2 H-Azirines as Potential Bifunctional Chemical Linkers of Cysteine Residues in Bioconjugate Technology

Article abstract of DOI:10.1021/acs.orglett.0c00415

2H-Azirine-2-caboxamides have been designed to perform as a new type of bifunctional thiol linker under very mild reaction conditions. The cleavage of a C-N double bond of 2H-azirine furnishes an amino amide functional group in situ through a thiol addition and ring-opening process. It works with a broad scope of thiols and 2H-azirines in the absence of any catalysts at room temperature. Cysteine-containing peptides have also been demonstrated to work efficiently in a completely water solution.

Full text of DOI:10.1021/acs.orglett.0c00415

pubs.acs.org/OrgLett
Letter
2H‑Azirines as Potential Bifunctional Chemical Linkers of Cysteine
Residues in Bioconjugate Technology
Yang Chen, Wenjie Yang, Jiamin Wu, Wangbin Sun, Teck-Peng Loh,* and Yaojia Jiang*
Cite This: https://dx.doi.org/10.1021/acs.orglett.0c00415
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: 2H-Azirine-2-caboxamides have been designed to
perform as a new type of bifunctional thiol linker under very mild
reaction conditions. The cleavage of a C−N double bond of 2H-
azirine furnishes an amino amide functional group in situ through a
thiol addition and ring-opening process. It works with a broad
scope of thiols and 2H-azirines in the absence of any catalysts at
room temperature. Cysteine-containing peptides have also been
demonstrated to work efficiently in a completely water solution.
ntibody drug conjugates (ADCs), known as peptides and
Aproteins conjugated through chemical linkers to drugs for
use in targeted therapy, have become an increasingly growing
research area and could potentially make a fundamental change
in cancer chemotherapy.1 Among them, cysteine-based
bioconjugation methods have evolved into a powerful
biotechnological tool, allowing varied chemical linkers to
have been designed via alkylation,2 reduction,3 oxidation,4 and
conjugate addition reactions.5 Maleimide is recognized as one
of the most popular chemical linkers. It has also been applied
in FDA-approved drugs such as Adcetris and Kadcyla (Figure
1, 1.a).6 Allenamide was developed by our group in 2014 as a
cysteine linker having excellent chemoselectivity under mild
conditions (Figure 1, 1.b).7 The Buchwald group has also
demonstrated the use of an organometallic palladium complex
as a cysteine arylation reagent (Figure 1, 1.c).8
In general, free thiols do not exist in antibodies, and all
cysteine residues are bound in disulfide bonds. To deal with
this issue, several new chemical linkers have been designed to
target dithiols instead.9 This approach would optimize the
drug-to-antibody ratio (DAR) value and decrease the risk of
off-target toxicity of drugs from antibodies.10 Along these lines,
dibromomaleimide-based reagents can be attacked by two
reduced cysteines generated from interchain disulfide bonds
with the elimination of bromide residues (Figure 1, 2.a).11
Recently, Griebenow’s group developed a photomediated
disulfide rebridging method based on a Thiol−yne coupling
reaction (Figure 1, 2.b).12 Pentelute and coworkers have
evaluated perfluoaromatic molecules as effective arylation
reagents under advanced mild reaction conditions (Figure 1,
2.c).13 Despite these advances, exploring new synthetic
methods to bind two thiols into a single linker is still a
developing field, especially in terms of atom economy, high site
selectivity, and mild reaction conditions.
2H-Azirines are the smallest unsaturated aza-heterocycles
and are useful in the preparation of important N-containing
compounds through opening the small ring due to their unique
ring strain.14 Most of the research has focused on the cleavage
of the 2H-azirine C−N single bond to generate 1,3-dipolar
intermediates.15 Similarly, C−C single-bond cleavage of 2H-
azirines has also been explored in limited examples.16
However, to the best of our knowledge, there is no example
of cleaving the 2H-azirine’s C−N double bond in a single step.
