Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells

Article abstract of DOI:10.1002/anie.201609607

The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol-containing molecules and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.

Full text of DOI:10.1002/anie.201609607

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201609607
German Edition: DOI: 10.1002/ange.201609607
Drug Delivery
Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in
Cells
Ester Jimꢀnez-Moreno, Zijian Guo, Bruno L. Oliveira, InÞs S. Albuquerque, Annabel Kitowski,
Ana Guerreiro, Omar Boutureira, Tiago Rodrigues, Gonzalo Jimꢀnez-Osꢀs, and
GonÅalo J. L. Bernardes*
Abstract: The cleavage of a protecting group from a protein or
drug under bioorthogonal conditions enables accurate spatio-
temporal control over protein or drug activity. Disclosed herein
is that vinyl ethers serve as protecting groups for alcohol-
containing molecules and as reagents for bioorthogonal bond-
cleavage reactions. A vinyl ether moiety was installed in a range
of molecules, including amino acids, a monosaccharide, a fluo-
rophore, and an analogue of the cytotoxic drug duocarmycin.
Tetrazine-mediated decaging proceeded under biocompatible
conditions with good yields and reasonable kinetics. Impor-
tantly, the nontoxic, vinyl ether duocarmycin double prodrug
was successfully decaged in live cells to reinstate cytotoxicity.
This bioorthogonal reaction presents broad applicability and
may be suitable for in vivo applications.
deprotection of a genetically encoded caged cysteine could be
[3]
used to reveal the active native protein in live cells.
[
4]
Similarly, palladium-mediated depropargylation,
phos-
[5]
phine-mediated Staudinger reduction, and tetrazine-trig-
gered inverse electron-demand Diels–Alder (IEDDA) elim-
[6]
ination reactions were successfully employed to restore the
activity of proteins bearing a caged lysine residue in the active
site. Bond-cleavage reactions are also attractive for drug-
delivery applications. Palladium-catalyzed deprotection of
a 5-fluoroacil prodrug was shown as a method for controlled
[7]
drug release in vivo. The IEDDA reaction between
a tetrazine and a caged doxorubicin derivative efficiently
[
8]
releases the cytotoxic drug. Strategies based on IEDDA
elimination reactions with tetrazines are particularly attrac-
tive for decaging relevant molecules in cells and interrogating
biology, because of the favorable kinetics and the abiotic
nature of tetrazines when compared to photo- and metal-
catalyzed reactions. One limitation, however, has been the
breadth of protecting groups available for stable, yet condi-
tionally reversible linkages. Typically, IEDDA elimination
reactions have been used with strained alkene protecting
groups connected through a carbamate, thus resulting in
B
ioorthogonal chemistry for covalently conjugating syn-
thetic molecules at a predefined protein residue has been
[1]
a major focus of research in the past two decades. Very
recently, focus has been placed on reactions which can instead
[
2]
cleave specific bonds under bioorthogonal conditions. This
strategy holds great potential for the precise spatiotemporal
[
1c,2]
control of protein function in vivo.
For example, photo-
[2,9]
a cascade release of a primary amine (Figure 1a).
Fur-
thermore, the reduced metabolic stability of strained alkenes
constitutes a major caveat for its utility. For instance, cis-
cyclooctene easily isomerizes to the non-reactive trans-cyclo-
octene, thus limiting the efficiency of the decaging process in
[
*] Dr. E. Jimꢀnez-Moreno, Z. Guo, Dr. B. L. Oliveira, Dr. O. Boutureira,
Dr. G. J. L. Bernardes
Department of Chemistry, University of Cambridge
Lensfield Road, CB2 1EW Cambridge (UK)
E-mail: gb453@cam.ac.uk
[
10]
cells.
