Development of a self-immolative linker for tetrazine-triggered release of alcohols in cells

Article abstract of DOI:10.1039/c9ob01167f

Bioorthogonal decaging reactions are a promising strategy for prodrug activation because they involve bond cleavage to release a molecule of interest. The trans-cyclooctene (TCO)-tetrazine inverse electron-demand Diels-Alder reaction has been widely applied in vivo for decaging of amine prodrugs, however, the release of alcohol-containing bioactive compounds has been less well studied. Here, we report a TCO-carbamate benzyl ether self-immolative linker for the release of OH-molecules upon reaction with a tetrazine trigger. The benzyl ether linker proved to be highly stable and can rapidly liberate alcohols under physiological conditions upon reaction with tetrazines. The mechanism and decaging yield were systematically examined by fluorescence and HPLC analysis by using a fluorogenic TCO-benzyl ether-coumarin probe and different 3,6-substituted tetrazine derivatives. This study revealed that decaging occurs rapidly (t_1/2 = 27 min) and the cycloaddition step happens within seconds (t_1/2 = 7 s) with reaction rates of ≈100 M^-1 s^-1. Importantly, the reaction is compatible with living organisms as demonstrated by the decaging of a prodrug of the antibacterial compound triclosan in the presence of live E. Coli, that resulted in complete cell killing by action of the released "OH-active drug". Overall, this work describes a new linker for masking alcohol functionality that can be rapidly reinstated through tetrazine-triggered decaging.

Full text of DOI:10.1039/c9ob01167f

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Development of a self-immolative linker for tetrazine-triggered
release of alcohols in cells
Sarah Davies,a Bruno L. Oliveira,a,b and Gonçalo J. L. Bernardes*a,b
Received 00th January 20xx,
Accepted 00th January 20xx
Bioorthogonal decaging reactions are a promising strategy for prodrug activation because they involve bond cleavage to
release a molecule of interest. The trans-cyclooctene (TCO)-tetrazine inverse electron-demand Diels–Alder reaction has
been widely applied in vivo for decaging of amine prodrugs, however, the release of alcohol-containing bioactive compounds
has been less well studied. Here, we report a TCO-carbamate benzyl ether self-immolative linker for the release of OH-
molecules upon reaction with a tetrazine trigger. The benzyl ether linker proved to be highly stable and can rapidly liberate
alcohols under physiological conditions upon reaction with tetrazines. The mechanism and decaging yield were
systematically examined by fluorescence and HPLC analysis by using a fluorogenic TCO–benzyl ether-coumarin probe and
different 3,6-substituted tetrazine derivatives. This study revealed that decaging occurs rapidly (t1/2 = 27 min) and the
cycloaddition step happens within seconds (t1/2 = 7 secs) with reaction rates of 100 M–1s–1. Importantly, the reaction is
compatible with living organisms as demonstrated by the decaging of a prodrug of the antibacterial compound triclosan in
the presence of live E. Coli, that resulted in complete cell killing by action of the released “OH-active drug”. Overall, this work
describes a new linker for masking alcohol functionality that can be rapidly reinstated through tetrazine-triggered decaging.
DOI: 10.1039/x0xx00000x
www.rsc.org/
dihydropyridazine. They demonstrated that this strategy could be
used to decage a TCO-carbamate-doxorubicin prodrug in cells17 and
later in vivo, by site-specifically releasing amine-containing drugs
Introduction
Inverse electron-demand Diels–Alder (IEDDA) reactions have
been exploited to ligate a variety of reporters (e.g. fluorophores,
affinity tags and PET isotopes) to biological entities for applications
such as cell imaging,1 PROTAC assembly in live cells,2 labelling of post-
PCR DNA3 and pretargeted imaging in vivo.1,4 Typically, these
ligations are performed between a tetrazine and a strained
dienophile,5 such as trans-cyclooctene (TCO) or its analogues d-TCO,6
s-TCO7 and oxoTCO8, with extremely fast kinetics (up to 3.3 x 106 M–
1s–1).9
from antibody-drug conjugates at the tumour site (doxorubicin18 and
monomethyl auristatin E19). In another approach, Mejia Oneto used
a tetrazine-modified hydrogel to site-selectively release Doxorubicin
from a TCO-carbamate prodrug at the tumour.20 Recently, prodrug
activation was achieved by using an enzymatic self-assembly
technique to install the tetrazine trigger inside cancer cells.21 These
approaches demonstrate the in vivo potential of the TCO-tetrazine
IEDDA reaction for prodrug activation of NH2-containing compounds.
