Photo-accelerated "click" reaction between diarylsydnones and ring-strained alkynes for bioorthogonal ligation

Article abstract of DOI:10.1039/c9cc02882j

We constructed a library of diarylsydnone (DASyd) candidates in search of a photoclickable reaction toward alkynes, enabling an ultra-accelerated reactivity, while suppressing the background cycloaddition in the dark. The in vitro and in vivo protein labe

Full text of DOI:10.1039/c9cc02882j

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
first methanofullerene derivatives of Sc N@D5h-C80
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DOI: 10.1039/C9CC02882J
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Photo-accelerated “Click” Reaction between Diarylsydnone and
Ring-strained Alkyne for Bioorthogonal Ligation
Received 00th January 20xx,
Xiaocui Zhang, Xueting Wu, Shichao Jiang, Jingshuo Gao, Zhuojun Yao, Jiajie Deng, Linmeng Zhang
and Zhipeng Yu*
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
a) Taran et al. : Click reaction of Monoarylsydnones with cycloalkynes (MASYC)
non-photo-induced, direct pathway
1,5-disubstituted pyrazole
We constructed a library of diarylsydnone (DASyd) candidates in
search of a photoclickable reaction toward alkynes, enabling an
ultra-accelerated reactivity, while suppressing the background
cycloaddition in dark. The in-vitro and in-vivo protein labelling
experiments revealed that the photo-accelerated DASyd-alkyne
cycloaddition exhibit robust selectivity.
R1
X
H
HO
R1
k2 in M-1s-1
PBS/DMSO (9/1)
X
+
N
H
H
N
X = F; k2 > 10
X = Ar; k2 < 2.9 X 10-3
N
℃
pH = 7.4, - 25
N
H
O
O
OH
ss
on
Pd-XPhos,
C-H
activation
coupling
ti
X = H
Ar-Br
reac
darkne
in
DASAC
Placed
backgroud
R2
Synthetic accessibility
405 nm photo-activated
Ultra-accelerated
R1
R2
Photo-accelerated
Bioorthogonal reactions are elaborate chemical tools of high
fidelity, not only for probing the dynamics but also
functionalization of biomolecules in their natural
environment.1 In the past two decades, plenty of efforts have
been made to enrich the library of available bioorthogonal
strategies meeting the stringent requirements, including:
nitrone-alkyne cycloaddition,2 traceless Staudinger ligation,3
tetrazine-dienophiles,4 Cu-catalyzed5 or Cu-free “azide-alkyne”
ligation,6 etc. In particular, the photo-click reactions,7 such as
nitrile imine (NI) mediated tetrazole-8 and sydnone-alkene9
conjugation, due to their smart response and high
spatiotemporal controllability, have become an appealing
research tool for ultra-fine ligation purpose. Although there
are several photo-click cycloadditions that could be utilized
selectively for olefins targeting ligation, like: photo-IEDDA,10
PQA,11 the study of alkyne centered strategy is still in infancy.
The photo-decarbonylation of dibenzocyclooctyne (DIBO)12
had been investigated by Popik et al. for bioconjugation, but
the relatively slow ligation rate retarded its use under ultra-
diluted condition.13 Therefore, the exploration of new photo-
initiated or photo-accelerated alkyne cycloaddition is still an
intriguing yet challenging task to harness superior
manipulation of the reaction process in 3-dimensional space.
Since Taran et al. reported the "Cu-catalyzed" regioselective
click reaction of monoarylsydnone (MASyd)-alkyne
cycloaddition,14 sydnone, an heterocyclic mesoionic dipole,15
had been extensively explored for bio-conjugation featuring
+
N
H
N
H
H
kobs= 0.650 s-1 for 1g
0.133 s-1 for 1c
N
N
O
O
OH
1,3-substituted pyrazole
Photo-click pathway
H
HO
R1
b) This work: Click reaction of Diarylsydnones with cycloalkynes (DASYC)
Scheme 1. Comparison of photo-activation versus DASAC strategy.
with both easy accessibility and decent stability. The "Cu-free"
version was achieved via ring-strain promotion with the
reaction rate up to 0.054 M-1s-1 and 1.46 M-1s-1 via the use of
bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) by Chin,16 and
dibenzoazacyclooctyne (DIBAC) by Murphy,17 respectively.
