Bioorthogonal Ligation and Cleavage by Reactions of Chloroquinoxalines with ortho-Dithiophenols

Article abstract of DOI:10.1002/anie.201913620

A bioorthogonal ligation and cleavage method via reactions of chloroquinoxalines (CQ) and ortho-dithiophenols (DT) is presented. Double nucleophilic substitutions of ortho-dithiophenols to chloroquinoxalines provide conjugates containing tetracyclic benzo[5,6][1,4]dithiino[2,3-b]quinoxaline with strong built-in fluorescence together with release of the other functional molecules. Three cleavable linkers were designed and successfully used in release of the molecules containing biotin from the protein conjugates. The CQ-DT bioorthogonal reactions can be applied for the bioorthogonal ligations, bioorthogonal cleavages, and trans-tagging of proteins, and show advantages of readily accessible unnatural orthogonal groups, appealing reaction kinetics (k₂≈1.3 m^?1 s^?1), excellent biocompatibility of orthogonal groups, and high stability of conjugates. This complements previous bioorthogonal reactions and is a new route for protein-fishing applications and in-gel fluorescence analysis.

Full text of DOI:10.1002/anie.201913620

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Both Bioorthogonal Ligations and Cleavages via Reactions of
Chloroquinoxalines with ortho-Dithiophenols
Authors: Youshan Li, Zhenbang Lou, Hongyun Li, Haijun Yang, Yufen
Zhao, and Hua Fu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201913620
Angew. Chem. 10.1002/ange.201913620
Link to VoR: http://dx.doi.org/10.1002/anie.201913620
http://dx.doi.org/10.1002/ange.201913620

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
Both Bioorthogonal Ligations and Cleavages via Reactions of
Chloroquinoxalines with ortho-Dithiophenols
Youshan Li, Zhenbang Lou, Hongyun Li, Haijun Yang, Yufen Zhao, and Hua Fu*
[*] Y. Li, Dr. Z. Lou, H. Li, Dr. H. Yang, Prof. Dr. Y. Zhao, Prof. Dr. H. Fu
Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education)
Department of Chemistry
Tsinghua University
Beijing 100084 (China)
E-mail: fuhua@mail.tsinghua.edu.cn
Supporting information for this article is given via a link at the end of the document.
Abstract: Here we report a novel kind of bioorthogonal ligation and
cleavage method via reactions of chloroquinoxalines (CQ) and
ortho-dithiophenols (DT), in which double nucleophilic
substitutions of ortho-dithiophenols to chloroquinoxalines provide
azide-alkyne cycloaddition,[11] the inverse electron-demand Diels-
Alder (IED-DA) reaction,[12] and the boronate formation.[13]
Compared with the diverse bioorthogonal ligations, the researches
on the bioorthogonal cleavages are limited. The most representative
examples include the light-induced bond cleavages,[14] the transition
metal-catalyzed deallylation or depropargylation,[15] the specific
IED-DA-induced ‘click and release’ reaction,[16] hydrazinolysis,[17]
reduction of diazo[18] and disulfide compounds,[19] acid-induced
hydrolysis of silyl esters.[20] Unfortunately, the conditions for some
of bioorthogonal cleavages are harsh, which may affect the activity
of biomolecules to some extent. In addition, the research of
literatures shows that the reactions suitable for both the
bioorthogonal ligations and cleavages are relatively rare. In 2013,
Robillard and coworkers developed the drug decaging and release
from antibodies through the tetrazine and trans-cyclooctene reaction,
in which the IED-DA cycloaddition reaction initiated release of the
drug attached to the trans-cyclooctene group (Figure 1a).[16a] The
approach is probably the most popular reaction to date for the
bioorthogonal ligation and release. Recently, Taran and co-workers
also described an intresting bioorthogonal method via the reaction
of iminosydnones and cyclooctynes allowing ligation and release of
functional molecules (Figure 1b).[21]
the
corresponding
conjugates
containing
tetracyclic
benzo[5,6][1,4]dithiino[2,3-b]quinoxaline with strong built-in
fluorescence together with release of the other functional molecules.
With this transformation, three cleavable linkers were designed and
successfully used in release of the molecules containing biotin from
the protein conjugates. The CQ-DT bioorthogonal reactions can be
applied as the bioorthogonal ligations, bioorthogonal cleavages and
trans-tagging of proteins, and show some advantages including
readily accessible unnatural orthogonal groups, appealing reaction
kinetics (k2 ≈ 1.3 M-1s-1), excellent biocompatibility of orthogonal
groups and high stability of conjugates. This work provides an
attractive complement to the previous bioorthogonal reaction
toolbox and opens a new route for protein-fishing applications and
in-gel fluorescence analysis.