Since many drugs do not contain an amine functional group that
is essential for their function, it is necessary to expand decaging
reactions to the release of other functional groups. Although there
are a large number of drugs that contain a hydroxyl group,22
bioorthogonal decaging for release of alcohols has been less
extensively reported than for amines. Several groups have reported
tetrazine-triggered release of alcohols from a vinyl ether handle
(Figure 1a).22–24 This reaction has been applied in live cells for
detection of RNA by using near-infrared fluorogenic probes23,
activation of a duocarmycin prodrug22 and release of doxorubicin-
conjugated nanoparticles.24 However, this reaction suffers from a
slow reaction rate (t1/2 = 58 min),22 which limits the application of this
reaction in vivo. A 3-isocyanopropyl handle for masking amines,
alcohols and thiols has also been reported (Figure 1a).25 The reaction
was successfully applied to trigger the fast release (t1/2 = 37 min in
50% PBS/serum) of an alcohol-containing fluorophore and amine-
containing drug (mexiletine) in zebra-fish embryos and cells,
respectively. However, this reaction is limited for in vivo applications
by the toxicity of the acrolein by-product. Therefore, additional
methods for fast decaging of protected-alcohols are required to
Bioorthogonal decaging reactions, in which a bond is broken to
release a molecule of interest, have increased the breadth of
applications for bioorthogonal chemistry10,11 beyond the study of
biomolecules through ligation reactions.12–14 Indeed, the cleavage of
a bond allows the removal of a protecting group and therefore the
selective activation of a protein, fluorophore or small molecule
drug.10,11,15,16 Although several decaging reactions have been
reported, many of them suffer from slow reaction rates that limit
their application in vivo.10,11 In seminal work, Robillard et al. reported
that the IEDDA reaction could be adapted for fast decaging by placing
an alcohol substituent in the allylic position, where it is appropriately
placed to eliminate upon tautomerisation of the 4,5-
a. Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW
Cambridge, United Kingdom. E-mail: gb453@cam.ac.uk
b. Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de
Lisboa. Av Prof Egaz Moniz, 1649-028 Lisboa, Portugal. E-mail:
gbernardes@medicina.ulisboa.pt
Electronic Supplementary Information (ESI) available: Supplementary Figures, Data
and Methods. See DOI: 10.1039/x0xx00000x
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greatly expand the scope of drugs that can be used for in vivo prodrug by a 1,6-elimination mechanism (Figure 1). Different TCO-benzyl
View Article Online
DOI: 10.1039/C9OB01167F
activation.
ether derivatives were prepared directly from trans-cyclooct-2-en-1-
ol (TCO-OH) via a convenient synthetic route without isomerisation
ocurring (Figure 1b and 1c). The proposed synthesis results in 100%
of trans-isomers and a late stage photochemical isomerisation step
is not required, enabling incorporation of a wider variety of payloads.
The TCO-carbamate benzyl ether linker is highly stable under
biological conditions and was shown to react rapidly with tetrazines
(cycloaddition complete within seconds and decaging half life 30
min). Importantly, the reaction is compatible with living organisms as
demonstrated by prodrug activation in the presence of live E. Coli
cells. The release of an antibacterial drug, triclosan, resulted in
complete cell death due to reinstation of the original bactericidal
activity.
