Synthesis and evaluation of photo-activatable β-diarylsydnone-L-alanines for fluorogenic photo-click cyclization of peptides

Article abstract of DOI:10.1039/c9ob00898e

Herein, we design and synthesize a series of photoactivatable β-diarylsydnone-l-alanines (DASAs), which have excellent photo-reactivity with high fluorescence turn-on toward alkenes in a biocompatible environment. The environmental sensing properties of t

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DOI: 10.1039/C9OB00898E
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Synthesis and Evaluation of Photo-activatable β-Diarylsydnone-L-
alanines for Fluorogenic Photo-click Cyclization of Peptides
Zhuojun Yao, Xueting Wu, Xiaocui Zhang, Qin Xiong, Shichao Jiang and Zhipeng Yu*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Herein, we designed and synthesized a series of photo-activatable proteins or peptides in living system, primarily through the
β-diarylsydnone-L-alanines (DASAs), which have excellent photo- supressing of the amber stop codon.16 Particularly, UAAs for
reactivity with high fluorescence turn-on toward alkenes in bio- photo-click ligation such as p-(2-tetrazole)-phenylalanine (p-
compatible environment. Environment sensing property of the Tpa)17
resulting fluorescent pyrazoline-alanine facilitates its probing (mPyTK)18
and
methyl-2-pyrrole-5-carboxy-tetrazolelysine
dipole;
Nε-(1-methylcycloprop-2-
as
capability. By introducing the DASA residue on side chain of linear enecarboxyamido)lysine (CpK)19 and spiro[2.3]hex-1-ene
peptides, the macrocyclic peptides resulting from the in-situ photo- modified lysine (SphK)20 containing unsaturated C-C bonds,
cyclization toward alkene residue exhibited its fluorogenic have been incorporated into proteins for subsequent live cell
translocation through live cell membranes.
dynamic study via bioorthogonal ligation.21 In spite of the
exciting developments in recent years, the discovery of
photoclickable UAAs are highly demanded, which would
provide a powerful chemical foundation for research on
proteins in vivo.
Diarylsydnone (DASyd), a class of mesoionic 5-membered
heterocycles, reacting with various dipolarophiles underwent
[3+2] cycloaddition affording pyrazoles (Pyes)22 or pyrazolines
(Pyis) by means of nitrile imine intermediation upon photo-
irradiation.23 We recently reported a photo-induced DASyd-
alkene cycloaddition reaction which provided fluorogenic
performance for protein labelling without tagging any
exogenous fluorophore.24 Although sydnone, as a remarkable
dipole, has attracted a lot of interests in the bioorthogonal
research field recently,25 DASyd cored amino acids are rarely
Photo-triggered chemoselective bioconjugation reactions
are landmark tools for tracing molecules in living cells,1
constructing biomaterials2 and perturbating drug potency,3 in
possession of spatiotemporal controllability. The photo-
induced ligation reactions currently have been investigated
including: photo-click 1,3-dipolar cycloaddition between alkene
and tetrazole4 or azirine,5 photo-induced o-naphthoquinone
methides-ene hetero-Diels-Alder coupling,6 photo-triggered
thiol-olefin7 or alkyne8 addition, photo-irradiated strain-
promoted azide–alkyne cycloaddition,9 photo-induced [4+2]
cycloaddition of phenanthrenequinone-alkene,10 photo-
oxidative inverse-electron-demand Diels-Alder ligation,11 and
strain-loadable alkene-azide photo-click chemistry.12 These
paired photo-reactive reagents with unique functionality were
introduced into proteins or peptides by either chemical
modification of natural amino acid residues13 or biological14
incorporation of synthetic unnatural amino acids (UAAs)15 to
exert their practicability. Due to the high abundance of
competitive reactivity from natural amino acids as well as
undesired perturbation resulted in non-specific modifications
on proteins, genetic incorporation of UAAs provided a more
versatile and precise way for site-selective modification of
O
O
OH
OH
MeO
F
NH2
NH2
N
N
5a
5b
N
N
O
O
O
O
DASAs on C4 terminal
OH
X
Y
O
C4
OH
O
N3
pBpa
Fusion
Design
H2
N
N
H2
N
N
O
O
O
DASAs on N3 terminal
OH
OH
OH
OH
O
O
O
O
CF3
F3C
H2
N
H2
N
H2
N
H2N
a. Key Laboratory of Green Chemistry and Technology of Ministry of Education,
College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064,
P. R. China, E-mail: zhipengy@scu.edu.cn
CF3
H
O
N
N
N
N
5d
5f
5e
5c
N
N
N
N
O
O
O
O
O
O
O
† Electronic Supplementary Information (ESI) available: Containing details on
experimental procedures, spectra property, and characterization of all new
compounds. See DOI: 10.1039/x0xx00000x
Figure 1. Design and structures of sydnone amino acids, 5a–f.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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a)
R1
O
O
O
modified phenylalanine, an alternative approach was developed.