Introduction
Bioorthogonal chemistry has become a powerful tool in chemical
biology[1] (the concept of ‘bioorthogonal chemistry’ was first
proposed by Bertozzi in 2003[2]), and it shows wide applications
such as chemical labelling of biomolecules in living cells,[3] post-
translational modification of proteins[4] and construction of antibody
drug conjugates.[5] Therefore, the development of highly efficient
bioorthogonal reactions is of great importance. The bioorthogonal
reactions should meet the following conditions including fast rate,
high yield, good solubleness and high stability of the reactants and
product(s) under physiological conditions, non-toxicity to the
biological system, minimizing steric interactions with the
biomolecule and facilitating incorporation by the endogenous
cellular machinery.[6]
A number of bioorthogonal reactions
including bioorthogonal ligations and cleavages have been
developed thus far. The most representative bioorthogonal ligations
include the native chemical ligation of proteins,[7] the protein
labeling through the reaction of bisarsenical dyes with a genetically
incorporated tetracysteine unit,[8] the biocompatible Staudinger
ligation reaction of azides with modified triphenylphosphines,[9] the
Cu(I)-catalyzed azide-alkyne cycloaddition,[10] the strain-promoted
Figure 1. a) Robillard’s click and release based on the tetrazine and trans-
cyclooctene reaction. b) Taran’s ligation and cleavage method via the
reaction of iminosydnones with cyclooctynes. c) Our ligation and cleavage
strategy via double nucleophilic substitutions of o-dithiophenols (DT) to
chloroquinoxalines (CQ).
1
This article is protected by copyright. All rights reserved.

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
Herein, we report a novel and highly efficient strategy: both
bioorthogonal ligations and cleavages via mild and selective
reactions of readily accessible chloroquinoxalines (CQ) and ortho-
dithiophenols (DT) (Figure 1c), in which double nucleophilic
substitutions of ortho-dithiophenols to chloroquinoxalines provide
(2a-2d), respectively, in water/DMF provided conugates 3a–3d
within 10 min in 94–97% yields (entries 1–4). Subsequently, 2,3-
dichloroquinoxaline-6-carboxylic acid (1e) was attempted as the
partner of 4-methylbenzene-1,2-dithiol (2b), and an excellent yield
(98%) was afforded (entry 5). We investigated reaction of 1e with
2b in PBS/DMF (entry 6), and the reaction also provided a high yield
(> 99%). Inspired by the results above, we attempted to use various
mono-substituted chloroquinoxalines as the partners of o-
dithiophenols. Reactions of chloroquinoxalines 1g, 1i and 1k
containing histidine, cysteine and tyrosine units, respectively, with
2d in PBS/DMF were surveyed, and the conjugate (3d) was obtained
in 84–98% yields (entries 7–9). We found that the order of reactivity
on the chloroquinoxalines containing three amino acid units was 1g
> 1k > 1i, and the yields obviously increased with ratio rise of 2d
(Table S7). Besides, reactions of chloroquinoxaline 1a or 1k with o-
dithiophenol (2d) were peformed in cell growth medium (RPMI-
1640, + 10% fetal bovine serum), respectively, and 2-13% efficiency
loss was observed relative to the efficiency in PBS (Figure S8).
Based on the results above, we think that couplings of o-
dichloroquinoxalines or mono-substituted chloroquinoxalines with
o-dithiophenols can be used as bioorthogonal ligations, and the
reactions of mono-substituted chloroquinoxalines with o-
dithiophenols provide opportunities for bioorthogonal cleavages,
and simultaneous occurrence of bioorthogonal ligation and cleavage
in one reaction.
the
corresponding
conjugates
containing
tetracyclic
benzo[5,6][1,4]dithiino[2,3-b]quinoxaline with strong built-in
fluorescence together with release of the other molecules.