Recently, a significant advance has emerged as a flash ligation
with the rate up to 104 M-1s-1, between halogen-substituted
MASyd and ultra-strained 3,3,6,6-tetramethylthiacycloheptyne
(TMTH) or BCN (SPSAC, Scheme 1), reported by Taran's team.18
Further developments of MASyd were accomplished in 2018
and 2019 by Friscourt19 and Taran,20 respectively, mainly
focusing on its utilization as biochemical tools affording
monoarylpyrazole. Nevertheless, due to the lack of promising
photo-reactivity of the MASyd, the utilization of this approach
is diminished in spatiotemporal maneuverability demanding
task where visible-light activation is preferred to reduce the
photo-toxicity, gain higher tissue penetration.
In light of the NI mediated photo-click reaction of DASyd-
alkene,9 we proposed the alkene could be replaced with highly
strained alkynes to harness an unexpected ligation-reactivity.
Drastically different from the planar MASyd, the adjacent
ortho-diaryl moieties on the DASyd exhibit a double-twisted
conformation around its core, which is extremely detrimental
toward the direct thermo-favored [3+2] pathway, hindering
the attacking on the π-orbital of the mesoionic aromatics (Fig.
S3a ESI†).21 Under irradiation, the rapid depletion of DASyd via
photoconversion into NI also constitutes a disadvantage
toward undesired direct DASyd-alkyne cycloaddition (DASAC),
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: Containing details on
experimental procedures, spectra property, characterization of all new compounds
and XRD analysis. See DOI: 10.1039/x0xx00000x
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DOI: 10.1039/C9CC02882J
a)
b)
3 - Terminal
MeO
N
Exclusive desired reaction
conversion >85%
O
Messy reaction
Conversion 20%- 80%
Conversion < 20%
OMe
F
O
S
OMe
OMe
Conversion > 80%, having a
small amount of hydrolysate
3a-3c
4d
1a-1n
3a
2a-2d
4a
5a-5c
4a-4d
d)
c)
HPLC conversion
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m 1n
2a
2b
2c
2d
3b
3c
4b
4c
5a
5b
5c
C
4 - Term
O
311 nm, 2 min
373 nm, 2 min
N
3 - Term
OMe
MeO
405 nm, 2 min
in dark, 12 h
N
N
1g
O
O
h
(2h)
82

77
50
isolated
81
67
83
78
88
88
75
91
42
79
81
99
71
73
76
89
71
96
75
91
74
91
86
68
73
63
yield % dark (>15d) 79
HO
e)
C
4 - Term
R1 = H, R2 = CF3: 1a, 4c
R1 = COOEt, R2 = H: 1b
O
R2
R1
S
R1 = CF3, R2 = H: 1c, 2a, 4a, 5b
R1 = F, R2 = H: 1h
O
MeO
N
S
1l
F
1j
1m
1n, 3b, 4b, 5a
R1 = R2 = H: 1i
1e
O
R2
1f, 2b, 4d
1d, 2c, 3a
R1 = CF3, R2 = F: 1k
1g, 2d, 3c, 5c
O
Fig. 1 Screening of DASyd-BCN combinations for photo-accelerated or non-photo-induced reaction. Experiments were carried out at 25 oC in acetonitrile (ACN)/H2O = 1/1,
irradiated with indicated light sources. [DASyd] = 100 µM, [BCN] = 500 µM. a) The colour codes for ligation characteristic analysed via HPLC-MS. b) The N3 moieties in this study. c)
Screening results. d) Structures of 1g to illustrate the aryl building blocks on N3 and C4 moieties. e) The C4 moieties involved.
forming 1,5-diarylpyrazole. In addition, the mechanism of
To investigate the influence of the dipolarophiles alkyne (a-
photo-activation strategy was completely different from the e), the cycloaddition reaction were also performed for 1g and
DASAC pathway (Fig. S3a ESI†).21 To demonstrate our 1c toward phenyl acetylene, electron-deficient EdU23 as well as
hypothesis, an elaborate library of DASyd was constructed via ring-strained DIBO,24 BCN,25 and TMTH (Tables 1 and S8
C-H activation coupling22 of MASyd (Scheme 1), placing ESI†).26 Unfortunately, terminal alkynes a and b reacted inertly
electron rich aryls on N3-terminal and chromophores on C4- under either photo-accelerated or dark condition with only
terminal to tune their optical properties.
