Tetrazine-Responsive Self-immolative Linkers

Article abstract of DOI:10.1002/cbic.201600560

Molecules that undergo activation or modulation following the addition of benign external small-molecule chemical stimuli have numerous applications. Here, we report the highly efficient “decaging” of a variety of moieties by activation of a “self-immolative” linker, by application of water-soluble and stable tetrazine, including the controlled delivery of doxorubicin in a cellular context.

Full text of DOI:10.1002/cbic.201600560

Accepted Article
Title: Tetrazine Responsive Self-Immolative Linkers
Authors: Kevin Neumann; Sarthak Jain; Alessia Gambardella; Sarah Walker; Elsa
Valero; Annamaria Lilienkampf; Mark Bradley
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Tetrazine Responsive Self-Immolative Linkers
Kevin Neumann, Sarthak Jain, Alessia Gambardella, Sarah E. Walker, Elsa Valero, Annamaria
[
a]
Lilienkampf and Mark Bradley*
Abstract: Molecules that undergo activation or modulation following
the addition of benign external small molecule chemical stimuli have
numerous applications. Here, we report the highly efficient “decaging”
of a variety of moieties by activation of a “self-immolative” linker by
application of a water soluble and stable tetrazine, including the
controlled delivery of Doxorubicin in a cellular context.
common acrylate and acrylamide-based thermally responsive
[17]
materials, and the lack of tissue specificity with respect to pH.
Tetrazines have recently been exploited in a variety of
inverse electron-demand Diels–Alder reactions (DAINV) as a
[
18],[19],[20]
means of conjugating reporters (e.g. fluorophores
and
[
21],[22]
PET isotopes
) to a variety of biological entities such as
[
23]
[24]
[25],[26]
DNA,
targeting peptides,
and antibodies.
Typically,
these bioconjugations are performed between a tetrazine and
Stimuli responsive compounds that undergo molecular
modulation, activation or that switch on a desired physical,
chemical or biological function upon the addition of an external
chemical or physical trigger such as pH, temperature or light,
strained dienophile, such as trans-cyclooctene (TCO), resulting
[27]
in a fast DAINV reaction. Thus Chen developed bioorthogonal
protein activation chemistry, with protection of the catalytic lysine
residue in firefly luciferase with TCO rendering the protein
inactive and treatment with a tetrazine restoring the protein
[
1],[2]
have enormous power and biomedical potential.
Light in
particular has been used, as a stimulus, for various applications
ranging from polymers containing azobenzene units that
undergo reversible cis/trans photoisomerisation resulting in
[28]
function. Tetrazine ligation with TCO has also been utilised for
prodrug activation using Doxorubicin conjugated via
a
carbamate linkage to the allylic position of TCO, with DAINV
[
3]
controllable polymer modulation,
to nanoparticles that
[29]
liberating the free drug.
reversibly contract (150 to 40 nm) upon photo-triggering and
In addition to the widely applied reactivity with strained
alkenes and alkynes, tetrazines have been shown to rapidly
react with cyclopentanone morpholine enamines and N-vinyl
[
4]
light-mediated antibody activation.
Numerous polymer
architectures have been generated where pH can reversibly
[
5]
alter polymer topography by changes in protonation state, or
[30]
pyrrolidinones to liberate amines and amides, respectively.
[
6],[7]
irreversibly via bond cleavage (resulting in cargo liberation).
Recently, it was shown that tetrazines react with phenyl vinyl
Many chemical moieties have been developed that
respond to a range of molecular triggers. For example, boronate
functionalised polymers have been designed to react with
[31]
ethers resulting in liberation of a phenol moiety. This reactivity
directed our attention to the application of tetrazines not only in
the decaging of phenols but also to 1,6-elimination chemistry via
a self-immolative linker approach. Herein, we demonstrate the
selective release of both caged fluorophores and the anticancer
drug Doxorubicin by a tetrazine-mediated vinyl ether based
dienophile, upon a reaction with a DAINV reaction (Figure 1).
[
8]
glucose thereby mediating insulin release and hydrogels that
respond to the presence of specific antigens via swelling and
[
9]
cargo liberation have been synthesised. Li reported the caging
of the catalytic lysine residue in OspF with
propargyloxycarbonyl group, which was cleaved by Pd catalysis
a
[
10]
switching on the protein function.
A key feature of many of
INSERT FIGURE 1 HERE
these activation systems is the integration of a “self-immolative
[
11]
safety-catch”
type linker, whereby remote functional group
Figure 1. A) Nanoparticles with an average diameter of 35 nm were fabricated
from the amphiphilic block-co-polymer PEG-b-Dox, with the methacrylate–
Doxorubicin conjugated segment forming the hydrophobic core. Upon reaction
with tetrazine, doxorubicin (red spheres) is liberated, driven by the 1,6-
elimination reaction of the self-immolative linker. B) The mechanism of
activation leads to subsequent 1,6-elimination and target/cargo
liberation. This includes the work of Urano who used 500 nm
[
12]
light to liberate BODIPY-caged histamine
whereas Springer
applied this approach to generate carboxypeptidase activated
2
tetrazine-mediated vinyl ether decaging and cargo liberation (here RNH ).
