Photoisomerization-enhanced 1,3-dipolar cycloaddition of carbon-bridged octocyclic azobenzene with photo-released nitrile imine for peptide stapling and imaging in live cells

Article abstract of DOI:10.1039/d0ob01027h

A photo-induced 1,3-dipolar cycloaddition between nitrile imine and highly ring-strained NN double bond as a dipolarophile was discovered. The photo-isomerization of carbon-bridged octocyclic azobenzene (CBOA) into its trans-configuration accelerates the ligation reaction at a very rapid rate (28 400 M-1 s-1). The CBOA-based photo-click reaction was proved to be bioorthogonal. In addition, the NoxaB peptide was successfully cross-linked by a CBOA stapler which plays a dual role: Photo-control of the conformation of the peptide and photo-conjugation of probes in live cells.

Full text of DOI:10.1039/d0ob01027h

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DOI: 10.1039/D0OB01027H
ARTICLE
Photoisomerization‐enhanced 1,3‐dipolar Cycloaddition of
Carbon‐bridged Octocyclic Azobenzene toward Photo‐released
Nitrile Imine for Peptide Stapling and Imaging in Live Cells
Received 00th January 20xx,
Accepted 00th January 20xx
a
Jiajie Deng, Xueting Wu, Guiling Guo, Xiaohu Zhao and Zhipeng Yu*
DOI: 10.1039/x0xx00000x
A photo‐induced 1,3‐dipolar cycloaddition between nitrile imine and highly ring‐strained N=N double bond as
dipolarophile was discovered. The photo‐isomeriaztion of carbon‐bridged octocyclic azobenzene (CBOA) into its trans‐
configuration accelerate the ligation reaction in a very rapid rate (28400 M‐1 s‐1). The CBOA based photo‐click reaction was
proved to be bioothorgonal. In addtion, the NoxaB peptide was successfully cross‐linked by CBOA stapler which plays a
dual role: photo‐control of the conformation of peptide and photo‐conjugation of
probes in live cells.
Nitrile Imine-Carbon-bridged Octocyclic Azobenzene Photo-click Reaction (NICBOAP)
R1
R2
N
Introduction
6
5
N
4
7
N
N
3
8
Click chemistry is a powerful tool for bio‐conjugation due to its
fast reaction rate, high specificity and biocompatibility.1 Photo‐
induced click reactions have received a considerable attention
because of the temporal and spatial controllability,2 including:
1,3‐dipolar cycloaddition reactions between alkenes3/alkynes4
and nitrile imines,5 cycolpropenone caged cycloalkynes with
azides;6 and Diels–Alder reactions of dihydrotetrazine,7 3‐
(hydroxymethyl)‐2‐naphthol8 or 9,10‐phenanthrene‐quinone9
with alkenes. In order to accelerate the cycloaddition, ring‐
strain was considered to be an important means of activating
alkenes or alkynes. For example, ring‐strained spiro[2.3]hex‐1‐
ene (Sph)3b and trans‐cyclooct‐4‐en‐1‐ol (TCO)10 elevated the
efficiency of photo‐click chemistry dramatically. Recently,
Weaver reported a cycloaddition of alkyl azides with strained
cycloalkenes via the reversibly formed trans‐isomer which
mediated by photo‐sensitizer to conjugate biotin onto
insulin.11 We are curious about seeking dipolarophiles in which
the ring‐strain can be tuned directly via photo‐isomerization to
enhance selectivity. Azobenzenes could undergo photo‐
switching between trans‐ and cis‐isomers as ideal
conformational regulators.12 Therefore, annular azobenzenes
are worthy of being explored to tune the performance of
cycloaddition because photo‐induced conformation variations
could be transformed into ring‐strain loadings.