I. S. Albuquerque, A. Kitowski, A. Guerreiro, Dr. T. Rodrigues,
Dr. G. J. L. Bernardes
Instituto de Medicina Molecular, Faculdade de Medicina
Universidade de Lisboa
Avenida Professor Egas Moniz, 1649-028 Lisboa (Portugal)
E-mail: gbernardes@medicina.ulisboa.pt
Dr. G. Jimꢀnez-Osꢀs
Departamento de Quꢁmica, Universidad de La Rioja
Centro de Investigaciꢂn en Sꢁntesis Quꢁmica
26006 LogroÇo (Spain)
and
Institute of Biocomputation and Physics of Complex Systems (BIFI)
University of Zaragoza, BIFI-IQFR (CSIC)
Zaragoza (Spain)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201609607.
ꢃ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
Figure 1. a) Tetrazine-mediated decaging of amines from strained
alkenes connected through a carbamate linker. b) Decaging of alcohols
from vinyl ethers triggered by c) tetrazines. d) Vinyl-ether-caged alco-
hols studied.
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
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Herein, we report the development of a vinyl ether/
tetrazine system as IEDDA reaction partners for the traceless
decaging of alcohol-containing molecules. We demonstrate
the broad applicability of this reaction on several chemotypes,
including the protected amino acids serine and tyrosine, an
tography; Table 1). Of note, the vinyl ether reagents were
generally stable under biocompatible conditions (PBS pH 7.4
at 378C) over 8 hours, as assessed by HPLC/UV (Table 1; see
the Supporting Information). While some degree of instability
was observed for 1c, for instance, the free hydroxy-containing
molecule was not detected.
1
,6-anhydro sugar, a fluorophore, and a drug. Importantly, the
reaction proceeds under physiological conditions (aqueous
buffer pH 7.4 and 378C) and was applied to activate a potent
toxic derivative of the drug duocarmycin in cancer cells.
To harness the current chemical biology toolbox and
develop a broadly applicable technology for the controlled
release of alcohol-containing chemotypes, we set out to
develop bioorthogonal bond-cleavage reactions. In particular,
we envisaged that vinyl ethers could efficiently mask both
aliphatic and aromatic hydroxy groups, and be used for
traceless release of alcohols through a tetrazine IEDDA
bond-cleavage reaction. In fact, the reactivity of the vinyl
After confirming the stability of the vinyl ether molecules
in pH 7.4 buffer and their successful tetrazine-mediated
decaging, we proceeded to study the reaction mechanism in
detail through quantum mechanics at the M06-2X/6-31 + G-
(d,p) level of theory (Figure 2a; see the Supporting Informa-
tion). Unlike tetrazine reactions with highly reactive strained
[
8]
alkenes, our data indicate that the first step, that is, the
IEDDA cycloaddition, is the rate-limiting step of the reaction
°
À1
(TS1, DG ꢀ 25 kcalmol ; reaction time ca. 3 days) followed
°
À1
by very fast retro-Diels Alder (TS2anti, DG ꢀ 7 kcalmol )
°
À1
and phenoxy group cleavage (TS3, DG ꢀ 11 kcalmol ).
Once the 4-phenoxy-4,5-dihydropyridazine (int2) is obtained,
it readily tautomerizes into 4-phenoxy-1,4-dihydropyridazine
(int4), which can swiftly decage through an elimination
reaction. Of note, other dihydropyridazine tautomers pre-
[11]
group with tetrazines has been detailed in organic synthesis
and such a reactive pair was very recently employed to
[12]
visualize and detect RNA under bioorthogonal conditions.
We first used commercial phenyl vinyl ether 1a as a model
compound, and tetrazine 2a to challenge our decaging
hypothesis (Figure 1b–d). After reaction with 2a, phenol
(3a) and 3,6-di(pyridin-2-yl)pyridazine (4a) were obtained in
4
9% and 61% yield, respectively (Table 1). The reaction was
performed in dichloromethane to ensure that all the reagents
were soluble and at room temperature with only two molar
equivalents of tetrazine. Increasing the amount of tetrazine
showed no significant improvement in reaction yield. To
assess the scope of the reaction we then synthetized vinyl
[13]
ether derivatives 1b–e. Tyrosine and serine residues play
a paramount role in the building of binding pocket architec-
ture and controlling catalytic cycles, for example, as in
tyrosine kinases and serine proteases. Indeed, bioorthogonal
decaging of catalytically crucial residues is emerging as
[
2]
a disruptive technology in chemical biology. Furthermore
chromone-based fluorophores and sugars can efficiently be
[
14]
used to interrogate biological systems. Remarkably, decag-
ing vinyl ether derivatives of such molecules 1b–e with 2a
gave the corresponding free hydroxy derivatives in good
yields (50–68% yields after purification by column chroma-
Table 1: Stability of vinyl ethers 1a-e and their decaging with tetrazine
2
a.