Iodo-alanine derived organo-zinc complex was treated with 4-
View Article Online
DOI: 10.1039/C9OB00898E
O
OH
O
O
O
HN
NH2
HN
, Pd(OAc)2
Boc
XPhos, K2CO3, DMF, 120 o
,
1. NaOH
Br
Boc
O
O
N
H
N
N
N
2. TFA
85%
C
N
N
bromophenyl-sydnone 3c to form monoarylsydnone amino acid
(MASAA) 4c via Negishi coupling,29 followed by Heck-coupling
reactions28 with the various aryl halides to generate DASAs 4d-f
successively (Scheme 1b). Finally, 5a-f were obtained stepwise by
deprotection with sodium hydroxide and TFA in high yields.
70-75%
O
O
R1
5a-b
: R1 = OMe, F
R1
: R1 = OMe, F
3a-b
4a-b
: R1 = OMe, F
O
b)
Br
O
O
IZn
O
HN
R
2-Br, Pd(OAc)2, XPhos
K2CO3, DMF, 120 o
59-65%
Boc
N
N
Boc
H
N
HN
Pd2(dba)3, Sphos, DMF, 60 o
78%
C
C
O
3c
4c
N
O
O
O
To study the photo-reactivity of SAAs, 4a-f were chosen to
examine the UV-Vis spectroscopic properties, because of their
protected α-amino acid framework mimicking the peptide side-
chain environment. All SAAs except MASAA 4c exhibited
maximum absorption wavelength around 332 nm (Figure S1).
Then, we explored their photo-reactivity toward methyl
methacrylate (MMA) or trans-cyclooct-4-en-1-ol (TCO) in
ACN:H2O = 1:1 under irradiation with a 311 nm UV lamp or 371
nm or 405 nm LED array by HPLC-MS analysis (Table 1, Figures
S2-3). While all six SAAs were consumed under 311 nm within 1
min photo-illumination, only DASAs 4a-b and 4d-f provided
≥99% conversions to generate the desired Pyi cycloadducts. In
addition, even though 371 or 405 nm photo-illumination leaded
to lower conversions compared to 311 nm, 4a still afforded
≥90% and ≥12% conversions, respectively, making it a more
suitable choice for chemo-selective bioconjugation propose. No
cycloaddition was observed for 12 h in dark (Figure S4).
Subsequently, all Pyi adducts were micro-prepared, isolated
and validated via NMR spectra analyses (Table 1, columns 9-10).
Since MMA is readily accessible (Table 1), we explored the
photophysical properties of the resulting Pyis 7a-b and 7d-f
(Table 2, Figures 2a and S6). Based on the fluorescence emission
spectra, 7a harboured a red-shifted emission maximum in
comparison with other Pyis, accompanied by the largest Stokes
shift (Table 2. entry 1 vs. others). More importantly, when
excited, 7a-b and 7d-f showed higher fluorescence quantum
yields (Table 2. F 0.71-0.90, Figure 2a) in EtOAc vs. those in
ACN:PB = 1:1, and varying degrees of fluorescence turn-on
during the photo-click process, ranging from 85-fold (7f) to
>460-fold (7e). Encouraged by these results, we next examined
the photo-ligation quantum yield (R) to evaluate the efficiency
1. NaOH
85% 2. TFA
5c
for MASAA
O
O
O
R2
R2
1.NaOH
Boc
O
N
N
N
N
HN
2.TFA
85%
NH2
O
O
OH
O
4d-f
5c-f
: R2 = H, p-CF3Ph, m-2CF3Ph, Ph
: R2 = p-CF3Ph, m-2CF3Ph, Ph
Scheme 1. Synthetic routes for the sydnone amino acids, 5a–f.
explored. Inspired by its attractive features, herein we report
the synthesis of a set of DASAs and the subsequent evaluation
of their photo-reactivity, photophysical properties of the
resulting Pyis, in search of its application as photo-linking tool
on peptide framework.