- N2
h
2
1
R2
R1
intermediate
slow k2cis
R1
cis, ring-strained on
C8-N1=N2 and N1=N2-C3
N
N
N-N
bridge-opening
N
N
[3+2]
+
N
N
532 nm
405 nm
or
R2
Nitrile Imine
fast k2trans
5
h
- CO2
7
tetraazacycloundeca-1,4-dien-2-ium-3-ide
(TACD) a stable 1,3-dipole
4
3
6
2
8
1
R2
R1
Ultra-fast cycloaddition, k2 up to 28400 M-1s-1
Ring-strain loading in-situ on trans-CBOA
trans, ring-strained on
N
C8-N1=N2-C3
dihedral
N
O
O
X-ray crytal structures of 1a
Scheme 1. Comparison of trans and cis isomer of CBOA in 1,3‐dipolar
cycloaddition with nitrile imine generated from photolysis of tetrazole or
sydnone.
octocyclic azobenzene (CBOA) with remarkable photo‐physical
properties.14 Recently, Herges presented oxygen‐, sulfur‐ and,
especially nitrogen‐bridged CBOAs that could be switched
within near‐infrared region in water.15 In addition, CBOA
exhibited the value for applications in photo‐regulation of
DNA,16 peptide cross‐linking to control helicity17 and
photochromic molecular with turn‐on fluorescence.18
Intriguingly, CBOA could also be regarded as a dipolarophile to
react with 1,3‐dipole, which has not been previously studied in
detail (Scheme 1). Opposite to uncyclic azobenzenes, CBOA 1a
produces a highly twisted ring‐strain in form of the large
torsion angle on C8‐N1=N2‐C3 atoms in trans‐isomer.
Astoundingly, the crystal structure of the trans‐1a with higher
energy was obtained, consisting with the optimized geometry
via theoretical predictions.19
Eight‐membered ring‐system featuring unsaturated bonds
has been studies for their aromaticity and shapes.13 Siewertsen
revealed the photo‐switchable nature of carbon‐bridged
Results and discussion
Photo‐physical characters
a. Key Laboratory of Green Chemistry and Technology of Ministry of Education,
College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu (610064),
China. E‐mail: zhipengy@scu.edu.cn.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
We designed and synthesized a series of CBOAs with various
substitutions, including cyano‐, acetyl‐ (electron withdrawing
groups, EWG) and N‐Boc‐piperazine (electron donating groups,
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Journal Name
Tetrazoles3a‐3e and sydnones3f, 4a, 20 were reported to generate
Table 1. Structures of CBOA and their photo‐physical characters under
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photo‐isomerization stimulationa
nitrile imine (NI) intermediate, which cycDloOaI:d1d0i.t1i0o3n9/wD0itOhBa0l1k0e2n7He
or alkyne to afford pyrazoline or pyrazole, respectively. To
investigate the influence of the ring‐strain and electron effects
on the reactivity of azobenzenes, the photo‐triggered ligations
λmax
ε405
PSSc
(%)
b
b
Cyclic Azobenzene
τd (s)
17580
396
Фe
0.73
1.2
(nm)
(M-1cm-1)
were performed for tetrazole 2b toward CBOA 1a‐1d or
azobenzene (Fig. S37‐41, ESI†). There was no generation of
cycloadducts toward azobenzene, while CBOA can be
398
532
75
71
transformed into an unprecedented eleven‐membered TACD
4
396
398
362
604
644
via cycloaddition followed by N‐N bridge opening step. The
CBOA with EWG substitutions (1b and 1c) apparently provided
favorable reactivities, resulting in up to 99% HPLC yield.
However, 1d substituted with piperazine only afforded trace
amount of cycloadduct due to its poor photoisomerization
performance (Fig. S1, ESI†), indicaꢀng its inability to load the
photo‐energy to promote the ligation. Therefore, the photo‐
induced ring‐strain loading facilitates the reactivity of N‐N
double bond in CBOA as dipolarophile, and its electron‐
deficiency further accelerate the cycloaddition toward HOMO‐
controlled dipole, e.g. NI.
54
418
0.84
N.B.
N.B.
N.B.
3064
aPhoto‐isomerization scheme. bUV‐Vis absorption characteristics of CBOA in mixed
solvent at 250 μM. cPhotostationary state (PSS) at 405 nm obtained in acetonitrile‐
d3 via 1H NMR spectra. dHalf‐life of thermo‐relaxation of trans‐CBOA at 298 K.
ePhoto‐isomerization quantum yields cis→trans of CBOA under 405 nm laser
stimulation. See Fig. S1‐8 and table S1 ESI† for details. N.B. = not observed.
