"Close-to-Release": Spontaneous Bioorthogonal Uncaging Resulting from Ring-Closing Metathesis

Article abstract of DOI:10.1021/jacs.9b07193

Bioorthogonal uncaging reactions offer versatile tools in chemical biology. In recent years, reactions have been developed to proceed efficiently under physiological conditions. We present herein an uncaging reaction that results from ring-closing metathesis (RCM). A caged molecule, tethered to a diolefinic substrate, is released via spontaneous 1,4-elimination following RCM. Using this strategy, which we term "close-to-release", we show that drugs and fluorescent probes are uncaged with fast rates, including in the presence of mammalian cells or in the periplasm of Escherichia coli. We envision that this tool may find applications in chemical biology, bioengineering and medicine.

Full text of DOI:10.1021/jacs.9b07193

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Communication
“Close-to-Release”: Spontaneous Bioorthogonal
Uncaging Resulting from Ring-Closing Metathesis
Valerio Sabatino, Johannes G. Rebelein, and Thomas R. Ward
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b07193 • Publication Date (Web): 10 Sep 2019
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Scheme 1. Selected biocompatible uncaging reactions
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Scheme 2. Functionalization of the naphthalene precursor 1 for the spontaneous
release of cargoes
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Table 1. Selected results for the aqueous RCM of naphthalene precursor 1.
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60
Entrya mol% HG-II
Solvent
pH
7.0 140
7.0 240
6.0
6.0 > 95
Conv%
TON
140
120
441
200
1
1.0
2.0
1.0
5.0
H2O:acetone 9:1
H2O:acetone 9:1
PBS:acetone 9:1
PBS:acetone 9:1
2
3b
4b
441
a [1] = 1.0 mM. b PBS buffer contains 50 mM MgCl2.
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Table 2. RCM-triggered 1,4-elimination of carboxylic acids
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6
7
8
9
10
11
12
13
14
15
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20
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25
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30
31
32
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mol%
HG-II
time
(h)
Entrya
1
Substrate
Conv%
TON
5.0
20
340
70
3a
2
0.5
0.2
20
370
430
740
3b
0.25
2142
3b
4b
0.2
0.1
0.25
0.25
521
262
2617
3c
3d
5b
26020
6c
7
1.0
1.0
5.0
1
1
1
376
644
61
376
644
10
3e
8d
9d
20.0
1
437
41
3f
10b
0.2
0.25
471
2355
3g
a
b
c
Substrate concentration: 1.0 mM. Substrate concentration: 2.5 mM in PBS buffer:acetone 3:1.
Reaction carried out at pH 7.4. d [3f] = 20 µM,1% DMSO, pH 7.4.
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Table 3. RCM-triggered 1,4-elimination of alcohols from ethers 4a-b.
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6
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8
9
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45
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60
Entry
1a
Substrate
pH
time (h)
Conv% TON
150
320
455
310
640
9010
7.4
0.5
2a
3b
6.0
6.0
48
4a
4b
0.5
4b
7.4
3
271
532
a [4a] = 0.5 mM, 5% DMSO. b [4b] = 1.0 mM, 10% acetone.
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Table 4. RCM-triggered 1,4-elimination of amines or alcohols from substrates
5-6
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8
9
10
11
12
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44
45
46
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57
58
59
60
Entrya
1
Substrate
mol% HGII
0.2
pH
6.0
Conv%
TON
100
490
5a
170
170
90
170
845
90
2
1.0
0.2
1.0
7.4
6.0
7.4
3b
4
5b
5c
10.0
7.4
400
40
5c
50
51
221
91
403
9711
221
442
6
7
8
9
0.2
6.0
6.0
6.0
6.0
0.05
1.0
5d
0.4
6
a substrate concentration: 1 mM. b [5b] = 2.5 mM, PBS buffer:acetone 3:1. c reaction carried out for 16
hours in PBS buffer containing 1% DMSO.