For the goal of identifying a fast photo-responsive DASyd strained alkynes, distinctly, displayed
trace amount of product detected (Figs. S17-19 ESI†). The
a
significant
with minimal background reaction with BCN, a total of 112 improvement. Although, the yield for ligation of 1g with DIBO
arrays were performed under illumination with various light was only 56.0% in comparison to 90.3% with BCN (Table 1,
sources (311 nm hand-held lamp, 373 and 405 nm LED arrays) entry c vs. d), the DASAC-ligation of DIBO was also 1.4 times
versus placing in dark for 12 h (Fig. 1 and Table S1 ESI†). The slower than that of BCN. The non-photo-controlled reaction of
electron-tuning chromophores bearing on the C4 (Fig. 1e) highly strained THTM was relatively fast (Table 1, entry e),
paired with electron-donating aryls (EDG) on N3 (Fig. 1b) of consistent with Taran's report.18 But, its mediocre photo-yield
DASyds would be preferred. Through coupling of featured aryl under 405 nm irradiation made it become inadequate for
substituents (1a-1n) at C4 on N3-p-methoxyphenyl MASyd, further study. Overall, as a light-accelerated approach, the
DASyds 1f, 1g and 1n, showed good conversions reacting with BCN showed obvious advantages over the other alkynes
BCN at 405 nm light, but side-reactions were also observed for (Tables 1 and S8, Figs. S17-S18 ESI†), ligated efficiently with 1g
1f and 1n at 311 nm. Under 311 nm irradiation, DASyd 1c was under
a relatively good choice because 1k and 1e were expensive
despite the fluorescent property of the later one. ‘Scaffold
hopping’ at the N3 position offers opportunities to elevate the
photo-reactivity. However, DASyds (2a-2d), with a piperonyl
moiety at N3 position, lead to many side-reactions. Installation
of a benzothiophen-5-yl at N3 (3a-3c) didn’t provide a
remarkable bathochromic shift in their absorption (Table S5
ESI†), accompanied with unsatisfactory photo-conversion in
UV range. With a 3,4,5-trimethoxyphenyl at N3 (4a-4d), the
overall photo-reactivity was decreased, but the DASAC was
completed within 9 h for DASyd 4c with a 3,5-(CF3)2phenyl at
C4. Lastly, 4-fluorophenyl (5a-5c) suppressed the photo-
conversion, but slightly increased the rate of the DASAC
pathway. Characterization of 1,5-diaryl (Table S7 ESI†) and 1,3-
diaryl pyrazoles validated corresponding ligation pathways of
Table 1. Photo-activation vs. DASAC reaction in dark for 1,3-Dipolar
Cycloaddition of 1g with various alkynesa
MeO
MeO
O
O
MeO
MeO
O
MeO
h, 25 °C, 2 min
25 °C, 12 h
N
OMe
N
ACN/H2O = 1/1
ACN/H2O = 1/1
+
in dark
CO2
1g-X-h
1,3-pyrazole
N
N
CO2
N
1g-X-dark
1,5-pyrazole
N
X
O
O
1g
Dark
120 s/12 h
Entry
Alkyne
311 nm
trace
373 nm
405 nm
trace
N.D.b
a
trace
trace
PhAce
O
N
OH
HN
O
N.D.b
b
OH
trace
trace
O
EdU
N.D./8.60c
N.D./11.6c
21.8/92.7
c
d
e
53.7c
88.6c
86.6
96.8
97.2
92.8
56.0c
90.3c,d
55.0c,e
DIBO
HO
H
OH
H
BCN
non-photo-cycloaddition
vs.
photo-transformation,
S
TMTH
a100 μM DASyd and 500 μM alkyne in ACN/H2O (1/1) was irradiated with corresponding light
sources for 120 s. Yields were determined by calibration curve. See Table S8 and Figs. S17-S18
in ESI† for details. bN.D. = not detected. cIncomplete transformation of DASyd. The DASAC
adduct was observed in dtrace or e7.75% yield.
respectively. These results indicated DASyds 1g shows
negligible background reaction within 2 min temporal scale
and is suitable for subsequent exploration.