EDG). Maximum absorption wavelength, photostationary state,
half‐life and isomerization quantum yield of CBOAs were
offered in Table 1. The UV‐Vis spectra showed that the trans
We investigated the distributions of products resulting from
the photo‐click reaction of CBOA with tetrazoles or sydnones
in quartz test tube under exposure to various irradiation,
isomer of 1a, 1b and 1c were most effectively produced by
irradiation at 398 nm, 396 nm and 398 nm respectively, the
wavelength where εcis was maximal. Irradiation of 1b with 405
nm LED produced the trans isomer, which exhibited an n‐π*
transition maximum near 490 nm, approximately 94 nm
redshifted in comparison with the cis form. In contrast, there
was no photoisomerization of 1d observed under 405 nm laser
stimulation (Fig. S1, ESI†).
which mainly affords the TACD
4 (Fig. 1). Based on HPLC‐MS
analysis, the experimental results can be summarized as
follows: (i) sydnones and tetrazoles with EDG (2g and 3g) on C‐
terminal showed poor reactivity toward CBOA with quenched
NI as side‐products; (ii) dual irradiation of 311 and 405 nm
light afforded higher HPLC yield for the desired
4 and 4+2H,
Photo‐click reaction
suppressing the formation of quenched NI; (iii) the EWG
COOMe
hydrazonyl chloride 5
MeO
NC
(a)
(b)
N
C-teminal
N
H
MeO
N
Cl
- HCl
R2
N
OMe
1a
N
1b
4ba
N
base
N
N
N
h
N
Tet 2
- N2
h
N
PSS of
R2
CN
N
intermediate
CBOA 1
N
N
F3C
MeO
OMe
X
2a or 3a: R2 = p-CF3C6H4
3h: R2
=
MeO
R2
[3+2]
2b or 3b: R2 = 3,5-(CF3)2C6H3
2c or 3c: R2 = p-COOMeC6H4
2d or 3d: R2 = p-BrC6H4
2e or 3e: R2 = 3,5-Br2C6H3
2f or 3f : R2 = p-FC6H4
O
h
N
N
N
90o
C-teminal
N
O
- CO2
R1
N
N
R1
R2
Nitrile Imine
4
Y
N
90o
stable 1,3-dipole
N
2g or 3g: R2 = p-MeOC6H4
Syd 3
O
O
NC
CN
(c)
(d)
Isolated yield of compound 4 (%)
78,51, 69,62, 63,52, 53,54, 71,52, 40,45, 65, 69, 71, 82, 52, 69, 59, 53, 65, 66, 56, Trace,30,
61, 60,
Fig. 1 Screening of photo‐activated 1,3‐Dipolar cycloaddition of tetrazoles or sydnones with CBOA. (a) Reaction scheme displaying the nitrile imine sources. (b) X‐ray
crystal structure of desired 4ba. (c) Percentage distribution of desired products 4, reactant and by‐products after photo‐triggered [3+2] reaction. 50 μM 2 or 3 and 100
μM 1a or 1b in acetonitrile /H2O (1:1) were irradiated with designated light sources for 60 s. Conversions and yields were determined by HPLC‐MS analysis. See Figs.
S21‐35 ESI† for details. Tetrazole 2e precipitated out from acetonitrile/H2O (1:1, v/v). (d) Isolated yield of 1 mM of 2 or 3 with 1.5 eq. of 1a or 1b in 100 mL of
dichloromethane (DCM) to collect the desired TACD 4 for NMR identification after exposure under 405 nm LED (33.6 mW/cm2) and 311 nm UV lamp (10.8 mW/cm2)
for 60 min.
