PhotoTag: Photoactivatable Fluorophores for Protein Labeling

Article abstract of DOI:10.1021/acs.orglett.0c00957

We report experimental studies on the development of photoactivatable fluorophores for rapid, light-induced synthesis of protein conjugates. Proof-of-concept studies demonstrated that electronic excitation of photoactivatable BODIPY-ArN₃ (1) in

Full text of DOI:10.1021/acs.orglett.0c00957

pubs.acs.org/OrgLett
Letter
PhotoTag: Photoactivatable Fluorophores for Protein Labeling
Rachael Fay, Anthony Linden, and Jason P. Holland*
Cite This: https://dx.doi.org/10.1021/acs.orglett.0c00957
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı Supporting Information
ABSTRACT: We report experimental studies on the development
of photoactivatable fluorophores for rapid, light-induced synthesis
of protein conjugates. Proof-of-concept studies demonstrated that
electronic excitation of photoactivatable BODIPY-ArN (1) in the
3
presence of different proteins leads to efficient labeling in less than
1
0 min. After synthesis and isolation of the fluorescently tagged
protein, photochemical conversion yields using human serum
albumin and onartuzumab were 47 ± 7% and 42 ± 5%,
respectively.
luorescent protein conjugates are vital tools in chemical
and biomedical research. For instance, fluorescently
Although the “click” approach is reliable and efficient, it
requires multistep chemistry involving premodification of the
protein with either an alkyne or azide group, followed by
bioorthogonal protein-functionalization with the fluoro-
F
tagged antibodies (mAbs) are used in a wide range of
applications, including substrate staining in gel-based assays,
biomarker detection in tissue sections using immunohisto-
chemistry and confocal fluorescence microscopy, cellular
labeling in fluorescence assisted cell-sorting (FACS), and
molecular imaging in vivo using fluorescence-mediated
17,18
phore.
Both Michael addition to sulfhydryl groups and
copper-mediated “click” reactions require the use of potentially
harsh, redox-active chemicals for the bioconjugation step
(TCEP, DTT, THTPA, CuSO
compromise the biological integrity and function of the protein
), which has the potential to
4
1
tomography (FMT). Although inherently fluorescent pro-
10,12
by disrupting the tertiary structure.
To expand the scope of
teins, such as green fluorescent protein (GFP), can be used for
imaging, extrinsic labeling of nonemissive proteins with organic
fluorophores provides access to a more diverse set of tools with
fluorescently labeled biomolecules, alternative strategies for
tagging proteins are required. Ideally, new methods should be
simple, efficient, and reproducible, allowing for the rapid, one-
step labeling of unmodified proteins with minimal intervention.
Here, we report a new method to incorporate the BODIPY
scaffold into biomolecules as another method to complement
the existing toolbox of inherently and tagged fluorescent
proteins.
2
−4
control over their biological function and optical properties.
Many organic small-molecule fluorophores have been
employed for fluorescence-based detection methods. Com-
monly used dyes include the cyanine, fluorescein, rhodamine,
5
,6
and BODIPY families. The 4,4-difluoro-4-bora-3a,4a-diaza-s-
indacene (BODIPY) scaffold offers several advantages for use
in biological systems, including vivid chromophores, good
fluorescence quantum yields, and high stability at physiological
Photochemistry offers a range of reagents and mechanisms
with potential for use in light-induced protein-functionaliza-
tion. For example, in 2015, Murale et al. reported the synthesis
of an asymmetric photoactivatable BODIPY bearing a modified
benzophenone motif attached to the 2- or 3-position of one of
7
pH. Auxochromic modifications can also be introduced to the
BODIPY scaffold, thereby allowing the characteristic absorp-
tion and emission bands to be modulated to the more
biologically suitable near-infrared (NIR) region. For instance,
the addition of thienyl groups on the pyrrole ring induces a
19
the pyridyl groups. Excitation of the aryl ketone likely
produces a triplet biradical species, which was reported to
allow photoinduced labeling of three different proteins,
including HSA, bovine serum albumin (BSA), and lysozyme
under UV irradiation. Although promising, the approach has
several drawbacks. Specifically, photoinduced reactions gave
8
,9
bathochromic shift.