Table 1: Investigations on couplings of different substituted
chloroquinoxalines (1) with o-dithiophenols (2).[a]
Entry
1
2
Condition[b]
3, Yield (%)
1
2
3
4
5
6
7
8
9
1a
1a
1a
1a
1e
1e
1g
1i
2a (1 eq)
2b (1 eq)
2c (1 eq)
2d (1 eq)
2b (1 eq)
2b (1 eq)
2d (5 eq)
2d (10 eq)
2d (10 eq)
Condition A
Condition A
Condition B
Condition B
Condition B
Condition C
Condition C
Condition C
Condition C
3a, 94
3b, 95
3c, 97
3d, 97
3m + 3′m, 98
3m + 3′m, 99
3d, 96
3d, 84
1k
3d, 98
[a] [1] = [2] = 50 mM for entries 1–5, isolated yields. [1] = 0.5 mM, [2] =
0.5–5 mM for entries 6–9, and the yields were determined by HPLC. [b]
Condition A: K2HPO4 (2 eq), H2O/DMF (1:4), room temperature (RT), 10
min. Condition B: K2HPO4 (3 eq), H2O/DMF (1:4), RT, 10 min. Condition C:
PBS (20 mM, pH 7.4)/DMF (1:1), RT, 60 min. See Tables S1–S7 for more
screening details.
Results and Discussion
As mentioned above, it is highly desirable to develop a general and
highly efficient method for bioorthogonal ligation and cleavage. We
realized that reaction of o-dichloroquinoxalines with o-
dithiophenols could be performed in organic solvents under the
irradiation of light[22] or base-promoted conditions.[23] We surmised
that the selective reactions of o-dichloroquinoxalines and their
derivatives with o-dithiophenols could be used in bioorthogonal
ligations and cleavages. Firstly, various chloroquinoxalines and o-
dithiophenols were prepared (Schemes S1 and S2), and then
couplings of chloroquinoxalines with o-dithiophenols were
attempted in water/N,N-dimethylformamide (DMF) or phosphate
buffer saline (PBS, pH 7.4)/DMF (Table 1). Wide investigations on
the reagents and reaction conditions were performed, and some
reactions of o-dichloroquinoxalines or mono-substituted
chloroquinoxalines with o-dithiophenols readily occured in high
yields with excellent selectivity (Tables S1–S7). As shown in Table
1, couplings of o-dichloroquinoxaline (1a) with four o-dithiophenols
Figure 2. a) Investigation on reactivity of dichloroquinoxaline (1a) with
different nucleophilic reagents. NR = No Reaction. b) Competitive reaction
of 1e with 2b in the presence of GSH (0–200 equiv relative to 1e and 0–40
equiv relative to 2b). The conversion rates were determined by HPLC. The
data are average from three replicate experiments.
To investigate the compatibility of orthogonal groups to
versatile functional groups or complex systems, we first surveyed
reactivity of o-dichloroquinoxaline (1a) with compounds 4a–4h
containing common functional groups including sulfydryl, hydroxyl,
2
This article is protected by copyright. All rights reserved.

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
amino, indolyl and imidazolyl at millimolar concentration in
water/DMF in the presence of two equiv of K2HPO4 for 2 h (Figure
2a). The experiments showed that only 4a and 4b containing
sulfydryl could treat with 1a, and no reaction occurred for phenol
(4c), indole (4d), imidazole (4e), alcohol (4f), amine (4g) and even
NaOH (4h). We found that 1a was highly selective to thiols and
tolerated a broad range of functional groups. Subsequently, reaction
of 1a with N-acetyl cysteine methyl ester (4a) was attempted in PBS
(pH 7.4)/acetonitrile (v/v, 1:1), and different ratios of 4a/1a (1:1, 5:1
and 10:1) led to conversion rates of 4%, 17% and 32% to mono-
substituted chloroquinoxaline 1h, respectively (Figure S5a).
Although some treatment of o-dichloroquinoxalines with
compounds containing sulfydryl such as cysteine derivative is
disadvantageous to the bioorthogonal ligation, fortunately, the
subsequent addition of o-dithiophenol can smoothly cleave the C-S
bond to release free cysteine derivative, such as reaction of 1i with
2d (Table 1, entry 8). Next, the competitive reaction of 1e with 2b
was investigated in the presence of different concentration of
glutathione (GSH). The reaction efficiency suffered slight impact
when GSH (0-10 mM) was 0‒100 equiv relative to 1e and 0‒20
equiv relative to 2b. However, the conversion rate decreased to 73%
when GSH (20 mM) was 200 equiv relative to 1e and 40 equiv
relative to 2b (Figures 2b and S6). In addition, we found that o-
dithiophenols were relatively stable in cell medium in air for 12 h
(Figure S9). One possible reason is that the formation of
intramolecular hydrogen bond between two sulfydryls of o-
dithiophenols inhibits their oxidation in air.
