Discovery of Fluorogenic Diarylsydnone-Alkene Photoligation: Conversion of ortho-Dual-Twisted Diarylsydnones into Planar Pyrazolines

Article abstract of DOI:10.1021/jacs.8b02493

A small library of diarylsydnones (DASyds) was constructed based on aryl-pairing combinations and subjected to click reaction toward alkenes under photoirradiation with high efficiency. We were able to demonstrate the utility of DASyds for highly fluorescent turn-on ligation targeting the trans-cyclooct-4-en-1-ol moieties on protein.

Full text of DOI:10.1021/jacs.8b02493

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Communication
Discovery of Fluorogenic Diarylsydnone-Alkene Photo-ligation:
Conversion of ortho-Dual-Twisted Diarylsydnones into Planar Pyrazolines
linmeng Zhang, Xiaocui Zhang, Zhuojun Yao, Shichao Jiang, Jiajie Deng, Bo Li, and Zhipeng Yu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02493 • Publication Date (Web): 05 Jun 2018
Downloaded from http://pubs.acs.org on June 5, 2018
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Discovery of Fluorogenic Diarylsydnone-Alkene Photo-ligation:
Conversion of ortho-Dual-Twisted Diarylsydnones into Planar Pyra-
zolines
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Linmeng Zhang, Xiaocui Zhang, Zhuojun Yao, Shichao Jiang, Jiajie Deng, Bo Li and Zhipeng Yu*
Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, 29
Wangjiang Road, Chengdu 610064, P. R. China
Supporting Information Placeholder
core, allowing for unique tuning of reactivity and selectivity of
these chemical ligation reagents in 3-D way, as reported herein.
ABSTRACT: A small library of diarylsydnones (DASyds) was
constructed based on aryl-pairing combinations and subjected to
click reaction toward alkenes under photo-irradiation with high
efficiency. We were able to demonstrate the utility of DASyds for
highly fluorescent turn-on ligation targeting the TCO moieties on
protein.
In order to identify a DASyd with excellent photo-response, we
consider the electronic effects on the diaryl moieties might be
vital to the photo-decarboxylation,12 including the auxochromes,
the polycyclic aromatics (PAr) and the electron donors (eD) or
acceptors (eA).13 Accordingly, a small library of DASyd bearing
various aryl substituents on C4 and N3 positions were designed
(1a-n, Figure 1a; S8-19, Table S8) and constructed.14 The UV-Vis
spectroscopic properties of representative DASyds were evaluated
with maximum absorption wavelengths and the top-three ranked
Photo-induced chemoselective covalent-bond forming reactions
(photo-click)1 which introduce probing functionalities are desired
tools for materials science2 to functionalize nanoparticles and
interfaces with real-time response, as well as for the labeling of
oligonucleotides, proteins, and oligosaccharides.3 Photon utiliza-
tion3a, 4 and the precision of the chemistry4 are both vital for offer-
ing better bioorthogonality5 accompanied with higher spatiotem-
poral resolution. In spite of this need, only a limited number of
photo-click methods have been investigated currently including
tetrazole-alkene photo-click chemistry (TAP),6 photo-initiated
thiol-ene,4c -yne,4d -quinone4e reactions, and photo-induced strain-
promoted azide-alkyne cycloaddition (photo-SPAAC).2b Since the
TAP reaction6 was reported as a bioorthogonal reaction, the pho-
to-click strategy, including [3+2]-type: TAP; photo-SPAAC and
the [4+2]-type: o-naphthoquinone methides (oNQMs)-ene cou-
pling,4f has been used to visualize proteins in living cells, to study
D(R)NA dynamics,3c-d and to track drug molecules.6c In spite of
the exciting developments over the past two decades, demand
remains high for the discovery of new and high-performing photo-
click reactions, especially those with the ability to site-specifically
hit the desired targets for use in theranostics.
maximum extinction coefficients (max
) were observed for
1k>1j>1h (Figure 1b, Table S1) implying a C4-PAr-eA is benefi-
cial. When the N3-Ph substituent was an eD, it could offer higher
 value at λmax than seen with an eA moiety (1c vs. 1d). This effect
on the  value was particularly evident when the N3-Ph-eD was
coupled with a C4-Ph-eA, in contrast to a C4-Ph-eD (1e vs. 1j)
which processes a bathochromic shifting effect. Intriguingly, the
absorption peak tail of the simple DASyd could be observed in the
visible region (>400 nm, e.g. 1e and 1f), which opens the window
for visible light-mediated triggering.