Established methods for labeling biologically active mole-
cules with derivatives of BODIPY (or other fluorophores) are
usually based on thermochemically initiated reactions,
including modification of lysine residues with N-hydroxysucci-
nimide (NHS) and isothiocyanate (NCS) activated BODIPY
agents, cysteine-specific functionalization with maleimide-
BODIPY, and copper-mediated “click” chemistry with alkyne-
Received: March 15, 2020
10−16
or azide-functionalized BODIPY.
Reactions at cysteine
residues often require the addition of a reducing agent to break
disulfide bridges and reveal the reactive sulfhydryl groups.
©
XXXX American Chemical Society
https://dx.doi.org/10.1021/acs.orglett.0c00957
A
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
a
Scheme 1. Synthesis of BODIPY-ArN (1)
3
a
Key: (i) acetic anhydride, cat. TFA, 2,4-dimethylpyrrole, DCM; (ii) DDQ, DCM/THF; (iii) NEt , BF OEt , DCM; (iv) K CO , MeOH.
3
3
2
2
3
very low efficiencies (typically 0.1−0.6%; with a maximum of
.5% with tyrosine) and protein labeling yields were not
3
reported, and photoexcitation required the use of UV light at
wavelengths that are likely to damage the protein. To
circumvent these issues, we report the synthesis of BODIPY-
ArN , in which the aryl azide (ArN ) group is distal to the
3
3
BODIPY chromophore and is photoactivated at wavelengths
365 nm) that are not absorbed by most proteins, thereby
(
avoiding detrimental photodegradation.
BODIPY-ArN (1) was synthesized in four steps starting
3
from 4-hydroxybenzaldehyde and isolated with an overall yield
20
of 22% (Scheme 1; Supporting Figures 1−14). The ArN3
group was installed in the final step by amide coupling with 4-
azidoaniline hydrochloride in a yield of 62%. The single-crystal
X-ray structure of compound 1 was also obtained (Figure 1
Figure 2. Electronic absorption (UV/vis) profile of (blue) BODIPY-
ArN (1) and (red) BODIPY-azepin-MetMAb.
3
28
aryl nitrenes is well-established. It has a reported half-life on
the order of a nanosecond and can react via several pathways,
including H-atom abstraction, (C−H or X−H) bond insertion,
or intramolecular rearrangements to form a 7-membered
ketenimine ring. This ketenimine species reacts readily with
nucleophiles (including the ε-NH2 group of lysine) in a
bimolecular reaction to form an azepine linkage between the
29−32
protein and the ligand.
Although productive bimolecular
Figure 1. Single-crystal X-ray structure of BODIPY-ArN (1) (see
3
also Supporting Figure 24 for a depiction of the thermal ellipsoids).
reactions with primary amine groups of proteins appears to be
favored kinetically, the ketenimine intermediate can also react
slowly with other nucleophiles, including nonproductive
27,33
and Supporting Table 4). Full experimental details and
quenching with water.
Density functional theory (DFT)
1
19
13
characterization data including H, F, and C NMR, high-
resolution mass spectrometry, electronic absorption (UV/vis)
spectroscopy, fluorescence spectroscopy, and X-ray crystallog-
raphy data are presented in the Supporting Information.
The maximum excitation and emission wavelengths of
compound 1 were determined to be 497.5 and 508.5 nm,
respectively (Supporting Figure 12). The small Stokes shift
calculations were performed to investigate the mechanism and
photochemical reaction coordinate of compound 1 (Support-
ing Table 3 and Supporting Figures 15−17). The DFT
calculations revealed that rearrangement of the open-shell
1
singlet nitrene ( A state) to give the ketenimine is feasible and
2
subsequent nucleophilic attack by primary amines is
thermodynamically favored compared with attack by carbox-
ylate groups.
(
∼11 nm) is characteristic of BODIPY compounds, which
21,22
undergo little change in geometry upon photoexcitation.
Before testing protein-conjugation reactions, the photo-
The quantum yield of compound 1 (Φ) was determined in 0.1
chemical reactivity of BODIPY-ArN (1) was studied by using
3
M NaOH relative to fluorescein and found to be 0.03
high-performance liquid chromatography (HPLC). A solution
of compound 1 (0.25 mM, in MeOH) was irradiated by using
a high-powered light-emitting diode (LED, 365 nm; 263 mW)
at room temperature. Aliquots of the photolysis reaction were
analyzed by HPLC at time points from 0 to 5 min (Figure 3A).