Compared with previous reported disulfide bridging linkers,
2H-azirines performed as a new type of bifunctional thiol linker
with the advantages of being metal-free and atom-economical,
having high site-selectivity, and requiring mild reaction
conditions. In our continuing interest in 2H-azirine chem-
istry,17 herein we describe a bioconjugation method making
use of the thiol-2H-azirine coupling reaction through a thiol
addition followed by a ring-opening process cleavage the C−N
double bond.
We commenced our study by exploring the proposed double
addition of thiophenol 1a to the 2H-azirine 2a in an aqueous
solvent system under mild reaction conditions (Table 1). To
our delight, the dithiol product 3aa was formed when an
organic cosolvent (Table 1, entries 1−4) was used with the
aqueous phosphate buffer saline (PBS) solution. EtOH turned
out to be an optimal cosolvent. The role of the cosolvent is
likely to improve the solubility of thiophenol 1a. Meanwhile,
Received: February 5, 2020
https://dx.doi.org/10.1021/acs.orglett.0c00415
© XXXX American Chemical Society
Org. Lett. XXXX, XXX, XXX−XXX
A

Organic Letters
pubs.acs.org/OrgLett
Letter
(Table 1, entries 4−6). Further study found that the thiol
loading had an important role in the reaction. When an eight-
fold excess of thiophenol 1a was used, the yield of 3aa further
improved to 85%. Finally, temperature had only a minor
influence on the reaction in the range from 20 to 50 °C (Table
1, entries 9−11). Thus the optimal reaction conditions were
established by using PhSH 1a (1.6 mmol, 8.0 equiv) with 2H-
azirines 2a (0.2 mmol, 1.0 equiv) in EtOH and PBS (1:1 v/v)
as a cosolvent system (3 mL) at 37 °C under a nitrogen
atmosphere (1 atm) until 2a was completely consumed (∼20
h).
With the optimized reaction conditions in hand, we then
screened a series of 2H-azirines to establish a range of potential
ADC linker candidates (Scheme 1). Initially, different 2-
a b
,
Scheme 1. Substrate Scope of 2H-Azirines
Figure 1. Modification of cysteines in bioconjugation.
a
Table 1. Optimization of Reaction Conditions
b
entry
temp. (°C)
pH
1a:2a
solvent
3aa yield (%)
1
2
3
4
5
6
7
8
37
37
37
37
37
37
37
37
37
20
50
37
7.0
7.0
7.0
7.0
6.0
7.4
7.4
7.4
7.4
7.4
7.4
7.4
6:1
6:1
6:1
6:1
6:1
6:1
4:1
8:1
10:1
8:1
8:1
8:1
CH3CN
THF
H2O
40
56
22
60
45
70
50
85
79
78
65
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
9
10
11
12
a
Unless otherwise noted, reactions were carried out using PhSH 1a
(1.60 mmol, 8.0 equiv) and 2H-azirines 2 (0.2 mmol, 1.0 equiv) in
EtOH (1 mL) and PBS (1 mL) solution at 37 °C for 20 h under a
c
78
a
Unless otherwise noted, the reactions were carried out using PhSH
1a and 2H-azirines 2a (0.2 mmol) in PBS (1 mL) and organic solvent
(1 mL) at 37 °C under a nitrogen atmosphere (1 atm). Yields were
determined by NMR versus CH2Br2 as internal standard. 10 mL of
b
nitrogen atmosphere (1 atm). Isolated yields.
b
c
substituted 2H-azirines, including carboxylic acid, esters, and
amides, were investigated. In all cases, the 2H-azirines gave the
corresponding functionalized thioacetals in moderate to
excellent yield (3aa−ae). Of particular interest to bioorthog-
onal chemistry are the 2-amide-substituted 2H-azirines, which
would be expected to be soluble in aqueous media. Thus we
screened a variety of different 2-amide-2H-azirines. The aryls
PBS and 10 mL of EtOH were used.