N-Phenyl-sydnones were discovered to be clickable with al-
kynes via copper complex mediation by Taran7 or strain-
promotion by Chin8a and Murphy;8b and tremendous enhancement
of cycloaddition rate achieved via introduction of fluorine substi-
tution by Taran,8c elevated this functionality as potential chemical
probes to study biomolecular dynamics in vivo. Inspired by early
sydnone photo-reactions,9-10 we investigated the photo-induced
diarylsydnone-alkene cycloaddition reaction, which affords pyra-
zoline fluorophores (Scheme 1), as a potentially bioorthogonal
conjugation strategy.5 Since nitrile imine (NI) is the reactive dipo-
lar intermediate,11 diarylsydnones (DASyd) demonstrate com-
mendable selectivity toward electron-deficient or ring-strained
alkenes through a photo-induced transformation sequence.12 Un-
like those in 2,5-diaryltetrazoles, the two ortho-substituted aro-
matic rings in the DASyd scaffold are twisted around the sydnone
Scheme 1. The flipping of the diaryl substitutions in the SAP versus TAP.
Subsequent analysis of the photochemistry of the DASyds to-
ward methyl methacrylate in quartz test tube was inquired under
exposure to a handheld UV lamp (311 nm, 10.8 mW/cm2) or a
LED array (371 nm, 15.4 mW/cm2 or 405 nm, 33.6 mW/cm2).
Based on HPLC-MS analysis, the experimental results can be
summarized as follows: (i) substrates with N3-Ph-eA groups (1b
& 1d) showed poor reactivity toward alkene (Table 1, entries 2 &
4); (ii) 311 nm irradiation afforded higher conversions for the
entire DASyd library (Table 1); (iii) surprisingly, a p-aniline moi-
ety on either the N3 or C4 positions (e.g., 1e & 1f) exhibited very
poor photo-response despite their strong absorption at 371 nm and
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405 nm (Table 1, entries 5 & 6; Figure S34). Therefore, N3-p-
Only a single regioisomer of 3ga was observed based on the 1H
anisole-C4-Ar-eA-Syds are a better choice for continued screening.
Partial hydrolysis of the active intermediate was observed for 1j
and 1k, accounting for the discrepancy between isolated yield and
conversion (Table 1, entries 10 & 11, Figure S11-12) despite their
higher conversion at 405 nm. Overall, the three best DASyds at
311 nm irradiation were 1g, 1h and 1i in descending order of reac-
tivity (Table 1, entries 7-9), and no competitive cycloadditions
were observed in the dark environment.15
NMR signals of the Pyr protons and XRD structure, thus verifying
the synergistic occurrence of geometric flipping and planarization
with the photo-reaction (Table 1; Figure S34). Compared to the
DASyd (1g, 1i, 1j), the corresponding 2,5-diaryltetrazole has an
hypsochromic shift of absorbance peak around 37-48 nm, result-
ing in its complete inertness under excitation by 371 nm LED
(Figure S23).
To expand the scope of reporters16 paired with 1g, we examined
the photo-induced ligation by incubating 1g with a range of repre-
sentative alkenes upon 311 nm irradiation (10.8 mW/cm2) (Table
2). As expected, electron-deficient alkenes, such as 2a and 2d
(diethyl fumarate, DEF), reacted efficiently with 1g within 2h,
furnishing high yields, despite concomitant formation of the cor-
responding pyrazole as an oxidative aromatization product, espe-
cially for DEF (Table 2, entry 4, page 153 in SI). Alternatively,
alkene 2c (norbornene, NOR) exhibited a relative low yield (66%,
Table 2, entry 3, Figure S16) mainly because of side-reactions.
