Synthetic methodology towards allylic: Trans-cyclooctene-ethers enables modification of carbohydrates: Bioorthogonal manipulation of the lac repressor

Article abstract of DOI:10.1039/d0sc03216f

The inverse electron-demand Diels-Alder (IEDDA) pyridazine elimination is one of the key bioorthogonal bond-breaking reactions. In this reaction trans-cyclooctene (TCO) serves as a tetrazine responsive caging moiety for amines, carboxylic acids and alcohols. One issue to date has been the lack of synthetic methods towards TCO ethers from functionalized (aliphatic) alcohols, thereby restricting bioorthogonal utilization. Two novel reagents were developed to enable controlled formation of cis-cyclooctene (CCO) ethers, followed by optimized photochemical isomerization to obtain TCO ethers. The method was exemplified by the controlled bioorthogonal activation of the lac operon system in E. coli using a TCO-ether-modified carbohydrate inducer. This journal is

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DOI: 10.1039/D0SC03216F
ARTICLE
Synthetic Methodology Towards Allylic Trans-Cyclooctene-Ethers
Enables Modification of Carbohydrates: Bioorthogonal
Manipulation of the Lac Repressor
Mark A.R. de Geus,a G.J. Mirjam Groenewold,a Elmer Maurits,a Can Araman*a and Sander I. van
Kasteren*a
The inverse electron-demand Diels-Alder (IEDDA) pyridazine elimination is one of the key bioorthogonal bond-breaking
reactions. In this reaction trans-cyclooctene (TCO) serves as a tetrazine responsive caging moiety for amines, carboxylic
acids and alcohols. One issue to date has been the lack of synthetic methods towards TCO ethers from functionalized
(aliphatic) alcohols, thereby restricting bioorthogonal utilization. Two novel reagents were developed to enable controlled
formation of cis-cyclooctene (CCO) ethers, followed by optimized photochemical isomerization to obtain TCO ethers. The
method was exemplified by the controlled bioorthogonal activation of the lac operon system in E. coli using a TCO-ether-
modified carbohydrate inducer.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
N
N
A
6
H
H
1
2

N
N
5
4
N
N
N
5
H
N
Introduction
NH
2
O
O
O
R
- N2
R
H
3
O
O
O
HO
R
R
R
O
O
R
+
Bioorthogonal bond cleavage reactions have garnered
significant interest in recent years.1–4 Amongst these new “click-
to-release” reactions, the inverse electron-demand Diels-Alder
(IEDDA) pyridazine elimination has shown particular promise for
bioorthogonal utilization.5 The method employs a trans-
cyclooctene (TCO) carrying an allylic substituent that upon [4 +
2] cycloaddition with a 1,2,4,5-tetrazine results in the formation
of a 4,5-dihydropyridazine.6 This 4,5-tautomer can rearrange to
form two new tautomers of which the 1,4-tautomer can release
the allylic payload, followed by rearomatization to the
pyridazine (Fig. 1A).7,8 The excellent biocompatibility and high
bimolecular reaction rate of the IEDDA pyridazine elimination9
has given rise to a multitude of in vitro and in vivo applications
such as the regulation of protein activity10–12 and the activation
of pro-drugs, in which spatiotemporal control is achieved by
antibody-drug conjugates (ADCs),13–15 nanoparticles,16
enzymatic supramolecular self-assembly17 or hydrogel
injection.18,19
R
H2N
O
HO
1
N
N
N
N
NH
4
N
H
H
B
Previous work
HO Ph
Mitsunobu
OH
O
R
hv (254 nm)
SiO2
O
1

AgNO3
R
O
Br
R
NaH
NHBoc
N
C O
HO Ph
3 steps
TCO-OH
O

O
DABCO,
100°C
NH
Ph
O
Br
O
Ph
C
O
This work
HO Ph
To date, nearly all applications for this reaction have relied
on the protection of (primary) amines as TCO-carbamates (Fig.