As a control, the intermediate compound, BODIPY-OEt (3),
which lacks the ArN3 group, was also irradiated under
equivalent conditions to determine if the BODIPY core was
susceptible to photodegradation at 365 nm (Figure 3B). HPLC
analysis indicated that after 4 min compound 1 reacted
(
Supporting Figure 13) which is within the wide range of
previously reported quantum yields for BODIPY com-
pounds.
2
3−26
The electronic absorption profile of compound
1
showed an intense band at the wavelength used for
−
1
−1
photoactivation, 365 nm (ε = 10500 M cm ; Figure 2;
Supporting Figure 14 and Supporting Tables 1 and 2).
Light-induced activation of the para-substituted ArN group
3
initially leads to the formation a highly reactive open-shell
27
singlet nitrene species after loss of N (g). The reactivity of
2
B
https://dx.doi.org/10.1021/acs.orglett.0c00957
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Figure 3. HPLC chromatograms showing photodegradation over
time of (A) BODIPY-ArN (1) and (B) BODIPY-OEt (3).
3
Figure 4. SDS PAGE analysis of (A) unmodified MetMAb and (B)
BODIPY-azepin-MetMAb analyzed by (left) fluorescence imaging
showing pseudocolored bands and (right) Coomassie staining.
completely to give one major species, which was more polar
than the starting material. Intermediate 3 produced only minor
impurities after 5 min of irradiation, which indicated that the
BODIPY scaffold is stable to the irradiation conditions and
that the light-induced reactivity of compound 1 is mediated by
whether the dye was covalently bound to the protein and to
exclude the possibility that the photo-BODIPY-ArN3 was
forming aggregates (or polymers) during irradiation that
coelute with the protein fraction, a control reaction where
compound 1 was irradiated in the absence of protein was
performed. After irradiation, the reaction products were
purified by PD-10 SEC and the fractions analyzed by
fluorescence emission intensity. Analysis of the high molecular
weight fraction from control reactions showed <8% of the total
fluorescence intensity found in the protein-conjugation
reactions with MetMAb (Supporting Figure 19). These data
the ArN group. Mass spectrometry data from the photolyzed
3
product were consistent with the loss of N and the formation
2
of an azepine or related isomer (Supporting Figures 18 and
1
9).
Next, we performed a series of experiments to determine if
light-induced activation of BODIPY-ArN (1) in the presence
3
of protein could lead to the formation of fluorescent protein-
conjugates. Two different proteins were studied: HSA and the
humanized, monovalent engineered antibody onartuzumab
confirmed that light-induced activation of BODIPY-ArN in
3
(
formulated as the pharmaceutical MetMAb), which targets
the human hepatocyte growth-factor receptor c-MET (Scheme
).
Aliquots of BODIPY-ArN (1, DMSO) were added to stock
the presence of protein leads to specific protein functionaliza-
tion.
2
Further confirmation of protein conjugation was obtained by
running samples of unfunctionalized MetMAb and photo-
conjugated BODIPY-azepin-MetMAb on SDS-PAGE in non-
reducing conditions (Figure 4. Gels were first visualized by
fluorescence imaging, followed by incubation with Coomassie
to stain protein bands. Coomassie-stained gels showed one
band for MetMab at approximately 100 kDa, which
corresponds with the intact one-arm antibody (Figure 4,
right lanes A and B). Fluorescent imaging showed a band that
migrated to the same position but corresponded with
BODIPY-azepin-MetMAb (Figure 4, left lane B). Gel electro-
phoresis results of BODIPY-azepin-HSA and BODIPY-azepin-
MetMAb run under reducing conditions are shown in
Supporting Figures 20 and 21, respectively. The reducing
SDS−PAGE of BODIPY-azepin-MetMAb showed that both
the light chain and the heavy chain are fluorescently labeled.
Notably, under the conditions used for the gel electrophoresis,
noncovalently bound dye molecules are highly unlikely to
remain bound to the protein.