The genetic encodable ring-strained trans-cyclooct-4-en-1-ol
(TCO)4a appears to be promising, undergoing the photo-click
reaction to form 3gb (Table 2, entry 2), superior not only in liga-
tion precision but also satisfying F (up to 42%). Unexpectedly,
2e (5-vinyl-2'-deoxyuridine, VdU)17 behaves as a good ligation
substrate with respect to 1g, affording a 68% yield of 3ge with
little oxidation. The slightly lower yield is caused by incomplete
conversion of 1g because of the extra optical filtering effect from
VdU (Table 2, entry 5).18 Significant bathochromic shift of ab-
sorption (λmax shifted from 33 to 66 nm), arising in emission max-
ima (λem, from 525 to 543 nm), accompanied with large Stokes’
shifts (Δ, from 158 to 163 nm) were spectroscopic characteristics
of fluorophores, 3ga-3ge, upon isolation (Table 2).
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Table 2. Photo-activated 1,3-Dipolar Cycloaddition of 1g with
Various Alkene Dipolarophilesa
Figure 1. (a) Structures of selected DASyds with the C-H activation cross-
coupling yield. (b) Absorption spectra and molar absorption coefficients at
designated wavelength for selected DASyds from library in acetonitrile/H2O
(1:1) mixed solvent at 30 μM (250-450 nm).
Table 1. Photo-activated 1,3-Dipolar Cycloaddition of 1a-1n
with Methyl Methacrylatea
Stoke
shift
Yieldc
(%)
Conv.f
(%)

max/λem
(nm)
b
Entry
1
Alkene
F/εmax
367/525
376/534
0.56/15.9
0.42/11.2
158 nm
83
94
θ1 55.1°
θ2 35.5°
2
158 nm
85
97
90o
3
4
396/533
380/543
0.35/18.9
0.22/17.1
137 nm
163 nm
66
91g
95
Conv.c
@371
nm
-
Yieldb
@311
nm
Conv.c
@311
nm
97%e
>95%e
92%f
90%f
14%
17%
95%
94%
92%
Conv.c
@405
nm
78d (9e)
DASyds
Entry
R1
R2
46g
1
2
3
4
5
6
7
8
9
10
11
12
13
1a
p-MeOPh
H
H
Ph
N.D.d
N.D.d
55%
Trace
Trace
Trace
83%g
71%
70%
75%
70%
-
-
-
5
381/534
0.16/15.2
153 nm
68
1b o-COOMePh
1c
p-MeOPh
1d p-COOMePh
-
aIrradiation 20 mg of 1g and 5 eq. of alkenes in 50 mL of EtOAc for 2h. bF
denote fluorescence quantum yield, max in 103 M-1cm-1. cIsolated yields. d20 eq.
DEF. eSide-product from oxidative aromatization. f100 μM 1g and 0.5 mM 2 in
ACN/H2O (1:1), 311 nm UV for 60s. Determined by LC-MS. See Figure S15-
18 for details. gNot peak-to-peak conversion.
47%f
80%f
11%
N.D.d
43%
48%
47%
61%h
77%h
79%h
51%
8%
Ph
65%f
7%
1e
1f
1g
1h
1i
p-MeOPh
p- Me2NPh
p-MeOPh
p-MeOPh
p-MeOPh
p-MeOPh
p-MeOPh
p-MeOPh
p-MeOPh
p-NH2Ph
p-CF3Ph
p-CF3Ph
m-2F-p-CF3Ph
m-2CF3Ph
p-COOEtPh
6-COOMeNaph
6-Fluorescein
p-SO2NH2Ph
N.D.d
1%
5%
5%
Encouraged by the high efficiency of the photo-reaction, we
continued to determine the magnitude of fluorescence turn-on
effect derived from preferred DASyds, 1g-1i, toward TCO under
irradiation at 371 nm (15.4 mW/cm2), appending corresponding
photophysical properties of the resultant Pyrs (Table 3). Likewise,
the chromophoric and fluorophoric nature of products 3gb-3ib in
the visible range allowed for facile visualization of their formation.