1A, green). Recently, Robillard8 and Bernardes20,21 showed that
release of other functional groups, such as carboxylic acids
(from TCO-esters) and alcohols (from TCO-carbonates or TCO-
ethers), is indeed possible (Fig. 1A, orange and red). Whilst TCO-
esters and carbonates suffer from reduced hydrolytic stability
in biological systems,8,20,21 TCO-ethers are particularly
appealing due to their high stability and surprisingly fast
O
Pd(Ph3)4 (cat.)
2
O R
hv (254 nm)
Ph-SO3-Ag
O

HN
CCl3
R
HO
R
O
Improved synthetic
accessibility
TfOH (cat.)
3
Fig. 1 A) Overview of the inverse electron-demand Diels-Alder (IEDDA) pyridazine
elimination reaction, including the current scope of this method to decage carbamates,
carbonates, ethers and esters to obtain amines (green), alcohols (red) and carboxylic
acids (orange), respectively. B) Overview of the current synthetic methods to obtain
allylic TCO ethers, including methods from Robillard and co-workers (top)8 and
Bernardes and co-workers (bottom).21 C) Novel synthetic methods described in this
work, including reagents 2 and 3.
a. Leiden Institute of Chemistry
and The Institute for Chemical Immunology, Leiden University
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
Email: m.c.araman@lic.leidenuniv.nl, s.i.van.kasteren@lic.leidenuniv.nl
Electronic
Supplementary
Information
(ESI)
available:
See DOI: 10.1039/x0xx00000x
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decaging kinetics when compared to vinyl ether analogues.8,22– trichloroimidate 3 can result in elimination and reVaierwraAnrtgicelemOnelinnet
24
30,31
DOI: 10.1039/D0SC03216F
Unfortunately, widespread use of TCO-ethers is constricted pathways, including the formation of ionic intermediates.
by their challenging synthesis. Formation of the crucial cis- Cyclooctene trichloroamide 8 (see ESI for characterization),
cyclooctene (CCO) ether bond, employing either Mitsunobu trichloroacetamide (9) and the desired cyclooctene ether can be
chemistry or nucleophilic substitution of a primary alkyl formed from these reactive intermediates. N-trifluoroacetyl-
bromide with 1, is followed by photochemical isomerization25 protected L-serine methyl ester 10 was alkylated using 2
to the TCO-ether and isolation of the desired axial isomer (Fig. equivalents of 3 under triflic acid catalysis (-35°C to 0°C) to
1B, top).8 One exception is the direct alkylation of axial TCO-OH obtain 11 in 46% yield (Scheme 2B, Table S2). Furthermore,
to form a benzylic TCO-ether.8 Additionally, Bernardes and co- reagents 2 and 3 were both synthesized from cis-cyclooctene
workers developed a self-immolative linker in which a TCO- (12) in 3 steps (~48% yield) with a common intermediate (1) and
carbamate is connected to a benzyl ether (Fig. 1B, bottom).21 a single chromatographic purification (Scheme 1, Table S1).
Although 1 was used as a Mitsunobu substrate with moderate
success,8 this method essentially limits the scope to phenolic
nucleophiles without including the formation of ethers from
aliphatic alcohols.26 Furthermore, direct nucleophilic
substitution with 1 is limited to primary positions,8 in which the
desired aliphatic alcohol requires an additional transformation
into a leaving group. The self-immolative linker21 requires at
least four synthetic steps from the substrate, which to date has
also been limited to phenols. Taken together, synthesis and
utilization of TCO-ethers derived from functionalized aliphatic
alcohols encountered in biological systems is currently
unfeasible. This led us to develop novel synthetic methods for
the (regioselective) installation of TCO-ethers in biomolecules
as caging moieties, based on reagents 2 and 3, which we present
here (Fig. 1C). Furthermore, we applied our method to enable
the synthesis and bioorthogonal decaging of a TCO-ether
modified carbohydrate, as no TCO-protected variants of these
biomolecules currently exist.
