Dissociative reactions of benzonorbornadienes with tetrazines: Scope of leaving groups and mechanistic insights

Article abstract of DOI:10.1039/c7ob02191g

Bioorthogonal dissociative reactions boast diverse potential applications in chemical biology and drug delivery. The reaction of benzonorbornadienes with tetrazines to release amines from carbamate leaving groups was recently introduced as a bioorthogonal bond-cleavage reaction. The present study aimed at investigating the scope of leaving groups that are compatible with benzonorbornadienes. Synthesis of several benzonorbornadienes with different releasable groups is reported, and the reaction of these molecules with tetrazine was found to be rapid and afforded high release yields. The tetrazine-induced release of molecules proceeds in a cascade of steps including inverse-electron demand cycloaddition and cycloreversion reactions that form unstable isoindoles/isobenzofuran intermediates and spontaneously eliminate a leaving group of interest. In the case of oxygen-bridged BNBDs at room temperature, we observed the formation of an unproductive byproduct.

Full text of DOI:10.1039/c7ob02191g

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Dissociative reactions of benzonorbornadienes
with tetrazines: scope of leaving groups and
mechanistic insights†
Cite this: DOI: 10.1039/c7ob02191g
M. Xu,
R. Galindo-Murillo,
T. E. Cheatham, III.
and R. M. Franzini
*
Bioorthogonal dissociative reactions boast diverse potential applications in chemical biology and drug
delivery. The reaction of benzonorbornadienes with tetrazines to release amines from carbamate leaving
groups was recently introduced as a bioorthogonal bond-cleavage reaction. The present study aimed at
investigating the scope of leaving groups that are compatible with benzonorbornadienes. Synthesis of
several benzonorbornadienes with different releasable groups is reported, and the reaction of these
molecules with tetrazine was found to be rapid and afforded high release yields. The tetrazine-induced
release of molecules proceeds in a cascade of steps including inverse-electron demand cycloaddition
and cycloreversion reactions that form unstable isoindoles/isobenzofuran intermediates and spon-
taneously eliminate a leaving group of interest. In the case of oxygen-bridged BNBDs at room tempera-
ture, we observed the formation of an unproductive byproduct.
Received 31st August 2017,
Accepted 9th November 2017
DOI: 10.1039/c7ob02191g
rsc.li/obc
2
0
(
Scheme 1).
In particular, 1-(aminocarbonyloxymethyl)-
Introduction
BNBDs with heteroatoms at the bridging position exhibited
Bioorthogonal chemistry has made it possible to perform reac- prolonged stability in human serum whereas reaction with Tz
1
,2
tions in cells and living organisms. Biocompatible reactions resulted in rapid and efficient release of amine cargo mole-
3
4
20
are widely used in bioconjugation chemistry, cell imaging,
cules.
Only few biorthogonal dissociative reactions are
5
6
21
material science, and drug delivery. Inverse-electron demand known, such as the inverse-electron demand Diels–Alder pyr-
Diels–Alder cycloadditions are among the most prominent idazine elimination reaction,
bioorthogonal transformations because of rapid reaction rates tions,
and high inertness of the reagents to biological functional- cyclooctene with p-azidobenzylcarbamates, and reactions of
1,2,4,5-Tetrazines (Tz) and other electron-deficient Tz with vinyl ethers.
dienes react readily under physiological conditions with con- nal release transformations will be of great significance
2
2,23
modified Staudinger reac-
2
4,25
strain-promoted 1,3-dipolar cycloaddition of trans-
2
6
7
,8
4
27–29
ities.
Expanding the scope of bioorthogo-
9
formationally strained or electron-rich alkenes (e.g., trans- because such reactions boast broad applicability in drug
1
0
11,12
13
20,30,31
19,24,29
cyclooctene,
cyclopropene,
enamine,
and acylaze- release strategies
biosensing probes,
regulated
1
4
32–34
tine ). For example, the cycloaddition reaction of norbornene release of gaseous mediators,
and Tz generates a polycyclic intermediate that rapidly con- protein activity.
and chemical control of
3
5–37
However, dissociative biorthogonal reac-
verts into the stable 1,4-tautomer, which can be exploited for tions have been mostly limited to the release of amines from
1
5,16
labelling purposes.
Benzonorbornadienes (BNBD) react carbamate derivatives. The rapid liberation of other molecules
1
7
similarly with Tz; however, in the case of BNBD derivatives has been explored less thoroughly.
containing nitrogen or oxygen atoms at the bridging position,
Here we investigated the scope of leaving groups and effects
the intermediate undergoes a cycloreversion reaction to gene- of experimental parameters on Tz-induced release from BNBD
1
8,19
rate isoindoles and isobenzofurans.
We previously
reported that this transformation can be used to chemically
induce the release of drugs and reporter groups at physiologi-
cal conditions from highly stable BNBD precursors
Department of Medicinal Chemistry, College of Pharmacy, University of Utah,
30 S 2000 E, Salt Lake City, UT-84112, USA. E-mail: Raphael.franzini@utah.edu
†
Electronic supplementary information (ESI) available: Detailed experimental
Scheme 1 Tetrazine-induced released of leaving groups (LG) from
procedures, supporting figures, and tables. See DOI: 10.1039/c7ob02191g
benzonorbornadiene (BNBD) derivatives (X: O, NAc).
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derivatives. The synthesis of BNBDs with several leaving pared from m-PEG4-acid (4a, 44%; 4b, 47%). Similarly, reac-
groups was successful, and the reaction with Tz triggered tion of 1a/b with benzylchloroformate produced the corres-
rapid and high-yielding releases. During the study, it became ponding carbonates (6a, 46%; 6b 36%) and with menthyl-
apparent that the release yields of O-bridged BNBDs upon reac- chloroformate the carbonate 7b (35%). One of the ester deriva-
tion with Tz showed an intriguing temperature dependence. tives and one of the carbonates contained bioactive cargo
We analyzed these effects and identified the competitive for- molecules (ibuprofen, 5b and menthol, 7b) to illustrate the
mation of a stable byproduct at room temperature.
broad applicability of the reaction. The reaction of 1a with
dibenzylphosphoryl chloride provided the corresponding
phosphotriester 8a (21%), whereas the analogous reaction with
the NAc-derivative 1b was unproductive. We also prepared a
carbamate derivative of 1-(1-hydroxyethyl)-9-(N-acetyl-aza)
BNBD (2c) to investigate the effect of alkyl substituents at this
Results and discussion
Synthesis
With the goal of testing whether the reaction with Tz can position on reaction rate and amine release (Scheme 3a).
efficiently release diverse leaving groups from BNBDs, we syn- Attempts to synthesize derivatives of BNBDs with alkylamines
thesized several example molecules with either oxygen or at the bridging position consistently resulted in the ring-
N-acetyl at the bridging position (Scheme 2). Tested release opened napthylamines for all tested conditions, indicating low
molecules included carboxylic acid, alcohol, and dialkylpho- stability of such molecules.
sphate that were linked to the BNBD molecule as ester (3a/b,
a/b and 5b), carbonate (6a/b and 7b), and trialkylphosphate attempted to introduce leaving groups at the BNBD 1-methyl
8a), respectively. The efficient liberation of amines from position by nucleophilic displacement of a tosyl group
corresponding molecules with carbamate linkers was pre- (Scheme 3b). For example, many fluorophores and bioactive
To further expand the range of releasable groups, we
4
(
2
0
viously demonstrated,
and these molecules (2a/b) were molecules contain phenols, whose reversible modification
included in the study for comparison and mechanistic ana- would be of great interest, and we anticipated that their intro-
lysis. BNBD derivatives were prepared in one step from the duction would be most easily achieved by nucleophilic displa-
2
0
reported 1-(hydroxymethyl)BNBD precursors 1a/b (Scheme 2).
cement. Reaction of 1a with tosyl chloride cleanly provided the
Esterification of 2-phenoxyacetic acid (EDC and DMAP in sulfonylated compound 9a, which however lacked stability to
DCM) afforded the desired products 3a/b in 54% and 47% and be isolated and was directly used for nucleophilic displace-
5
b in 45% yield. Water-soluble ester derivatives 4a/b were pre-
Scheme 3 Synthesis of BNBDs substituted at methylene position and
introduction of leaving groups by nucleophilic substitution. (a) Synthesis
of p-nitrophenylcarbamate of 1-(1-hydroxyethyl)-9-(N-acetyl-aza)
BNBD. (b) Attempted nucleophilic displacement reactions at 1-methyl
position. Reagents and conditions: (d) 2-(Trimethylsilyl)phenyl imidazol-
sulfonate, CsF, dry MeCN, 45 °C, 8 h. (e) 4-Nitrophenyl isocyanate,
Scheme 2 Synthesis of benzonorbornadiene (BNBD) derivatives.