2 | J. Name., 2012, 00, 1-3
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a) in phosphate buffer (PB), pH = 6.0
b)
in E. coli lysate
lyso
lyso-BCNn+1g, dark, 5 h
a)
b)
View Article Online
lyso-BCNn+1g, 405 nm, 3 min
lyso-BCNn
0.4
0.3
0.2
0.1
0.0
DOI: 10.1039/C9CC02882J
1g-hν 373 nm
lyso-(1,3)-Pye1
0.8
1g-dark
kobs = 4.20E-6 s-1
R2 = 0.999
lyso-(1,3)-Pye2
0.4
t
= 1.91 day
½
kobs = 0.650 s-1
15 kDa
R2 = 0.998
0.0
15 kDa
t
= 1.07 s
½
lyso-(1,5)-Pye1
n = 1 n = 2 n = 3
Fluo: Cy3 channel, CB: coomassie blue channel, WB: HRP-Avidin, enhanced chemiluminescence (ECL)
2x105 4x105 6x105
Time /s
0
2 4 6 8
c)
d)
14000
14400
14800
15200
Biotin
Mass / Da.
Cy3
Lyso-(1,3)-pye6a1
Lyso-BCN1
Lyso
Fig. 2 Comparison of apparent kinetics and in-vitro protein labelling under irradiation
versus in dark for 1g with BCN. a) Real-time monitoring of absorption evolution, and
exponential decay fitting results, 20 µM of 1g, 1 mM of BCN in ACN/H2O = 1/1; b)
Deconvoluted HPLC-MS spectra for chemical modification of lysozyme and subsequent
comparison of labelling efficiency, 5 µM of lysozyme, 20 eq. of 1g in phosphate buffer
(PB, pH = 6.0).
3
O
O
HN
O
Lyso-6a1
Lyso-BCN2
N
N
O
Dark, 1 h
405 nm, 3 min
O
6a
405 nm irradiation with undetectable DASAC (Table 1, entry d).
Besides, the synthetic availability and compact size of BCN
facilitate our research on its fast bioorthogonal applications.
Preliminary study on photo-accelerated cycloaddition
kinetics (kobs-p, the exponential decay) was obtained via tracing
time-resolved absorbance evolution for DASyd 1g at 350 nm
and 1c at 335 nm (λmax in 250-400 nm range, but faint
absorbance of the product) (Fig. S2 ESI†). The apparent
kinetics were determined to be 0.65 ± 0.010 and 0.13 ± 0.002
s-1 (the [3+2] cycloaddition step, k2 = 3.3 × 104 M-1s-1, Fig. S4
ESI†), respectively (Figs. 2a and S3b-3c ESI†), which were
independent neither on the illumination intensity nor the
concentration of BCN.9 In contrast, the kobs-t for non-photo-
induced reaction was also examined under identical
concentration of reactants, DASyd/BCN = 1/50, (1g and 1c, 20
µM), resulting in only 4.2 ± 0.17 × 10-6 and 3.9 ± 0.51 × 10-6 s-1
(k2 = 4.9 × 10-3 M-1s-1, Fig. S3d ESI†), which was about 5 orders
of magnitude slower than that of the photo-ligation reaction
(Figs. 2a and S3b ESI†). Varying the BCN concentration, the
kobs-t responded in positively linear correlation, proving that
the DASAC-ligation was rate-limited by the initial [3+2]
cycloaddition step (Fig. S3d ESI†).18 Furthermore, the photo-
conversion quantum yields of 1g and 1c were measured to be
0.24 (373 nm) and 0.21 (373 nm), respectively (Fig. S6 ESI†).