We designed two types of sydnone amino acids (SAAs) in which
phenylalanine moiety, as one of the aryl components, was linked at
C4- (5a-b) or N3-terminal (5d-f) of the sydnone core, respectively
(Figure 1). The N3-π-EDG was essential for excellent photo-reactivity,
including N3-p-MeOPh with polarized electron density, while
trifluoromethyl modified C4-aryl ring exhibited outstanding
fluorescence turn-on according to our recent study.24 Notably, 5b
and 5f which are akin to p-benzoyl-L-phenylalanine (pBpa),26
a
genetic encodable photo-cross-linker, will be the target substrates
for directed evolution of tRNA synthetase to incorporate DASAs into
proteins in future research. Therefore, two synthetic routes were
explored to construct six SAAs (Scheme 1). First, monoarylsydnones
(MASyds) 3a-c were obtained through
a
straightforward
transformation from N-aryl glycine via N-nitrosylation and
intramolecular cyclization promoted by trifluoroacetic anhydride
(TFAA).27 Then the protocol to obtain DASAs 4a-b were proved to be
easily achieved by Heck-type cross-coupling28 between C4-H MASyd
and a protected 4-bromophenylalanine (Scheme 1a). For N3-sydnone
Table 1. Photo-induced 1,3-Dipolar Cycloaddition of 4a-4f with MMA or TCO
h, 25°C, 1 min
in ACN/H2O = 1/1
R1
N
R4
R3
R2
R4
4a
4b
4c
4d
4e
4f
: R1 = p-MeOPh; R2 = Boc-L-Phe-Ome
: R1 = p-FPh; R2 = Boc-L-Phe-Ome
: R1 = Boc-L-Phe-OMe; R2 = H
: R1 = Boc-L-Phe-OMe; R2 = p-CF3Ph
: R1 = Boc-L-Phe-OMe; R2 = m-2CF3Ph
: R1 = Boc-L-Phe-OMe; R2 = Ph
+
N
N
R1
R3
6a-b
O
R2
O
N
CO2
311 nma
OMe
4a-f
7a-f, 8a-f
371 nma
405 nma
Isolated yieldb
Entry
No.
H
H
H
H
OMe
OMe
OMe
6a
H
H
H
H
Me
Me
Me
Me
HO
HO
HO
HO
O
O
O
O
6a
6a
6a
6b
6b
6b
6b
1
2
3
4
5
6
4a
4b
4c
4d
4e
4f
>99c
>99c
U.P.d
>99c
>99c
>99c
>99c
>99
92.6c
81.5c
N.R.e
56.8
52.0
70.9c
97.7c
90.7
N.R.e
70.1
66.3
81.6c
12.9
8.1
-
4.6
0.4
3.7
12.4
11.9
-
6.4
0.5
7.3
75
71.3
-
72
70
71
80.4
56.6
-
80
67.9
79.8
U.P.d
>99c
>99
>99c
aHPLC-MS conversion (%). 50 μM SAAs and 500 μM alkenes in ACN:H2O = 1:1 were photo-irradiated with corresponding light sources for 1 min. See Figures S2-3 in
ESI for details. bMicropreparations were conducted. 30 mg of SAAs and 20 equiv. MMA (6a) or 5 equiv. TCO (6b) in 50 mL ethyl acetate (EtOAc) with 311 nm UV light
for 2 h. cTrace amount of by-products formed. dU.P. = All converted into undesired products. eN.R. = No photo-reaction.