Attempts to apply the promising TCO-tetrazine IEDDA reaction to
release other functional groups have recently been reported. Both
our group26 and that of Robillard27 described TCO-ester prodrugs that
were decaged with tetrazine to release carboxylic acids. Additionally,
Robillard also showed that the TCO handle could be cleaved from
carbonates and ethers to release alcohol-containing molecules
(Figure 1a).27 However, the reported approach is limited by the
synthesis of the TCO-molecule that involves photoisomerization of
the cis-cyclooctene-ether to the trans-isomer by using UV light. This
final step is very low yielding (312% after 1.3–7 days under flow, to
obtain, for example 144 mg from 5.5 g of cis-product) and requires a
specialized flow set-up.27 Although this route is achievable on the
reported model compounds, it is not always feasible to obtain such
a large quantity of cis-product, particularly if this reaction is to gain
more widespread use in the area of drug activation. In addition, the
synthesis of the ether bond is challenging and attempts to form the
ether bond from trans-cycloocten-1-ol resulted in isomerisation to
the cis-form.27
Results and Discussion
Initially, taking inspiration from the reported TCO-carbamate linker,
we proposed an analogous carbonate linker for the release of
alcohols. Fluorescent compound 7-hydroxycoumarin (2) was chosen
as the molecule of interest to enable the kinetics of release and the
stability of the linker to be easily assessed by fluorescence. Model
compound 1 was synthesised by activation of TCO-OH with para-
nitrochloroformate followed by reaction with 2 (See the ESI). Next,
the reaction of model compound 1 with tetrazine in 50% DMSO/H2O
at 30 °C was studied under second-order conditions by measuring
the fluorescence intensity (λex = 325 nm, λem = 460 nm, Figure 2a).
Fig. 2. TCO-carbonate linker for the release of alcohols a. Tetrazine-
triggered release of 7-hydroxycoumarin (2) from carbonate 1. b.
Release of 2 in 50% H2O/DMSO at 30 °C monitored by following the
increase in fluorescence (λex = 320, λem = 465 nm). c. Fittings of the
stability of 1 in PBS, complete cell culture media (DMEM) and 20%
plasma/PBS, followed by the increase in fluorescence. Compound 1
was shown to be stable in 50% H2O/DMSO over the time period of
the decaging reaction (See Figure S1).
Fig. 1. a. Reported decaging reactions for the release of alcohols:
vinyl ether (limited by its slow reaction rate), TCO-ether (fast release,
requires low yielding photoisomerisation in the final step), TCO
carbonate (unstable), 3-isocyanopropyl handle (toxic by-product). b.
This work: TCO-carbamate benzyl ether linker for the release of
alcohols. c. Proposed mechanism of decaging for the TCO-carbamate
benzyl ether linker: Release of the amine from the TCO-carbamate as
previously reported,17 followed by 1,6-elimination to release the
alcohol.
The increase in coumarin fluorescence was complete within 90
minutes (t1/2 = 19 min, Figure 2b), which is a similar order of
magnitude to, but slower than, the release of doxorubicin from the
carbamate linker (complete within 16 min).17 Importantly it was
significantly faster than the previously reported vinyl ether decaging
[with 350 fold excess of vinyl ether, the fastest tetrazine had a
pseudo first-order rate constant (kobs) of approximately 2x10–4 s–1
that corresponds to a half-life of 58 min].22 Carbonate 1 was,
Here we report a novel strategy for the release of “alcohol-
molecules” in which a TCO-carbamate is connected to a benzyl ether
self-immolative linker. We rationalized that upon tetrazine reaction
the formed aniline derivative could drive the release of the alcohol
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addition, use of the proposed TCO-carbamate benzyl ether linker for
DOI: 10.1039/C9OB01167F
decaging should address the instability of the carbonate linker.
however, highly unstable in 20% plasma (t1/2 = 5 min) and cell media
(t1/2 = 45 min) and presented moderate stability in PBS (t1/2 = 193
min, Figure 2c), which prevents its application in biological systems.
This result was consistent with the work of Robillard who recently
reported a TCO-carbonate linker and showed that it underwent 100%
fragmentation after 5 h in 50% mouse serum at 37 °C.27
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Initially, the proposed synthetic route attempted to attach the
model compound, 7-hydroxycoumarin (2) in the final step (Figure 3,
route 1), in order to minimise the amount of payload that is required.