[
c]
[d]
Vinyl
ethers
Yield
Conv.
[%]
Stability
[%]
[
a]
Alcohol
Pyridazine
[
b]
[e]
1
1
1
1
1
a
61
68
57
50
65
49
65
72
47
50
100
73
100
56
100
Figure 2. a) Proposed mechanism based on quantum mechanics for
the IEDDA cycloaddition of 1a and 2a, followed by in situ alcohol
b
c
d
e
100
77
°
release. Only the relevant activation free energies (DG ) are shown.
100
n.d.
The initial cycloaddition is the rate-limiting step. After very fast
nitrogen cleavage, different dihidropyridazine tautomers int2–int4
equilibrate before irreversibly decaging to the experimentally obtained
products (3a and 4a). See Figure S4 in the Supporting Information for
[f]
100
[
a] The reactions were performed in dichloromethane at room temper-
ature with 1 equiv of vinyl ether (100 mm) 1a–e and 2 equiv of tetrazine
a (200 mm) for 72 hours. [b] The reaction was complete after 40 hours.
c] Yield of isolated product. [d] The stability (as % of remaining starting
1
2
[
the whole calculated minimum energy pathway. b) H NMR release
studies of 1a upon reaction with 2a. The reaction was performed at
3 mm of 1a and 2a in 10% D O/CD CN. The reaction was monitored
compound) was assessed by HPLC in PBS pH 7.4 at 378C with
a concentration of vinyl ether of 200 mm using acetophenone as an
internal standard. [e] 10% H O in DMF. [f] 1e does not absorb in the UV.
2
3
for 96 h. While the reaction was not always complete at 96 h, the
results obtained were consistent with the mechanism supported by the
theoretical calculations. Ph=phenyl, Py=pyridine.
2
n.d.=not determined.
2
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[6,8]
viously reported to undergo decaging (int3), are unreactive
in this reaction. To support the theoretical data, we mixed 1a
1
and 2a in 10% D O in CD CN, and recorded the H NMR
2
3
spectra at selected times to gain insight into the reaction
mechanism (Figure 2b). Peaks in the aromatic region
assigned to the final products could be identified after
2
4 hours. Conversely, no peaks in the d = 2.5–6 ppm region
could be attributed to int2, and the intermediate species int3
and int4 were observed over the course of the experiment.
Given that no intermediate species were identified through
1
[8]
H NMR analysis, unlike amines decaging from carbamates,
our data clearly supports that the Diels–Alder cycloaddition
is rate-limiting in this case. A fast and irreversible decaging
step after IEDDA cycloaddition is thus responsible for the
experimental observations, and fully in line with the predicted
reaction coordinate diagram (see Figure S4 in the Supporting
Information). Furthermore, our spectral data also confirm
that no reaction intermediates are trapped, and that all of
them evolve to a common tautomer before the decaging step
Figure 3. a) General protocol for 2c-mediated intracellular decaging of
(
Figure 2b).
fluorogenic coumarin 1d. b) Cytotoxicity dose-response curves of 2c
and 1d in HepG2 cells, obtained after 48 hours of exposure. c) Detec-
tion of fluorescent coumarin (blue) upon tetrazine decaging inside
HepG2 cells by confocal microscopy. Cells were incubated for 5 hours
with 25 mm 1d and then for 4 hours with 10 mm of 2c (bottom panel)
or equivalent vehicle control (top panel). Before image acquisition,
nuclei were stained with Syto61 (red). Scale bar represents 20 mm.
With a detailed assessment of the reaction mechanism, we
then performed kinetic studies by following the decrease of
the tetrazine absorbance, that is, the rate-limiting step, in the
visible region. We used 1a and screened tetrazines 2a–c,
which included different substituents (Figure 1 and Table 2).