the monothiol adducts could also be detected and charac-
terized as enamine 4aa and 2H-aziridine 5aa. To improve the
selectivity of the thioacetals, we then screened the reaction pH
and found that the yield of 3aa improved to 70% at pH 7.4
B
https://dx.doi.org/10.1021/acs.orglett.0c00415
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
with electron-donating substituents (3af−ag) worked effi-
ciently, whereas electron-withdrawing (3ah) groups failed to
react. The halogen substituents (F, Cl, Br, and I) on the para-
site of the aryl ring reacted smoothly and afforded the desired
products in 40 to 72% yield (3ai−al). The halogen-
functionalized thioacetal adducts are useful because they can
be applies in further cross-coupling reactions.18 Heterocyclic-
(3am−an) and naphthalenyl-substituted (3ao) substrates also
proceeded smoothly, albeit in fluctuant yield ranging from 50
to 83%. Cyclohexyl-substituted 2H-azirine also performed well
and gave the corresponding adduct in 51% yield (3ap). Finally,
a 2H-azirine bearing a quaternary carbon also reacted
efficiently and gave the thioacetal product in 68% yield (3aq).
Subsequently, we explored different thiols under the
standard conditions (Scheme 2). In general, the reaction
tolerated a broad array of thiols carrying useful functionalities
including halogen, NO2, NH2, and OH groups. Considering
the electronic effects, thiophenols with electron-rich groups
reacted more efficiently and gave the thioacetal adduct
products in 92% to 94% yield (3ba−ca), whereas electron-
deficient groups resulted in moderate to good yield (3da−ga).
Importantly, thiophenols containing active hydrophilic groups
such as amino and hydroxyl groups underwent the reaction
smoothly and offered the desired products with excellent
chemoselectivity (3ha−ia). No N- or O-addition products
were observed. Naphthalenyl- (3ka) and heterocyclic-sub-
stituted (3la−ma) thiols also reacted smoothly in excellent
yield. Also, alkyl dithiol reacted smoothly, albeit in slightly
decreased yield (3oa).
To further demonstrate the utility of our method in
biological chemistry, we explored the reactivity of cysteine
derivatives with 2H-azirines. Cysteine 1p reacted with the
simple 2H-azirine 2a under the standard reaction conditions to
give the expected thioacetal product in a 42% yield (Scheme
3). We also noted that the ethanol cosolvent was not
Scheme 3. Optimization of Reaction Conditions for
a
Cysteine
a b
,
Scheme 2. Substrate Scope of Thiols
a
Unless otherwise noted, the reactions were carried out using cysteine
1p (1.6 mmol, 8.0 equiv) and 2H-azirine 2 (0.2 mmol, 1.0 equiv) in
PBS (1 mL) at 37 °C for 20 h under a nitrogen atmosphere (1 atm),
b
and the yields were determined by NMR. Isolated yields.
necessary, and a 65% yield of the cysteine adduct was obtained
in just the buffer solution alone. This further underlines the
biocompatibility of our proposed linker method. Finally, we
examined different 2H-azirines with MeO, Cl, and CN
functional groups. The 4-methoxyl-2H-azirine 2g reacted
with cysteine in excellent yield (95% crude yield and up to
87% by isolation).
Having established the desired linker mode for cysteines, we
then explored different peptides containing free thiol moieties
to react with the best performing methoxy 2H-azirine 2g
(Scheme 4). Peptides with two cysteines resulted in the
formation of cyclic thioacetal adduct bridges (Scheme 4,
entries 3−6). On the contrary, peptide sequences with single
cysteine residues resulted in the linker working in a linchpin
mode, forming a dimeric thioacetal (Scheme 4, entries 1 and
2). It should be noted that in both cases, the reaction was
carried out in an aqueous solution at room temperature and
tolerated several other amino acid residues including NH2,
OH, and COOH groups.