First, 4-aminobenzyl alcohol (3) was reacted with tert-butyl
dimethylsilyl chloride (TBSCl) to give 4 in 77% yield. This was followed
by reaction with triphosgene to generate isocyanate 5 and then
reaction with TCO-OH to give desired carbamate 6 (10% yield over 2
steps). This yield is comparable to the previously reported one-step
reaction of TCO-OH with commercial benzyl isocyanate, which gave
yields of 2137% with reaction times of  3 days.17,28
Given these results, we considered the design of an alternative
decaging reaction for the release of alcohols, in which TCO is
connected to a self-immolative benzyl ether linker through a
carbamate (Figure 1b). This carbamate was expected to eliminate
CO2 and the free amine of the self-immolative linker, which can then
undergo 1,6-elimination to release the free alcohol (Figure 1c). In
Fig. 3. Synthetic routes to TCO-carbamate benzyl ether linkers. Route 1. Conditions: a. TBSCl (1.1 equiv.), imidazole, CH2Cl2, rt, 24 h; b.
triphosgene (0.4 equiv.), NEt3 (1.1 equiv.), toluene, 70 °C, 3 h; c. TCO-OH (0.8 equiv.), NEt3 (0.9 equiv.), toluene, rt, 16 h; d. HCl (1 M), MeOH,
rt, 1 h; e. PBr3 (0.8 equiv.), Et2O, 0 °C, 16 h; f. 7-hydroxycoumarin (2) (1.5 equiv.), Cs2CO3 (2.0 equiv.), MeCN, rt, 10 min. Route 2. Conditions:
g. Boc2O (1.1 equiv.), N,N-diisopropylethylamine (1.0 equiv.), THF, 75 °C, 20 h; h. PBr3 (0.4 equiv.), Et2O, 0 °C, 20 h; i. 7-hydroxycoumarin (2)
(1.5 equiv.), Cs2CO3 (4.5 equiv.), MeCN, rt, 30 min or triclosan (1.1 equiv.), Cs2CO3 (2.0 equiv.), MeCN, rt, 24 h; j. HCl (4 M in dioxane), rt, 15
h; k. triphosgene (0.5 equiv.), dioxane, 60 °C, 516 h; l. TCO-OH (0.5 equiv.), DABCO (3.0 equiv.), toluene, 100 °C, 16 h.
TBS deprotection resulted in 7 with no observable isomerisation (See Figure S3). With these results in hand we decided that this route
of the double bond. However, in the following bromination step with was not synthetically useful since it required the use of a large
PBr3, the double bond isomerised to give the bromide as 70% cis- amount of expensive TCO to give the final compound in low yield
isomer. Isomerisation occurred after 5 min at 0 °C and no further predominantly as the less active cis-isomer, which would then
isomerisation occurred after the reaction was left for 12 h. This require separation by chiral HPLC.26
highlights the difficulty of synthesis involving TCO because the
In a second route, 4-aminobenzyl alcohol (3) was first protected
double bond is highly unstable and readily isomerises. This behaviour
with a tert-butyloxycarbonyl (Boc) group to give 9 in 96% yield (Figure
interferes with the decaging kinetics because the cis-isomer is 7
3, route 2). Reaction with PBr3 resulted in bromide 10, which was
orders of magnitude less reactive towards tetrazines than the trans-
subsequently reacted with 7-hydroxycoumarin (2) and caesium
isomer.29 It was not possible to separate the cis- and trans-isomers
carbonate to give 11a in 20% yield over 2 steps. Next, Boc
of the bromide (owing to its instability on silica), so the mixture of
deprotection was attempted by using bromotrimethylsilane.
isomers was used in the subsequent step. Reaction with 7-
Unfortunately, after removal of the Boc group, the self-immolative
hydroxycoumarin (2) and caesium carbonate gave final product 8 in
linker can undergo 1,6-elimination and although complete
5% yield (over 3 steps) as a mixture cis:trans 7:3. It has been reported
consumption of 11a occurred, no free aniline was observed.
that the trans-isomer selectively binds to AgNO3-impregnated silica
Alternatively, we found that it was possible to generate isocyanate
and this is used to separate the trans-isomer during the
13a from 11a without isolating the free aniline intermediate. This
photochemical synthesis of TCO-OH.30 However, attempts to
step involved the reaction of 11a with HCl (4 M in dioxane) to
separate the isomers of 8 by trapping onto AgNO3-coated silica were
generate intermediate 12a, followed by reaction with triphosgene to
unsuccessful. Interestingly, the product mixture is stable to further
give isocyanate 13a.31 The formation of the anilinium chloride salt
isomerisation for 3 weeks in the light in CDCl3 at room temperature
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proved crucial to prevent elimination and generate the isocyanate obtained for pH 47.4, however no reaction occurs atVpieHw 9Ar,tiwclehOicnhlinies
from the Boc-protected amine, which may be a useful strategy in consistent with previous reports.33
DOI: 10.1039/C9OB01167F
self-immolative linker synthesis. Finally, isocyanate 13a was reacted
with TCO-OH. Dibutyltin dilaurate, a catalyst commonly used for
isocyanate reactions, caused isomerisation of TCO. However, by
using 1,4-diazabicyclo[2.2.2]octane (DABCO) as a catalyst, final
compound 8 was obtained in 8% yield over 3 steps. Again, although
this yield is fairly low, it is similar to previous reactions of TCO-OH
with isocyanates17,28 and is an improvement on the one-step
photochemical isomerisation (312% after 1.3–7 days under flow).27
In addition, it does not require the alcohol in gram-scale, which is
essential when working with expensive drug payloads. Importantly,
the product was obtained entirely as the axial, trans-isomer.