2 | J. Name., 2012, 00, 1‐3
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substituted CBOA 1b could react more cleanly with less by‐ experiment of a chemically tagged lyDsoO‐I(: 10.1039/D0OB01027H
1e
)
(Fig. 2a). To
n
products; (iv) sydnone 3h offered superior reactivity and visualize the covalent modification via SDS‐PAGE analysis, a
selectivity toward both CBOAs under single 405 nm irradiation FITC‐PEG‐DASyd
6 was synthesized and protein labelling
(Fig. 1, and Fig. S21‐35, ESI†). experiments were performed at 5 µM concentration under
Reactivity with nitrile imine. Irradiating 1a or 1b with diode 405 nm photo‐induction. There was an exclusively labelling
laser at 405 nm produced photostationary states (PSS) that band of lyso‐(1e)n in first the lane from the in‐gel fluorescence
comprise approximately 75% and 71% trans‐isomer with the imaging. In control lanes in which native lysozyme was subject
quantum efficiency of Z→E determined to be 0.73 and 1.2, to the identical photo‐labelling conditions, there was no
respectively (Table 1, Fig. S8, ESI†). Based on the higher fluorescence signal detected, suggesting a high selectivity of
selectivity of CBOA photo‐ligation promoted by the addition of the NI‐CBOA photo‐click system. The photo‐conjugation of
405 nm irradiation, we propose that the ring‐strain of trans‐ lyso‐(1e)n were also confirmed by LC‐MS analysis (Fig. 2b).
CBOA boosts the rate of 1,3‐ dipolar cycloaddition. To compare
Peptide stapling
the reactivity of trans with cis isomer of CBOA in the
The photoisomerization of CBOA has been used to regulate the
cycloaddition step, we synthesized hydrazonyl chloride to
conformation of peptide FK‐11 in vitro17 via stapling on a pair
serve as a readily accessible NI precursor,21 whose reactivity
of L‐Cys residues through thiol‐alkylation. We investigated
CBOA‐cross‐linked NoxaB peptide, p2 and p4 (Figs. S13‐14,
ESI†), because their α‐helix might be precisely photo‐
controlled for inhibition of Mcl‐1 22
an anti‐apoptotic member
of Bcl‐2 family for cancer cell survival. The circular dichroism
(CD) spectra showed that distance‐matched23 CBOA‐cross‐
can be released without photo‐stimulation. Therefore,
hydrazonyl chloride
5 and 3 eq. 1a or 1b were dissolved in
DCM in a pressure‐resistant tube, supplied with Et3N to adjust
the pH to be basic, and divided into two portions, one of which
was exposed to shining of 405 nm LED, and the other was kept
,
in dark. Based on HPLC‐MS analysis, desired cycloadducts
4
was formed rapidly in the presence of 405 nm irradiation,
which was about 10‐fold more than that in dark over the
reacꢀon course (Fig. S36, ESI†).
a)
cis-1a
trans-1a
The rates of cycloaddition step. To demonstrate the potential
of CBOA for photo‐bioconjugation that is relevant to many
biological applications, we investigated the second order
reaction rate of the ligation step between 1b and photo‐
released NI, which must be fast enough to be useful at low
concentration. Through the one‐pot competition experiment
cis-1a
85.1
6.2
trans-1a
35.8
4ba
28.9
7.9
4ba
dihedral angle across two
phenyls of CBOA(°)
end-to-end distance (Å)
8.5
b)
12.7 Å
1
13.0 Å
10.7 Å
analysed via H NMR tracing (Fig. S9, ESI†), we observed that
the photo‐ligation cycloadduct 4bb from 5 eq. CBOA and
tetrazole 2b at 311 nm irradiation was approximately 15‐fold
more than that from 15.8 eq. methacrylamide (MAA). Given
that the second‐order rate constant between MAA and NI was
obtained via real‐time measurement, the cycloaddition rate
between 1b and 2b was determined to be k2 = 28400 ± 2300
M‐1 s‐1 (Fig. S9, ESI†), which enables possible live cell photo‐
conjugation.