Table 2: Kinetics of the reaction of 1a with the tetrazines 2a–c.
[
a]
À4 À1 À1
k2 ꢄ10
2
Dienophile
m s
HepG2 cells at the concentrations used (Figure 3b; see the
Supporting Information for A549 cells). In short, after
incubation with 25 mm of 1d for 5 hours, 2c (10 mm) was
added to the cells for 4 hours. At this time, cells were imaged
using confocal microscopy and the turn-on fluorescence of the
released coumarin was recorded as a result of the successful
tetrazine decaging of the vinyl ether protecting group
installed in 1d (Figure 3c). Importantly, this study highlights
the biocompatibility of this approach for turn-on live cell
imaging applications.
2
2
2
2
a
b
c
a
1a
1a
1a
3.92Æ0.11
0.063Æ0.013
5.37Æ0.13
1890Æ40
5-norbornen-2-ol
[a] The reactions were performed in 10% H O in DMF and were followed
2
by UV through the decay of UV absorption of the tetrazines. An excess of
50–350 fold of 1a was used. In the case of 5-norbornen-2-ol the kinetic
rate was determined using the same solvent system with 2a with a 20- to
00-fold excess of 5-norbornen-2-ol.
1
1
The targeted delivery of drugs to diseased tissues remains
a topic of intensive study, and an unsolved issue in modern
drug discovery. Currently, alcohol-containing drugs account
for approximately 50% of all small FDA-approved chemical
entities (cf. DrugBank v5.0; see the Supporting Information),
thus providing ample opportunity for the design of innovative
drug delivery constructs. As a proof-of-concept for our
technology we assessed the spatiotemporal delivery of
a duocarmycin-like natural product (5; Figure 4a). Duorca-
Stability studies of 1a in the system solvent used (10% H O in
DMF) showed no significant degradation after 8 hours. As
expected, tetrazines bearing electron-withdrawing groups
2
(2a) led to faster reactions compared to the one bearing
electron-donating ones (2b). Finally, 2c proved to have the
fastest kinetics, probably because of the reduced steric
hindrance brought about by hydrogen-substituted tetra-
[
15]
zines. We also compared the kinetics of the vinyl ether/
tetrazine decaging with a very reactive, strained alkene as
reference, 5-norbornen-2-ol. The second-order rate constant
[
17]
mycins are isolated from Streptomyces spp. bacteria and
have attracted considerable attention as payloads in anti-
À1 À1
[18]
determined (k = 0.189m s ) in 10% H O in DMF, although
body–drug conjugates, given their potent cytotoxic activity.
2
2
lower, compares well with literature values for the same
Interestingly, halogen-bearing duorcamycin cytotoxics
undergo a Winstein spirocyclization reaction to afford the
bioactive cyclopropanyl, a DNA-alkylating species. This
feature has been explored in antibody-directed enzyme
prodrug therapy, where a nontoxic glycosydic derivative of
duocarmycin is activated by a conjugate of an enzyme and
a tumor-specific antibody. To demonstrate that our tetra-
zine-mediated IEDDA cleavage of vinyl ethers could be
applied for the traceless release of an alcohol-containing drug,
À1 À1
[16]
reaction (k = 1.3m
s
in H O at 208C). The differences
2
2
observed are attributed to the faster IEDDA reactions in
polar protic solvents.
To show the potential bioorthogonality of this bond-
cleavage reaction, we next studied its use for the decaging of
a vinyl ether fluorogenic probe in live cells (Figure 3a). To
this end, we used a vinyl ether nonfluorescent coumarin
derivative (1d) and 2c, as both were shown to be nontoxic to
[
19]
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4
6.5 hour period (twice the doubling time of these cells and
comparable to our cytotoxicity studies; Figure 4b,e). Satisfy-
ingly, we observed that at 10 mm, the product formed upon
tetrazine-decaging of 5 is as toxic as 7 alone (Figure 4 f; see
the Supporting Information for identical study on HepG2
cells), thus suggesting complete drug activation in cells.
Hence, this data advocates that tetrazine-mediated bond
cleavage of vinyl ethers may be used for the traceless release
of alcohol-containing drugs.