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Table 5. Close-to-release in biological media
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Entry
1a
Substrate
mol% HG-II Media
Conv%
14±1
5±0.5
32±0
26±1
32±0
20±1
50±2
27±0
TON
69±2
10±1
32±0
26±1
32±0
20±1
10±0
5±0
3d
3d
4a
4a
4a
4a
4a
4a
4a
0.2
0.5
1.0
1.0
1.0
1.0
5.0
5.0
1.5c
DMEM
9
10
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25
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2a
cell lysate
DMEM
3b
4b
cell lysatec
E. coli, PBS
HeLa, DMEM
HeLa, DMEM
5b
6b
7b
8b
HeLa, DMEM
FBS10%
9b
E. coli periplasm 27±6
17±1
a [3d] = 500 µM. b [4a] =100 µM. All reactions included 1% or less DMSO, pH = 6-7.4. c lyophilized E.
coli cell lysate solution in PBS buffer. c Estimated uptake of biotinylated HG-II catalyst from 10 µM
incubation.11
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“Close-to-Release”: Spontaneous Bioorthogonal Uncaging Resulting
from Ring-Closing Metathesis
Valerio Sabatino, Johannes G. Rebelein and Thomas R. Ward*
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Department of Chemistry, University of Basel, Building 1096, Mattenstrasse 24a, Biopark Rosental, 4058 Basel, Switzerland
ABSTRACT: Bioorthogonal uncaging reactions offer versatile tools in chemical biology. In recent years, reactions have been devel-
oped to proceed efficiently under physiological conditions. We present herein an uncaging reaction that results from ring-closing
metathesis (RCM). A caged molecule, tethered to a di-olefinic substrate, is released via spontaneous 1,4-elimination following RCM.
Using this strategy, which we term “close-to-release”, we show that drugs and fluorescent probes are uncaged with fast rates, includ-
ing in the presence of mammalian cells or in the periplasm of E. coli. We envision that this tool may find applications in chemical
biology, bioengineering and medicine.
Scheme 1. Selected biocompatible uncaging reactions
Bioorthogonal chemistry provides tools to scrutinize and
modulate biological processes. Bertozzi pioneered the field by
capitalizing on the Staudinger ligation,1 which paved the way to
numerous bioorthogonal ligations.2 The possibility of triggering
organic reactions in vivo provides tools for targeted therapy.3
Such reactions occur with high selectivity and turnover num-
bers (TONs) and proceed with fast kinetics under high dilution.
There are several examples of dissociative reactions, mainly
based on transition-metal catalysis and/or pericyclic reactions.3
The inverse-electron demand Diels-Alder reaction between te-
trazine and trans-cyclooctene (TCO) is one of the fastest uncag-
ing reactions.4 Versteegen et al. reported the cycloaddition be-
tween tetrazine and TCO bearing a carbamate to form 4,5-dihy-
dropiridazine, which readily tautomerizes in aqueous media,
Scheme 1a.5 This strategy, coined “click-to-release”, leads to
the uncaging of the amine via the formation of the 1,4-tautomer.
Antibody-drug conjugates are developed relying on the click-
to-release approach to activate prodrugs in the extracellular
space of tumors.6 In the context of metal-catalyzed biocompat-
ible reactions, precious metals including ruthenium, gold and
palladium occupy a place of choice. Indeed, these have been
shown to catalyze the de-allylation and de-propargylation of
caged substrates in cellulo and in vivo, Scheme 1b.7
Recently, olefin metathesis has gained attention as a
bioorthogonal tool in chemical biology.8 As Ru-based catalysts
tolerate water and oxygen, a number of biocompatible protocols
have been reported.9 Davis used olefin metathesis for
bioorthogonal ligation via cross-metathesis. Schultz performed
cross-metathesis of olefin-bearing proteins using the Hoveyda-
Grubbs catalyst (HG-II).10 Our group reported the directed evo-
lution of an artificial metathase in the periplasm of E. coli.11 To
expand on this work, we set out to develop a strategy that capi-
talizes on RCM, leading to the uncaging of a substrate. We con-
templated the possibility that RCM may trigger the release of a
cargo via an elimination reaction, driven by the generation of
an aromatic moiety.