With the obtained results in hand, we propose a possible
reaction pathway for the 2H-azirine thioacetalization (Scheme
5a). Upon thiol 1a’s initial attack of 2H-azirine 2a, two possible
products (enamide 4aa or aziridine 5aa) can be envisioned. To
test our hypothesis, we prepared compounds 4aa and 5aa
independently and allowed them to react with 1a under
standard reaction conditions (Scheme 5b). In this control
a
Unless otherwise noted, the reactions were carried out using thiol
compounds 1 (1.6 mmol, 8.0 equiv) and 2H-azirines 2a (0.2 mmol,
1.0 equiv) in EtOH (1 mL) and PBS (1 mL) solution at 37 °C for 20
b
h under a nitrogen atmosphere (1 atm). Isolated yield.
C
https://dx.doi.org/10.1021/acs.orglett.0c00415
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Scheme 4. Reactions of Peptide Sequences*
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c00415.
Experimental procedures and characterization data for
new compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Yaojia Jiang − Institute of Advanced Synthesis, School of
Chemistry and Molecular Engineering, Nanjing Tech University,
Nanjing 211816, P. R. China; orcid.org/0000-0003-0695-
0069; Email: iamyjjiang@njtech.edu.cn
Teck-Peng Loh − Institute of Advanced Synthesis, School of
Chemistry and Molecular Engineering, Nanjing Tech University,
Nanjing 211816, P. R. China; Division of Chemistry and
Biological Chemistry, School of Physical and Mathematical
Sciences, Nanyang Technological University, Singapore 637616
Singapore; orcid.org/0000-0002-2936-337X;
*
Email: teckpeng@ntu.edu.sg
Unless otherwise noted, the reactions were carried out using peptide
(1.6 mmol, 8.0 equiv) and 2H-azirine 2g (0.2 mmol, 1.0 equiv) in
Authors
PBS (1 mL) at 37 °C for 20 h under a nitrogen atmosphere (1 atm).
a
Conversion was quantitative in each case (determined by LC−MS).
Yang Chen − Institute of Advanced Synthesis, School of
Chemistry and Molecular Engineering, Nanjing Tech University,
Nanjing 211816, P. R. China
Scheme 5. Mechanism Study
Wenjie Yang − Institute of Advanced Synthesis, School of
Chemistry and Molecular Engineering, Nanjing Tech University,
Nanjing 211816, P. R. China
Jiamin Wu − Institute of Advanced Synthesis, School of
Chemistry and Molecular Engineering, Nanjing Tech University,
Nanjing 211816, P. R. China
Wangbin Sun − Institute of Advanced Synthesis, School of
Chemistry and Molecular Engineering, Nanjing Tech University,
Nanjing 211816, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c00415
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge funding from the National Natural
Science Foundation of China (21971112), Jiangsu Province
Funds Surface Project (BK 20161541), and the Starting
Funding of Research (39837107) from Nanjing Tech
University.
experiment, only aziridine 5aa reacted completely to the
desired product thioacetal 3aa in 78% yield. This result
supports the path-B pathway involving an aziridine inter-
mediate 5aa.
REFERENCES
■
In summary, we have demonstrated the use of 2H-azirine-2-
caboxamides as a new type of biocompatible bifunctional thiol
linker through a thiol addition and ring-opening cascade
process. The linking reaction works with a broad substrate
scope under very mild reaction conditions without any
catalysts with excellent chemoselectivity. The method also
tolerates the presence of other nucleophilic functional groups,
such as NH2 and OH. Cysteine-containing peptides have also
been demonstrated to work efficiently in aqueous solutions.
The developed method has potential for use in bifunctional
chemical linkers for constructing antibody−drug conjugates.
(1) (a) Hughes, B. Antibody−Drug Conjugates for Cancer: Poised
to Deliver? Nat. Rev. Drug Discovery 2010, 9, 665−667. (b) Teicher,
B. A.; Chari, R. V. J. Antibody Conjugate Therapeutics: Challenges
and Potential. Clin. Cancer Res. 2011, 17, 6389−6397. (c) Scott, A.