O
O
O
d
O
NO2
O
NO2
4
5
c
f
O
Br
18
OH
O
O
a
e
b
1
12
2
CCl3
NH
3
Scheme 1. Synthesis of 1, 2, 3, 4 and 5 from cis-cyclooctene (12). Reagents/conditions:
(a) NBS, AIBN, cyclohexane, reflux, 71%; (b) acetone, H2O, NaHCO3, reflux, 83%; (c) 4-
nitrophenyl chloroformate, pyridine, DCM, 0°C to rt, 83%; (d) Pd(PPh3)4, toluene, 50°C,
92%; (e) NaHMDS, Boc-ON, THF, 0°C to rt, 81%; (f) trichloroacetonitrile, DBU, NaHCO3,
DCM,
0°C
to
rt,
~
80%
(Table
S1).
A
O
O
O
HO Ph
L2Pd(0)
HO Ph
O
O
Ph
-CO2
-tBuOH
PdL2
2
Results and discussion
We envisioned a two-step procedure in which the use of
electrophilic cyclooctene reagents secured formation of a CCO-
ether bond under mild conditions, followed by photochemical
isomerization25 to the desired TCO-ether. Palladium catalysis
enables mild, decarboxylative conversion of allyl carbonates to
allyl ethers via a reactive π-allyl cation species.27,28 We found a
ring strained variant of this reaction was feasible for phenols by
transforming para-nitrophenyl carbonate 4 into cyclooctene
ether 5 under Pd(PPh3)4 catalysis (50°C) in 92% yield (Scheme
1). Based on this initial observation, we reasoned the palladium-
catalyzed method for direct allylation by Grover and co-
workers29 would enable cyclooctene ether formation in a single
step. We designed cyclooctene tert-butyl carbonate reagent 2,
which decarboxylates upon coordination with a palladium
catalyst (Scheme 2A). The spectator tert-butoxide formed in this
step29 ensures rapid deprotonation of the phenol nucleophile,
which can subsequently attack the π-allyl electrophile to form
the ether bond. We examined the cyclooctene ether formation
with N-trifluoroacetyl-protected L-tyrosine methyl ester 6 using
reagent 2 (1.2 equivalents) under Pd(PPh3)4 catalysis (80°C)
instead of the previously reported Mitsunobu procedure (12%
yield),8 obtaining cyclooctene ether 7 in 80% yield (Scheme 2A).
In parallel, we designed a second reagent (3) for the
synthesis of cyclooctene ethers from aliphatic alcohols (Scheme
2B). Lewis acid triggered activation of cyclooctene
Example
O
O
O
O
a
O
O
HN
CF3
HN
CF3
O
HO
7
6
B
Tf-O
Tf-O
O
CCl3
O
CCl3
TfOH
HO
O
CCl3
NH2
R
N
NH
H
R
H
3
HO
R
HO
O
CCl3
NH2
O
O
HN
R
9
8
CCl3
O
O
Example
HO
O
O
b
O
O
O
HN
CF3
HN
10
CF3
11
Scheme 2. A) Design of cyclooctene tert-butyl carbonate reagent 2 for the palladium
catalyzed installation of cyclooctene ethers on phenols, including the proposed
mechanism based on the work of Grover and co-workers.19 Reagents/conditions: (a) 2,
Pd(PPh3)4, dioxane, 80°C, 80%; B) Design of cyclooctene trichloroimidate reagent 3 for
the Lewis acid triggered formation of cyclooctene ethers from aliphatic alcohols.
Proposed mechanism and products observed upon treating 3 with catalytic triflic acid
are shown. Reagents/conditions: (b) 3, TfOH, DCM, -35°C to 0°C, 46% (Table S2).