We were encouraged by reports of RCM leading to a transient
2,5-cyclohexadien-1-ol moiety, followed by spontaneous 1,4-
elimination of water.12 We hypothesized that a leaving group
may be subject to such a 1,4-elimination, Scheme 1c. This hy-
pothesis was confirmed by subjecting the naphthalene precursor
1 to RCM in water. It led to good yields in the presence of var-
ious Ru-based metathesis catalysts. The HG-II performed best
(See SI, Table S2), and was selected for all subsequent studies.
The reactions were carried out in water or in buffered solutions
at pH 6 – 7, with 10% acetone. Having identified that the 1,4-
elimination of water proceeds under "near physiological"
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conditions (Table 1), we sought to equip the di-olefinic sub-
strate with a caged-cargo is released upon 1,4-elimination.
Table 2. RCM-triggered 1,4-elimination of carboxylic acids
1
2
3
Table 1. Selected results for the aqueous RCM of naphtha-
lene precursor 1
4
5
6
7
8
mol%
HG-II
time
(h)
Conv
%
Entrya Substrate
TON
9
mol%
HG-II
Entrya
Solvent
pH
Conv% TON
1
5.0
20
340
70
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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44
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47
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60
3a
1
1.0
H2O:acetone 9:1 7.0
H2O:acetone 9:1 7.0
PBS:acetone 9:1 6.0
PBS:acetone 9:1 6.0
140
240
441
> 95
140
120
441
200
2
0.5
0.2
20
370
430
740
3b
0.25
2
2.0
1.0
5.0
2142
3b
3b
4b
4b
0.2
0.1
0.25
0.25
521
262
2617
3c
a [1] = 1.0 mM. b PBS buffer contains 50 mM MgCl2.
For this purpose, we substituted the hydroxy functionality by
leaving groups amenable to release a cargo. Starting from the
naphthalene precursor 1-(2-allylphenyl)prop-2-en-1-ol 1, four
functional groups were evaluated, Scheme 2.
5b
26020
3d
6c
7
1.0
1.0
5.0
1
1
1
376
644
61
376
644
10
Scheme 2. Functionalization of the naphthalene precursor 1 for the
spontaneous release of cargoes
3e
8d
9d
20.0
1
437
41
3f
10b
0.2
0.25
471
2355
3g
a Substrate concentration: 1.0 mM. b Substrate concentration: 2.5
mM in PBS buffer:acetone 3:1. c Reaction carried out at pH 7.4. d
[3f] = 20 µM, 1% DMSO, pH 7.4.
These data highlight that both products are formed concur-
rently and in equimolar amounts, suggesting that the release of
the acid-bearing cargo does not occur prior to the RCM event.
We additionally monitored the stability of the ester substrates
in the reaction media at 37 °C: no spontaneous hydrolysis was
detected by GC-MS or NMR within 24 hours, (see SI, Figures
S1 and S2). With the long-term goal of complementing the me-
tabolism,13 we contemplated the possibility of uncaging nico-
tinic acid, a precursor of NAD(P)H, starting from substrate 3a.
Gratifyingly, the RCM of substrate 3a led to the release of nic-
otinic acid with 34% yield (Table 2, entry 1). As the uncaging
reaction required 5% or higher catalyst loading, we set out to
improve the nucleofugacity by introducing electron-withdraw-
ing groups on the aromatic cargo. Accordingly, halogen-substi-
tuted substrates 3b and 3c afforded 214 and 261 TONs at 0.2%
catalyst loading, respectively (Table 2, entries 3 and 4). Intro-
duction of p-trifluoroacetyl group 3d afforded 260 TONs using
a 0.1% catalyst loading (Table 2, entry 5). With medical
The results of the close-to-release uncaging of seven di-ole-
finic substrates 3a-g bearing an ester moiety are collected in Ta-
ble 2. In order to ensure that the release of the cargo occurs only
as a result of the ring-closing event, the formation of both naph-
thalene and the cargo were monitored by NMR and GC-MS (see
SI, Figure S3 and S5).