In summary, we described a vinyl ether/tetrazine pair as
IEDDA reaction partners for the efficient traceless decaging
of alcohol-containing molecules in live cells. Considering the
wealth of hydroxy groups in chemical probes and drugs,
coupled to the need of circumventing adverse drug reactions,
the spatiotemporal delivery method disclosed herein may find
broad applicability in chemical biology and molecular med-
icine by unraveling new biology and leveraging the controlled
modulation of (patho)physiological events. Additionally, and
in combination with strategies for the genetic encoding of
vinyl-ether-protected tyrosine and serine derivatives, this
tetrazine IEDDA decaging reaction is likely to find use for
precise control of protein function in vivo.
Figure 4. a) The N-Ac CBI double prodrug 5 reacting with 2c leading
to the formation of the intermediate 6 which undergoes a Winstein
spirocyclization to afford the bioactive cyclopropanyl 7. b) General
protocol for 2c-mediated intracellular decaging of 5. c) HPLC time-
course of the reaction between 5 and 2c. d) Half maximal inhibitory
concentration (IC ) of 2c, 5, and 7 in A549 and HepG2 cells.
5
0
e) Cytotoxicity fitted dose-response curves of 2c, 5, and 7 in A549
cells, obtained after 46 hours 30 min of exposure. f) Cytotoxic effects
of intracellular activation of the prodrug 5 by 2c inside A549 cells. For
data on HepG2 cells, see the Supporting Information.
Experimental Section
Decaging of vinyl ether duocarmycin prodrug in vitro: The N-Ac
CBI prodrug 5 was diluted in PBS pH 7.4 to a final concentration of
100 mm from a 10 mm stock in acetonitrile. Then the benzoic acid
tetrazine 2c was added to a final concentration of 500 mm from
a 50 mm stock in DMSO. The reaction was performed at 378C and was
monitored by HPLC/UV at different times until completion.
Decaging of vinyl ether duocarmycin double prodrug 5 in cells:
Cells were incubated with increasing concentrations of 5 or equiv-
alent vehicle controls for 24 h. The culture medium was then
exchanged to complete medium supplemented with increasing
concentrations of tetrazine 2c, drug or equivalent vehicle controls.
Cells were incubated for another 46.5 h until proceeding with the
CellTiter-Blue Cell Viability Assay (Promega). Relative fluorescence
units (R.L.U.) were normalized to the values obtained for the
appropriate vehicle controls. Bars represent the average of 3
independent experiments and error bars represent standard error of
the mean (SEM).
we synthetized the N-Ac-double prodrug 5 in three steps from
the N-Boc-protected 1,2,9,9a-tetrahydrocyclopropa[1,2-c]-
benz[1,2-e]-indol-4-one (CBI) starting material (see the
[20]
Supporting Information for details). We chose this simple
duocarmycin analogue featuring only an acetyl group at-
tached to the DNA-alkylating CBI core because it has been
[
21]
shown to be very toxic to rapidly replicating cells.
We
envisioned that upon tetrazine IEDDA deprotection of the
vinyl ether, the halogen prodrug 6 would be readily formed
and undergo a rapid Winstein spirocyclization reaction to
afford active drug 7. This tetrazine-triggered cascade forma-
tion of an active drug through an intermediate prodrug is
[
22]
known as the double prodrug concept.
Acknowledgments
Next we studied the stability of the double prodrug 5 in
PBS pH 7.4 at 378C using HPLC. While species 6 and active
drug 7 were not formed, we detected some degree of
degradation of 5 over time (of note, formation of neither 6
or 7 was observed). Importantly, 5 was found to be less toxic
when compared with the active drug 7 in both HepG2 and
A549 cells (Figure 4d,e; see the Supporting Information).