3
solutions of HSA (reconstituted in sterile saline; 69.08 kDa, 60
Lys, ε280 = 36500 M− cm ) or MetMAb (containing the
1
−1
native formulation buffer; 99.16 kDa, 66 Lys, ε = 161465
M
8
2
80
−
1
−1 34
cm ). The pH of the protein solution was adjusted to
.0−8.5 by using aliquots of Na CO . Solutions were mixed
2
3
thoroughly before irradiation at 365 nm for 10 min at room
temperature. Full experimental details are presented in the
Supporting Information. After irradiation, proteins were
purified from the small-molecule components by size-exclusion
chromatography (SEC) using PD-10 columns eluted with PBS
buffer (pH7.4) (Supporting Figure 20). Purified proteins were
collected in the high molecular weight fraction and analyzed by
electronic absorption spectroscopy (Figure 2 (red)), gel
electrophoresis (Figure 4 and Supporting Figures 21 and
2
2), and SEC coupled to HPLC (Supporting Figure 23).
HPLC analysis and visual analysis confirmed that the protein
fraction (detected at λ = 280 nm) coeluted with a peak
observed in the 515 nm channel, which was assigned to the
absorption of the BODIPY dye. However, to determine
After confirming the successful protein conjugation, we next
evaluated the photochemical conjugation efficiency (PCC; or
Scheme 2. Photochemical Conjugation of BODIPY-ArN (1) to the Monovalent Antibody Onartuzumab (Formulated as
3
MetMAb)
C
https://dx.doi.org/10.1021/acs.orglett.0c00957
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
yield) and the degree of labeling (DOL) by using different
stoichiometric equivalents of compound 1 (Table 1). To avoid
friendly and unlike other modification chemistries requires no
additional protein handling steps, such as prepurification or
prefunctionalization. We continue to expand on these proof-of-
concept studies by developing photoactivatable dyes (Photo-
Tags) using a range of fluorophore classes and different
photoactive groups.
Table 1. Degree of Labeling and Photochemical Conversion
Yields for the Light-Induced Protein-Conjugation Reaction
BODIPY-ArN (1) with MetMAb and HSA versus Varying
3
Equivalents of Compound 1
ASSOCIATED CONTENT
sı Supporting Information
■
[
protein]
(μM)
1
DOL avg ± sd PCC yield ± sd (%)
*
protein
(equiv)
(n = 3)
(n = 3)
MetMAb
168
2.5
5
10
2.5
4.8
10
0.82 ± 0.07
2.21 ± 0.24
4.06 ± 0.34
1.17 ± 0.18
1.62 ± 0.28
1.84 ± 0.44
33 ± 3
42 ± 5
41 ± 3
47 ± 7
34 ± 6
28 ± 4
The Supporting Information is available free of charge on the
ACS Publications Web site. The Supporting Information is
available free of charge at https://pubs.acs.org/doi/10.1021/
acs.orglett.0c00957.
1
1
68
01
HSA
208
95
1
Details on the synthesis and characterization of all
compounds; further experimental data on the reactivity
9
6
of BODIPY-ArN ; DFT data; further details of the
3
single-crystal X-ray structure of compound 1 (PDF)
precipitation of the protein and to ensure that the DMSO
content remained <10%, reactions using 10 equiv of
compound 1 were performed at lower protein concentrations.
The DOL was calculated from calibrated UV/vis spectra of the
purified protein conjugates by using eq 1. From the
experimental DOL, the yield could be calculated by division
with the theoretical maximum yield.
Accession Codes
CCDC 1950789 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
Amaxεprotein
A280 − AmaxC280)εmax
DOL = (
(1)
AUTHOR INFORMATION
Corresponding Author
■
where
A280(dye)
Jason P. Holland − Department of Chemistry, University of
C280 = A
Zurich, CH-8057 Zurich, Switzerland; orcid.org/0000-
max(dye)
0
002-0066-219X; Phone: +41446353990;
Email: jason.holland@chem.uzh.ch; www.hollandlab.org
Photochemical conversion efficiencies did not reach
quantitative yields since other competing reactions exist. It
has been reported that the ketenimine species can react slowly
Authors
3
2,33
Rachael Fay − Department of Chemistry, University of Zurich,
CH-8057 Zurich, Switzerland
Anthony Linden − Department of Chemistry, University of
Zurich, CH-8057 Zurich, Switzerland; orcid.org/0000-
with water and O2.