The stability of 8 was then assessed by monitoring the
fluorescence intensity (λex = 325 nm, λem = 460 nm) over 15 h. The
compound proved to be stable for 15 h in PBS, complete cell culture
media (DMEM), LB media and 20% plasma/H2O, with no increase in
fluorescence observed (See Figure S4). Next, we studied the reaction
of 8 with tetrazines 1520 (Figure 4a and b). Quantitative NMR
(qNMR) with benzoic acid as an external standard was used to
accurately determine the concentration of stock solutions of each
reagent (See Figure S5). This was shown to be important for the
determination of the reaction rates by using second-order kinetics in
which the reagents must be equimolar. qNMR can also be used to
determine the concentration of saturated solutions, which is useful
for compounds of low solubility (eg. tetrazine 16). With exact
concentrations determined, the reactants were mixed in a 1:1 ratio
in 50% H2O/DMSO and the reaction mixture was analysed after 24 h
Fig. 4. Kinetics and yields of decaging. a. Tetrazine-triggered release
of alcohol 2 from TCO-coumarin 8. b. Structures of tetrazines 1520
used in this study. c. Decaging yield of the reaction of TCO-coumarin
by High-Performance Liquid Chromatography (HPLC). The highest
decaging yield (39%) was observed with tetrazine 20. Tetrazines 16
and 19 showed particularly low yields (<10%) whereas 15, 17 and 18
resulted in yields of 22, 25 and 32%, respectively (Figure 4c). For this 8 with tetrazines 1520 assessed by HPLC/UV. Concentration of the
reason, along with its higher stability relative to other tetrazines (See
Figure S6), tetrazine 20 was chosen for subsequent studies. It should
be mentioned that decaging yields are often obtained from
monitoring the fluorescence intensity and comparing it to the
maximum obtained after complete decaging of the protected
fluorophore.32 In our case we found that the reaction mixture can
quench the coumarin fluorescence and therefore obtaining a yield by
this method is unreliable (See Figure S7).
released coumarin was determined by using a calibration curve with
known concentrations of coumarin and benzoic acid as an internal
standard (See Figure S11). d. Rate of consumption of tetrazine upon
reaction of 8 with 20, determined by following the decrease in
absorbance (λ = 530 nm) by stopped-flow. e. Release of 2 by reaction
of 8 with 20, determined by following the increase in fluorescence
(λex = 320, λem = 465 nm). f. Decaging yield of the reaction of TCO-
coumarin 8 with 20 determined by HPLC under different conditions
after reaction for 24h.
The kinetics of the reaction of 8 with tetrazine 20 (addition step)
were then assessed by monitoring the decrease of the tetrazine
absorbance at 530 nm using stopped-flow spectrometry (Figureꢀ4d).
The second-order rate constant was found to be 96.4 ± 12.3 M–1s–1
(t1/2 = 7 s) in DMSO. In addition, the rate of decaging was determined
by following the increase in fluorescence over time (Figure 4e). These
studies revealed that the release of coumarin was complete within
120 minutes (t1/2 = 27 min), which is faster than the previously
reported vinyl ether22 and similar to the release yield reported by
Robillard (initial release is complete within 30 min and an additional
10% release occurs after 20 h).27 Importantly, the reaction was also
shown to occur in cell media (t1/2 = 120 min, See Figure S9). The
release of coumarin was also monitored by HPLC coupled to a
fluorescence detector (See Figure S10). Finally, the yield of the
reaction was assessed under different conditions by HPLC analysis.