Rapid reaction of lower concentration of reactants is of great
concern for modification of biomolecules under physiological
conditions. Here, reaction of 2,3-dichloroquinoxaline-6-carboxylic
acid (1e) with 4-methylbenzene-1,2-dithiol (2b) was used as the
example to investigate the corresponding second-order rate constant
in PBS (pH 7.4)/MeCN (Figure S11). The second order rate constant
k2 ≈ 1.30.1 M-1s-1 was calculated from the slope of the graph using
the relationship: kobs = [2b]k2.
UV-vis spectra of reactants 1e, 2b and their products 3m, 3'm
were recorded, and the tetracyclic heteroacenes (3m and 3'm)
showed strong absorption peak at 398 nm. Importantly, the
conjugates 3a, 3c, 3d, 3m and 3'm with different substituents
exhibited strong photoluminescence (PL) (Figure S12) in the visible
light spectral region.[23]
Figure 3. Bioorthogonal ligations of o-dichloroquinoxalines containing protein C2A(S83C) with different o-dithiophenols. a) Reaction routes. b) Four o-
dithiophenols 2d, 7-9. Reaction conditions: C2A(S83C) (13 μM), 5 (65 μM), 2d or 7-9 (130 μM), TCEP (2.6 mM), PBS (20 mM, pH 7.4) at room temperature
for 60 or 90 min (Note: The regioselectivity during formation of two C-S bonds is ignored). c) HPLC traces of C2A(S83C), in-situ formed 6 and 11. d) ESI-
MS spectrum of 6 (Theoretical molecular weight: 15680.7. Calculated molecular weight: 15685.1). e) ESI-MS spectrum of 11 (Theoretical molecular weight:
17958.1. Calculated molecular weight: 17961.9). f) Gel analysis of C2A(S83C) (lane 1), o-dichloroquinoxaline functionalized C2A(S83C) 6 (lane 2) and
bioorthogonal products 10–13 (lane 3–6). Fluorescence signals were acquired using EB channel (upper panel). Coomassie staining was used to assess protein
loading (lower panel)
3
This article is protected by copyright. All rights reserved.

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
Proteins are a kind of important biopolymers, and their chemical
modification can provide a variety of purposes in chemical biology
and medicine[24] such as installing a fluorophore[8,25] or binding
partner[26] on proteins. Before investigating bioorthogonal reaction
for protein modification, we first attempted to use GSH as a model
peptide to appraise the CQ-DT reactions. The experiments showed
that the reactions worked well in 60 min and almost quantitative
To evaluate efficiency of the CQ-DT reactions in a more
complex system, we investigated conjugation of o-dithiophenols
containing cell transmembrane peptide (7) or fluorescein (9) with o-
dichloroquinoxaline-functionalized His-C2A(S105C) (14) (14 was
prepared via Michael addition of sulfydryl of cysteine residue on
His-tagged
C2A(S105C)
to
maleimide-functionalized
chloroquinoxaline 5) in E. coli cell lysate (Figure 4b). The
conjugates were observed by HPLC and ESI-MS (Figures S13‒S40). experimental results showed that the reactions proceeded smoothly
With these encouraging small molecules results in hand, we
examined the CQ-DT reactions outlined above with a model protein
to evaluate their potential as a novel bioorthogonal methodology.
We chose an engineered variant of the C2A domain of
Synaptotagmin-I, C2A(S83C) (molecular weight 15680 Da) with
single and surface-exposed cysteine residue[27] as the model protein.
As shown in Figure 3a, we first modified C2A(S83C) with
maleimide-functionalized o-dichloroquinoxaline 5 to form 6 (HPLC
and ESI-MS determination, see Figures 3c,d) via Michael addition
of free cysteine residue in C2A(S83C) to maleimide group of 5. To
our delight, treatment of o-dichloroquinoxaline 6 containing protein
C2A(S83C) with various o-dithiophenols (2d, 7–9) (Figure 3b) in
PBS (pH 7.4) at room temperature resulted in rapid and complete
conversions of 6 to the corresponding ligation products (10–13)
(Figures 3a, S47–S51) via analysis of ESI-MS (such as Figure 3e),
HPLC (such as Figure 3c) and gel analysis (Figure 3f). It is
noteworthy that o-dithiophenols containing cell transmembrane
peptide (7),[28] biotin (8) and fluorescein (9) all provided complete
conversions (Note: tBuS-deprotection was carried out with tris(2-
carboxyethyl)phosphine (TCEP) in PBS buffer for tBuS-protected o-
dithiophenols).
and were almost not interfered by free thiol in the vicinity of
complex aqueous system, and the modified His-C2A(S105C)
protein could be visualize by fluorescence imaging at concentration
of around 15 M through the CQ-DT reactions in cell lysate.