Reagents and conditions: (a) Chloroformate, DMAP, dry DCM, rt, over-
night. (b) Carboxylic acid, EDC, DMAP, dry DCM, rt,
Dibenzylphosphoryl chloride, DMAP, THF, rt, overnight.
3
h. (c)
DMAP, dry THF, 50 °C, 10 h. (f) Tosyl chloride, Et
3
N, DMAP, dry DCM, rt,
2 h. (g) 4-(trifluoromethyl)benzenethiol, Et N, dry DCM, rt, overnight.
3
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ment. Attempts to etherify 9a with phenols were unproductive. Table 1 Yield of release and second-order rate constants (k
2
) for reac-
a
tions of benzonorbornadiene derivatives and tetrazines
Test reactions with highly nucleophilic 4-(trifluoromethyl)thio-
1
phenol converted 9a in to a new product. H NMR characteriz-
ation results were however inconsistent with the phenyl sulfide
a
2
k ]
[M−1 s−1
b
Compound
Release [%]
c
1
0a and suggested the formation of 11a instead (Scheme 3). 2a
80.0 ± 7.9
87.5 ± 0.4
15.0 ± 2.2
71.2 ± 7.2
96.0 ± 0.8
68.2 ± 14.8
82.3 ± 2.1
92.9 ± 4.8
76.7 ± 11.1
94.7 ± 4.9
96.8 ± 2.0
41.2 ± 1.2
0.058 ± 0.0003
0.020 ± 0.0007
0.017 ± 0.0005
0.065 ± 0.00081
0.027 ± 0.00008
0.086 ± 0.0007
0.025 ± 0.011
0.015 ± 0.003
0.057 ± 0.00031
0.017 ± 0.0011
c
2
2
3
b
c
a
Apparently, the steric bulk of the BNBD moiety precludes
nucleophilic attack at this position, and benzenethiols react
3
8,39
preferentially at the alkene.
These results show that it is 3b
4
4
5
a
b
b
possible to introduce diverse leaving groups via electrophilic
addition, whereas addition of groups via nucleophilic displace-
ment is ineffective.
6a
6
7
8
b
b
a
0.0092 ± 0.0026
0.053 ± 0.0016
Tetrazine-induced release
Having successfully established that BNBD with several leaving
a
The release of the leaving groups was measured at 6 h by integration
1
groups are synthetically accessible (Scheme 2), we aimed to of characteristic peaks in H NMR spectra. c(BNBD) = 6 mM, c(DPTz) =
b
1
8 mM, T = 37 °C, (Fig. S1–8 in the ESI). Reaction rates were deter-
investigate whether they could efficiently release these groups
by reactions with Tz. We investigated the reaction of the
mined from the concentration dependence of the pseudo-first order
rate constants obtained from time-dependent absorbance measure-
BNBD-derivatives
with
3,6-di-(2-pyridyl)-1,2,4,5-tetrazine ments at λabs = 525 nm and T = 37 °C (Fig. S1 in the ESI; c(BNBD) =
c
2
.5 mM, 3.75 mM. 5 mM or 6.25 mM; c(PEG-Tz) = 0.25 mM). Values
(
DPTz, Chart 1) and quantified the release of leaving groups by
taken from ref. 20.
1
H NMR (Fig. 1 and Table 1). BNBDs (6 mM) were incubated at
3
2
7 °C with DPTz (18 mM) in DMSO containing 10% H O. The
release of the leaving groups was followed by integration of
characteristic peaks in H NMR spectra (Fig. 1 and Fig. S1–S8
in ESI†). All NAc-BNBD derivatives (3b–7b) liberated the
leaving groups in high yields (82–96%; Fig. 1, Table 1). The
release from O-BNBDs 3a, 4a and 6a was consistently lower
although still in the range of 68–77% yield (Table 1). We also
1
2
tested the release for 4a in pure D O with water-soluble
PEG-Tz (Chart 1) and the release was 74% (Fig. S9 in ESI†).
The measurements are in agreement with our previous obser-
vation that carbamate-modified BNBDs 2a/b upon reaction
2
0
with DPTz release p-nitroaniline in >80% yield.
Reaction kinetics of benzonorborandienes with tetrazines
We further aimed at understanding how the different leaving
groups affect the reaction rate of BNBDs with Tz. The kinetics
of the bimolecular reaction step of BNBD derivatives and
water-soluble PEG-Tz (37 °C, 90% DMSO, 10% water) was
determined by measuring the consumption of the PEG-Tz
absorbance signal at λabs = 525 nm. In the presence of excess
BNBD derivatives (2.5 to 6.25 mM), PEG-Tz (0.25 mM) dis-
appeared exponentially within few hours (Fig. S1 in ESI†). The
concentration dependence of the pseudo-first order rate con-
stants agreed with a second order rate law consistent with the
Chart 1 Structures of tetrazines used in this study.
2
0
results for the carbamate derivatives 2a/b. The second order
rate constants under these conditions were in the range of k =
2
.0092–0.086 M−
1
s
−1
(Table 1).
0
BNBD derivatives with bridging oxygens (2a–6a) reacted
–3.5-fold faster with PEG-Tz than NAc-BNBDs (Table 1). The
2
effect of leaving groups on reaction rates was modest, except
for the menthyl carbonate 7b, whose reaction with Tz was unu-
sually slow. The BNBD-derivative 2c, containing a p-nitrophe-
nylcarbamate group attached to a bridgehead 1-hydroxyethyl
group (Scheme 3a), reacted 3.4-fold slower than the corres-
ponding methylene compound 2a. Additional steric bulk at
this position evidently interferes with the approach of the Tz
to the alkene dienophile. 2c also showed an unusually low
p-nitroaniline release of 15.0% (Fig. S2†).
1
Fig. 1 H NMR spectra of reaction of 6a and 6b with DPTz at t = 5 min
2
8,40
Water can accelerate cycloaddition reactions,
including
and t = 6 h. Legend: ▲, 6a/6b; ●, benzyl alcohol. (Full spectra of as well
as spectra for release reactions with other BNBD precursors are shown
in the ESI.†)
2
0
the reaction of 2a/b with Tz. To quantitatively determine the
effect of water over the entire range of solvent compositions,
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2
Fig. 2 Dependence of second order rate constants (k ) of the bimole-
cular reaction between 4a and PEG-Tz on water content. (c(4a) = 2 mM,
c(PEG-Tz) = 0.2 mM, T = 37 °C).
Fig. 3 Temperature dependence of the reactions of 2a, 2b and DPTz.
