijk, A. M. C. H.; Leeuwenburgh, M.
A.; van der Marel, G. A.; Kessler, B. M.; Overkleeft,
H. S.; Kisselev, A. F. Bioorg. Med. Chem. Lett. 2007,
17, 3402; (b) Berkers, C. R.; Verdoes, M.; Lichtman,
E.; Fiebiger, E.; Kessler, B. M.; Anderson, K. C.;
Ploegh, H. L.; Ovaa, H.; Galardy, P. J. Nat. Methods
2005, 2, 357; (c) Ovaa, H.; van Swieten, P. F.; Kessler,
B. M.; Leeuwenburgh, M. A.; Fiebiger, E.; van den
Nieuwendijk, A. M. C. H.; Galardy, P. J.; van der
Marel, G. A.; Ploegh, H. L.; Overkleeft, H. S. Angew.
Chem., Int. Ed. Engl. 2003, 42, 3626; (d) Kessler, B.
M.; Tortorella, D.; Altun, M.; Kisselev, A. F.; Fiebiger,
E.; Hekking, B. G.; Ploegh, H. L.; Overkleeft, H. S.
Chem. Biol. 2001, 8, 913; (e) Bogyo, M.; McMaster, J.
S.; Gaczynska, M.; Tortorella, D.; Goldberg, A. L.;
Ploegh, H. L. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
6629.
Figure 1. In-gel readout of fluorescently labeled proteasome subunits
in EL4 lysates (A–C) and living EL4 cells (D–F). (A–C) EL4 lysates
(10 lg total protein) were incubated with the indicated concentrations
of 11a (A), 11b (B), or 11c (C) for 1 h at 37 ꢁC and resolved by SDS–
PAGE. (D–F) EL4 cells (1 · 106 cells) were exposed to the indicated
concentrations of 11a (D), 11b (E), or 11c (F) for 2 h at 37 ꢁC. The cells
were harvested and lysed and the proteins were resolved by SDS–
PAGE.
Acknowledgments
This work was supported by The Netherlands Organiza-
tion for Scientific Research (NWO) and The Nether-
lands Proteomics Centre. Thanks to Kees Erkelens
and Fons Lefeber for assistance with NMR.
10. See for recent publications on proteasome inhibitors: (a)
Marastoni, M.; Baldisserotto, A.; Trapella, C.; Gavioli,
R.; Tomatis, R. Bioorg. Med. Chem. Lett. 2006, 16, 3125;
(b) Fu, Y.; Xu, B.; Zou, X.; Ma, C.; Yang, X.; Mou, K.;
Fu, G.; Lu, Y.; Xu, P. Bioorg. Med. Chem. Lett. 2007, 17,
1102; (c) Baldisserotto, A.; Marastoni, M.; Trapella, C.;
Gavioli, R.; Ferretti, V.; Pretto, L.; Tomatis, R. Eur.
J. Med. Chem. 2007, 42, 586; (d) Verdoes, M.; Florea, B.
I.; van der Linden, W. A.; Renou, D.; van den Nie-
uwendijk, A. M.; van der Marel, G. A.; Overkleeft, H. S.
Org. Biomol. Chem. 2007, 5, 1416; (e) Ling, T.; Macherla,
V. R.; Manam, R. R.; McArthur, K. A.; Potts, B. C. Org.
Lett. 2007, 9, 2289.
Supplementary data
Experimental procedures and analytical data for all
compounds are available. Supplementary data associ-
ated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2007.09.025.
References and notes
1. For some recent examples see: (a) Sexton, K. B.; Witte, M.
D.; Blum, G.; Bogyo, M. Bioorg. Med. Chem. Lett. 2007,
17, 649; (b) Yee, M. C.; Fas, S. C.; Stohlmeyer, M. M.;
Wandless, T. J.; Cimprich, K. A. J. Biol. Chem. 2005, 280,
29053.
11. (a) van Swieten, P. F.; Leeuwenburgh, M. A.; Kessler, B.
M.; Overkleeft, H. S. Org. Biomol. Chem. 2005, 3, 20;
(b) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol.
2005, 1, 13.