Having a suitable masked vinyl ether double prodrug in hand,
we performed a decaging reaction under physiological con-
ditions (PBS pH 7.4 at 378C) with 2c. Remarkably, close to
complete formation of 7 was achieved after 7 hours at 378C in
PBS pH 7.4, with the short-lived species 6 as an intermediate
We thank the European Commission (Marie Skłodowska-
Curie ITN Protein Conjugates; Marie Skłodowska-Curie IEF
to E.J.M. and B.L.O.; Marie Curie IEF to O.B.), China
Scholarship Council (PhD studentship to Z.G.), FCT Portugal
(FCT Investigator to G.J.L.B.), MINECO (CTQ2015-70524-
R and RYC-2013-14706 to G.J.O.), and the EPSRC for
financial support. We also thank BiFi (Memento cluster) for
computer support. G.J.L.B. is a Royal Society University
Research Fellow and the recipient of a European Research
Council Starting Grant (TagIt).
(
Figure 4c). After successful demonstration of decaging of 5
under physiologically relevant conditions, we next proceeded
to evaluate the feasibility of this approach for the tetrazine-
mediated drug-delivery. A549 cells were first incubated with 5
for 24 hours, after which time 2c was added for an additional
Conflict of interest
The authors declare no conflict of interest.
4
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Communications
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Keywords: caged compounds · ethers · heterocycles ·
drug delivery · fluorescent probes
Seitz, H. Meier, C. Methfessel, J. Med. Chem. 2001, 44, 47 – 57;
e) D. R. Soenen, J. M. Zimpleman, D. L. Boger, J. Org. Chem.
003, 68, 3593 – 3598; f) A. Hamasaki, R. Ducray, D. L. Boger, J.
2
Org. Chem. 2006, 71, 185 – 193.
[
12] H. Wu, S. C. Alexander, S. Jin, N. K. Devaraj, J. Am. Chem. Soc.
2016, 138, 11429 – 11432.
[
13] a) Y. Okimoto, S. Sakaguchi, Y. Ishii, J. Am. Chem. Soc. 2002,
[
1] a) E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48,
974 – 6998; Angew. Chem. 2009, 121, 7108 – 7133; b) K. Lang,
1
6
24, 1590 – 1591; b) M. Blouin, R. Frenette, J. Org. Chem. 2001,
6, 9043 – 9045.
6
J. W. Chin, Chem. Rev. 2014, 114, 4764 – 4806; c) N. Krall, F. P.
da Cruz, O. Boutureira, G. J. L. Bernardes, Nat. Chem. 2016, 8,
[
14] a) S. T. Laughlin, C. R. Bertozzi, Proc. Natl. Acad. Sci. USA
009, 106, 12 – 17; b) L. Xue, I. A. Karpenko, J. Hiblot, K.
2
103 – 113.
Johnsson, Nat. Chem. Biol. 2015, 11, 917 – 923.
15] M. R. Karver, R. Weissleder, S. A. Hilderbrand, Bioconjugate
Chem. 2011, 22, 2263 – 2270.
16] K. Lang, L. Davis, J. Torres-Kolbus, C. Chou, A. Deiters, J. W.
Chin, Nat. Chem. 2012, 4, 298 – 304.
[
[
2] J. Li, P. R. Chen, Nat. Chem. Biol. 2016, 12, 129 – 137, and
references therein.
[
[
[
3] D. P. Nguyen, M. Mahesh, S. J. Elsꢀsser, S. M. Hancock, C.
Uttamapinant, J. W. Chin, J. Am. Chem. Soc. 2014, 136, 2240 –
2243.
17] I. Takahashi, K. Takahashi, M. Ichimura, M. Morimoto, K.
Asano, I. Kawamoto, F. Tomita, H. Nakano, J. Antibiot. 1988, 41,
[
4] J. Li, J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M. Yang,
S. Jia, X. Zhang, P. R. Chen, Nat. Chem. 2014, 6, 352 – 361.
5] J. Luo, Q. Liu, K. Morihiro, A. Deiters, Nat. Chem. 2016, 8,
1915 – 1917.
[
[
18] R. C. Elgersma, R. G. E. Coumans, T. Huijbregts, W. M. P. B.
Menge, J. A. F. Joosten, H. J. Spijker, F. M. H. de Groot,
M. M. C. van der Lee, R. Ubink, D. J. van den Dobbelsteen,
D. F. Egging, W. H. A. Dokter, G. F. M. Verheijden, J. M. Lem-
mens, C. M. Timmers, P. H. Beusker, Mol. Pharm. 2015, 12,
1027 – 1034.