M.; Wolchok, J. D.; Old, L. J. Antibody Therapy of Cancer. Nat. Rev.
Cancer 2012, 12, 278−287. (d) Perez, H. L.; Cardarelli, P. M.;
Deshpande, S.; Gangwar, S.; Schroeder, G. M.; Vite, G. D.; Borzilleri,
R. M. Antibody−Drug Conjugates: Current Status and Future
Directions. Drug Discovery Today 2014, 19, 869−881. (e) Diamantis,
N.; Banerji, U. Antibody-Drug ConjugatesAn Emerging Class of
Cancer Treatment. Br. J. Cancer 2016, 114, 362−367. (f) Yu, C.;
Tang, J.; Loredo, A.; Chen, Y.; Jung, S. Y.; Jain, A.; Gordon, A.; Xiao,
H. Proximity-Induced Site-Specific Antibody Conjugation. Bioconju-
gate Chem. 2018, 29, 3522−3526. (g) Li, Z.-Z.; Czechowicz, A.;
D
https://dx.doi.org/10.1021/acs.orglett.0c00415
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
Scheck, A.; Rossi, D. J.; Murphy, P. M. Hematopoietic Chimerism and
Donor-Specific Skin Allograft Tolerance after Non-Genotoxic CD117
Antibody-Drug-Conjugate Conditioning in MHC-Mismatched Allo-
transplantation. Nat. Commun. 2019, 10, 616−622. (h) Cohen, D. T.;
Zhang, C.; Fadzen, C. M.; Mijalis, A. J.; Hie, L.; Johnson, K. D.;
Shriver, Z.; Plante, O.; Miller, S. J.; Buchwald, S. L.; Pentelute, B. L. A
Chemoselective Strategy for Late-Stage Functionalization of Complex
Small Molecules with Polypeptides and Proteins. Nat. Chem. 2019,
11, 78−85.
Cysteine Based Corrosion Inhibitor Formulations for Steel Protection
in 15% HCl Solution. J. Mol. Liq. 2017, 246, 112−118.
(6) (a) Gregory, J. D. The stability of N-Ethylmaleimide and Its
Reaction with Sulfhydryl Groups. J. Am. Chem. Soc. 1955, 77, 3922−
3923. (b) Chalker, J. M.; Bernardes, G. J. L.; Lin, Y. A.; Davis, B. G.
Chemical Modification of Proteins at Cysteine: Opportunities in
Chemistry and Biology. Chem. - Asian J. 2009, 4, 630−640. (c) Webb,
S. Back on Target. Nat. Biotechnol. 2013, 31, 191−193. (d) Lutzke, A.;
Neufeld, B. H.; Neufeld, M. J.; Reynolds, M. M. Nitric Oxide Release
from A Biodegradable Cysteine-Based Polyphosphazene. J. Mater.
Chem. B 2016, 4, 1987−1998. (e) Shen, B.-Q.; Xu, K.-Y.; Liu, L.;
Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.;
Yu, S.-F.; Mai, E.; et al. Conjugation Site Modulates the in vivo
Stability and Therapeutic Activity of Antibody-Drug Conjugates. Nat.
(2) (a) Jiang, Y.; Park, C.-M.; Loh, T.-P. Transition-Metal-Free
Synthesis of Substituted Pyridines via Ring Expansion of 2-Allyl-2H-
Azirines. Org. Lett. 2014, 16, 3432−3435. (b) Sun, M. M. C.; Beam,
K. S.; Cerveny, C. G.; Hamblett, K. J.; Blackmore, R. S.; Torgov, M.
Y.; Handley, F. G. M.; Ihle, N. C.; Senter, P. D.; Alley, S. C.