1
Analysis by H NMR. (c(2a/2b) = 6 mM, c(DPTz) = 18 mM, T = 22, 37
and 50 °C, samples measured at 2 h, DMSO-d6/D
2
O, 9 : 1, v/v). Legend:
2
a or 2b: ▲; p-nitroaniline: ●; byproduct: ★; tentative I5: △.
traced back to a brief exposure of the reaction mixture to room
temperature. We were therefore interested to analyze the effect
Chart 2 Effect of boronic acid substituent on reaction rates of norbor-
nene and tetrazines. Structures of norbornene and 12 and second order of temperature on the reaction. Solutions of 2a and DPTz were
rate constants for the reaction with PEG-Tz.
incubated at 22 °C, 37 °C, and 50 °C in DMSO/H O (9 : 1) and
reactions were analyzed by H NMR at 2 h (Fig. 3, Fig S10 and
2
1
11 in ESI†). Formation of p-nitroaniline from 2a increased
we measured the rates of the reaction of the water-soluble notably with temperature. At 50 °C, the reaction resulted in a
BNBD-derivative 4a and PEG-Tz in solutions varying from 10% release of 78%, whereas at 22 °C only traces of p-nitrophenyl-
H O in DMSO to pure H O (Fig. 2). In agreement with the aniline were formed. Instead a byproduct was generated for 2a
2
2
2
0
results for 2a/b, the measured rate constants increased sub- at 22 °C. Heating a solution containing the byproduct to 50 °C
−
1 −1
stantially from 10% H
2
O (k
). Additional increase of the water content only shown), indicating that the byproduct irreversibly traps the
modestly enhanced the reaction rate, which reached k
= 0.264 p-nitroaniline molecule. In contrast, 2b efficiently released
2
= 0.086 M
s
) to 40% H
2
O (k
2
=
failed to provide substantial quantities of p-nitroaniline (not
1
−1
0
.199 M−
s
2
−
1 −1
M
s
in pure H
2
O (rates were comparable in PBS buffer; not p-nitroaniline even at room temperature, and a byproduct
corresponding to the one formed for 2a was absent (Fig. 3).
shown).
To further accelerate the reaction of BNBDs with Tz, we Conclusively, while NAc-bridged BNBDs appear to be generally
evaluated whether introduction of boronates could increase applicable for release reactions, care should be taken with the
the bimolecular kinetics. This experiment was based on a oxygen derivatives because cargo release from these molecules
recent report that vinyl boronates react significantly faster depends on the reaction conditions.
4
1
with Tz than the corresponding olefins. To test this possi-
bility, we compared the rate of the reaction of PEG-Tz with
Formation of byproduct
norbornene and 2-norbornene-2-boronic acid (12, generated We were interested to identify the byproduct observed in the
−
1 −1
in situ from the pinacol ester; Chart 2). 12 (k
2
= 0.041 M
s
)
room-temperature reaction of 2a and DPTz (Fig. 3). This
reacted slower than norbornene (k2 = 0.615 M−
1
s
−1
). product proved unstable to HPLC purification. Instead, we
Possibly, the increased steric repulsion in the transition state studied the molecule in the reaction mixture by a series of
of the cycloaddition step overcompensates the electron- NMR experiments that included gCOSY, HSQC, and HMBC
1
donating properties of the boronic acid in the context of (Fig. 4, Fig. S12†). Four H NMR signals were visible in the
norbornenes.
4–5 ppm spectral range (Fig. 4a): H (δH = 4.01 ppm, 1H), Hb
a
Conclusively, the O-BNBDs reacted faster than the NAc-BNBDs (δH = 4.16 ppm, 1H), H (δ = 4.79 ppm, 1H), and Hd (δH
and the substituents had little impact on the reaction rate 4.97 ppm, 1H). H– H gCOSY correlations were present
=
c
H
1
1
except in the case of significant steric bulk.
between H and H as well as H and H (Fig. 4b). According
a
b
c
d
to the HSQC spectrum, H
a
and H
b
were linked to a single
Effect of temperature on tetrazine-induced release
carbon (δ = 68.8 ppm, C ), and H and H were linked to
C
1 c d
During the analysis of Tz-induced release reactions, we carbons C (δ = 36.7 ppm) and C (δ = 93.7 ppm), respectively
2
C
3
C
observed in some instances unusually low release yields for (Fig. 4c). HMBC results showed coupling between H
BNBDs with oxygen-bridges and the presence of unexpected well as H and C . We considered several possible structures
peaks in the H NMR spectra. This discrepancy could be (Chart 3) and checked whether they would agree with the
c 3
and C as
d
2
1
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Fig. 4 NMR analysis of formed side product of the bioorthogonal bond-cleavage reaction of 2a and DPTz at 22 °C. Four peaks (a–d) associated
1
with the observed product are highlighted by ● and assigned to protons in the proposed structures. (a) H NMR spectra at different time points.
(
b) gCOSY spectrum. (c) HSQC spectrum. (d) HMBC spectrum.
proton shift from intermediate I2, and this conversion has
been documented for other reactions of norbornene deriva-
1
5,18,31
tives with Tz.
Analysis of the reaction mixture by LC-MS
revealed a prominent +18 peak (564.8, Fig S16†) in addition to
the expected 1,4-tautomer mass (546.8, Fig S16†). It is concei-
vable that opening of the oxygen bridge follows formation of
the 1,4-tautomer considering the highly stabilized carbocation
intermediate of such a reaction. Differentiating structures 14
and 15 based on the NMR data is difficult. Although the avail-
able information is insufficient to determine the byproduct
with certainty, we consider the formation of the 1,4-tautomer
Chart 3 Hypothetical structures of byproduct of room temperature
reaction of 2a and DPTz.
(
14) as the major byproduct with possible rearrangement to
observed NMR peak patterns. Considered structures included the ring-opened product (15) as the most plausible outcome
the putative intermediate I2 (13), the corresponding 1,4-tauto- based on the mechanistic information from prior studies of
1
5,17,18,31
mer (14), the ring-open 1,4-tautomer (15) and a derivative in norbornene-derivatives with Tz
which a proton-shift resulted in C–C bond cleavage and pyrida- spectral data (Fig. 4).
zine formation (16) similar to a recently reported example.
as well as the recorded
4
2
Transition states of retro Diels–Alder reactions have an elev-
The number of four observed protons in the 4–5 ppm spectral ated entropy of activation, which could explain the observed
range is incompatible with intermediate I2, which was to be dependence of the product ratio on temperature: the rate of
expected because the 1,3-proton shift is generally rapid. For the retro Diels–Alder reaction increases significantly with
1
6, the protons Ha,b should couple to a single hydrogen temperature and becomes dominant at higher temperature
whereas the fourth proton should be a singlet. However, no whereas at room temperature the 1,3-proton shift outcompetes
such interaction is discernible by gCOSY, and protons Ha,b do the cycloreversion reaction. The difference between the formed
2 3
not interact with neither C nor C
, which excludes 16 as the product for the O- and NAc-BNBDs at 22 °C can be rationalized
possible byproduct. The most plausible structure in agreement by the higher dienofugacity of isoindoles relatives to
4
3
with the NMR data is the 1,4-tautomer (14) formed by 1,3- isobenzofurans.
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The lack of product release from the observed byproduct
upon heating also agrees with the formation of 14 and 15.
Based on a computational analysis (Fig. S13 and Tables S1, S2
in ESI†), the cycloreversion reaction of intermediates I2 is
expected to be thermodynamically favorable (ΔΔG(2a) =
4.7 kcal mol− ; ΔΔG(2b) = −8.9 kcal mol ), whereas conver-
1
−1
−
sion of the 1,4-tautomers to I3 is predicted to be endergonic
−1
−1
1
(
ΔΔG(2a) = 2.3 kcal mol ; ΔΔG(2b) = 6.6 kcal mol ).
Fig. 6 H NMR analysis of reaction between 2a and DPTz in CDCl
Conditions: c(2a) = 6 mM, c(DPTz) = 18 mM, T = 37 °C, measurements
taken at t = 2 h, in CDCl
. Legend: ▲: 2a; ●: intermediate of putative
structure I5; ■: DPPz; □: DPTz.
3
.
Decomposition intermediates
3
Finally, we were interested to assess the intermediates formed
after the cycloreversion reaction as well as the final byproducts
of the reaction cascade. The design of the BNBDs is such that
isoindole/isobenzofuran intermediates are expected to form
upon reaction with Tz (I3, Scheme 4) and eliminate leaving
2
0
p-nitrophenylaniline. The instability of I3 prevents its iso-
lation and individual characterization. We hypothesized that
the transition state of the elimination step (I3 to I4) would be
charged, and the reaction rate would depend on solvent
2
0
groups. Release of benzylic substituents from 5-membered
4
4–46
aromatic rings has been reported,
although electron
3
polarity. Indeed, incubation of 2a and DPTz in CDCl near-
donating-substituents are normally needed, and elimination
4
7
quantitatively transformed the BNBD into a product with a
single peak at 5.56 ppm (t = 2 h, 2 H), and only trace quantities
of p-nitroaniline were formed (Fig. 6). The observed solvent
dependence of the elimination step agrees with the proposed
mechanism (Scheme 4). We postulate that the elimination of
leaving groups from I3 forms high-energy intermediates of the
structure I4. A computational analysis (Fig. S13 and Table S1
in ESI†) predicted that the ΔΔGs for the formation of I4 are
of carbamates from furan is slow. Comparing the consump-
tion of 2b and DPTz with the appearance of p-nitroaniline
revealed that no accumulation of I3 occurs (Fig. 5, see also
ref. 20). In contrast, for 2a release was delayed, and a singlet was
visible at 5.59 ppm, whose integration (assuming two protons)
accounts for the difference between consumed 2a and formed
1
−1
2
2.6 kcal mol− and 19.2 kcal mol , for 2a and 2b respectively.