It is possible that reactive nucleophiles
(
i.e., histidine) present in the formulation buffer of MetMAb
compete with lysine groups on the protein. In addition to
intramolecular rearrangement to the ketenimine, the aryl
0
002-9343-9180
nitrene species produced after photoinduced loss of N can
2
undergo intersystem crossing to give a triplet species or
protonation to form a nitrenium ion which is unreactive
toward amino acids.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c00957
28
Photochemical conjugation to MetMAb and HSA proceeded
Author Contributions
efficiently with slightly varying yields that were dependent on
R.F. conducted and designed all experiments, analyzed the
data, and wrote the first draft. A.L. collected and analyzed the
crystallography data. J.P.H. supervised the project, designed
experiments, analyzed data, and wrote the manuscript. All
authors corrected and approved the final version of this work.
the initial equivalents of BODIPY-ArN added. The PCC was
3
calculated as the average (n = 3) ± the standard deviation (sd).
HSA was labeled the most efficiently with 2.5 equiv of
compound 1 (PCC = 47 ± 7%). MetMAb could be labeled
slightly more efficiently with a larger initial excess of
compound 1 (5−10 equiv). Our previous studies involving
immunoreactivity measurements indicated that irradiation of
Notes
The authors declare no competing financial interest.
32
MetMAb at 365 nm does not compromise protein function.
ACKNOWLEDGMENTS
Light-induced protein conjugation offers several advantages
over existing coupling methods. First, reactions are extremely
fast and run to completion in just a few minutes. Increased
■
J.P.H. thanks the Swiss National Science Foundation (SNSF
Professorship PP00P2_163683 and PP00P2_190093), the
European Research Council (676904, ERC-StG-2015, Nano-
SCAN), the Swiss Cancer League (KLS-4257-08-2017), and
the University of Zurich for financial support.
31
irradiation flux can also reduce reaction times. Functionaliza-
tion requires only one-step from unmodified proteins and
tolerates standard formulation buffers. The reaction is
reproducible and proceeds in good PCC yields. Photo-
activatable ArN3 dyes are easy to synthesize, can be
crystallized, are bench stable, and are not susceptible to
hydrolysis. Finally, the conjugation process is simple and user
REFERENCES
(1) Hoffman, R. M. Nat. Rev. Cancer 2005, 5 (10), 796.
(2) Crivat, G.; Taraska, J. W. Trends Biotechnol. 2012, 30, 8−16.
■
D
https://dx.doi.org/10.1021/acs.orglett.0c00957
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
(
3) Fujiki, Y.; Tao, K.; Bianchi, D. W.; Giel-Moloney, M.; Leiter, A.
B.; Johnson, K. L. Cytometry, Part A 2008, 73 (2), 11.
4) Bozhanova, N. G.; Baranov, M. S.; Klementieva, N. V.; Sarkisyan,
(
K. S.; Gavrikov, A. S.; Yampolsky, I. V.; Zagaynova, E. V.; Lukyanov,
S. A.; Lukyanov, K. A.; Mishin, A. S. Chem. Sci. 2017, 8 (10), 7138.
(
4
(
5) Kowada, T.; Maeda, H.; Kikuchi, K. Chem. Soc. Rev. 2015, 44,
953−4972.
6) Suzuki, T.; Matsuzaki, T.; Hagiwara, H.; Aoki, T.; Takata, K.
Acta Histochem. Cytochem. 2007, 40, 131−139.
(
(
7) Ni, Y.; Wu, J. Org. Biomol. Chem. 2014, 12, 3774−3791.
8) Leushina, E. A.; Usol’Tsev, I. A.; Bezzubov, S. I.; Moiseeva, A.
A.; Terenina, M. V.; Anisimov, A. V.; Taydakov, I. V.; Khoroshutin, A.
V. Dalt. Trans. 2017, 46 (48), 17093.
(
9) Li, G.; Otsuka, Y.; Matsumiya, T.; Suzuki, T.; Li, J.; Takahashi,
M.; Yamada, K. Materials 2018, 11 (8), 1297.