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applications in mind, we evaluated the possibility of uncaging occurs as a result of the RCM event: 44 TONs at 0.2% catalyst
1
2
3
4
5
6
7
8
drugs as a result of RCM. The substrate 3e led to the release of
valproic acid, an anti-neuroleptic drug, which gave the highest
yield (64%) at pH 6 and 37% yield at physiological pH (Table
2, entries 6 and 7). The improved close-to-release activity ob-
served at lower pH may be an asset for chemotherapy, leading
to higher concentrations of drugs in acidic environments, typi-
cally present in hypoxic cancer cells.14 RCM of substrate 3f trig-
gers the uncaging of Alofanib, a tyrosine-kinase inhibitor. The
drug release was achieved with yields up to 43% albeit at high
catalyst loading (Table 2, entry 9). Uncaging of myristic acid
from 3g gave 235 TONs at 0.2% catalyst loading (Table 2, entry
10). The RCM of esters is characterized by fast rates: 80% of
the observed 3-bromo-4-nitrobenzoic acid from substrate 3c is
produced within 10 minutes (see SI, Figure S7).
loading (Table 4, entries 8 and 9).
Table 4. RCM-triggered 1,4-elimination of amines or alco-
hols from substrates 5-6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mol%
HG-II
Entrya
1
Substrate
pH Conv% TON
Next, we tested the RCM of ether substrates 4a and 4b. De-
spite the fact that ether substrates might benefit from the allylic
chalcogen effect,15 they proved more challenging than esters,
which we trace-back to the poor nucleofugacity of alcoholates,
Table 3.16 Accordingly, the uncaging efficiency of pentafluoro-
phenol (pKa = 5.5) from derivative 4b was highest (Table 3,
entries 3 and 4). Subjecting the pro-fluorogenic substrate 4a to
RCM, led to the immediate appearance of fluorescence, result-
ing from the uncaging of umbelliferone (64 TONs, 32% yield
at 0.5 mol% HG II, Table 3, entry 2).
0.2
6.0
100
490
5a
2
1.0
0.2
1.0
7.4
6.0
7.4
170
170
90
170
845
90
3b
4
5b
Table 3. RCM-triggered 1,4-elimination of alcohols from
ethers 4a-b
5c
10.0
7.4
400
40
5c
6
7
8
0.2
6.0
6.0
6.0
50
403
9711
221
0.05
1.0
51
5d
time
(h)
Entry
1a
Substrate
pH
7.4
Conv%
TON
221
0.5
150
310
9
0.4
6.0
91
442
2a
6.0
48
320
640
6
4a
4b
a substrate concentration: 1 mM. b [5b] = 2.5 mM, PBS buffer:ac-
etone 3:1. Reaction carried out for 16 hours in PBS buffer con-
taining 1% DMSO.
3b
4b
6.0
7.4
0.5
3
455
271
9010
532
c
Despite the tolerance of Ru-based metathesis catalysts to-
wards a variety of functional groups,17 these are irreversibly
poisoned in the presence of a few equivalents of glutathione,11
nature’s ubiquitous redox buffer, present in mM concentrations
in the cytoplasm of aerobic cells. To minimize the exposure of
the soft-metal catalyst to thiols, we evaluated the viability of the
close-to-release strategy under the following conditions: i) in
culture media, ii) in the presence of E. coli cells and cell lysates,
iii) in the presence of HeLa cells and iv) in the periplasm of E.
coli. The results are collected in Table 5.
a [4a] = 0.5 mM, 5% DMSO. b [4b] = 1.0 mM, 10% acetone.
Carbamates are frequently used as linkers to liberate
amines.16 Table 4 summarizes the results with carbamate sub-
strates 5a-d. Decreasing the catalyst loading led to a marked
increase in TONs. The RCM of substrate 5a, 5b and 5d gave
respectively 49, 84 and 40 TONs at 0.2% (Table 4, entries 1,3
and 6). Decreasing the catalyst loading to 0.05 mol%, (i.e. 0.5
µM HG-II) led to 97 TONs for the production of 4-chloro-3-
(trifluoromethyl)aniline from substrate 5d (Table 4, entry 7).