The in-vitro protein labelling experiment were conducted via
a chemically tagged lysozyme-BCNn with DASyd 1g (Fig. S9
ESI†). Comparing the efficiency of 1g under 405 nm LED vs. in
dark condition, the results showed that Lyso-BCNn was
completely transformed into corresponding Lyso-(1,3-
pyrazole)n within 3 min, while less than 2% into Lyso-(1,5-
pyrazole)n for 5 h (Figs. 2b and S10 ESI†), respectively. The
results of sharp contrast inspired us to investigate DASyd-BCN
photoclick ligation under more stringent conditions.
Fig. 3 Synthesis and application of Biotin-suflo-Cy3-DASyd 6a. All protein labelling
experiments were performed at 5 µM of Lyso-(BCN)n, 20 eq. 6a, 3 min. a) Images of
SDS-PAGE and Western Blot assay for selective Lyso-(BCN)n-6a photo-ligation in various
channel were performed in PB buffer, b) or E. coli lysate. c) The structure of 6a. d)
Deconvoluted mass spectra for analysis of the photo-acceleration vs. cycloaddition in
dark.
signal band from unexposed lane was caused by 1 h incubation
o
and denaturation process at 80 C (Figs. 3b and S11-S12 ESI†),
leading to background cycloadduct. The selectivity of the
photo-ligation was also confirmed via biotin targeting Western
Blot assay in imaging through ECL channel and deconvoluted
HPLC-MS analyses with exclusively matched mass (Fig. 3d).
Collectively, all these data strongly supported the unique
DASyd-BCN photo-reactivity with high specificity toward BCN
reporter on proteins via the photo-pathway (Figs. 3 and S11-
S12 ESI†).
To introduce the BCN reporter onto live cells, we modified
the chimeric antibody Cetuximab with BCN-OCOO-PNP (Fig.
S13 ESI†) and incubated the resulting Cetu-BCN with EGFR
positive cancer cells, A549, through the specific binding on the
outer membrane surface (Fig. 4a).11 Because the 405 nm
visible-light induced ligation of DASyd (6a)-BCN, an image
containing spindle-shaped circles in Cy3 channel could be
observed, colocalized with signal in the green channel (stained
with membrane-embedding dye, DIO) on the cell surface after
irradiation for 30 s. Meanwhile, almost no Cy3 signal can be
seen for unexposed reaction for 30 min. Control group using
unmodified Cetuximab also barely showed any signal after
photo-stimulation, suggesting the excellent selectivity (Fig. 4b).
Time-tracing for the DASAC conjugation and controlled
imaging shed light on background interference until 150 min
incubation (Figs. S14-S16 ESI†). These results together
demonstrated that the fast and successful labelling of living
cells within 30 s can be achieved via ligation of 6a with BCN
reporter by photo-acceleration instead of the process in dark.
Therefore, the photo-induced DASyd-BCN ligation could be
utilized as bioorthognal labelling tool.
To demonstrate the utility of DASyd-BCN photoclick ligation,
allowing visualization of covalent modification of Lyso-(BCN)n
on SDS-PAGE, a bifunctional Biotin-suflo-Cy3-DASyd (6a, Fig
3c)27 was synthesized from 1g. Protein labelling experiments
were performed at 5 µM concentration. From the in-gel
fluorescence imaging, it can be clearly seen that the exclusively
labelling band of Lyso-pyen was detected in the lane where all
the essential requirement for 405 nm photo-click was fulfilled
in either PB buffer (Fig. 3a) or E. coli lysate (Fig. 3b). The weak
Conclusively, via screening a library of DASyds, we were able
to fully demonstrate the fast photo-responsive “click” reaction,
realizing the covalent-bond ligation within 3 mins under 405
nm light exposure with only highly ring-strained alkynes. The
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Journal Name
7
8
9
M. A. Tasdelen and Y. Yagci, Angew. Chem. Int. Ed., 2013, 52,
View Article Online
5930-5938.
Ar2
N
P. An, T. M. Lewandowski, T. G. Erbay,DPO. IL:i1u0.a10n3d9Q/C.9LCinC,02J.8A82mJ.
Chem. Soc., 2018, 140, 4860-4868.