Three DASyds show excellent degrees of fluorescence turn-on
effect, ranging from 122-fold for 3gb to 321-fold for 3ib (Table
3).15 As displayed in Figure 2, complete photo-conversion to the
desired Pyr 3hb was achieved within 520s as evidenced by the
1j
95%h
92%h
95%h
90%
10%h
24%h
18%
N.D.d
1k
1m
1n
69%g
a5 mg of 1 and 20 eq. of MMA in 10 mL of ethyl acetate (EtOAc) with 311 nm
UV for 2 h unless noted otherwise. bIsolated yield. 100 μM 1 and 2 mM 2a in
c
ACN/H2O (1:1) was irradiated with designated light source for 60s. Deter-
mined by LC-MS. See Figure S2-14 for details. dN.D. = not detected. eAll
f
converted into undesired products. By-products formed. g30 mg of 1 in 50 mL
of EtOAc scale. hHydrolysis as a predominate side-reaction.
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iterative increase in a new absorption peak at 397 nm and a broad
sfGFP-Pyr-1g were estimated to be 40%, 32% and 14% (Figure
emission peak centered at 553 nm simultaneously. Complete con-
version to 3gb requires longer time than 3hb, but still proceeds
within a similar scale (for turn-on ratio in ACN:PBS = 1:2,
pH~7.4, also see Figure S19-S21). Overall, DASyds 1g-1i hold
great potential for fluorogenic imaging.
S26-32, S35-36) with 1.8%, 4.4% and 8.1% of non-specific reac-
tions respectively. The LC-MS/MS analysis revealed that the
photo-ligation reaction of 1g has a highly spatial preference to-
ward K97-TCO residue of lyso-TCO (Figure S33). In-gel fluores-
cence imaging indicated obvious fluorescent turn-on bands only
for lanes meeting all requirements for the photo-ligation (Figure
3a-b).
4
5
6
7
8
9
The plausible mechanism of the DASyd–alkene photo-reaction
was proposed,12 but the reaction rate as a ligation method has not
been investigated. We probed the rate profile by capturing time-
dependent fluorescence evolution under LED irradiation (371nm,
28.0 mW/cm2, Figure S1) via monitoring the λem signal of corre-
sponding Pyr product. Unexpectedly, the apparent first-order rate
constant, kobs, detected for 1g-1i with either TCO or NOR were
almost equal (kobs1g-TCO = 0.045 s−1 vs. kobs1g-NOR = 0.044 s−1, Fig-
ure S1), independent on the alkene type or concentration but de-
lyso‐TCO
sfGFP‐TCOK
sfGFP‐BocK
M.W.
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(a)
1g
hν
15 kDa
10 kDa
(c)
lyso
lyso‐TCO
lyso
-
-
-
+
+
+
-
-
-
+
-
+
+
lyso
+
-
+
lyso‐Pyr
sfGFP‐Pyr
M.W.
(b)
1g
hν
35 kDa
sfGFP‐BocK sfGFP‐TCOK
-
-
-
+
-
+
+
-
-
-
+
-
+
+
+
+
pendent on the DASyd itself (kobs1g-TCO = 0.045 s−1 vs. kobs1i-TCO
=
0.034 s−1, Table 3), implying that the decarbonylation of 4,5-
diaryl-2-oxa-1,5-diazabicyclo[2.1.0]pentan-3-one12b (Figure S1)
might become the rate determining step instead of the ultra-fast
[3+2] cycloaddition11 (Figure S1). The quantum yields of the
photo-chemical reaction for 1g, 1h, and 1i were determined to be
0.20, 0.10, and 0.12, respectively (for yield in ACN:PBS = 1:2,
pH~7.4, also see Figure S25).
lyso‐NR.
25 kDa
sfGFP‐NR.