The fluorescent 4-methyl-7-aminocoumarin was uncaged from
the pro-fluorogenic probe 5c at pH 7.4 in up to 40% yield (Table
4, entries 4 and 5). We also showed that carbonates, despite
their hydrolytic propensity, lead to the release of alcohols. In
PBS buffer at pH 7.4, GC-MS and NMR monitoring of the re-
action using the activated p-nitrophenyl carbonate 6 (see SI,
Figure S27), confirms that the release of the p-nitrophenol only
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Table 5. Close-to-release in biological media metal-catalyzed bioorthogonal reactions. The close-to-release
1
2
3
4
5
6
7
8
strategy leads to the uncaging of metabolites, drugs and fluores-
cent probes under physiological conditions. Some of the most
notable features include: i) low catalyst loading and concentra-
tions, ii) no inert atmosphere required, iii) multiple turnovers in
the periplasm of bacterial cells, in the presence of mammalian
cells and in serum. This proof-of-concept study paves the way
towards various applications such as metabolic engineering, in
vivo imaging and prodrug activation, etc.
Entry Substrate mol%
HG-II
Media
Conv
%
TON
1a
2a
3b
4b
3d
3d
4a
4a
0.2
0.5
1.0
1.0
DMEM
14±1
5±0.5
32±0
26±1
69±2
10±1
32±0
26±1
cell lysate
DMEM
cell lysatec
9
5b
6b
4a
4a
1.0
1.0
E. coli, PBS 32±0
32±0
20±1
ASSOCIATED CONTENT
Supporting Information
The Supporting Information (general information, experimental
section, Figures S1-S30 and Tables S1-S19) is available free of
charge via the Internet at http://pubs.acs.org
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HeLa,
DMEM
20±1
50±2
27±0
7b
8b
4a
4a
5.0
5.0
HeLa,
DMEM
10±0
5±0
AUTHOR INFORMATION
HeLa,
DMEM
FBS10%
Corresponding Author
* Thomas.Ward@unibas.ch
9b
4a
1.5c
E. coli
27±6
17±1
ORCID
periplasm
Valerio Sabatino: 0000-0002-0424-813X
Johannes G. Rebelein: 0000-0003-2560-716X
Thomas R. Ward: 0000-0001-8602-5468
a [3d] = 500 µM. b [4a] =100 µM. All reactions included 1% or
less DMSO, pH = 6-7.4. c lyophilized E. coli cell lysate solution in
PBS buffer. c Estimated uptake of biotinylated HG-II catalyst from
10 µM incubation.11
ACKNOWLEDGMENT
Substrates 3d and 4a were tested in biological media such as
E. coli cells (or cell lysates) and Dulbecco Modified Eagle’s
Medium (DMEM, solution with high concentrations of glucose,
amino acids and vitamins). RCM of ester 3d afforded 69 TONs
in DMEM at low catalyst loading (0.2%, corresponding to 1
µM, Table 5 entry 1). The pro-fluorogenic ether 4a afforded 32
TONs in both DMEM and PBS in the presence of E. coli cells,
and 26 TONs in cell lysate at 1% catalyst loading, respectively
(Table 5 entries 3 and 5). Next, we evaluated the uncaging of
substrate 4a in media containing HeLa cells. Gratifyingly, we
observed up to 50% uncaging of umbelliferone and up to 20
TONs within 90 minutes, (Table 5 entries 6 and 7, see SI Figure
S22 and S23). The reaction in the presence of serum retained up
to 64% of the activity compared to the reaction in DMEM, (Ta-
ble 5, entry 8). The effect of substrate, products and catalyst on
HeLa cells viability was evaluated using the MTT assay. After
24-hour incubation, the cells present excellent viability at the
concentrations of catalyst and substrate (and the products um-
belliferone and naphtalene) used in the activity assays (see SI,
Figure S24 and S25). In the event that the generated naphtha-
lene proved toxic, it may be replaced or decorated to minimize
its harmfulness.
TRW thanks the University of Basel, the NCCR Molecular Sys-
tems engineering, the SNF (grant 200020_182046) and the ERC
(the DrEAM, advanced grant 694424). JGR thanks the European
Molecual Biology Organisation for a long-term fellowship (ALTF-
194-2017). We thank Prof. Dr. Yaakov Benenson (d-bsse, ETHZ)
for access to the mammalian cell culture facility
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TOC GRAPHIC
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caged cargo
active cargo
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fluorescent probe
biomolecule
drug
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