p5’ (2c locked)
trans-p4
cis-p4
p4 in dark (helicity 62 %)
p4 PSS 405 nm
(helicity 80 %)
p4 PSS 514 nm)
(helicity 62 %)
30
20
10
0
c)
p5' in ACN/PB = 1/1
(helicity 76 %)
-10
-20
190
200
210
220
230
240
wavelength (nm)
Protein labelling
d)
Then, we performed the in‐vitro protein photo‐labelling
2h
1f
peptide
Sequencea
Charge
Cell Viabilityb (%)
97.3 ± 5.9
+3
+3
0
p1
p2
p3
p4
p5
p5'
AACLRRIGDCVNLRQKLLN-NH2
AAC’LRRIGDC’VNLRQKLLN-NH2
AACLRAIGDAVNLCQALLNK-NH2
25 μM
Lyso-1en
Lyso-1en
250 μM
6, 405 nm, 2 min
3h, 405 nm, 2 min
a)
b)
c
80.1 ± 6.3
101.0 ± 3.7
105.0 ± 3.8
49.8 ± 4.9
45.7 ± 4.9
+
+
photo-labelling conditions:
1.0
c
0
0
AAC’LRAIGDAVNLC’QALLNK-NH2
AAC1’’LRAIGDAVNLC1’’QALLNK-NH2
AAC2’’LRAIGDAVNLC2’’QALLNK-NH2
d
e
0
0.5
Lyso-1e
Lyso
Fig. 3 a) The detailed structural information of cis‐1a 14
from X‐ray crystal structure. b) AMBER Molecular Mechanics optimized
conformations of p4 stapled with either cis‐ or trans‐CBOA and photo‐
clicked p5’. c) Multiple rounds of photo‐switching of CBOA stapler
,
trans‐1a and 4ba
0.0
Lyso-(1e-6)n
6
+
+
+
-
-
+
-
-
+
+
+
-
-
+
-
-
n = 1
hν 405 nm
fluorescein
Lyso-(1e-3h)n
Lyso-1en
n = 2
n = 1
n = 2
n = 1
Lyso
15 KDa
accompanied by reciprocation of α‐helicity of p4 at 298 K, comparison of
corresponding CD spectra of p4 and p5’ in single diagram; d) Sequence
14000
14500
15000
Mass /Da.
15500
16000
Coomassie blue
and biological activity of the chemical modified peptides. See Fig. S17
b
ESI† for details. aAcetylated N‐terminal Ala; Measured via ATP assay by
Fig. 2 a) Images of SDS‐PAGE in various channel and b) deconvoluted MS
spectra for selective Lyso‐(1e)n‐6 photo‐ligation performed in PB buffer, pH =
7.4. See Figs. S10‐12 ESI† for details. The mass signals of desired protein
conjugate were marked in circles.
treating Hela cells with 20 μM peptide for 42 h. cC’ = 1f‐linked L‐cysteine.
dp4 photo‐clicked with 2h to produce p5
produce p5’ (Fig. S16, ESI†).
.
ep4 photo‐clicked with 2c to
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ARTICLE
Journal Name
helicity calculated to be 75% (Fig. 3b‐c). In addition, peptide p5’
View Article Online
a)
displayed consistent tumor cell suppressDioOnI:e10ff.e10c3t9w/Dit0hOBp0510(F27igH.
3d). Thus, it is reasonable to conclude that the better tumour
cell suppressing activity of p5 and p5’ was due to its enhanced
α‐helicity in concert with a promising cell permeability as
charge neutral NoxaB peptide (Fig. 3d).24
Live cell imaging
b)
DIC
FITC
Hoechst
PI
Merged
Lin reported a bisaryl cross‐linked NoxaB peptide with
substantially strengthened α‐helicity and cell‐killing activity but
pre‐tagging of a fluorophore on its N‐terminal was required.24
Herein, we are attempting to trigger the activation of the
NoxaB, ligate a fluorescent probe and then track its subcellular
distribution simultaneously via the NI‐CBOA photo‐click system
in‐vivo (Fig. 4a).
0.5 h
1 h
To test in‐vitro performance, we first utilized cis‐1f clamped
p4, and verified its reactivity to form activated p6 by LC‐MS
analysis, which can be induced just under 405 nm LED in PB
buffer (Fig. S18, ESI†). Hela cells were treated with 10 μM p4
2 h
4 h
and 50 μM 6, then photo‐irradiated under a single 405 nm LED
for 1 min. Fluorescence localization and morphology of the
cells were identified at specified time, after double staining
with Hoechst 33342 and propidium iodide (PI) to distinguish
phases of apoptosis. Initially, accumulated fluorescence within
the cytoplasmic region was observed in FITC channel during
the first 2 h after photo‐triggering. As incubation time
exceeded 4 h, strong fluorescein signals developed even in the
nucleus with concomitant appearance of PI signals in red
channel, indicating a rapid progression of late‐stage apoptosis
(Fig. 4b). In control groups without photo‐stimulation, NoxaB
p4 was kept inactive because there were negligible
fluorescence signals in both green and red channels (Fig. S19a,
ESI†). Regardless of whether photo‐activation was applied or
12 h
Fig. 4 Fluorescence imaging of live HeLa cells incubated for specified
time with peptide p4 and DASyd after 405 nm LED irradiation for 1
min. a) Photo‐click reaction scheme. b) Cells were stained with both
Hoechst 33342 and PI, imaged in various channels. Scale bar represents
20 μm.