Figure 3. Selective photo-induced fluorogenic labeling of proteins by 1g in
vitro. Coomassie blue stain (top panel) and in-gel fluorescence (bottom panel)
of the same protein gel after resolved by SDS-PAGE (a) lyso (b) sfGFP. (c)
Deconvoluted mass spectra of control protein (red) and the TCO-protein mix-
ture (blue) showing the formation of the Pyr adduct (green). NR. = non-specific
reaction (purple).
Table 3. Photophysical Properties of the TCO-Pyrs and Fluo-
In summary, with a small library of photo-activatable DASyds
in hand, we were able to identify 1g-1i as excellent candidates for
photo-clickable fluorogenic ligation. The 1g showed high reactivi-
ty toward TCO and TCO-containing proteins when exposed to
near-UV light, giving rise to fluorescent Pyr cycloadducts with
outstanding ratio of fluorescence turn-on effect. We will consider
the applications of DASyd derivatives as light-activable reagents
for ligation reaction in living systems.
rescence Turn-On Efficiencya
b
ASSOCIATED CONTENT
Supporting Information
kobs
Fluorescence
turn-onf

max/em
(nm)
c
c
d
e
Comp.
max
400nm
F
R
(s-1)
1g
3gb
1h
3hb
1i
331/-
376/532
334/-
396/552
331/-
383/543
13,573
11,156
15,333
18,533
11,853
13,178
347
8,100
213
18,400 0.33
187 N.D.
N.D.
0.42
N.D.
0.20 0.045
0.10 0.043
0.12 0.034
122-fold
205-fold
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
321-fold
Experimental details, characterization of all new compounds, and
computational results (PDF).
X-ray crystal structure of 1f (CCDC-1556441) (CIF)
X-ray crystal structure of 1g (CCDC-1556442) (CIF)
X-ray crystal structure of 1m (CCDC-1823237) (CIF)
X-ray crystal structure of 3ga (CCDC-1556443) (CIF)
3ib
11,611 0.28
b
c
a25 μM 1g-1i in ACN/H2O (1:1). λmax in the 300-500 nm region. M-1cm-1.
dFluorescence quantum yield in EtOAc. eReaction quantum yield. fObtained by
comparing the emission intensity of Pyr to that of the initial reactants mixture
at λem; λex = 400 nm.
AUTHOR INFORMATION
Corresponding Author
zhipengy@scu.edu.cn.
ORCID
Zhipeng Yu: 0000-0001-6411-6078
Figure 2. Time course of photo-ligation of TCO-DASyd as monitored by UV-
vis (dashed lines) and fluorescence (solid lines). 1h in ACN/H2O (1:1) to derive
concentrations of 25 μM, and the activation wavelength was set at 365 nm
(0.95 mW/cm2). For fluorescence, λex = 400 nm.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
To demonstrate the utility of the DASyds for bioconjugation,
we first appended the TCO moiety to lysozyme (lyso-TCO, M.W.
~14.3 kDa) via chemical tagging and to sfGFP via genetic incor-
poration (sfGFP-Q204TCOK, M.W. ~27.9 kDa).19 Then, the resi-
due-specific protein modification was investigated by reacting the
resultant proteins with 1g and 1k under irradiation of 311 nm and
405 nm light for 2 min, respectively. The resultant mixture was
profiled by in-gel fluorescence imaging (Figure 3a-b) and LC–MS
(Figure 3c). The conversions into lyso-Pyr-1g or lyso-Pyr-1k or
We thank Dr. Jason J. Chruma (Sichuan University) for valuable
help and suggestions in manuscript preparation. We thank Dr.
Wenshe Liu (Texas A&M) and Dr. Qing Lin (SUNY-UB) for
generously providing us the plasmids pEvol-PylT-PylRS and
pET-sfGFP-Q204TAG. 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 Pro-
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18721. (d) Yagci, Y.; Jockusch, S.; Turro, N. J. Macromolecules 2010, 43,
6245–6260.