Reduction−Alkylation Strategies for the Modification of Specific
Monoclonal Antibody Disulfides. Bioconjugate Chem. 2005, 16, 1282−
1290. (c) Fairlie, D. P.; Dantas de Araujo, A. Stapling Peptides Using
Cysteine Crosslinking. Biopolymers 2016, 106, 843−852. (d) Lee, B.;
̈
Biotechnol. 2012, 30, 184−189. (f) Henkel, M.; Rockendorf, N.; Frey,
A. Selective and Efficient Cysteine Conjugation by Maleimides in the
Presence of Phosphine Reductants. Bioconjugate Chem. 2016, 27,
2260−2265.
(7) Abbas, A.; Xing, B.-G.; Loh, T.-P. Allenamides as Orthogonal
Handles for Selective Modification of Cysteine in Peptides and
Proteins. Angew. Chem., Int. Ed. 2014, 53, 7491−7494.
́
Sun, S.; Jimenez-Moreno, E.; Neves, A. A.; Bernardes, G. J. L. Site-
Selective Installation of An Electrophilic Handle on Proteins for
Bioconjugation. Bioorg. Med. Chem. 2018, 26, 3060−3064.
(8) (a) Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute,
B. L.; Buchwald, S. L. Organometallic Palladium Reagents for
Cysteine Bioconjugation. Nature 2015, 526, 687−691. (b) Rojas, A.
J.; Zhang, C.; Vinogradova, E. V.; Buchwald, N. H.; Reilly, J.;
Pentelute, B. L.; Buchwald, S. L. Divergent Unprotected Peptide
Macrocyclisation by Palladium-Mediated Cysteine Arylation. Chem.
Sci. 2017, 8, 4257−4263. (c) Kubota, K.; Dai, P.; Pentelute, B. L.;
Buchwald, S. L. Palladium Oxidative Addition Complexes for Peptide
and Protein Cross-Linking. J. Am. Chem. Soc. 2018, 140, 3128−3133.
(9) (a) Zhang, Z.; Burns, D. C.; Kumita, J. R.; Smart, O. S.; Woolley,
G. A. A Water-Soluble Azobenzene Cross-Linker for Photocontrol of
Peptide Conformation. Bioconjugate Chem. 2003, 14, 824−829.
(b) Lotti, L.; Coiai, S.; Ciardelli, F.; Galimberti, M.; Passaglia, E.
Thiol-ene Radical Addition of L-Cysteine Derivatives to Low
Molecular Weight Polybutadiene. Macromol. Chem. Phys. 2009, 210,
1471−1483. (c) Jafari, M. R.; Lakusta, J.; Lundgren, R. J.; Derda, R.
Allene Functionalized Azobenzene Linker Enables Rapid and Light-
responsive Peptide Macrocyclization. Bioconjugate Chem. 2016, 27,
509−514. (d) Wang, T.; Riegger, A.; Lamla, M.; Wiese, S.; Oeckl, P.;
Otto, M.; Wu, Y.; Fischer, S.; Barth, H.; Kuan, S. L.; Weil, T. Water-
Soluble Allyl Sulfones for Dual Site-Specific Labelling of Proteins and
Cyclic Peptides. Chem. Sci. 2016, 7, 3234−3239.
(10) (a) Xu, K.-Y.; Liu, L.; Dere, R.; Mai, E.; Erickson, R.;
Hendricks, A.; Lin, K.; Junutula, J. R.; Kaur, S. Characterization of the
Drug-to-Antibody Ratio Distribution for Antibody−Drug Conjugates
in Plasma/Serum. Bioanalysis 2013, 5, 1057−1071. (b) Badescu, G.;
Bryant, P.; Bird, M.; Henseleit, K.; Swierkosz, J.; Parekh, V.;
Tommasi, R.; Pawlisz, E.; Jurlewicz, K.; Farys, M.; Camper, N.;
Sheng, X.-B.; Fisher, M.; Grygorash, R.; Kyle, A.; Abhilash, A.;
Frigerio, M.; Edwards, J.; Godwin, A. Bridging Disulfides for Stable
and Defined Antibody Drug Conjugates. Bioconjugate Chem. 2014, 25,
1124−1136. (c) Tsuchikama, K.; An, Z. Antibody-Drug Conjugates:
Recent Advances in Conjugation and Linker Chemistries. Protein Cell
2018, 9, 33−46.