Assuming that the transition state energy of the elimination
step is close to the ΔΔG of the reaction, the higher stability of
I3 for 2a relative to 2b agrees with the lower predicted energy
barrier for the NAc-compound relative to that of the O-BNBD
compound. The calculations also support the observed solvent
dependence because the predicted ΔΔGs increases drastically
with decreasing solvent polarity (not shown). Isoindoles and
isobenzofurans are reactive species, and the elimination
product I4 will eventually convert into downstream molecules
(
Fig. 5). In the analysis of the reaction of 2b with DPTz, an
intermediate (I5) emerged in parallel with the formation of
p-nitroaniline (Fig. 4a and 5). The intermediate shows three
Scheme 4 Proposed mechanism of tetrazine-inducible cargo release.
singlet peaks at δ
peak is shifted upfield from δH = 1.88 ppm to 2.33 ppm. HSQC
analysis (Fig. 7) revealed that the δ = 5.90 and 5.24 ppm
H
= 6.45, 5.90, and 5.24 ppm, and the acetyl
H
1
3
protons are linked to a common carbon with a C NMR signal
at δC = 92.3 ppm. The δH = 6.45 ppm proton couples with a
carbon at δ
carbon at δ
C
= 84.8 ppm and the acetyl protons couple with a
= 24.6 ppm. The possible formation of 3-methyl-
C
eneisoindolin-1-ol (I5 in Scheme 4) generated by elimination
of the carbamate and subsequent addition of H O to the tran-
2
sient structure I4 would agree with the spectral data.
With longer incubation time, I5 disappears, which is
accompanied by a loss of DPTz and formation of two new pro-
ducts (Fig. 5). Two equivalents of DPTz are consumed for each
molecule of 2b whereas only one equivalent of 3,6-di-(2-
pyridyl)-1,2-pyridazine (DPPz) is formed (Fig. 5). It appears as
if the intermediate I5 reacts with DPTz generating a mixture of
two products visible by NMR and HPLC (Fig. 5, S14 in ESI†).
Fig. 5 Concentrations of starting material (2b) and reaction intermedi-
ates (I5, Scheme 4; IB, IC: putative tetrazine adducts) and products
(
DPPz: 3,6-di-(2-pyridyl)-1,2,4,5-tetrazine; pNA: p-nitroaniline) as a
function of time.
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Fig. 8 Summary the different substituents and leaving groups investi-
gated and their effect on the reaction rate and release yield.
An unanticipated outcome of this study was the formation
of an unproductive byproduct for oxygen-bridged BNBDs.
Fig. 7 HSQC spectrum of the bioorthogonal bond-cleavage reaction of
2
b and DPTz at T = 37 °C, from 2 h–6 h. c(2b) = 6 mM, c(DPTz) = Based on mechanistic considerations and the available spec-
1
8 mM.
tral data, we postulate the formation of the 1,4-tautomer of the
addition product. It is established that the fate of the initial
cycloaddition adduct after nitrogen expulsion of norbornene
1
8
derivatives with Tz depends on the atom at the bridging site.
The observed masses for these products in LC-MS analysis
were 379.8 m/z and 338.8 m/z (Fig. S15 in ESI†), which sup-
ports the formation of DPTZ-adducts (in the case of the lower
mass, adduct formation would be associated with loss of acetyl
group). It is tempting to speculate that Tz can react with the
electron-rich double bond of I5 although product decompo-
sition during solvent evaporation prevented analysis of isolated
products. Formation of downstream products will likely
depend on experimental conditions and the structure of the
BNBD molecules.
In summary, the proposed mechanism (Scheme 4) involves
the bimolecular reaction of Tz and modified BNBDs, generat-
ing polycyclic intermediates (I1) that release N
adducts of the general structure I2. A retro Diels–Alder reaction
results in a isobenzofuran/isoindole intermediate (I3), which
eliminates the leaving group at the methyl position and in
the presence of water forms 3-methyleneisoindolin-1-ol (I5).
Most norbornene derivatives (including BNBDs) with carbons
at the bridging position form 1,4-tautomer adducts, whereas
BNBDs with oxygen or nitrogen atoms at this position were
1
7,18
reported to undergo cycloreversions.
To the best of our
knowledge, the observed side product would be the first
example of a reaction of an oxygen-bridged BNBD derivative
with Tz that forms a 1,4-tautomer (or a decomposition product
thereof) rather than the isobenzofuran. The production com-
position depends on reaction conditions, and isobenzofuran
formation with subsequent payload release is predominant at
37 °C, whereas the byproduct is the major species formed at
room temperature. No byproduct was observed for NAc-
bridged BNBDs, possibly because of the higher dienofugacity
2
forming
4
3
of isoindole relative to isobenzofuran. Based on NMR and
HPLC results, the isoindole derivatives decompose through a
cascade of products including 3-methyleneisoindolin-1-ol and
ultimately forms adducts of elusive structures with Tz.
3
-Methyleneisoindolin-1-ol then reacts with an additional Tz
The obtained results provide valuable information on con-
siderations for BNBD release designs, especially with respect
to whether to use O- or NAc-BNBDs (Fig. 8). BNBDs with
oxygen bridging groups provide faster reaction rates than the
NAc-derivatives (approx. 3-fold) and are easier to synthesize.
molecule forming adducts of elusive structures.
Conclusions
The present study established that BNBD derivatives are Tz- However, NAc-BNBDs provide higher release yields and
activated traceless-release molecules for several leaving groups. product formation is independent of temperature. Generally,
Synthetic strategies were developed for esters, carbonates, and steric bulk at the bridgehead position should be avoided as it
phosphotriesters, and each of these moieties (including can considerably decrease both the rate of reaction and the
examples of bioactive molecules) could be released in high release yield. In conclusion, the reaction of BNBDs is a poss-
yields as previously reported for carbamates. The synthesis of ible avenue to chemically release active molecules in biological
BNBDs with leaving groups introduced by nucleophilic substi- systems with potential applications in biosensing, in cellulo
tutions (e.g. phenols) was unsuccessful. Bimolecular reaction activity control, and drug delivery.
rates as well as release yields were comparable among the
2
0
different leaving groups (Fig. 8). As previously found, reac-
tion rates increased with water content but the effect plateaued
Experimental section
Materials and instrumentation
−1
−1
at H O > 40%. Reaction rates up to k = 0.264 M
s
were
2
2
observed, which is respectable for a biorthogonal reaction.
Attempts to further enhance the rate by the introduction of All chemical reagents and solvents were obtained from com-
boronate groups did not provide the desired result.
mercial sources (Sigma-Aldrich, Alfa-Aesar, Combi-Blocks,
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Acros-Organic, TCI) and used without further purification. dry DCM (2 mL) at 0 °C was added a mixture of 1a (57 mg,
Thin-layer chromatography (TLC) analysis was used to monitor 0.33 mmol) and DMAP (51 mg, 0.42 mmol) in dry DCM
reactions. Purification of compounds was achieved by silica gel (2 mL). The resulting solution was kept for 30 min at 0 °C
1
13
(
300–400 mesh) column chromatography. H NMR and
C
before EDC·HCl (103 mg, 1.2 mmol) was slowly added. The
NMR spectra for compound characterization were recorded on ice-water bath was removed and the reaction mixture was
a Varian Mercury-400 spectrometer and release and mecha- stirred for 3 h at room temperature. The reaction mixture was
nism studies were recorded on a Varian Mercury-500 spectro- diluted with DCM (50 mL) and washed with brine (3 × 50 mL).
meter with chemical shifts expressed as ppm (in CDCl3, The organic layer was dried over Na SO , filtrated, and concen-
2
4
MeOD-d
standard.