L. Zhang, X. Zhang, Z. Yao, S. Jiang, J. Deng, B. Li and Z. Yu, J.
Am. Chem. Soc., 2018, 140, 7390-7394.
a)
b)
O
O
h, 405 nm, 30 s
O
Cetuximab-BCN
DASyds (6a)
EGFR(+)-cells
N
Ar1
DIC
Cy3
DIO
NB
Merge
10 H. Zhang, W. S. Trout, S. Liu, G. A. Andrade, D. A. Hudson, S.
L. Scinto, K. T. Dicker, Y. Li, N. Lazouski, J. Rosenthal, C.
Thorpe, X. Jia and J. M. Fox, J. Am. Chem. Soc., 2016, 138,
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11 J. Li, H. Kong, L. Huang, B. Cheng, K. Qin, M. Zheng, Z. Yan
and Y. Zhang, J. Am. Chem. Soc., 2018, 140, 14542-14546.
12 A. A. Poloukhtine, N. E. Mbua, M. A. Wolfert, G. J. Boons and
V. V. Popik, J. Am. Chem. Soc., 2009, 131, 15769-15776.
13 (a) M. Bjerknes, H. Cheng, C. D. McNitt and V. V. Popik,
Bioconjug. Chem., 2017, 28, 1560-1565; (b) D. A. Sutton and
V. V. Popik, J. Org. Chem., 2016, 81, 8850-8857; (c) B. C.
Sanders, F. Friscourt, P. A. Ledin, N. E. Mbua, S. Arumugam, J.
Guo, T. J. Boltje, V. V. Popik and G. J. Boons, J. Am. Chem.
Soc., 2011, 133, 949-957.
14 S. Kolodych, E. Rasolofonjatovo, M. Chaumontet, M. C.
Nevers, C. Creminon and F. Taran, Angew. Chem. Int. Ed.,
2013, 52, 12056-12060.
15 (a) A. Rodriguez and W.J. Moran, Synthesis, 2009, 4, 650-654.
(b) D. L. Browne, J. P. A. Harrity, Tetrahedron, 2010, 66, 553-
568.
Fig. 4 Fluorescence microscopy imaging to characterize the localization of the DASyd
6a-Alkyne photo-click labelling on living A549 cells. a) Schematic illustration of the
procedure; b) Colocalization of photo-click labelling of Cetu-BCN on the cell surface
(Cy3) and 3,3'-Dioctadecyloxacarbocyanine (DIO) versus DASAC reaction and
background control, Scale bar: 20 µm, Exposure time: 2 s; NB = NucBlue™ Probe.
kinetic and protein photo-conjugation study provided strong
evidence for us to distinguish the distinct photo-pathway from
the background DASAC-reaction. Bifunctional DASyd 6a was
successfully applied to live cell surface labelling study with high
fidelity. Therefore, the DASyd-BCN cycloaddition has emerged
16 S. Wallace and J. W. Chin, Chem. Sci., 2014, 5, 1742-1744.
17 M. K. Narayanam, Y. Liang, K. N. Houk and J. M. Murphy,
Chem. Sci., 2016, 7, 1257-1261.
18 (a) H. Liu, D. Audisio, L. Plougastel, E. Decuypere, D. A.
Buisson, O. Koniev, S. Kolodych, A. Wagner, M. Elhabiri, A.
Krzyczmonik, S. Forsback, O. Solin, V. Gouverneur and F.
Taran, Angew. Chem. Int. Ed., 2016, 55, 12073-12077. (b) L.
Plougastel, O. Koniev, S. Specklin, E. Decuypere, C. Créminon,
D. Buisson, A. Wagner, S. Kolodych, F. Taran,
as
a
promising
photo-click
tool
for
detecting
biomacromolecules in living system. To avoid the irreversible
damage of high-energy photon to live cells, the introduction of
Si or S atom on sydnone core might be one way to elongate
the wavelength of photoexcitation.
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.
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Conflicts of interest
The authors declare no competing financial interests.
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