iSPAAC: Isomer-Free Generation of a Bcl-xL-Inhibitor in Living Cells

Article abstract of DOI:10.1002/chem.201803032

Strain-promoted azide–alkyne cycloadditions (SPAAC) have proven extremely useful for labeling of biomolecules, but typically produce isomeric mixtures. This is not appropriate for the formation of bioactive molecules in living cells. Here, the first use o

Full text of DOI:10.1002/chem.201803032

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Title: iSPAAC: isomer-free generation of a Bcl-xL-inhibitor in living cells
Authors: Christian Lis, Stefan Rubner, Corinna Gröst, Ralf Hoffmann,
Daniel Knappe, and Thorsten Berg
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iSPAAC: isomer-free generation of a Bcl-xL-inhibitor in living cells
Christian Lis[a], Stefan Rubner[a], Corinna Gröst[a], Ralf Hoffmann[b], Daniel Knappe[b], Thorsten Berg* [a]
Abstract: Strain-promoted azide-alkyne cycloadditions (SPAAC)
have proven extremely useful for labeling of biomolecules, but
typically produce isomeric mixtures. This is not appropriate for the
formation of bioactive molecules in living cells. Here, we report the
first use of SPAAC for the isomer-free synthesis of a bioactive
molecule, both in vitro and inside cultured cells. We developed the
symmetrical cyclooctyne SYPCO and used it for the generation of a
chemically uniform triazole inhibitor of protein-protein interactions
mediated by Bcl-xL via isomer-free SPAAC (iSPAAC). Tumor cells
treated with the reactants of the iSPAAC reaction contained higher
concentrations of triazole, and displayed higher apoptosis levels, than
cells treated with pre-synthesized triazole. We envision iSPAAC as a
broadly applicable method for modulating intracellular targets with
organic molecules with molecular weights prohibitively large for
strain-promoted azide-alkyne cycloaddition (iSPAAC) by
pyrrolocyclooctynes is achieved by placement of the substituent
R1, which is required for functionalization, at the nitrogen atom of
the pyrrole ring annulated at the 5,6-positions of the cyclooctyne.
Substituents at the -positions of the pyrrole have to be placed
symmetrically. iSPAAC is anticipated to enable the intracellular
generation of bioactive molecules, whose molecular weight is
prohibitively large for cellular uptake, by two smaller and thus
more cell-permeable building blocks (Figure 1c).[8] Intracellular
assembly of bioactive molecules by iSPAAC would help to
reconcile the requirement of organic modulators of protein-protein
interactions for a higher molecular weight in order to target large
protein-protein interfaces effectively,[9] with the superior cell
permeability and/or oral bioavailability of smaller compounds.[10]
cellular uptake, via smaller and thus more cell-permeable components. Unfortunately, the sterically demanding tert-butyl groups of
PYRROC precluded its use for creating bioactive molecules by
iSPAAC. In addition, the conformational flexibility conferred by the
Strain-promoted azide-alkyne cycloadditions (SPAAC) have
alkyl linker on the pyrrole nitrogen of PYRROC is typically not
proved extremely useful for bioorthogonal chemistry. While the
beneficial for placement of protein-binding substituents.
reaction between cyclooctyne and phenyl azide was first
described more than half a century ago,[1] the recent application
of SPAAC by Bertozzi’s group[2] for the detection of cell-surface
azido sugars via a biotinylated cyclooctyne has opened up a new
research field. A number of strained cycloalkynes have been
developed for the labeling of biomolecules with fluorophores and
other reporter molecules in living cells.[3] While SPAAC obviates
the need for the toxic copper ions required for copper-catalyzed
azide-alkyne cycloadditions (CuAAC),[4] a significant drawback of
SPAAC is the lack of a preferred relative orientation of the azide
and the cycloalkyne in the transition state. SPAAC with
unsymmetrical cycloalkynes generates regioisomers, typically in
similar ratios[2, 5], while SPAAC with the symmetrical cycloalkyne
BCN[6] generates stereoisomers (Figure 1a). While this inherent
lack of regio- or stereoselectivity is typically considered to be less
relevant for the labeling of biomolecules, it precludes the use of
SPAAC for the intracellular synthesis of bioactive molecules.