2 | J. Name., 2012, 00, 1-3
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Carbohydrates orchestrate a diverse array of biological executed with the general flow setup described by Fox and co-
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processes and as such, spatiotemporal control over these workers25 using silver (I) exchanged tosDicOaI:c1i0d.10s3il9ic/Da0gSCel03(2T1A6gF
biological activities would be a powerful addition to the “click- silica) as the stationary phase.41 Irradiation (λ = 254 nm) of
to-release” toolkit. A classic example of a biological process mixture of 23 and methyl benzoate in Et2O for ± 24 h, whilst
directed by glycans is the switching of the lac operon.32,33 It is a continuously circulating the reaction mixture over the
regulatory element that is used to control nutrient-dependent stationary phase, was followed by treatment of the stationary
transcription in E.coli. When lactose concentration is low, the phase with NH3 in MeOH to obtain (partially deacetylated) 24.
activity of the operon is inhibited by a repressor element.34 Zemplén deacetylation of 24 afforded 25 after extractive
When the concentration of lactose increases, it will bind to the desalting in ≥ 30% yield over 2 steps. This final product, 3-TCO-
repressor, leading to a decreased affinity of the lactose- IPG (25), was exclusively obtained as the desired axial TCO
repressor complex for the operon.35 This results in enhanced isomer and its purity was optimized by carefully increasing the
gene transcription for proteins under control of this promotor. polarity of the photoisomerization reaction mixture with
This system has been extensively used for the expression of isopropanol (2 – 5%; Fig. S1).
recombinant proteins in E. coli and the synthetic lac operon
We next evaluated whether the TCO-modified IPG 25 could
inducer isopropyl β-D-1-thiogalactopyranoside (IPTG, 13) is a be used to control the function of the parent molecule (15) in a
key reagent here. Control over its activity has previously been biological setting (Fig. 2A). For this we transformed E. coli to
attempted using both photochemical36 and hypoxia-triggered37 express the model protein ovalbumin (OVA) under control of
induction strategies. We therefore deemed it an excellent the lac operon. Overnight cultures were inoculated in LB
model carbohydrate to test IEDDA pyridazine elimination on medium supplemented with 1% glucose to inhibit leaky
carbohydrates. Initial experiments with peracetylated IPTG (14) expression. Next, cultures were grown in fresh LB medium (no
under conditions typically used for photochemical
AgNO3
AcO
OAc
isomerization of cyclooctenes confirmed the high affinity of the
thioacetal functionality towards the silver nitrate used for
enrichment of the TCO-isomers upon photoisomerization
(Scheme 3). We therefore designed a novel inducer for the lac
operon, substituting the sulfur with oxygen to obtain an O-
glycoside, isopropyl β-D-1-galactopyranoside (IPG, 15, Scheme
4).
O
S
AcO
OAc
14 • AgNO3
HO
AcO
OAc
b
OH
O
a
O
S
S
HO
AcO
c
OH
OAc
13
14
AcO
OAc
O
S
AcO
OAc
14
Starting from peracetylated β-D-galactopyranoside 16,
installation of the beta isopropyl group on the anomeric center
was achieved in a one-pot, two-step procedure in 70% yield.
The ‘disarmed’ pentaacetate was first transformed into an
anomeric bromide, which was subsequently activated with
stoichiometric I2 in the presence of isopropanol to obtain 17,
according to a method reported by Field and colleagues.38
Deacetylation gave IPG (15) in 90% yield. Stannylene acetal-
mediated alkylation of IPG (15) with (Z)-3-bromocyclooct-1-ene
(18) in the presence of CsF or TBAI as an additive at 105°C was
initially investigated (Table S3). A mixture of 3-CCO-IPG (19, ~
20% yield) and 6-CCO-IPG (20, ~ 20% yield) was obtained and
purified via (laborious) chromatographic separation.
Decomposition of 18 at high temperature into 1,3-
cyclooctadiene and hydrogen bromide39 limited its use.
Attempts to alkylate 6-TBS and 4,6-DTBS functionalized
derivatives of 15 in a regioselective manner with 18 were
unsuccessful (Table S4).