4
or DMSO-d
6
) using Me
4
Si (TMS) as the internal trated under reduced pressure. The crude product was purified
by column chromatography (hexane : ethyl acetate = 6 : 1, v/v)
Mass spectra were measured by the University of Utah to afford 3a as a yellow oil in a yield of 55 mg (54%). (R = 0.40
f
Chemistry Mass Spectrometry Facility. Analytical tests were in hexane : EA = 4 : 1, v/v).
1
performed by reverse-phase HPLC (Thermo Scientific, USA)
3
H NMR (400 MHz, CDCl ) δ 7.31–7.25 (m, 3H), 7.14–7.12
using a Luna C18 column (5 μM, 250 × 10 mm, Phenomenex, (m, 1H), 7.09 (dd, J = 2.0 Hz, J = 5.6 Hz, 1H), 7.02–6.96 (m,
1
2
USA). UV-VIS photospectrometic kinetic measurements were 3H), 6.92–6.88 (m, 2H), 6.81 (d, J = 5.6 Hz, 1H), 5.74 (d, J =
performed on a microplate reader SpectramaX M5 (Molecular 2.0 Hz, 1H), 5.13 (d, J = 12.8 Hz, 1H), 5.03 (d, J = 12.8 Hz, 1H),
1
3
Device, USA) in 96-well plates.
4.72 (s, 2H). C NMR (100 MHz, CDCl ) δ 169.1, 157.8, 150.0,
3
1
1
47.5, 145.2, 141.8, 129.7, 125.5, 125.3, 121.9, 120.6, 119.5,
Photospectrometric analysis of reaction kinetics
14.8, 91.0, 82.5, 65.2, 61.9 ppm. HRMS (ESI): calcd for
+
Reactions of BNBDs and PEG-Tz were monitored at λabs
=
C H O [M + Na] 331.0946, found 331.0956.
19
16 4
5
25 nm, which is a local absorbance maximum of PEG-Tz. All
(9-Acetyl-1,4-dihydro-1,4-epiminonaphthalen-1-yl)methyl
kinetic experiments were performed in triplicates. For pseudo- 2-phenoxyacetate (3b). 3b was prepared using the procedure
first order curve fitting Origin 8.0 software was used with the described for 3a, affording the desired compound as a yellow
kt
exponential formula: y = A
25 nm; A
stant). Results are expressed as the mean ± standard deviation 2.0 Hz, 1H), 7.18 (dd, J = 2.0 Hz, J = 5.6 Hz, 1H), 7.03–6.96
1
× e + y
0
(t: time; y: absorbance at oil in a yield of 47%. (R
f
= 0.15 in hexane : EA = 4 : 1, v/v).
1
5
1
: absorbance at t = 0; k: pseudo-first order rate con-
H NMR (400 MHz, CDCl ) δ 7.30–7.28 (m, 2H), 7.24 (d, J =
3
1
2
(
n = 3).
(m, 4H), 6.93–6.90 (m, 2H), 6.84 (d, J = 5.6 Hz, 1H), 5.49 (s,
13
3
H), 4.73 (s, 2H), 1.93 (s, 3H). C NMR (100 MHz, CDCl
3
) δ
Kinetics in different systems
168.8, 168.6, 157.7, 148.8, 148.1, 144.4, 142.6, 129.6, 125.3,
Kinetic tests were prepared in 96-well plates and thoroughly 121.7, 120.3, 114.6, 75.1, 67.1, 65.2, 61.9, 22.9. HRMS (ESI):
+
mixed at 37 °C for UV-Vis measurements. Final solutions calcd for C21
contain tetrazine (0.25 mM) and BNBDs (2.5 mM, 3.75 mM, Benzyl-((1,4-dihydro-1,4-epoxynaphthalen-1-yl)methyl) car-
mM, 6.25 mM) in DMSO/H O system (9 : 1, v/v) or tetrazine bonate (6a). To a solution of 1a (53 mg, 0.32 mmol) in dry
0.2 mM) and 2a (2 mM) in DMSO/H O gradient system (from DCM (6 mL) was added DMAP (0.5 mmol, 70 mg) and benzyl
0% H O–100% H O, v/v). Stock solutions of pinacol ester of chloroformate (71 mg, 0.35 mmol) at 0 °C. The ice-water bath
4
H19NO [M + Na] 372.1212, found 372.1211.
5
2
(
2
1
2
2
1
2 (60 mM) and tetrazine (4 mM) in DMSO were prepared. Pre- was removed and the reaction was kept at 25 °C overnight. The
incubated solutions of pinacol ester of 12 in PBS were prepared reaction mixture was diluted with DCM (50 mL) and washed
and has been completely hydrolyzed into boronic acid (12) with brine (3 × 50 mL). The organic layer was dried over
after 6 h verified by HPLC. Final solutions contain 12 (5 mM, Na
.25 mM, 7.5 mM) with tetrazine (0.5 mM), and norbornene The crude product was purified by column chromatography
5 mM) tetrazine (0.5 mM) at 23 °C for measurements in (hexane : ethyl acetate = 6 : 1, v/v) to afford 6a as a yellow oil in
2 4
SO , filtrated, and concentrated under reduced pressure.
6
(
DMSO/0.01 M PBS system (1 : 3, v/v).
a yield of 48 mg (46%). (R
f
= 0.50 in hexane : EA = 4 : 1, v/v).
H NMR (400 MHz, CDCl ) δ 7.41–7.32 (m, 5H), 7.25–7.23
m, 1H), 7.18–7.16 (m, 1H), 7.08 (dd, J = 1.6 Hz, J = 5.6 Hz,
1
3
Tetrazine-induced release studies
(
1
2
1
Release was quantified by integration of characteristic H NMR 1H), 6.99–6.96 (m, 2H), 6.88 (d, J = 5.6 Hz, 1H), 5.72 (d, J =
peaks. Solutions containing BNBD (18 mM) and DPTz (6 mM) 1.6 Hz, 1H), 5.21 (s, 2H), 5.06 (d, J = 12.0 Hz, 1H), 4.93 (d, J =
1
13
were incubated at 37 °C and H NMR spectra recorded at t = 12.0 Hz, 1H). C NMR (100 MHz, CDCl ) δ 155.2, 149.9, 147.6,
3
6
h. 18-crown-6-ether or furan was used as internal standard 144.9, 141.7, 135.0, 128.6, 128.4, 125.3, 125.1, 120.3, 119.4,
for peak integration. Studies were performed in triplicates and 90.7, 82.4, 82.3, 70.0, 64.9. HRMS (ESI): calcd for C19H O
16 4
+
results are expressed as the mean ± standard deviation (n = 3).
[M + Na] 331.0946, found 331.0946.
9-Acetyl-1,4-dihydro-1,4-epiminonaphthalen-1-yl)methyl
benzyl carbonate (6b). 6b was prepared using the procedure
(
Synthetic procedures
Compounds PEG-Tz, 1a/b and 2a/b were prepared as described for 6a, affording the desired compound as a yellow
2
0
described.
1,4-Dihydro-1,4-epoxynaphthalen-1-yl)methyl 2-phenoxyace-
oil in a yield of 36%. (R
f
= 0.15 in hexane : EA = 4 : 1, v/v).
1
(
H NMR (400 MHz, CDCl ) δ 7.43–7.34 (m, 5H), 7.27–7.23
3
tate (3a). To a solution of 2-phenoxyacetic acid (0.3 mmol) in (m, 2H), 7.02 (dd, J = 2.4 Hz, J = 5.6 Hz, 1H), 6.98–6.94 (m,
1
2
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13
3
H), 5.44 (brs, 3H), 5.23 (s, 2H), 1.95 (s, 3H). C NMR (5 mL) added at 0 °C. The mixture was allowed to warm to
(
100 MHz, CDCl ) δ 168.5, 155.0, 148.9, 148.1, 144.3, 142.3, ambient temperature and then stirred at 45 °C overnight. The
3
1
6
3
35.1, 128.6, 128.4, 125.3, 125.2, 120.5, 120.2, 74.9, 69.6, 67.1, mixture was diluted with ethyl acetate (50 mL) and washed
+
4.9, 29.7, 22.8. HRMS (ESI): calcd for C21
72.1212, found 372.1221.