[
13]
prodrugs of Doxorubicin (Dox).
Numerous other examples
exist where the 1,6-elimination process has been used as a
response to a variety of analytes and redox states leading to
The designed system comprised a tetrazine (the stimulus) that
decages a phenol(ate) prompting the subsequent release of
Doxorubicin via 1,6-elimination. Additional functionalisation of
[
14],[15],[16]
polymer “depolymerisation”.
Even though these triggers
and materials show huge potential there are still major
challenges, including problems associated with light stimulated
materials due to high tissue absorbance, neurotoxicity for
the linker with
a methacrylate allowed the formation of
amphiphilic PEG-b-Dox co-polymer nanoparticles that, upon
reaction with tetrazine, released Doxorubicin resulting in the
“
switch-on” of cytotoxicity. In the absence of the stimulus, these
[
a]
Kevin Neumann, Sarthak Jain, Alessia Gambardella, Dr. Sarah E.
Walker, Dr. Elsa Valero, Dr. Annamaria Lilienkampf and Prof. Mark
Bradley
EaStCHEM School of Chemistry, University of Edinburgh
Joseph Black Building, David Brewster Road, Edinburgh, EH9 3FJ,
UK.
PEG-b-Dox nanoparticles have low cytotoxicity and therefore
have the potential to improve drug efficacy. Vinyl groups have
been shown to be good dienophiles in DAINV reactions and have
been used to efficiently label and subsequently image 5-vinyl-2’-
[
32]
E-mail: mark.bradley@ed.ac.uk
deoxyuridine modified DNA.
Our hypothesis was that vinyl
ethers could act as masking groups for phenols and that
tetrazine-mediated activation via the incorporation of a self-
Supporting information for this article is given via a link at the end of
the document.
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immolative liker would allow the “switch-on” of fluorophores as
well as enabling targeted drug release. The phenolic groups of
fluorescein 1 and resorufin 2 were readily converted to vinyl
ethers using the vinyl boronic anhydride pyridine complex
To further demonstrate the potential of external small-molecule
controlled cargo release, the activated linker 11 was conjugated
to the anti-cancer agent Doxorubicin (Figure 4A), a DNA
interchelating anthracycline antibiotic used for the treatment of
malignancies including breast and ovarian tumours, sarcomas,
[
33]
reported by O’Shea to give the quenched fluorophores bis-O-
vinyl fluorescein 3 and O-vinyl resorufin 4 (Figure S1–S2).
Incubation with dipyridyl tetrazine 5 allowed efficient removal of
the vinyl groups via the DAINV with the reaction being readily
[
34]
and acute leukemias.
The Doxorubicin monomer 14 showed
>90% conversion to the free drug after 5 day incubation with
tetrazine 5 (Figure 4B). The methacrylate moiety of 14 was
1
-
monitored by H NMR and regeneration of fluorescence, giving a
n
polymerised with the RAFT reagent PEG CTA (M 10,000 g mol
1
23-fold increase in fluorescence with bis-O-vinyl fluorescein 3
)
using APS/TMEDA as the redox initiator to give the
amphiphilic PEG-b-Dox co-polymer 15 (M 13,000 g mol ).
n
-1
and 99-fold increase with O-vinyl resorufin 4 (Figure 2, Figure
S3–S4).
These mild reaction conditions were required as thermally or UV
initiated polymerisations led to co-reaction of the vinyl ether
groups. Once placed in water, the PEG-b-Dox co-polymer 15
formed nanoparticles with a diameter of 35 nm (Figure 4C,
Figure S7), with the hydrophobic core of each particle,
INSERT FIGURE 2 HERE
1
Figure 2. A) Tetrazine 5 triggered decaging of bis-O-vinyl fluorescein 3 and O-
vinyl resorufin 4 at 37 °C in PBS (pH 7.4) led to fluorescence “switch on”. B)
DAINV reaction between tetrazine 5 (200 µM) and 4 (35 µM) led to a 99-fold
increase in fluorescence (λEx/Em 530/590 nm) whereas the same reaction with 3
consisting of four Doxorubicin units (determined by H NMR)
linked to the methacrylate backbone, surrounded by
hydrophilic PEG shell.
a
(35 µM) led to a 23-fold increase (λEx/Em 485/528 nm). C) Monitoring of the
reaction between bis-O-vinyl fluorescein 3 and tetrazine S1 (See Figure S5) in
1
DMSO by H NMR showed the initial formation of the mono O-vinyl fluorescein
with full conversion to 1 after 48 h at 37 °C.
INSERT FIGURE 4 HERE
Figure 4. Synthesis and characterisation of Doxorubicin conjugated
nanoparticles (PEG-b-Dox). A) i) Doxorubicin hydrochloride, Et N, 51 %; ii)
3
To broaden this approach,
immolative linker was designed consisting of a 4-hydroxymethyl
phenyl vinyl ether scaffold, thereby allowing cargo conjugation
a
tetrazine responsive self-
PEG CTA, APS, TMEDA, DMSO, 30 °C. B) Release profile of Doxorubicin
from monomer 14 (2.3 mM) by tetrazine 5 (23 mM) at 37 °C in PBS/ACN as
monitored by HPLC (λAbs 495 nm). C) Size analysis (by dynamic light
scattering) of the PEG-b-Dox derived nanoparticles showing an average
diameter of 35 nm in PBS (pH 7.4) at 37 °C.