2 | J. Name., 2012, 00, 1-3
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O
a)
Table 2. Photophysical properties of the MMA-Pyisa
R1
b)
View Article Online
O
NH
N
O
N
O
Me
DOI: 10.1039/C9OB00898E
O
OMe
2x104
R2
0s
1.6
O
h, 25 °C
N
1s
3s
5s
7s
Me
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
+
OMe
Ac Val N
H
CLeu Aib Val
N
H
CLeu NH2
O
N
N
O
R2
R1
7a-b
O
O
N
P1
CO2
4a-b
4d-f
6a
7d-f
and
and
h, 25 °C
9s
23s
CO2
1x104
c
F
O
DASAs

(nm)
ex/em
43s
b
d
Entry No.
R EtOAc/ACN:PB
F. E.
R1
R2
diastereomers
O
N
N
(1:1)
NH
0
1
2
3
4
5
7a
p-MeOPh Boc-L-Phe-OMe 362/497 0.26
p-FPh Boc-L-Phe-OMe 356/459 0.32
p-CF3Ph 376/484 0.15
7e Boc-L-Phe-OMe m-2CF3Ph 380/497 0.17
7f Boc-L-Phe-OMe Ph 360/461 0.29
0.90/0.09
0.71/0.27 >270
0.87/0.44 220
0.72/0.46 >460
0.88/0.34 85
>90
300
400
500
600
Wavelength(nm)
7b
Ac Val N
H
CLeu Aib Val
N
CLeu NH2
H
O
O
P2
7d Boc-L-Phe-OMe
c)
DIC
Blue channel
DIO
Merge
P1
+ UV
a25 μM 7a-b and 7d-f in ACN:PB = 1:1. bReaction quantum yield in ACN:PB = 1:1.
cFluorescence quantum yield. dFluorescence enhancement by comparing the
fluorescence quantum yields of Pyis vs. DASAs (Figure S7) in ACN:PB = 1:1.
P3
+ UV
of the photochemistry. In general, the photo transformation
quantum yields of DASAs were determined to be 0.26-0.32 with
EDG on N3-terminal better than EWG on C4-terminal (Table 2.
entries 1, 2, 5 vs. 3, 4). Since Pyis were solvatochromic
fluorophores,30 we investigated the solvent dependent
fluorescence variation (Figure 2b, Figure S5). To our satisfaction,
all Pyi fluorophores were sensitive to the micro-environment
change that lied in the dielectric constant of the solvent (Figure
Figure 3. (a) Cyclization of peptide P1 bearing 1,3-dipolar cycloaddition in
positions i and i+4. (b) Time-dependent changes in absorption (dashed lines) and
fluorescence emission spectra (solid lines) of 150 μM linear peptide P1 in ACN:PB
= 1:1 (pH = 7.4) under irradiation of UV light. (c) Fluorescence images of live A549
cells for in-situ cyclization of 150 μM P1 in the medium after 371 nm irradiation
P3 as the control group.
.
crosslink the side-chain via the photo-induced 1,3-dipolar
cycloaddition under 311 nm UV light or 371 nm LED array in a
variety of solvents including organic or buffer solution. To our
surprise, the side chains of the peptide showed desired
cyclization only in the presence of PB containing solvents with a
time-dependent fluorescence generation under continuous
illumination (Figures 3b, S9), while other solvents, including
ACN:H2O = 1:1, only leaded to side-reactions (solvolysis or
nucleophilic additions, Figure S8). By inquiring the circular
dichroism (CD) spectra of peptide P1 in different solvents, we
found that the degree of α-helicity was reduced in ACN:PB = 1:1,
therefore the phosphate probably behaved as a unique
conformational relaxer of the peptide P1, causing the proximity
of alkene residue toward DASA 5a in the disorder
conformational dynamics (Figure S10a). After purification by
S5). Significantly, 7a displayed
a sharp increasing of
fluorescence intensity in non-protonic solvent, like:
dichloromethane (DCM), EtOAc and acetonitrile (ACN); the ratio
of fluorescence elevation effect from protonic methanol
(MeOH) to EtOAc was 18-fold (Figure 2b). Therefore, it was
beneficial to probe the micro-environment of the cycloadduct
when on peptides or proteins via comparing the relative
fluorescence brightness.