H
19NO
4
[M + Na]
with brine (3 × 40 mL). The organic layer was dried over
Na SO , filtrated and concentrated under reduced pressure.
2
4
(
1,4-Dihydro-1,4-epoxynaphthalen-1-yl)methyl-2,5,8,11 tetra- The mixture was purified by column chromotography
oxatetradecan-14-oate (4a). To a solution of m-PEG4 acid (hexanes: ethyl acetate = 1 : 1, v/v) and the crude product was
71 mg, 0.3 mmol) in dry DCM (2 mL) was added the mixture repurified over reversed-phase HPLC to afford 8a as yellow oil
(
of 1a (57 mg, 0.33 mmol) and DMAP (51 mg, 0.42 mmol) in in a yield of 30 mg (21%). (The mobile phase A was 0.1% TFA
dry DCM (2 mL) at 0 °C. After the resulting solution was kept in water and mobile phase B was acetonitrile. A gradient of
for another 30 min at 0 °C, EDC·HCl (103 mg, 1.2 mmol) was 0–100% B ranging from 1–20 min was run at a flow rate of
−
1
slowly added. The ice-water bath was removed and the reaction 4.0 mL min .) (R
mixture was stirred for 3 h at room temperature. The reaction
f
= 0.25 in hexane : EA = 2 : 1, v/v).
H NMR (400 MHz, CDCl ) δ 7.38–7.24 (m, 9H), 7.28–7.25
mixture was diluted with DCM (50 mL) and washed with brine (m, 1H), 7.18–7.16 (m, 1H), 7.07 (dd, J = 1.6 Hz, J = 4.4 Hz,
1
3
1
2
(
3 × 50 mL). The organic layer was dried over Na
and concentrated under reduced pressure. The crude product 5.12–5.08 (m, 4H), 4.90–4.86 (m, 2H), 4.79–4.76 (m, 2H).
was purified by column chromatography (ethyl acetate : exane = NMR (100 MHz, CDCl ) δ 149.9, 147.4, 144.7, 141.7, 135.7,
2 4
SO , filtrated, 1H), 7.02–6.96 (m, 2H), 6.88 (d, J = 4.4 Hz, 1H), 5.73 (brs, 1H),
1
3
C
3
1
0 : 1, v/v) to afford 4a as a yellow oil in a yield of 53 mg (44%). 135.6, 128.6, 128.0, 125.3, 125.1, 120.2, 119.6, 91.3, 91.2, 82.3,
(
R
f
= 0.45 in EA : hexane = 6 : 1, v/v).
69.6, 64.7, 64.6.
(3-((4-(Trifluoromethyl)phenyl)thio)-1,2,3,4-tetrahydro-1,4-
1
H NMR (400 MHz, CDCl ) δ 7.24–7.22 (m, 1H), 7.17–7.15
3
(
m, 1H), 7.06 (dd, J
1
= 1.6 Hz, J
H), 6.84 (d, J = 5.6 Hz, 1H), 5.70 (d, J = 2.0 Hz, 1H), 5.02 (d, J = To a solution of 1a (68 mg, 0.4 mmol) in dry DCM (3 mL) was
2.8 Hz, 1H), 4.84 (d, J = 12.8 Hz, 1H), 3.76 (t, J = 6.4 Hz, 2H), added TsCl (79 mg, 0.4 mmol) and DMAP (cat, 5 mg). After the
.63–3.59 (m, 10H), 3.53–3.51 (m, 2H), 3.35 (s, 3H), 2.67 (t, J = resulting solution was cooled to 0 °C, Et N (122 mg, 1.2 mmol)
) δ 171.5, 149.9, 147.7, was slowly added. The ice-water bath was removed and the
2
= 5.6 Hz, 1H), 6.98–6.96 (m, epoxynaphthalen-1-yl)methyl 4-methylbenzenesulfonate (11a).
2
1
3
6
1
7
3
1
3
.0 Hz, 2H). C NMR (100 MHz, CDCl
3
44.8, 142.0, 125.3, 125.1, 120.3, 119.4, 90.9, 82.3, 71.9, 70.6, reaction mixture was stirred for 3 h at room temperature. 4-(tri-
0.5, 70.4, 70.3, 66.4, 61.4, 61.3, 59.0, 58.9, 34.9. HRMS (ESI): fluoromethyl)benzenethiol (155 mg, 0.8 mmol) was added and
+
calcd for C21
H
28
O
7
[M + Na] 415.1733, found 415.1740.
the mixture kept for 12 h. The reaction mixture was diluted
(
9-Acetyl-1,4-dihydro-1,4-epiminonaphthalen-1-yl)methyl with DCM (50 mL) and washed with brine (3 × 50 mL). The
2
,5,8,11-tetraoxatetradecan-14-oate (4b). 4b was prepared organic layer was dried over Na SO , filtrated, and concen-
2
4
using the procedure described for 4a, affording the desired trated under reduced pressure. The crude product was purified
compound as a yellow oil in a yield of 40%. (R = 0.20 in by column chromatography (hexane : EA = 4 : 1, v/v) to afford
f
EA : hexane = 6 : 1, v/v).
9a as a yellow oil in a yield of 51 mg (60%). (R
f
= 0.80 in
1
H NMR (400 MHz, CDCl
3
) δ 7.27–7.23 (m, 2H), 7.03–7.00 hexane : EA = 2 : 1, v/v).
1
(m, 1H), 6.99–6.96 (m, 2H), 6.92 (d, J = 5.6 Hz, 1H), 5.47 (brs,
H NMR (400 MHz, CD OD-d ) δ 7.83 (d, J = 8.4 Hz, 2H),
3 4
1
3
3
1
1
7
2
H), 5.37 (s, 2H), 3.79 (t, J = 6.4 Hz, 2H), 3.62–3.59 (m, 10H), 7.60 (d, J = 8.4 Hz, 2H), 7.46–7.42 (m, 4H), 7.38–7.35 (m, 1H),
.53–3.51 (m, 2H), 3.35 (s, 3H), 2.68 (t, J = 6.4 Hz, 2H), 1.94 (s, 7.25–7.18 (m, 2H), 7.14 (dd, J = 2.0 Hz, J = 6.4 Hz, 1H), 5.19
H). C NMR (100 MHz, CDCl ) δ 171.4, 169.5, 168.4, 149.2, (s, 1H), 4.78 (d, J = 11.2 Hz, 1H), 4.69 (d, J = 11.2 Hz, 1H),
1
2
1
3
3
48.2, 144.6, 142.2, 133.7, 132.2, 129.0, 127.9, 127.5, 126.5, 3.52–3.49 (m, 1H), 2.44 (s, 3H), 2.07–2.02 (m, 1H), 1.74 (dd, J
1
1
3
26.0, 125.3, 124.2, 122.2, 120.8, 120.4, 120.2, 75.1, 71.8, 70.5, = 4.0 Hz, J
2
= 12.0 Hz, 1H). C NMR (100 MHz, CDCl
3
) δ 145.2,
0.4, 70.3, 67.0, 66.5, 66.4, 61.4, 59.0, 58.9, 35.1, 35.0, 24.0, 143.6, 143.5, 141.6, 132.3, 129.9, 128.1, 127.8, 127.7, 125.9,
+
7
2.8. HRMS (ESI): calcd for C23H31NO [M + Na] 456.1998, 119.5, 118.9, 86.3, 83.2, 83.1, 67.6, 46.7, 37.4, 21.7. HRMS
+
found 456.1996.
(ESI): calcd for C25
((1,4-dihydro-1,4-epoxynaphthalen-1-yl)methyl) 529.0735.