Importantly, the reactions exhibited remarkable selectivity without
detectable background signals for fluorescence labeling in the cell
lysate. In addition, o-dichloroquinoxaline-functionalized His-
C2A(S105C) 14 was incubated in cell lysate or cell medium for the
indicated time, then treated with o-dithiophenol 7, and the
conjugates were determined by Gel analysis. We found that more
than 80% efficiency was maintained in cell lysate or cell growth
medium relative to PBS (Figure 4c and Figure S52). Therefore, the
CQ-DT reactions are compatible with different biomolecules and
can be used for protein labeling in PBS, cell lysate or cell growth
medium. We believe that the conjugates containing tetracyclic
benzo[5,6][1,4]dithiino[2,3-b]quinoxaline
with
built-in
fluorescence will provide important applications for antibody-free
Western Blot analysis.
Furthermore, we investigated practicability of the CQ-DT
reactions as versatile cleavable methods for protein-fishing
applications. The previous conditions for the desorption of the
strong biotin-avidin affinity usually are harsh such as heating, which
cause the unspecific release of background proteins through random
adsorption to the avidin surface, and the fact hampers the subsequent
analysis. Therefore, it is of significance to develop an efficient
method for gentle release of biotin-protein complexes.[1e]
Encouraged by our results above, we here investigated
bioorthogonal ligations and cleavages using pre-targeted model
protein bearing a biotin moiety. Our strategy is shown in Figure 5a:
Michael addition of sulfydryl of cysteine residue on the protein to
maleimide-functionalized chloroquinoxaline (I) linking with biotin
leads to II, then treatment of II with 2-(3,4-dimercaptophenyl)acetic
acid (2d) provides ligation product III and cleavage product IV, and
the obtained conjugates are analyzed by gel analysis and ESI-MS.
We designed and synthesized three maleimide-functionalized
chloroquinoxalines with cleavable linkers, and their coupling with
C2A(S83C) afforded pre-targeted proteins 17–19. Firstly,
chloroquinoxaline derivative 17 containing C2A(S83C) and a biotin
moiety through linkage of thiol ether was used for the bioorthogonal
ligation and cleavage. Incubation of 17 with 2d in PBS (pH 7.4) at
room temperature for 90 min provided conjugate 20 in almost
quantitative yield (Figure 5b) via analysis of HPLC, ESI-MS (Figure
5e) and gel analysis (Figure 5f) releasing cleavage product 21. Next,
treatment of previously prepared chloroquinoxaline derivative 18
containing C2A(S83C) and a biotin moiety through linkage of 4-
(hydroxymethyl)phenol with 2d under the similar conditions gave
20 in almost quantitative yield (Figure 5c) via analysis of HPLC,
ESI-MS and gel analysis releasing 22, in which formation of 22
underwent a cascade process including cleavage of a C-O bond and
Figure 4. Efficiency in a crude cell lysate or cell medium on the CQ-DT
reactions. a) Reaction routes. b) Gel analysis of the CQ-DT conjugates in the
cell lysate. c) o-Dichloroquinoxaline-functionalized His-C2A(S105C) 14
was incubated in cell lysate or cell medium for the indicated time, then
treated with o-dithiophenol 7, and the conjugates were determined by Gel
analysis. Fluorescence signals were acquired using EB channel.
4
This article is protected by copyright. All rights reserved.

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
Figure 5. Both bioorthogonal ligations and cleavages. a) Our strategy on biorthogonal ligations and cleavages. b) Reaction of chloroquinoxaline derivative
17 with 2d. c) Reaction of chloroquinoxaline derivative 18 with 2d. d) Reaction of chloroquinoxaline derivative 19 with 2d. Reaction conditions: 17–19 (13
μM), 2d (130 μM), PBS (10 mM, pH 7.4) at room temperature for 90 min. e) ESI-MS of 20. f) Gel analysis of C2A(S83C) (lane 1) and 20 (lanes 2–4 from
reactions of 17–19 with 2d, respectively). g) Reactions of 17–19 with 7. Reaction conditions: 17–19 (13 μM), 7 (130 μM), PBS (10 mM, pH 7.4) at room
temperature for 90 min. h) Gel analysis of C2A(S83C) (lane 1) and 23 (lanes 2–4 from reactions of 17–19 with 7, respectively). Fluorescence signals were
acquired using EB channel (upper panel). Coomassie staining was used to assess protein loading (lower panel). (Note: The regioselectivity during formation
of two C-S bonds is ignored.)