10) Matsumoto, T.; Urano, Y.; Shoda, T.; Kojima, H.; Nagano, T.
Org. Lett. 2007, 9 (17), 3375.
11) Chauhan, D. P.; Saha, T.; Lahiri, M.; Talukdar, P. Tetrahedron
Lett. 2014, 55 (1), 244.
12) Cheng, M. H. Y.; Savoie, H.; Bryden, F.; Boyle, R. W.
Photochem. Photobiol. Sci. 2017, 16 (8), 1260.
13) Tyagarajan, K.; Pretzer, E.; Wiktorowicz, J. E. Electrophoresis
003, 24 (14), 2348.
14) Albrecht, M.; Lippach, A.; Exner, M. P.; Jerbi, J.; Springborg,
M.; Budisa, N.; Wenz, G. Org. Biomol. Chem. 2015, 13 (24), 6728.
15) Rezende, L. C. D. D. L.; Emery, S.; Emery, F. Orbital Elec. J.
Chem. 2013, 5 (1), 62.
16) Ono, M.; Watanabe, H.; Ikehata, Y.; Ding, N.; Yoshimura, M.;
Sano, K.; Saji, H. Sci. Rep. 2017, 7 (1), 1−11.
17) Pickens, C. J.; Johnson, S. N.; Pressnall, M. M.; Leon, M. A.;
(
(
(
(
2
(
(
(
(
Berkland, C. J. Bioconjugate Chem. 2018, 29, 686−701.
(
(
18) Lang, K.; Chin, J. W. Chem. Rev. 2014, 114, 4764−4806.
19) Murale, D. P.; Hong, S. C.; Yun, J. H.; Yoon, C. N.; Lee, J. S.
Chem. Commun. 2015, 51 (30), 6643.
20) Sampedro, A.; Villalonga-Planells, R.; Vega, M.; Ramis, G.;
Fernandez De Mattos, S.; Villalonga, P.; Costa, A.; Rotger, C.
Bioconjugate Chem. 2014, 25 (8), 1537.
21) Zhu, X. Y.; Yao, H. W.; Fu, Y. J.; Guo, X. F.; Wang, H. Anal.
Chim. Acta 2019, 1048, 194.
22) Thakare, S. S.; Chakraborty, G.; More, A. B.; Chattopadhyay,
S.; Mula, S.; Ray, A. K.; Sekar, N. J. Lumin. 2018, 194, 622.
23) Kuhn, H. J.; Braslavsky, S. E.; Schmidt, R. Pure Appl. Chem.
004, 76 (12), 2105.
(
́
(
(
(
2
(
(
24) Brouwer, A. M. Pure Appl. Chem. 2011, 83, 2213−2228.
25) Boens, N.; Leen, V.; Dehaen, W. Chem. Soc. Rev. 2012, 41 (3),
1
(
(
130.
26) Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891−4932.
27) Preston, G. W.; Wilson, A. J. Chem. Soc. Rev. 2013, 42 (8),
3
(
289.
28) Gritsan, N. P.; Platz, M. S. Chem. Rev. 2006, 106 (9), 3844.
(
29) Voskresenska, V.; Wilson, R. M.; Panov, M.; Tarnovsky, A. N.;
Krause, J. A.; Vyas, S.; Winter, A. H.; Hadad, C. M. J. Am. Chem. Soc.
009, 131 (32), 11535.
30) Eichenberger, L. S.; Patra, M.; Holland, J. P. Chem. Commun.
019, 55, 2257.
31) Patra, M.; Eichenberger, L. S.; Fischer, G.; Holland, J. P. Angew.
Chem., Int. Ed. 2019, 58, 1928.
32) Fay, R.; Gut, M.; Holland, J. P. Bioconjugate Chem. 2019, 30,
814.
2
(
2
(
(
1
(
33) Platz, M. S. Acc. Chem. Res. 1995, 28 (12), 487.
(
34) Bateman, A. Nucleic Acids Res. 2019, 47 (D1), D506.
E
https://dx.doi.org/10.1021/acs.orglett.0c00957
Org. Lett. XXXX, XXX, XXX−XXX