6
linked p4 containing two cysteines at i and i+11 positions with
increasing in α‐helicity at PSS 405 nm and completely reversed
after 514 nm irradiation (Fig. 3a‐c, α‐helicity = 80%405nm vs.
62%514nm). While the helicity of p2 with two L‐cys at i and i+7
positions could not be controlled by alternated stimulation of
405 and 514 nm LED light (Fig. S15, ESI†).
not, cells supplied with only DASyd
6 scarcely showed any
subcellular fluorescence (Fig. S19d‐e, ESI†), which reflects the
benign nature of both DASyd and NI dipole in‐vivo.
A unique advantage of the CBOA stapler is that it integrates
the dual functions of a photo‐modulator and a dipolarophile to
adjust and extend the efficacy of the peptide. Comparing the
X‐ray crystal structure of photo‐ligation product 4ba with
trans‐1a and cis‐1a14 (Fig.3a, Table S3‐4, ESI†), both the
dihedral angle between the two phenyls of dipolarophile and
the distance within two acyls on the stapled L‐cys by NI‐CBOA
cycloadduct linker were akin to those of the trans‐1a which
constrains the NoxaB with an enhanced α‐helicity (Fig. 3a and
3b). To demonstrate this feature, FITC‐tetrazole 2h was
prepared and photo‐clicked with 1f‐modified p4 under both
405 and 311 nm irradiation to obtain fluorescein‐labelled p5
(Fig. 3d). In cell viability measurement via ATP assay, the
Conclusions
Collectively, the highly twisted ring‐strain on N=N double bond
of CBOA loaded via photo‐isomerization has been
demonstrated to be effective in promoting the cycloaddition
with photo‐released NI with very rapid rates. The α‐helicity of
CBOA stapled NoxaB peptide has been successfully
strengthened under 405 nm photo‐control. In live cell studies,
the combination of a α‐helicity regulator and a bioorthogonal
ligation has been realized to enhance the efficacy of
pharmaceutical peptides via the NI‐CBOA photo‐click reactions.
parent peptide p1, p3, 1f‐stapled p2 and p4 showed poor or
no activity in prevention of Hela cells propagation, while
fluorescein tagged p5 showed a significant efficacy (Fig. 3d). In
order to provide stronger evidence, we took tetrazole 2c
whose structure was closely related with 2h to photo‐react
with p4 to afford peptide p5’ in which the FITC fluorophore
was omitted for the sake of better quality in CD spectra
acquisition. The CD spectra of p5’ indicated a very similar
curve in comparison with that of p4 at PSS 405 nm with the α‐
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
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ARTICLE
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Röhricht, R. Höppner and R. Herges, J. A c.
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We thank Dr. Pengchi Deng (Analytical & Testing Center, SCU)
for help on NMR spectra collection. Financial support was
provided by the National Natural Science Foundation of China
(21502130), the “1000‐Youth Talents Program” and the
Fundamental Research Funds for the Central Universities. We
thank the Xiaoming Feng laboratory (SCU) for access to
equipment.
D
m
OI
.
: 1
C
0
h
.1
e
0
m
39
.
/D
S
0
O
B
,
0
2
10
0
2
16,
7
H
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Organic & Biomolecular Chemistry
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DOI: 10.1039/D0OB01027H
stapled NoxaB
FITC-PEG-DASyd
PEG
+
PEG
=
A novel photo-click ligation reaction between nitrile imines and photo-switchable
octocyclic azobenzenes was established to both tune the conformation of NoxaB peptide
and conjugate probes with enhanced efficacy in cell apoptosis.