Instead, IPG (15) was regioselectively benzylated using
organotin chemistry followed by acetylation to obtain 2,4,6-
OAc-3-OBn-IPG (21) in 91% yield over two steps. Hydrogenation
of 21 in the presence of Pearlman’s catalyst afforded 2,4,6-OAc-
IPG (22) in 86% yield. Alkylation of 22 could be achieved in the
timespan of hours at low temperature (≤ 0°C), typically
employing 4 equivalents of trichloroimidate 3 and 10 mol% of
triflic acid (Table S5). The crude reaction mixture was washed
with aqueous NaOH (to remove 9)40 before purification by silica
gel chromatography to obtain 23 in ≥ 30% yield. Singlet
sensitized photoisomerization of 23 to 24 (Table S6) was
Scheme 3. Evaluation of the fate of peracetylated IPTG (14) during photoisomerization
conditions. Reagents/conditions: (a) Ac2O, pyridine, rt, 100%; (b) methyl benzoate, hv
(254 nm), Et2O, heptane, rt; (c) NH4OAc (aq), DCM, rt, 61%.
AcO
AcO
HO
OH
OAc
O
OAc
O
a
b
O
OAc
O
O
O
AcO
AcO
HO
OAc
16
OAc
OH
17
15
HO
O
HO
OH
O
O
c
O
O
+
HO
d
OH
OH
19
20
AcO
OAc
O
AcO
HO
OAc
O
e
AcO
O
OAc
O
f
O
O
O
O
BnO
O
g
OAc
OAc
OAc
22
21
23
HO
O
AcO
O
OH
O
OAc
O
h
Ph-SO3-Ag
OH
OAc
24
25
Scheme 4. Synthesis of IPG (15), 3-CCO-IPG (19), 6-CCO-IPG (20), and 3-TCO-IPG (25)
from peracetylated β-D-galactopyranoside (16). Reagents/conditions: (a) i. HBr, AcOH,
DCM, 0°C to rt; ii. 2-propanol, I2, DCM, 0°C to 4°C, 70% over 2 steps; (b) NaOMe, MeOH,
DCM, rt, 90%; (c) i. Bu2SnO, toluene, 105°C; ii.18, CsF, toluene, 105°C, ~20% (19), ~ 20%
(20) (Table S3); (d) i. Bu2SnO, toluene, 105°C; ii. benzyl bromide, TBABr, toluene, 70°C;
iii. Ac2O, pyridine, rt, 91% over 2 steps; (e) Pd(OH)2/C, H2, EtOAc, rt, 86%; (f) 3, TfOH,
DCM, -40°Cto rt, ~ 30% (Table S5); (g) methyl benzoate, hv (254 nm), Et2O/isopropanol,
rt; (h) NaOMe, MeOH, rt, ~ 30% over 2 steps (Table S6).
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/D0SC03216F
Fig. 2. Recombinant expression of proteins with caged IPG variants. A) Schematic representation of the chemical control over lac operon activity, mediated by 3-TCO-IPG (25), which
does not induce expression. IEDDA pyridazine elimination with 3,6-dimethyl-tetrazine (26) liberates IPG (15), which induces overexpression of the protein of interest. B) Recombinant
OVA expression, comparing the effects of DMSO (negative control, 1% v/v), IPG (15, positive control, 1 mM), 3-CCO-IPG (19, 1 mM) and 6-CCO-IPG (20, 1 mM). The respective
compounds were added at t = 0 h after which protein expression was followed over time. C-D) Temporal control of OVA expression via addition of tetrazine 26 (2.5 mM) in the
presence of 3-TCO-IPG (25, 1 mM). C) Direct addition of 25 and 26 at t = 0 h (left) or addition of 25 at t = 0 h (right). D) Addition of 26 1 h (left) or 2 h (right) after adding 25 at t = 0
h. E-F) Replicate expression experiments for OVA (2E; N = 3) and eGFP_A206K (2F; N = 4) comparing decaging conditions (1 mM 25 at t = 0 and 2.5 mM 26 at 1 = 1 h) with addition
of 25 (1 mM), 15 (1 mM), DMSO (1% v/v) and a negative control. Mean quantification values (Coomassie) were plotted with SD as error bars. Representative gel sections are shown
to serve as an example. Full gels for all replicate experiments are shown in the ESI (Fig. S6-12). Relative band intensity was measured via densitometry for all experiments. For eGFP
(2F), quantification was based on the Coomassie signal of the monomeric protein. *Significant difference based on an unpaired, two-tailed t-test (P < 0.05).