(11) (a) Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L.
M.; Papaioannou, D.; Waksman, G.; Caddick, S.; Baker, J. R. Protein
Modification, Bioconjugation, and Disulfide Bridging Using Bromo-
maleimides. J. Am. Chem. Soc. 2010, 132, 1960−1965. (b) Jones, M.
W.; Strickland, R. A.; Schumacher, F. F.; Caddick, S.; Baker, J. R.;
Gibson, M. I.; Haddleton, D. M. Polymeric Dibromomaleimides as
Extremely Efficient Disulfide Bridging Bioconjugation and Pegylation
Agents. J. Am. Chem. Soc. 2012, 134, 1847−1852. (c) Robin, M. P.;
O’Reilly, R. K. Fluorescent and Chemico-Fluorescent Responsive
Polymers from Dithiomaleimide and Dibromomaleimide Functional
Monomers. Chem. Sci. 2014, 5, 2717−2723. (d) Grison, C. M.;
Burslem, G. M.; Miles, J. A.; Pilsl, L. K. A.; Yeo, D. J.; Imani, Z.;
Warriner, S. L. M.; Webb, E.; Wilson, A. J. Double Quick, Double
Click Reversible Peptide “Stapling. Chem. Sci. 2017, 8, 5166−5171.
(3) (a) Zimmerman, E. S.; Heibeck, T. H.; Gill, A.; Li, X.; Murray,
C. J.; Madlansacay, M. R.; Tran, C.; Uter, N. T.; Yin, G.; Rivers, P. J.;
Yam, A. Y.; Wang, W. D.; Steiner, A. R.; Bajad, S. U.; Penta, K.; Yang,
W.; Hallam, T. J.; Thanos, C. D.; Sato, A. K. Production of Site-
Specific Antibody−Drug Conjugates Using Optimized Non-Natural
Amino Acids in A Cell-Free Expression System. Bioconjugate Chem.
2014, 25, 351−361. (b) Shinmi, D.; Taguchi, E.; Iwano, J.;
Yamaguchi, T.; Masuda, K.; Enokizono, J.; Shiraishi, Y. One-step
Conjugation Method for Site-Specific Antibody−Drug Conjugates
through Reactive Cysteine-Engineered Antibodies. Bioconjugate Chem.
2016, 27, 1324−1331. (c) Guo, J.-X.; Kumar, S.; Chipley, M.; Marcq,
O.; Gupta, D.; Jin, Z.-W.; Tomar, D. S.; Swabowski, C.; Smith, J.;
Starkey, J. A.; Singh, S. K. Characterization and Higher-Order
Structure Assessment of An Interchain Cysteine-Based ADC: Impact
of Drug Loading and Distribution on the Mechanism of Aggregation.
Bioconjugate Chem. 2016, 27, 604−615. (d) Thompson, P.; Fleming,
R.; Bezabeh, B.; Huang, F.-Y.; Mao, S.-L.; Chen, C.; Harper, J.;
Zhong, H.-H.; Gao, X.-Z.; Yu, X.-Q.; Hinrichs, M. J.; Reed, M.;
Kamal, A.; Strout, P.; Cho, S.; Woods, R.; Hollingsworth, R. E.; Dixit,
R.; Wu, H.; Gao, C.-S.; Dimasi, N. Rational Design, Biophysical and
Biological Characterization of Site-Specific Antibody-Tubulysin
Conjugates with Improved Stability, Efficacy and Pharmacokinetics.