Regio- and stereoisomers of bioactive organic molecules
generally have substantially different binding affinities for their
biological targets, and may even display affinities for different
targets altogether; therefore, the interpretation of cell-based data
generated with isomeric mixtures is difficult.
We recently presented the pyrrolocyclooctyne PYRROC as
the first cycloalkyne that, in the reaction with azides, forms
triazoles in an isomer-free manner (Figure 1b).[7] Isomer-free
[a]
M.Sc. C. Lis, M.Sc. S. Rubner,[#] Dr. C. Gröst,[#] Prof. Dr. T. Berg
Leipzig University, Institute of Organic Chemistry,
Johannisallee 29, 04103 Leipzig, Germany
E-mail: tberg@uni-leipzig.de
http://home.uni-leipzig.de/tberg/
[b]
Prof. Dr. R. Hoffmann, Dr. D. Knappe
Figure 1. SPAAC between strained cycloalkynes and azides. a) SPAAC with
non-symmetrical cyclooctynes generates regioisomers; SPAAC with BCN is
expected to generate stereoisomers. b) Isomer-free SPAAC with symmetrical
pyrrolocyclooctynes. c) Concept of intracellular formation of large bioactive
ligands from two smaller fragments by iSPAAC.
Leipzig University, Institute of Bioanalytical Chemistry and Center
for Biotechnology and Biomedicine (BBZ)
Deutscher Platz 5, 04103 Leipzig, Germany
These authors contributed equally.
[#]
Supporting information for this article is given via a link at the end of
the document.
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To facilitate isomer-free generation of bioactive molecules by
iSPAAC, we designed and synthesized the symmetrical
pyrrolocyclooctyne SYPCO (1, Figure 2a). SYPCO, like
PYRROC,[7] does not form isomers in the reaction with azides
(Figure 1b). However, unlike PYRROC, SYPCO does not display
sterically demanding substituents in the -positions of the pyrrole
ring, and allows for direct placement of the functional substituent
R1 on the pyrrole nitrogen. Synthesis of SYPCO started with the
pyrrolocyclooctene 2, which can be prepared on a multigram
scale as previously described.[7] Reduction of its pyrrole moiety
[11]
with NaCNBH3
and re-installation of the tert-butoxycarbonyl
(Boc) group afforded the 3-pyrroline 3, which was tetrabrominated
to yield 4. Treatment with KOtBu in THF recreated the pyrrole
system in the vinyl bromide 5. A second elimination step with
KOtBu in diethylether / n-hexane and catalytic amounts of 18-
crown-6 provided SYPCO (1) in 5.5 % total yield over 5 synthetic
steps based on 2.
SYPCO (1) displayed a second-order rate constant of 0.020
± 0.004 M-1s-1 in the reaction with benzyl azide in acetonitrile as
1
analyzed by H-NMR (Figure S1 in the Supporting Information),
which is three-fold lower than the rate constant of PYRROC
(k = 0.060 ± 0.004 M-1s-1).[7] A drop in rate constant could be
advantageous in this context, increasing the likelihood that the
reactants cross the cell membrane before cycloaddition takes
place. Only one peak was observed for the benzylic protons of the
resulting triazole, confirming the absence of regioisomers. Further
support for isomer-free triazole formation in SPAAC with SYPCO
(1) arises from its reaction with a chiral azide derived from N-tert-
butoxycarbonyl protected L-alanine, which showed only one
singlet for the tert-butyl group in the 1H-NMR spectrum (Figure S1
in the Supporting Information). In contrast, the tert-butyl group of
the triazole obtained by reacting the same chiral azide with BCN
1
(Figure 1a) accounts for two singlets in the H-NMR spectrum,
consistent with the formation of diastereoisomers (Figure S1 in
the Supporting Information).