glucose) before adding (caged) inducers (1 mM) and tetrazine together, it appears this chemical method can be utilized as a
(2.5 mM) in DMSO. We first tested whether IPG (15) could general tool for temporally controlled gene expression in E.coli.
induce expression, which it did in a comparable level to IPTG
(13; Fig. 2B, S2 and Table S7). Furthermore, up to 10% DMSO
was tolerated for IPG (15) induced OVA expression (Fig. S3 and
Conclusions
Table S8).
To assess the functionality of the TCO-protecting group, we
In conclusion, we report the synthesis of TCO-ethers using two
novel reagents (2-3), providing facile access to TCO-protected
phenols and aliphatic alcohols, respectively. This methodology
enables access to the modification of complex biomolecules as
TCO-ethers. A carbohydrate probe, 3-TCO-IPG (25), made in this
manner was successfully used to chemically modulate lac
operon activity, thereby providing a first example of control
over glycan activity by IEDDA pyridazine elimination. The
spatiotemporal control available to this method also bodes well
for other applications in which carbohydrate/receptor
interactions direct biological processes.
confirmed 3-TCO-IPG (25) did not affect expression levels in
absence of a deprotection agent (Fig. S4 and Table S9).
Subsequently, we analyzed whether deprotection with the
prokaryote-compatible 3,6-dimethyl-tetrazine (26) could be
used to switch on recombinant protein expression.12,42 We
found that 26 caused a minor delay on IPG (15) induced OVA
expression without reducing overall expression levels (Fig. S5
and Table S10).
Therefore, deprotection of 3-TCO-IPG (25) with 26 was
studied in a time course experiment (Fig. 2C-D). Simultaneous
addition of both 25 and 26 at the same time did not result in a
controlled overexpression. However, a preincubation of 1 or 2
hours with 25 before addition of 26 did result in tightly
controlled overexpression of OVA. This was particularly striking
at late time points (Fig. 2D and Table S11). Replicate
experiments (N = 3) confirmed these findings (Fig. 2E and S6-8,
Table S12-15). Control compounds 3-CCO-IPG (19) and 6-CCO-
IPG (20) failed to induce overexpression (Fig. 2B and Table S16).
A reduction in expression compared to the DMSO background
was even observed for 19 (Fig. S4 and Table S9), but this was
not a property of the CCO-moeity in general.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Jessica E. Pigga and Joseph M. Fox (University of
Delaware) for useful discussions concerning the TAg silica gel.
S.I.V.K. was the recipient of an ERC Starting Grant of the
European Research Council (ERC-2014-StG-639005). C.A.
acknowledges the Netherlands Organization for Scientific
Research (NWO)-CW-ECHO Grant program 2016 for financial
support.
The method was next applied to the expression of two other
proteins, eGFP (Fig. 2F and S9-12, Table S17-21; N = 4) and
DsRed2 (Fig. S13). Again, overexpression after addition of 26
was observed for both proteins based on Coomassie
quantification (Fig. 2F and Table S22). For eGFP, a significant
overexpression was found after 5 hours (Fig. 2F). Taken
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25
H. Wu, S. C. Alexander, S. Jin and N. K. Devaraj, J. Am.
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Chem. Soc., 2016, 138, 11429–11432.
Notes and references
DOI: 10.1039/D0SC03216F
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DOI: 10.1039/D0SC03216F