A portion of the tosyl-protected intermediate (9a) was iso-
21 3 2 4
H F S O [M + Na] 529.0731, found
Dibenzyl
phosphate (8a). To a solution of N-chlorosuccinimide (812 mg,
.1 mmol) in dry toluene (6 mL) was added dibenzyl phosphite lated, purified, and characterized by H NMR. H NMR
1665 mg, 6 mmol) at ambient temperature. After the resulting (400 MHz, CDCl ) δ 8.27 (brs, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.43
1
1
6
(
3
solution was kept for 3 h, the reaction mixture was filtrated (d, J = 8.4 Hz, 2H), 7.31–7.29 (m, 1H), 7.22–7.20 (m, 1H), 7.12
and filtrate was diluted with hexane (50 mL) and washed. The (dd, J = 2.0 Hz, J = 6.0 Hz, 1H), 7.05–7.03 (m, 2H), 6.90 (d, J =
organic layer was dried over Na SO to afford dibenzylpho- 5.2 Hz, 1H), 6.59 (brs, 1H), 4.96 (d, J = 11.2 Hz, 1H), 4.82 (d, J =
1
2
2
4
sphoryl chloride as ayellow oil in a yield of 1490 mg (84%). 11.2 Hz, 1H), 3.09 (s, 3H), 2.52 (s, 3H).
Dibenzylphosphoryl chloride was used immediately without
further purification.
1-(1,4-Dihydro-1,4-epoxynaphthalen-1-yl)ethanol (1c). To a
solution of 2-(trimethylsilyl)phenyl imidazolsulfonate (600 mg,
To a well-stirred solution of dibenzylphosphoryl chloride 2 mmol) and furan-2-ylmethanol (316 mg, 3 mmol) in dry
444 mg, 1.5 mmol) in dry THF (5 mL) was a solution of 1a MeCN (5 mL) was added cesium fluoride (600 mg, 4 mmol).
52 mg, 0.3 mmol) and DMAP (184 mg, 1.5 mmol) in dry THF The reaction mixture was heated to 45 °C and maintained at
(
(
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this temperature for 8 h. The mixture was diluted with ethyl (brs, 1H), 5.39–5.31 (m, 2H), 4.61–4.55 (m, 1H), 2.11–2.07 (m,
acetate (250 mL) and washed with water (2 × 100 mL). The 1H), 1.95 (brs, 4H), 1.74–1.68 (m, 2H), 1.51–1.38 (m, 2H),
organic layer was again washed with brine (2 × 100 mL), dried 1.16–1.01 (m, 2H), 0.96–0.90 (m, 7H), 0.83 (t, J = 7.2 Hz, 3H).
1
3
over Na
pressure. The crude was purified by column chromatography 148.4, 143.6, 142.8, 142.7, 125.1, 125.0, 124.8, 120.2, 119.7,
hexane : ethyl acetate = 2 : 1, v/v) to provide 1c as a yellow oil 119.6, 78.4, 75.1, 75.0, 40.5, 40.4, 33.8, 31.3, 26.1, 26.0, 23.2,
in a yield of 150 mg (40%). 20.9, 19.6, 19.5, 15.4. MS (ESI): calcd for C24 [M +
2
SO
4
, filtrated, and concentrated under reduced
4
C NMR (100 MHz, MeOD-d ) δ 168.3, 154.8, 148.8, 148.5,
(
H
31NO
4
1
+
H NMR (400 MHz, CDCl ) δ 7.31–7.21 (m, 2H), 7.07–7.05 Na] 420.21, found 420.14.
3
(
(
m, 1H), 7.00–6.96 (m, 2H), 6.89 (d, J = 5.6 Hz, 1H), 5.72–5.71
m, 1H), 4.67 (m, 1H), 1.56–1.49 (m, 3H). C NMR (100 MHz,
1
3
CDCl ) δ 150.8, 148.2, 147.8, 144.6, 142.6, 141.6, 125.0, 124.9, Conflicts of interest
3
1
20.3, 120.2, 120.1, 96.1, 82.0, 66.2, 65.5, 19.2, 18.8. HRMS
+
There are no conflicts to declare.
(ESI): calcd for C12
12 2
H O [M + Na] 211.0735, found 211.0736.
1
-(1,4-Dihydro-1,4-epoxynaphthalen-1-yl)ethyl (4-nitrophe-
nyl)carbamate (2c). To a solution of 1c (75 mg, 0.4 mmol) and
DMAP (98 mg, 0.6 mmol) in THF (10 mL) was added 4-nitro-
phenylisocyanate (58 mg, 0.48 mmol). The reaction was stirred
at 50 °C for 12 h. The mixture was diluted with ethyl acetate
Acknowledgements
We gratefully acknowledge financial support from the
University of Utah, the Huntsman Cancer Institute, and the
Utah Science and Technology Research Initiative. We thank
Jack Skalicky for help with NMR experiments and the Centre
for High Performance Computing at the University of Utah for
computational resources.
(150 mL) and washed with water (2 × 100 mL), and brine (3 ×
1
00 mL). The organic layer was dried over Na SO , filtrated,
2
4
and concentrated under reduced pressure. The crude was puri-
fied by column chromotography (hexane : ethyl acetate = 4 : 1,
v/v) to afford 2c as a yellow solid in a yield of 50 mg (36%).
f
(R = 0.55 in hexane : EA = 2 : 1, v/v).
1
H NMR (400 MHz, CDCl
3 1 2
) δ 8.22 (dd, J = 2.0 Hz, J =
Notes and references
7
.2 Hz, 2H), 7.57 (dd, J = 2.0 Hz, J = 7.2 Hz, 2H), 7.27–7.25
1
2
(
J
6
3
1
9
m, 2H), 7.21–7.09 (m, 1H), 7.17 (brs, 1H), 7.08 (dd, J
= 5.6 Hz, 1H), 7.02–7.00 (m, 2H), 6.91 (d, J = 5.6 Hz, 1H),
.01–5.96 (m, 1H), 5.77 (d, J = 2.0 Hz, 1H), 1.56 (d, J = 6.4 Hz,
1
= 2.0 Hz,
1 E. M. Sletten and C. R. Bertozzi, Acc. Chem. Res., 2011, 44,
666–676.
2 P. Shieh and C. R. Bertozzi, Org. Biomol. Chem., 2014, 12,
9307–9320.
3 C. S. McKay and M. G. Finn, Chem. Biol., 2014, 21, 1075–
1101.
4 H. E. Murrey, J. C. Judkins, C. W. Am Ende, T. E. Ballard,
Y. Fang, K. Riccardi, L. Di, E. R. Guilmette, J. W. Schwartz,
J. M. Fox and D. S. Johnson, J. Am. Chem. Soc., 2015, 137,
11461–11475.
2
1
3
3
H). C NMR (100 MHz, CDCl ) δ 152.2, 150.1, 147.6, 144.8,
43.6, 143.2, 142.2, 125.4, 125.3, 125.2, 120.3, 119.8, 117.7,
4.5, 82.1, 69.7, 16.1. HRMS (ESI): calcd for C H N O [M +
1
9
16 2 5
+
Na] 379.0957, found 379.0963.
9-Acetyl-1,4-dihydro-1,4-epiminonaphthalen-1-yl)methyl 2-
4-isobutylphenyl)propanoate (5b). 5b was prepared using the
procedure described for 3a, affording the desired compound
as a yellow oil in a yield of 45%. (R = 0.30 in hexane : EA =
(
(
f
5 M. K. Rahim, R. Kota, S. Lee and J. B. Haun, Nanotechnol.
Rev., 2013, 2, 215–227.
6 T. Reiner and B. M. Zeglis, J. Labelled Compd. Radiopharm.,
2014, 57, 285–290.
2
: 1, v/v).
1
H NMR (400 MHz, MeOD-d
4
) δ 7.27 (dd, J
1
= 2.4 Hz, J
2
=
7
.2 Hz, 1H), 7.23–7.18 (m, 2H), 7.10–7.05 (m, 3H), 7.03–6.99
(
m, 1H), 6.96–6.93 (m, 1H), 6.92–6.78 (m, 1H), 6.64 (d, J = 5.6
7 A. C. Knall and C. Slugovc, Chem. Soc. Rev., 2013, 42, 5131–
5142.