release of an functional molecule (it means a decaged process).[29]
The decaged process was confirmed through reaction of S80 with 2b
in PBS (pH 7.4)/acetonitrile (v/v, 1:1) at room temperature (Figure
S54a). Further, we made chloroquinoxaline derivative 19 containing
mercaptoethanol, and reaction of 19 with 2d also led to a complete
conversion of 19 to ligation product 20 releasing 22 via a cascade
process including cleavage of a C-S bond and release of an
functional molecule[30] (Figure 5d). The decaged process was also
verified through reaction of S81 with 2b in PBS (pH 7.4)/acetonitrile
C2A(S83C) and
a biotin moiety through linkage of 2-
5
This article is protected by copyright. All rights reserved.

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
[4] a) Y. Y. Yang, J. M. Ascano, H. C. Hang, J. Am. Chem. Soc. 2010, 132,
3640-3641; b) W. P. Heal, B. Jovanovic, S. Bessin, M. H. Wright, A. I.
Magee, E. W. Tate, Chem. Commun. 2011, 47, 4081-4083.
[5] a) J. Y. Axup, K. M. Bajjuri, M. Ritland, B. M. Hutchins, C. H. Kim,
S. A. Kazane, R. Halder, J. S. Forsyth, A. F. Santidrian, K. Stafin, Y.
Lu, H. Tran, A. J. Seller, S. L. Biroc, A. Szydlik, J. K. Pinkstaff, F.
Tian, S. C. Sinha, B. Felding-Habermann, V. V. Smider, P. G. Schultz,
Proc. Natl. Acad. Sci. USA 2012, 109, 16101-16106; b) A. Beck, L.
Goetsch, C. Dumontet, N. Corvaia, Nat. Rev. Drug Discovery 2017, 16,
315-337; c) B. Oller-Salvia, G. Kym, J. W. Chin, Angew. Chem., Int.
Ed. 2018, 57, 2831-2834; Angew. Chem. 2018, 130, 2881–2884.
[6] C. R. Bertozzi, Acc. Chem. Res. 2011, 44, 651-653.
[7] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science 1994,
266, 776-779.
[8] B. A. Griffin, S. R. Adams, R. Y. Tsien, Science 1998, 281, 269-272.
[9] E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007-2010.
[10] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew.
Chem. Int. Ed. 2002, 41, 2596-2599; Angew. Chem. 2002, 114, 2708-
2711.
[11] a) S. T. Laughlin, J. M. Baskin, S. L. Amacher, C. R. Bertozzi, Science
2008, 320, 664-667; b) N. J. Agard, J. A. Prescher, C. R. Bertozzi, J.
Am. Chem. Soc. 2004, 126, 15046-15047.
(v/v, 1:1) at room temperature (Figure S54b). Therefore, the
experiments in Figures 5b-5d and S54 showed that the present
method could be used in release of biologically active molecules.
Finally, we attempted reactions of different substituted
chloroquinoxalines 17–19 containing C2A(S83C) with 12, and
conjugate 23 was observed in almost quantitative yields (Figure 5g)
determined by HPLC, ESI-MS and gel analysis (Figure 5h).
Therefore, the present method provides a novel strategy for
bioorthogonal ligations and cleavages, and it will be widely applied
in chemical labelling of biomolecules, purification or enrichment of
chemically modified proteins, construction of antibody drug
conjugates and other fields.
Conclusion
In summary, we have developed a novel kind of bioorthogonal
reactions of readily available chloroquinoxalines (CQ) and ortho-
dithiophenols (DT) incorporated with functional molecules such as
amino acids, biotin, fluorescein, peptides and protein, and the
[12] a) M. L. Blackman, M. Royzen, J. M. Fox, J. Am. Chem. Soc. 2008,
130, 13518-13519; b) H. Wu, N. K. Devaraj, Acc. Chem. Res. 2018, 51,
1249-1259; c) B. L. Oliveira, Z. Guo, G. J. L. Bernardes, Chem. Soc.
Rev. 2017, 46, 4895-4950.
[13] a) J. P. M. Antonio, R. Russo, C. P. Carvalho, P. M. S. D. Cal, P. M. P.
Gois, Chem. Soc. Rev. 2019, 48, 3513-3536; b) B. Akgun, D. G. Hall,
Angew. Chem. Int. Ed. 2018, 57, 13028-13044; Angew. Chem. 2018,
130, 13210-13228.