The yield was shown to be highest in 50% H2O/DMSO, although there
was no clear correlation between water content and yield (Figure 4f).
It should be noted that it was not possible to study the reaction in
>50% H2O owing to the limited solubility of 8. The reaction was also
shown to be pH dependent (Figure 4f); similar yields (2530%) were
Next, we applied this linker for drug release. For proof of concept
studies, we chose antibacterial drug triclosan (TCS, 21). Compound
14 was synthesised according to the previously reported protocol
(Figure 3, route 2). Bromination and coupling of the drug gave 11b in
56% yield and conversion of 11b to 14 in the final 3 steps was
achieved in 18% yield. Again, the product was obtained as the 100%
axial, trans-isomer. The reaction was then studied by HPLC by using
an internal standard and a decaging yield of 22% was observed (See
Figures S12 and S13). In addition, detection of intermediate peaks by
Liquid Chromatography-Mass Spectrometry (LC-MS) analysis
confirmed our proposed mechanism of decaging (See Figure S14).
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flow. The linker was shown to be highly stable in media (DMEM and
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DOI: 10.1039/C9OB01167F
LB) and 20% plasma. Reaction with tetrazine was shown to be
complete within 120 min and through observation of intermediates,
the proposed 1,6-elimination mechanism from the aniline benzyl-
ether derivative was confirmed. Finally, triggered release of an
alcohol-containing antibacterial drug, triclosan (21), was carried out
in the presence of live E. Coli cells and the bactericidal activity was
reinstated, which resulted in complete cell killing. Overall this work
provides a new linker for the release of alcohol-containing drugs. The
significant improvement in kinetics from the previous vinyl ether
handle22 suggests this reaction may hold potential for in vivo prodrug
activation.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Fig. 5. Cell decaging experiments a. Representation of decaging in the
presence of live E. Coli, which results in cell death from release of
antibacterial drug, triclosan (21). b. Representative IC50 curves of
triclosan (21) and the reactant pair 14 + 20 obtained by measuring
the viability using the cell titre blue assay. The experiment was
repeated 3 times and similar results were obtained each time.
Average values of IC50 were found to be 122 ± 10 nM (21) and 298 ±
20 nM (reactant pair 14 + 20) c. Cell viability after treatment of E. Coli
with either 21, 20, 14, 14 + 20. Treatment with bioorthogonal
reactant pair 14 + 20 resulted in complete cell killing. The experiment
was repeated 3 independent times.
We thank the EPSRC (PhD studentship to S.D.), the European
Commission (Marie Sklodowska-Curie Fellowship to B.L.O., GA
No. 702574) and FCT Portugal (iFCT IF/00624/2015 and
02/SAICT/2017, Grant 28333). G.J.L.B. is a Royal Society
University Research Fellow (URF\R\180019). The authors thank
Dr Vikki Cantrill for her help with the editing of this manuscript.
Notes and references
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Finally, the decaging reaction was carried out in the presence of live
bacteria [E. Coli BL21(DE3), Figure 5a]. First, the bactericidal activity
of triclosan (21) was determined by assessing the cell viability at
concentrations of 50 nM1 μM and the IC50 was found to be 122 ±
10 nM (Figure 5b). The bioorthogonal reactant pair, TCO-triclosan 14
+ tetrazine 20 (10 equiv.), was shown to be 3 times less active (IC50
= 298 ± 20 nM) than triclosan (21) alone. This lower activity is due to
the non-quantitative decaging yield. Both the prodrug TCO-triclosan
14 and tetrazine 20 were shown to be non-toxic at all these
concentrations (See Figure S15). After the initial assessment of
toxicity, the decaging reaction was then carried out and viability was
assessed by both cell titre blue assay (Figure 5c) and by measuring
OD600 (See Figure S16). Consistent results were obtained by both
assays. At a concentration of 1 μM, triclosan (21) resulted in
complete cell killing whereas cells treated with either TCO-triclosan
14 or tetrazine 20 were 100% viable. Complete cell death occurred
upon treatment with the bioorthogonal reactant pair 14 + 20.
Therefore, the reinstation of the bactericidal activity of triclosan (21)
was achieved upon decaging in the presence of live cells.
9
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