ABT-737 (Figure 2b) is a potent inhibitor of the protein-
protein interactions mediated by Bcl-xL and other anti-apoptotic
Bcl-2 family proteins.[12] It binds to the BH3 domain of Bcl-xL over
an extended surface area and has a high molecular weight of 813
g/mol. While it is still cell-permeable in tissue culture, its low oral
bioavailability has precluded its further development as an anti-
tumor agent.[13] We chose ABT-737 as a model system to explore
the concept of creating a large triazole inhibitor from two smaller
components, i.e. a functionalized cycloalkyne and an azide, in
vitro and in living cells (Figure 1c), and to assess whether
assembly of the inhibitor by iSPAAC increases the intracellular
concentration as compared to treatment of cells with pre-
synthesized triazole. We chose to replace the central N-phenyl
piperazine moiety of ABT-737 by the fused ring system created
by the SPAAC reaction with SYPCO, resulting in the design of the
ABT-737 mimetic 6 (Figure 2b). Functionalization of SYPCO (1)
with the 4-chloro biphenyl group required for 6 was achieved by
deprotonation of the pyrrole nitrogen of 1 and subsequent
acylation with the methyl ester 7 to afford 8 (Figure 2c). The azide
9 required for the generation of 6 was synthesized from
sulfonamide 9h, which was obtained in a 8-step synthesis in a
modified procedure for the acylsulfonamide part of ABT-737
(Figure 2c, Scheme S1 in the Supporting Information).[14] iSPAAC
between 8 and 9 afforded the ABT-737 mimetic 6 (Figure 2b,c).
Figure 2. SYPCO facilitates the rational design of a Bcl-xL inhibitor by iSPAAC.
a) Synthesis of SYPCO (1). b) Structures of ABT-737 and its triazole-based
mimetic 6. c) Synthesis of the iSPAAC reactants 8 and 9.
Docking of 6 into the co-crystal structure of the Bcl-xL/ABT-
737 complex[15] with AutoDock Vina[16] indicated a very similar
binding mode to that of ABT-737 (Figure 3a). 6 was found to
inhibit the interaction between Bcl-xL and the Bak BH3 domain
with an IC50 of 37.7 ± 3.5 µM as analyzed in a competitive binding
assay based on fluorescence polarization (Figure 3b). The azide
9 and, especially, the cycloalkyne 8, displayed significantly
weaker effects (Figure 3b). In order to investigate whether
iSPAAC between 8 and 9 is templated by Bcl-xL, we analyzed the
reaction rate in the absence or presence of Bcl-xL by reversed
phase (RP)-HPLC. The second-order rate constant at room
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binding of a fluorescent-labeled Bak-derived peptide to Bcl-xL analyzed by
fluorescence polarization (n = 3). c) RP-HPLC analysis of the reaction between
8 and 9 in the absence of Bcl-xL (n=3), in the presence of Bcl-xL (n=4), and in
the presence of both Bcl-xL and ABT-737 (n = 3).
temperature in the absence of protein was determined as 1.14 ±
0.19 M-1 s-1 (Figure 3c, Figure S2 in the Supporting Information),
which represents a 50 to 60-fold rate acceleration as compared to
the reaction of 1 with benzyl azide in acetonitrile (k = 0.020 ±
0.004 M-1s-1, Figure S1 in the Supporting Information). Previous
reports of rate accelerations in SPAAC caused by shifting from a
In the presence of Bcl-xL, the rate constant for formation of
6 (k = 2.10 ± 0.23 M-1 s-1, Figure 3c, Figure S2 in the Supporting
Information) was significantly higher than in the absence of Bcl-xL
[p = 0.0044 (t-test, two-tailed, unpaired)]. To exclude the
possibility that the increased rate constant in the presence of
protein was caused by non-specific effects, we also analyzed the
rate in the presence of both Bcl-xL and ABT-737, which occupies
the Bcl-xL BH3 domain.[15] Under these conditions, the rate
constant was virtually the same as in the absence of protein (k =
1.19 ± 0.08 M-1s-1, Figure 3c, Figure S2 in the Supporting
Information). These data indicate that iSPAAC between 8 and 9
is templated by the BH3 domain of Bcl-xL, an effect which is
expected to support formation of 6 by intracellular iSPAAC (Figure
1c). Although the observed template effect is weak, it opens up
the possibility of creating more active derivatives of 6 by kinetic
target-guided synthesis.[20] iSPAAC between 8 and 9 at the
physiological temperature of 37 °C using 40 µM or 80 µM of
reactants in the absence of Bcl-xL provided the triazole 6 in yields
of 64 ± 9 % and 84 ± 5 % respectively after 24 h (Figure S2 in the
Supporting Information).