[
[
6] J. Li, S. Jia, P. R. Chen, Nat. Chem. Biol. 2014, 10, 1003 – 1005.
7] J. T. Weiss, J. C. Dawson, K. G. Macleod, W. Rybski, C. Fraser, C.
Torres-Sꢁnchez, E. E. Patton, M. Bradley, N. O. Carragher, A.
Unciti-Broceta, Nat. Commun. 2014, 5, 3277.
8] R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen, M. S.
Robillard, Angew. Chem. Int. Ed. 2013, 52, 14112 – 14116;
Angew. Chem. 2013, 125, 14362 – 14366.
9] a) R. Rossin, S. M. J. van Duijnhoven, W. ten Hoeve, H. M.
Janssen, L. H. J. Kleijn, F. J. M. Hoeben, R. M. Versteegen, M. S.
Robillard, Bioconjugate Chem. 2016, 27, 1697 – 1706; b) G.
Zhang, J. Li, R. Xie, X. Fan, Y. Liu, S. Zheng, Y. Ge, P. R.
Chen, ACS Cent. Sci. 2016, 2, 325 – 331; c) J. M. Mejia Oneto, I.
Khan, L. Seebald, M. Royzen, ACS Cent. Sci. 2016, 2, 476 – 482.
1813 – 1835.
[
[
[
[
19] L. F. Tietze, J. M. von Hof, M. Mꢂller, B. Krewer, I. Schuberth,
Angew. Chem. Int. Ed. 2010, 49, 7336 – 7339; Angew. Chem.
2010, 122, 7494 – 7497.
[
20] D. M. Tercel, S. M. Stribbling, H. Sheppard, B. G. Siim, K. Wu,
S. M. Pullen, K. J. Botting, W. R. Wilson, W. A. Denny, J. Med.
Chem. 2003, 46, 2132 – 2151.
21] a) P. A. Aristoff, P. D. Johnson, D. Sun, L. H. Hurley, J. Med.
Chem. 1993, 36, 1956 – 1963; b) M. Tercel, S. M. Stribbling, H.
Sheppard, B. G. Siim, K. Wu, S. M. Pullen, K. J. Botting, W. R.
Wilson, W. A. Denny, J. Med. Chem. 2003, 46, 2132 – 2151.
22] H. Bundgaard, Adv. Drug Delivery Rev. 1989, 3, 39 – 65.
[
10] R. Rossin, S. M. van den Bosch, W. ten Hoeve, M. Carvelli,
R. M. Versteegen, J. Lub, M. S. Robillard, Bioconjugate Chem.
[
2013, 24, 1210 – 1217.
[
11] a) S. Sueur, M. Lagrenee, F. Abraham, C. Bremard, J. Hetero-
cycl. Chem. 1987, 24, 1285 – 1289; b) D. L. Boger, S. M. Sakya, J.
Org. Chem. 1988, 53, 1415 – 1423; c) J. Sauer, D. K. Heldmann, J.
Hetzenegger, J. Krauthan, H. Sichert, J. Schuster, Eur. J. Org.
Chem. 1998, 2885 – 2896; d) D. Che, T. Wegge, M. T. Stubbs, G.
Manuscript received: September 30, 2016
Revised: November 8, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Drug Delivery
E. Jimꢁnez-Moreno, Z. Guo, B. L. Oliveira,
I. S. Albuquerque, A. Kitowski,
A. Guerreiro, O. Boutureira, T. Rodrigues,
G. Jimꢁnez-Osꢁs,
G. J. L. Bernardes*
&&&& — &&&&
Vinyl Ether/Tetrazine Pair for the
Traceless Release of Alcohols in Cells
Caged alcohols: The spatiotemporal
release of chemical matter under bio-
compatible conditions remains a chal-
lenging task. Disclosed herein is a tech-
nique that can be broadly applied to
decage alcohol-containing moieties in
cells. A proof-of-concept for the release of
a fluorophore and a potent cytotoxic drug
is described.
6
www.angewandte.org
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!