Hz, 1H), 5.68 (brs, 1H), 5.27–5.16 (m, 2H), 3.84–3.79 (m, 1H),
2
7
.46–2.42 (m, 2H), 1.91 (s, 3H), 1.85–1.79 (m, 1H), 1.49 (d, J =
.2 Hz, 3H), 0.89–0.85 (m, 6H). C NMR (100 MHz, MeOD-d4)
8 H. Wu and N. K. Devaraj, Top. Curr. Chem., 2016, 374, 3.
9 X. Ji, K. Ji, V. Chittavong, R. E. Aghoghovbia, M. Zhu and
B. Wang, J. Org. Chem., 2017, 82, 1471–1476.
1
3
δ 174.4, 168.0, 148.9, 148.3, 143.7, 143.6, 142.6, 142.3, 140.4,
1
1
3
40.3, 137.8, 128.9, 127.0, 126.9, 124.8, 124.7, 124.6, 120.0, 10 M. L. Blackman, M. Royzen and J. M. Fox, J. Am. Chem.
19.9, 119.8, 75.0, 74.8, 66.8, 61.5, 61.2, 44.8, 44.6, 44.5, 30.1, Soc., 2008, 130, 13518–13519.
0.0, 21.3, 17.2, 17.0. MS (ESI): calcd for C26
H
29NO
3
[M + 11 D. M. Patterson, L. A. Nazarova, B. Xie, D. N. Kamber and
+
Na] 426.20, found 426.13.
J. A. Prescher, J. Am. Chem. Soc., 2012, 134, 18638–18643.
(
9-Acetyl-1,4-dihydro-1,4-epiminonaphthalen-1-yl)methyl (2-iso- 12 J. Yang, J. Seckute, C. M. Cole and N. K. Devaraj, Angew.
propyl-5-methylcyclohexyl) carbonate (7b). 7b was prepared Chem., Int. Ed., 2012, 51, 7476–7479.
using the procedure described for 6a but with 2.5 eq. of 13 S. Lai, W. Mao, H. Song, L. Xia and H. Xie, New J. Chem.,
DMAP, affording the desired compound as a yellow oil in a
yield of 35%. (R = 0.70 in hexane : EA = 1 : 1, v/v).
H NMR (400 MHz, MeOD-d ) δ 7.34–7.32 (m, 1H),
.28–7.25 (m, 1H), 7.13–7.10 (m, 1H), 7.03–6.95 (m, 3H), 5.75
2016, 40, 8194–8197.
f
14 S. B. Engelsma, L. I. Willems, C. E. van Paaschen, S. I. van
Kasteren, G. A. van der Marel, H. S. Overkleeft and
D. V. Filippov, Org. Lett., 2014, 16, 2744–2747.
1
4
7
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View Article Online
Organic & Biomolecular Chemistry
Paper
1
5 N. K. Devaraj, R. Weissleder and S. A. Hilderbrand, 31 R. Rossin, S. M. van Duijnhoven, W. Ten Hoeve,
Bioconjugate Chem., 2008, 19, 2297–2299.
6 A. C. Knall, M. Hollauf and C. Slugovc, Tetrahedron Lett.,
H. M. Janssen, L. H. Kleijn, F. J. Hoeben, R. M. Versteegen
and M. S. Robillard, Bioconjugate Chem., 2016, 27, 1697–
1706.
1
2
014, 55, 4763–4766.
17 R. N. Warrener and P. A. Harrison, Molecules, 2001, 6, 353–369. 32 A. K. Steiger, Y. Yang, M. Royzen and M. D. Pluth, Chem.
1
8 G. M. Priestley and R. N. Warrener, Tetrahedron Lett., 1972,
3, 4295–4298.
Commun., 2017, 53, 1378–1380.
1
33 D. Wang, E. Viennois, K. Ji, K. Damera, A. Draganov,
Y. Zheng, C. Dai, D. Merlin and B. Wang, Chem. Commun.,
2014, 50, 15890–15893.
1
2
9 H. Wu, B. T. Cisneros, C. M. Cole and N. K. Devaraj, J. Am.
Chem. Soc., 2014, 136, 17942–17945.
0 M. Xu, J. Tu and R. M. Franzini, Chem. Commun., 2017, 53, 34 W. Wang, X. Ji, Z. Du and B. Wang, Chem. Commun., 2017,
6
271–6274.
53, 1370–1373.
2
2
1 J. Li and P. R. Chen, Nat. Chem. Biol., 2016, 12, 129–137.
2 R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen
35 J. Li, S. Jia and P. R. Chen, Nat. Chem. Biol., 2014, 10, 1003–
1005.
and M. S. Robillard, Angew. Chem., Int. Ed., 2013, 52, 36 J. Luo, Q. Liu, K. Morihiro and A. Deiters, Nat. Chem., 2016,
4112–14116. 8, 1027–1034.
3 X. Fan, Y. Ge, F. Lin, Y. Yang, G. Zhang, W. S. Ngai, Z. Lin, 37 G. Zhang, J. Li, R. Xie, X. Fan, Y. Liu, S. Zheng, Y. Ge and
1
2
S. Zheng, J. Wang, J. Zhao, J. Li and P. R. Chen, Angew.
Chem., Int. Ed., 2016, 55, 14046–14050.
4 R. M. Franzini and E. T. Kool, J. Am. Chem. Soc., 2009, 131,
P. R. Chen, ACS Cent. Sci., 2016, 2, 325–331.
38 D. I. Davies, L. T. Parfitt, C. K. Alden and J. A. Claisse,
J. Chem. Soc. C, 1969, 1585–1590.
2
2
2
1
6021–16023.
39 N. Cengiz, T. N. Gevrek, R. Sanyal and A. Sanyal, Chem.
Commun., 2017, 53, 8894–8897.
40 A. C. Knall, M. Hollauf, R. Saf and C. Slugovc, Org. Biomol.
Chem., 2016, 14, 10576–10580.
5 K. Gorska, A. Manicardi, S. Barluenga and N. Winssinger,
Chem. Commun., 2011, 47, 4364–4366.
6 S. S. Matikonda, D. L. Orsi, V. Staudacher, I. A. Jenkins,
F. Fiedler, J. Chen and A. B. Gamble, Chem. Sci., 2015, 6, 41 S. Eising, F. Lelivelt and K. M. Bonger, Angew. Chem., Int.
212–1218. Ed., 2016, 55, 12243–12247.
7 K. Neumann, S. Jain, A. Gambardella, S. E. Walker, 42 A. Kaya, E. Salamci, A. Menzek, S. Erdem, E. Sahin and
1
2
2
E. Valero, A. Lilienkampf and M. Bradley, ChemBioChem,
017, 18, 91–95.
8 E. Jimenez-Moreno, Oliveira,
K. Ecer, Tetrahedron, 2017, 73, 5381–5388.
43 D. Margetic, D. Butler and R. Warrener, Synlett, 2013,
2609–2613.
2
Z.
Guo,
B.
L.
I. S. Albuquerque, A. Kitowski, A. Guerreiro, O. Boutureira, 44 Z. Li, X. Gao, W. Shi, X. Li and H. Ma, Chem. Commun.,
T. Rodrigues, G. Jimenez-Oses and G. J. Bernardes, Angew.
Chem., Int. Ed., 2017, 56, 243–247.
9 H. Wu, S. C. Alexander, S. Jin and N. K. Devaraj, J. Am.
Chem. Soc., 2016, 138, 11429–11432.
0 J. M. Mejia Oneto, I. Khan, L. Seebald and M. Royzen, ACS
Cent. Sci., 2016, 2, 476–482.
2013, 49, 5859–5861.
45 Z. Li, X. Li, X. Gao, Y. Zhang, W. Shi and H. Ma, Anal.
Chem., 2013, 85, 3926–3932.
46 X. Ao, S. A. Bright, N. C. Taylor and R. B. P. Elmes, Org.
Biomol. Chem., 2017, 15, 6104–6108.
47 S. Tekkam and M. G. Finn, Org. Lett., 2017, 19, 2833–2836.
2
3
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