purely organic solvent to a mixture of organic solvent and water
5b, 5c, 17]
have involved up to 28-fold increases,[3b,
while rate
accelerations exceeding 100-fold have been predicted for
changing from an entirely organic solvent to an entirely aqueous
environment.[17] Our previous work also revealed a dramatically
accelerated conversion of an identical pair of SPAAC reactants
by changing the solvent from acetonitrile to an entirely aqueous
buffer.[7] Nevertheless, it is also conceivable that the higher rate
of reaction between 8 and 9, as compared to the reaction between
1 and benzyl azide, is partially caused by the electronic
differences between the reagents.[18] As expected, triazole 6
eluted as a single peak in the HPLC (Figure S2 in the Supporting
Information), consistent with its isomer-free formation from 8 and
the chiral azide 9.
Inhibition of Bcl-xL by ABT-737 has been reported to induce
apoptosis in K562 human leukemia cells.[21] Similarly, K562 cells
treated with pre-synthesized 6 at concentrations of 40 and 80 µM
resulted in dose-dependent induction of apoptosis (Figure 4a,
Figure S3 in the Supporting Information). Treatment of K562 cells
with an equimolar mixture of cycloalkyne 8 and azide 9 (at 40 µM
and 80 µM each) for 24 h resulted in a higher induction of
apoptosis than treatment of cells with pre-synthesized 6 at the
same concentration. Cells treated with the mixture of 8 and 9 at
40 µM showed the same degree of apoptosis as cells treated with
pre-synthesized 6 at 80 µM. Either 8 or 9 alone did not
significantly increase the apoptotic rate, excluding the possibility
that the induction of apoptosis by the mixture of 8 and 9 was
caused by the individual iSPAAC components. While further
biological studies would be required to validate the Bcl-xL-
dependency of the induction of apoptosis in leukemia cells by 6,
the data are consistent with the notion that, at the same molarity,
the concentration of 6 inside K562 cells is higher in cells treated
with a mixture of 8 and 9 than in cells treated with pre-synthesized
triazole 6.
To investigate this possibility further, K562 cells were
treated with either the mixture of 8 and 9, or with pre-synthesized
6 (all compounds at 80 µM) for 12 h. This shorter incubation
period was chosen in order to reduce the degree of membrane
instability and apoptotic cell death induced by 6, which would
hamper data analysis. Subsequently, cells were lysed, and the
intracellular content of 6 was analyzed via RP-HPLC. Lysates of
cells treated with the mixture of 8 and 9 contained 2.2-fold higher
concentrations of 6 than lysates of cells treated with pre-
synthesized 6, in parallel experiments carried out at the same time
under otherwise identical conditions (p = 0.004, Fig. 4b, c). The
chemical identity of 6 in lysates from cells treated with 8 and 9
was confirmed by RP-HPLC coupled mass spectrometric (MS)
analysis (Fig. S4 in the Supporting Information). The more than
Figure 3. a) Docking of 6 into the ABT-737-binding pocket of Bcl-xL. The position
of ABT-737 (yellow) in the co-crystal structure (PBD ID 2YXJ)[15] is shown for
comparison. The figure was created with PyMol.[19] b) Effect of 6, 8, and 9 on
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two-fold higher concentrations of 6 found in cells treated with the
reactants 8 and 9, compared to cells treated with pre-synthesized
6, indicate that over half of the molecules of 6 in lysates of cells
treated with the mixture of 8 and 9 must be formed by intracellular
iSPAAC. In the hypothetical scenario where 8 and 9 undergo
iSPAAC exclusively in the tissue culture medium, with a 100 %
yield, the maximum possible intracellular concentration of 6 would
be equal to that observed in cells treated with pre-synthesized 6.