[14] a) J. Wang, Y. Liu, Y. Liu, S. Zheng, X. Wang, J. Zhao, F. Yang, G.
Zhang, C. Wang, P. R. Chen, Nature 2019, 569, 509-513; b) L. Donato,
A. Mourot, C. M. Davenport, C. Herbivo, D. Warther, J. Léonard, F.
Bolze, J.-F. Nicoud, R. H. Kramer, M. Goeldner, A. Specht, Angew.
Chem. Int. Ed. 2012, 51, 1840-1843; Angew. Chem. 2012, 124, 1876-
1879.
[15] a) T. Volker, F. Dempwolff, P. L. Graumann, E. Meggers, Angew.
Chem. Int. Ed. 2014, 53, 10536-10540; Angew. Chem. 2014, 126,
10705-10710; b) R. M. Yusop, A. Unciti-Broceta, E. M. Johansson, R.
M. Sanchez-Martin, M. Bradley, Nat. Chem. 2011, 3, 239-243; c) J. Li,
J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M. Yang, S. Jia, X.
Zhang, P. R. Chen, Nat. Chem. 2014, 6, 352-361.
[16] a) R. M. Versteegen, R. Rossin, W. ten Hoeve, H. M. Janssen, M. S.
Robillard, Angew. Chem. Int. Ed. 2013, 52, 14112-14116; Angew.
Chem. 2013, 125, 14362-14366; b) R. Rossin, R. M. Versteegen, J. Wu,
A. Khasanov, H. J. Wessels, E. J. Steenbergen, W. ten Hoeve, H. M.
Janssen, A. H. A. M. van Onzen, P. J. Hudson, M. S. Robillard, Nat.
Commun. 2018, 9, 1484. c) R. Rossin, S. M. van Duijnhoven, W. Ten
Hoeve, H. M. Janssen, L. H. Kleijn, F. J. Hoeben, R. M. Versteegen,
M. S. Robillard, Bioconjug. Chem. 2016, 27, 1697-1706; d) J. M. Mejia
Oneto, I. Khan, L. Seebald, M. Royzen, ACS Cent. Sci. 2016, 2, 476-
482.
corresponding
conjugates
containing
tetracyclic
benzo[5,6][1,4]dithiino[2,3-b]quinoxaline were formed in excellent
yields in organic solvents, PBS buffer, cell medium, or cell lysate
together with release of the other functional molecules in direct or
indirect ways. The obtained conjugates are highly stable in the cell
medium (Figure S10) and show strong fluorescence. The built-in
fluorescence avoids active alteration of biomolecules for secondary
derivatization with a fluorophore, which is very useful for probe
development and screening. The CQ-DT bioorthogonal reactions
can be used as the bioorthogonal ligations, bioorthogonal cleavages
and the trans-tagging of proteins under physiological conditions. We
believe that the CQ-DT bioorthogonal reactions with multifunctions
of bioorthogonal ligations, cleavages and built-in fluorescence
should provide a new strategy for bioorthogonal chemistry and will
find a wide range of applications.
Acknowledgements
We are grateful for the assistance of Zi Yang on the protein
expression and purification in Protein Preparation and Identification
Facility at Technology Center for Protein Science of Tsinghua
University. This research was supported by the National Natural
Science Foundation of China (Grant No. 21772108).
[17] P. P. Geurink, B. I. Florea, N. Li, M. D. Witte, J. Verasdonck, C. L.
Kuo, G. A. van der Marel, H. S. Overkleeft, Angew. Chem. Int. Ed.
2010, 49, 6802-6805; Angew. Chem. 2010, 122, 6954-6957.
[18] S. H. Verhelst, M. Fonovic, M. Bogyo, Angew. Chem. Int. Ed. 2007,
46, 1284-1286; Angew. Chem. 2007, 119, 1306-1308.
[19] C. A. Gartner, J. E. Elias, C. E. Bakalarski, S. P. Gygi, J. Proteome Res.
2007, 6, 1482-1491.
Keywords: bioorthogonal ligations • bioorthogonal cleavages •
chloroquinoxalines • ortho-dithiophenols • protein modification
[20] a) K. Qin, Y. Zhu, W. Qin, J. Gao, X. Shao, Y.-l. Wang, W. Zhou, C.
Wang, X. Chen, ACS Chem. Biol. 2018, 13, 1983-1989; b) J.