(3) (a) Herner, A.; Lin, Q. Top. Curr. Chem. 2016, 374, 1–31. (b)
Whitehead, S. A.; McNitt, C. D.; Mattern-Schain, S. I.; Carr, A. J.; Alam,
S.; Popik, V. V; Best, M. D. Bioconjugate Chem. 2017, 28, 923–932. (c)
Nainar, S.; Kubota, M.; McNitt, C.; Tran, C.; Popik, V. V.; Spitale, R. C. J.
Am. Chem. Soc. 2017, 139, 8090–8093. (d) Li, J.; Huang, L.; Xiao, X.;
Chen, Y.; Wang, X.; Zhou, Z.; Zhang, C.; Zhang, Y. J. Am. Chem. Soc.
2016, 138, 15943–15949.
(4) (a) Zhang, H.; Trout, W. S.; Liu, S.; Andrade, G. A; Hudson, D. A.;
Scinto, S. L.; Dicker, K. T.; Li, Y.; Lazouski, N.; Rosenthal, J.; Thorpe, C.;
Jia, X.; Fox, J. M. J. Am. Chem. Soc. 2016, 138, 5978–5983. (b) Yu, Z.;
Ohulchanskyy, T. Y.; An, P.; Prasad, P. N.; Lin, Q. J. Am. Chem. Soc.
2013, 135, 16766–16769. (c) Killops, K. L; Campos, L. M.; Hawker, C. J.
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V. A.; Chan, J. W.; Patton, D. L. J. Am. Chem. Soc. 2009, 131,
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(15) The competitive thermal cycloaddition reactions with TCO and
MMA in dark are extremely slow at 25 oC (Figure S22).
(16) (a) Grammel, M.; Hang, H. C. Nat. Chem. Biol. 2013, 9, 475–484.
(b) Patterson, D. M.; Nazarova, L. A.; Xie, B., Kamber, D. N.; Prescher, J.
A. J. Am. Chem. Soc. 2012, 134, 18638–18643.
(17) (a) Rieder, U.; Luedtke, N. W. Angew. Chem., Int. Ed. 2014, 53,
9168–9172. (b) Nguyen, K.; Fazio, M.; Kubota, M.; Nainar, S.; Feng, C.;
Li, X.; Atwood, S. X.; Bredy, T. W.; Spitale, R. C. J. Am. Chem. Soc.
2017, 139, 2148–2151. VdU is a surrogate for thymidine both in vitro and
vivo.
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Turn‐On Fluorescent
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Syd
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Scheme 1. The flipping of the diaryl substitutions in the SAP versus TAP.
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Figure 1. (a) Structures of selected DASyds with the C-H activation cross-coupling yield.
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Figure 1. (b) Absorption spectra and molar absorption coefficients at designated wavelength for selected
DASyds from library in acetonitrile/H2O (1:1) mixed solvent at 30 µM (250-450 nm).
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Table 1. Photo-activated 1,3-Dipolar Cycloaddition of 1a-1n with Methyl Methacrylate
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Table 2. Photo-activated 1,3-Dipolar Cycloaddition of 1g with Various Alkene Dipolarophiles
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Table 3. Photophysical Properties of the TCO-Pyrs and Fluorescence Turn-On Efficiency
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Figure 2. Time course of photo-ligation of TCO-DASyd as monitored by UV-vis (dashed lines) and
fluorescence (solid lines). DASyd in ACN/H2O (1:1) to derive concentrations of 25 µM, and the activation
wavelength was set at 365 nm (0.95 mW/cm2). For fluorescence, λex = 400 nm.
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Figure 3. Selective photo-induced fluorogenic labeling of proteins by 1g in vitro. Coomassie blue stain (top
panel) and in-gel fluorescence (bottom panel) of the same protein gel after resolved by SDS-PAGE (a) lyso
(b) sfGFP. (c) Deconvoluted mass spectra of control protein (red) and the TCO-protein mixture (blue)
showing the formation of the Pyr adduct (green). NR. = non-specific reaction (purple).
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