(
such as fluorophores or drugs) via carbamate formation, with
remote activation (based on a 1,6-elimination reaction) following
liberation of the phenolate by the tetrazine-mediated removal of
the vinyl ether group (Figure 1B). Furthermore, the phenyl ring
contained a C3 spacer linked to a methacrylate moiety for
polymerisation chemistries. The tetrazine responsive self-
Tetrazine-mediated controlled drug release was explored using
HEK273T cells. The PEG-b-Dox nanoparticles (loading
equivalent to 8 µM of Dox) showed no cytotoxicity after 48 h
immolative linker
tetrahydropyranyl
6
was synthesised by selective para
(
MTT assay), whereas 1 µM “free” Doxorubicin resulted in
(THP) ether protection of 2,4-
complete cell death (Figure S8–S9). Tetrazine 5 showed no
toxicity at a concentration of 35 µM. When the cells were treated
with 1 µM PEG-b-Dox nanoparticles (equivalent to 4 µM of Dox),
the addition of tetrazine 5 (35 µM) triggered cytotoxicity with
dihydroxybenzaldehyde 7 (to give compound 8) and subsequent
ortho etherification using 3-bromopropyl methacrylate to give 9
(
Figure 3A). Following THP deprotection, vinylation of the free
phenol was achieved using the vinyl boronic anhydride pyridine
80% cell death after 48 h incubation (Figure 5, Figure S10).
[
31]
complex
as described above. Finally, aldehyde 10 was
and the resulting linker 6 transformed into
Comparable results were obtained with prostate cancer cell line
PC3, with cytotoxicity of PEG-b-Dox triggered only in
combination with tetrazine (Figure S11). This indicates that the
nanoparticles underwent efficient tetrazine triggering, even in a
complex cellular environment, leading to a controlled switch-on
of cytotoxicity.
reduced with NaBH
4
the nitrophenyl activated carbonate 11. Nile Blue was coupled to
the linker to give the Nile Blue carbamate 12 with quenched
fluorescence (Figure 3B), which upon reaction with tetrazine 5
under aqueous conditions underwent vinyl group removal with
subsequent 1,6-elimination, loss of CO
2
and switch-on of
fluorescence of Nile Blue 13 (Figure 3, Figure S6).
INSERT FIGURE 5 HERE
INSERT FIGURE 3 HERE
Figure 5. Tetrazine triggered release of Doxorubicin. HEK273T cells were
grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
1
0 % FBS. Nanoparticles, tetrazine 5 and/or free Doxorubicin were incubated
with cells at 37 °C with 5 % CO for 48 h. A) Control (just cells); B) PEG-b-Dox
5 nanoparticles (1 µM equiv. of Dox); C) Tetrazine 5 (35 µM); D) Tetrazine 5
35 µM) and PEG-b-Dox 15 nanoparticles (1 µM). The samples were stained
with propidium iodide (2 µM) and analysed by flow cytometry (λEx 488 nm with
00–554 nm broad pass filter). Forward versus side scatter (SSC-A) profiles
were used to gate intact cellular materials and determine membrane integrity
PI).
Figure 3. Synthesis and activation of the tetrazine cleaved self-immolative
linker. a) i) 2,4-Dihydro-2H-pyran, PPTS, DCM, 60 %; ii) 3-Bromopropyl
2
1
(
methacrylate, Cs
Cs CO , vinyl boronic anhydride pyridine complex, Cu(OAc)
NaBH , MeOH, quant.; v) Phenylchloroformate, Et N, 83 %.; vi) Nile Blue 13,
Et N, DCM/THF, rt, 16 %; vii) DAINV of 12 (40 µM) and tetrazine 5 (100 µM) led
2 3
CO , DMF, 50 °C, 77 %; iii) (1) 1M HCl (aq.), MeOH, (2)
2
3
2
, DCM, 55 %; iv)
4
3
5
3
to an 8-fold increase in fluorescence in PBS (pH 7.4) at 37 °C (see Figure S6).
b) Fluorescence spectra of Nile Blue 13 and the fully quenched Nile Blue
carbamate 12 (λEx/Em 590/645 nm).
(
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COMMUNICATION
Kevin Neumann, Sarthak Jain, Alessia
Gambardella, Dr. Sarah E. Walker, Dr.
Elsa Valero, Dr. Annamaria Lilienkampf
and Prof. Mark Bradley*
Page No. – Page No.
Tetrazine Responsive Self-Immolative
Linkers
Doxorubicin was conjugated to PEG-b-Dox nanoparticles via a self-immolative linker, comprised of a
vinyl ether caged phenol that undergoes a 1,6-elimination upon DAINV reaction with a tetrazine,
resulting in cargo release and switch-on of cytotoxicity.
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Fig5
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