Szychowski, A. Mahdavi, J. J. Hodas, J. D. Bagert, J. T. Ngo, P.
Landgraf, D. C. Dieterich, E. M. Schuman, D. A. Tirrell, J. Am. Chem.
Soc. 2010, 132, 18351-18360.
[1] a) R. D. Row, J. A. Prescher, Acc. Chem. Res. 2018, 51, 1073-1081; b)
E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974-
6998; Angew. Chem. 2009, 121, 7108-7133; c) O. Boutureira, G. J. L.
Bernardes, Chem. Rev. 2015, 115, 2174-2195; d) J. Li, P. R. Chen, Nat.
Chem. Biol. 2016, 12, 129-137; e) G. C. Rudolf, W. Heydenreuter, S.
A. Sieber, Curr. Opin. Chem. Biol. 2013, 17, 110-117.
[2] H. C. Hang, C. Yu, D. L. Kato, C. R. Bertozzi, Proc. Natl. Acad. Sci.
USA 2003, 100, 14846-14851.
[3] a) X. Fan, J. Li, P. R. Chen, Natl. Sci. Rev. 2017, 4, 300-302; b) D. M.
Patterson, J. A. Prescher, Curr. Opin. Chem. Biol. 2015, 28, 141-149;
c) C. P. Ramil, Q. Lin, Chem. Commun. 2013, 49, 11007-11022.
[21] S. Bernard, D. Audisio, M. Riomet, S. Bregant, A. Sallustrau, L.
Plougastel, E. Decuypere, S. Gabillet, R. A. Kumar, J. Elyian, M. N.
Trinh, O. Koniev, A. Wagner, S. Kolodych, F. Taran, Angew. Chem.
Int. Ed. 2017, 56, 15612-15616; Angew. Chem. 2017, 129, 15818-
15822
[22] A. B. Pierini, M. T. Baumgartner, R. A. Rossi, J. Org. Chem. 1987, 52,
1089-1092.
[23] J. Nafe, S. Herbert, F. Auras, K. Karaghiosoff, T. Bein, P. Knochel,
Chem. Eur. J. 2015, 21, 1102-1107.
[24] E. A. Hoyt, P. M. S. D. Cal, B. L. Oliveira, G. J. L. Bernardes, Nat. Rev.
Chem. 2019, 3, 147-171.
6
This article is protected by copyright. All rights reserved.

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
[25] B. N. Giepmans, S. R. Adams, M. H. Ellisman, R. Y. Tsien, Science
2006, 312, 217-224.
[26] J. M. Chalker, G. J. Bernardes, B. G. Davis, Acc. Chem. Res. 2011, 44,
730-741.
[27] I. S. Alam, A. A. Neves, T. H. Witney, J. Boren, K. M. Brindle,
Bioconjug. Chem. 2010, 21, 884-891.
[28] U. Soomets, M. Lindgren, X. Gallet, M. Hällbrink, A. Elmquist, L.
Balaspiri, M. Zorko, M. Pooga, R. Brasseur, Ü. Langel, Biochim.
Biophys. Acta. 2000, 1467, 165-176.
[29] a) K. Neumann, S. Jain, A. Gambardella, S. E. Walker, E. Valero, A.
Lilienkampf, M. Bradley, Chembiochem 2017, 18, 91-95; b) R.
Weinstain, E. N. Savariar, C. N. Felsen, R. Y. Tsien, J. Am. Chem. Soc.
2014, 136, 874-877; c) S. Ye, J. J. Hu, D. Yang, Angew. Chem. Int. Ed.
2018, 57, 10173-10177; Angew. Chem. 2018, 130, 10330–10334.
[30] a) M. H. Lee, J. L. Sessler, J. S. Kim, Acc. Chem. Res. 2015, 48, 2935-
2946; b) N. Huguenin-Dezot, D. A. Alonzo, G. W. Heberlig, M.
Mahesh, D. P. Nguyen, M. H. Dornan, C. N. Boddy, T. M. Schmeing,
J. W. Chin, Nature 2019, 565, 112-117.
7
This article is protected by copyright. All rights reserved.

10.1002/anie.201913620
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Bioorthogonal reactions of chloroquinoxalines (CQ) with ortho-dithiophenols (DT) under the physiological conditions provided corresponding
conjugates with strong built-in fluorescence releasing functional molecules with direct or indirect manners. With this transformation, three cleavable
linkers were designed and successfully used in release of the molecules containing biotin from the protein conjugates
8
This article is protected by copyright. All rights reserved.