In order to utilize the DASA-alkene photo-click ligation for
the fluorogenic tracing of peptides in live cell via imaging, we
selected methacrylamide modified lysine and DASA 5a to
replace i and i+4 sites of a Balaram's 310-helix31 peptide,
respectively (Figure 3a). Both Grubbs32 and Qin33 have studied
the stapling of this α-helical peptide model via chemically
modified lysine residues. Different from previous studies, the
incorporation of DASA 5a into the peptide sequence with more
deeply embedded DASyd moiety would arouse more rigidity in
resulting macrocyclic peptide after photo-click reaction, which
might provide strengthened control on the conformation of the
peptide. With the peptide P1 synthesized, we attempted to
reverse-phase HPLC,
a
pair of macrocyclic peptidic
diastereomers P2 were characterized by CD spectrum analysis
in trifluoroethanol (TFE) or ACN:PB = 1:1. Peptide P3 (Ac-Val-
Lys-Leu-Aib-Val-5a-Leu-NH2) was selected as a control in which
the lysine residue was unmodified to avoid conformational
perturbation during spectral scanning (190-250 nm). We
observed that the macrocyclic peptide P2 lost the α-helical
conformation, comparing to the peptide P3 (Figure S10b),
presumably due to the tight control of the macrocyclic space
(Figure 3a).
To study the membrane permeability of peptide P2 via in-
situ cyclization from P1 in live cells, A549 cells culturing with 150
μM P1 vs. P3 were photo-irradiated under 371 nm LED array for
1 min and then incubated for 3 h, followed by DIO staining to
visualize the cell membrane. Contrast to the control group
treating with P3 (without alkene residue) which scarcely
presented any fluorescence signal in blue channel (lower panel,
Figure 3c), accumulated fluorescence was observed
a)
b)
7a 7b 7d 7e 7f
600k
500k
400k
300k
200k
100k
0
1.0
0.8
0.6
0.4
0.2
0.0
500k
400k
300k
200k
100k
0
EtOAc
DCM
7a
7a
7b
7d
7e
7f
ACN
ACN:PB = 1:1
ACN:H2O = 1:1
MeOH
400
450
500
550
600
650
300
400
500
600
Wavelength (nm)
Wavelength (nm)
Figure 2. Characterization of the absorbance and fluorescence properties of the MMA-
Pyis. (a) Absorption (dashed lines) and fluorescence emission spectra (solid lines) of 30
μM Pyis 7a-f in ACN:PB = 1:1. λex = 360 nm for 7a, 7b, 7f and 378 nm for 7d, 7e. Inset:
Images of the fluorescence of MMA-Pyis in ACN:PB = 1:1 with irradiation at 365 nm. (b)
Fluorescence spectra for 30 μM 7a in various solvents. λex = 360 nm.
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12 K. Singh, C. J. Fennell, E. A. Coutsias, R. Latifi, S. Hartson and J.
intermittently in the cytoplasm for P1 cyclization via
fluorescence microscopy imaging (upper panel, Figures 3c and
S11). This pattern of distribution indicated that the cyclized P1
might penetrate the cell membrane through the pinocytotic
pathway.34 The cell viability after photo-cyclization of peptide
P1 in-situ was determined to be 91 ± 1.7% (Figure S12).
Furthermore, we treated the A549 cells with preformed cyclic
peptide P2 vs. the linear peptide P4 (Ac-Val-Lys-Leu-Aib-Val-
Pyis-Leu-NH2) which possesses the fluorescent Pyis residue as
the control. Intriguingly, preformed P2 displayed the
distribution of blue fluorescence signal akin to the in-situ
cyclized P1, while the linear P4 still showed negligible signal
(Figure S13), indicating the in-situ photo-cyclization of P1 is a
valid mean for tuning the properties of the peptide.
View Article Online
D. Weaver, Chem, 2018, 4, 124.
DOI: 10.1039/C9OB00898E
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In summary, we reported the design and synthesis of DASAs with
minimal spatial occupation. By investigating the properties of
corresponding Pyis products formed with MMA under photo-
irradiation, prominent fluorescence turn-on was observed. Among
them, 5a harbouring the high photo-reactivity was introduced into
an α-helical peptide in combination with MMA-modified lysine to
generate fluorescent cyclopeptide via photoclick ligation. The
fluorogenic photo-macrocyclization demonstrated the utilization of
DASAs as UAAs to explore the biological activity of macromolecules
in live cells.
19 Z. Yu, Y. Pan, Z. Wang, J. Wang and Q. Lin, Angew. Chem. Int.
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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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