The results of the cell-based assays can only be explained by the
reactants 8 and 9 having a superior cell permeability as compared
to 6, with a major proportion of iSPAAC occuring inside the cell.
possibilities to label biomolecules in living cells. The isomer-free
variant iSPAAC, using symmetrical strained cycloalkynes such as
SYPCO, could similarly become widely used for the intracellular
generation of bioactive molecules with higher molecular weights,
via smaller and thus more cell-permeable components. Potential
applications of this methodology include the isomer-free,
intracellular synthesis of large bioactive molecules such as
protein-protein interaction inhibitors, protein dimerizers,[22]
,
bivalent ligands that target two separate domains within the same
protein,[23] and PROTACS consisting of both a ligand of E3
ubiquitin ligases and of the protein targeted for degradation.[24]
Since the concept of iSPAAC doubles most of the cut-off
parameters defined by the Lipinski rules for orally bioavailable
drugs, it may enable fundamentally new avenues in the
development of drugs against challenging molecular targets.[10]
Future challenges will involve the development of iSPAAC
reagents with optimized steric demands, electronic properties,
and inherent reactivities for the respective applications.
Acknowledgements
This work was generously supported by the Deutsche
Forschungsgemeinschaft (BE 4572/2-1), and the European Union
and the Free State of Saxony, European Regional Development
Fund. We thank the Core Unit Fluorescence-Technologies of the
Interdisciplinary Centre for Clinical Research (IZKF) Leipzig at the
Faculty of Medicine of the University of Leipzig (Kathrin Jꢀger and
Andreas Lꢁsche). We extend our thanks to the NMR core facility
(Lothar Hennig) for support with the NMR-based kinetic analysis.
Keywords: apoptosis • bioorthogonal chemistry • cyclooctynes •
inhibitors • protein-protein interactions
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Figure 4. a) Rate of apoptosis in K562 cells treated with the individual SPAAC
reactants 8 or 9, the ABT-737 mimetic 6, or a mixture of 8 and 9 (n = 3). Error
bars represent standard deviations (SD). ** p < 0.01 (t-test, two-tailed, paired).
b) HPLC analysis of lysates from cells treated with either 8, 9, 6, or a mixture of
8 and 9 at 80 µM of each component. c) Relative concentrations of 6 in lysates
from cells treated with either pre-synthesized 6 (80 µM) or a mixture of the
iSPAAC reactants 8 and 9 (80 µM each) in parallel experiments (n = 4). Error
bars represent standard deviations (SD). **p < 0.01 (t-test, two-tailed, paired).
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generate
a rationally designed inhibitor of protein-protein
interactions mediated by Bcl-xL, both in vitro and in living cells, via
isomer-free SPAAC. To our knowledge, our data represent the
first isomer-free generation of a bioactive molecule by SPAAC.
The first application of SPAAC to living systems by the
Bertozzi group[2] triggered a large number of research studies in
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Entry for the Table of Contents
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Isomer-free zone: The formation of
isomers in strain-promoted azide-
alkyne cycloadditions (SPAAC) has
precluded their use for the formation
of bioactive molecules in living cells.
Here, we present the first isomer-
free generation of a bioactive
Christian Lis, Stefan Rubner, Corinna
Gröst, Ralf Hoffmann, Daniel Knappe,
Thorsten Berg*
Page No. – Page No.
iSPAAC: isomer-free generation of a
Bcl-xL-inhibitor in living cells
molecule by SPAAC. The
cycloalkyne SYPCO facilitated the
intracellular synthesis of a rationally
designed inhibitor of protein-protein
interactions mediated by Bcl-xL via
isomer-free SPAAC (iSPAAC).
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