J. Controlled Release 2016, 236, 100−116.
(4) (a) Lin, S.; Yang, X.; Jia, S.; Weeks, A. M.; Hornsby, M.; Lee, P.
S.; Nichiporuk, R. V.; Iavarone, A. T.; Wells, J. A.; Toste, F. D.;
Chang, C. J. Redox-Based Reagents for Chemoselective Methionine
̈
Bioconjugation. Science 2017, 355, 597−602. (b) Bar, R. M.;
̈
Kirschner, S.; Nieger, M.; Brase, S. Alkyl and Aryl Thiol Addition
to [1.1.1]Propellane: Scope and Limitations of A Fast Conjugation
Reaction. Chem. - Eur. J. 2018, 24, 1373−1382. (c) Tossounian, M.-
A.; Wahni, K.; Van Molle, I.; Vertommen, D.; Astolfi Rosado, L.;
Messens, J. Redox-Regulated Methionine Oxidation of Arabidopsis
Thaliana Glutathione Transferase Phi9 Induces H-Site Flexibility.
Protein Sci. 2018, 28, 56−67. (d) Wang, Y.; Yang, J.; Yi, J. Redox
Sensing by Proteins: Oxidative Modifications on Cysteines and the
Consequent Events. Antioxid. Redox Signaling 2012, 16, 649−657.
(5) (a) Yu, J.; Yang, X.; Sun, Y.; Yin, Z. Highly Reactive and
Tracelessly Cleavable Cysteine-Specific Modification of Proteins via
4-Substituted Cyclopentenone. Angew. Chem., Int. Ed. 2018, 57,
11598−11602. (b) Kalia, D.; Malekar, P. V.; Parthasarathy, M.
Exocyclic Olefinic Maleimides: Synthesis and Application for Stable
and Thiol-Selective Bioconjugation. Angew. Chem., Int. Ed. 2016, 55,
1432−1435. (c) Smith, N. J.; Rohlfing, K.; Sawicki, L. A.; Kharkar, P.
M.; Boyd, S. J.; Kloxin, A. M.; Fox, J. M. Fast, Irreversible
Modification of Cysteines through Strain Releasing Conjugate
Additions of Cyclopropenyl Ketones. Org. Biomol. Chem. 2018, 16,
2164−2169. (d) Ituen, E. B.; Akaranta, O.; Umoren, S. A. N-Acetyl
E
https://dx.doi.org/10.1021/acs.orglett.0c00415
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
(12) Griebenow, N.; Dilmac, A. M.; Greven, S.; Brase, S. Site-
Specific Conjugation of Peptides and Proteins via Rebridging of
Disulfide Bonds Using the Thiol-Yne Coupling Reaction. Bioconjugate
Chem. 2016, 27, 911−917.
(13) Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.-T.; Lin, Y.-S.;
Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach
to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135, 5946−
5949.
(14) (a) Okamoto, K.; Shimbayashi, T.; Yoshida, M.; Nanya, A.;
Ohe, K. Synthesis of 2H-Azirines by Iridium-Catalyzed Decarbox-
ylative Ring Contraction of Isoxazol-5(4H)-ones. Angew. Chem., Int.
Ed. 2016, 55, 7199−7202. (b) Rieckhoff, S.; Titze, M.; Frey, W.;
Peters, R. Ruthenium-Catalyzed Synthesis of 2H-Azirines from
Isoxazolinones. Org. Lett. 2017, 19, 4436−4440. (c) Okamoto, K.;
Nanya, A.; Eguchi, A.; Ohe, K. Asymmetric Synthesis of 2H-Azirines
with a Tetrasubstituted Stereocenter by Enantioselective Ring
Contraction of Isoxazoles. Angew. Chem., Int. Ed. 2018, 57, 1039−
1043. (d) Qi, X.; Jiang, Y.; Park, C.-M. Divergent Reactivity of α-
Oximino Carbenoids: Facile Access to 2-Isoxazolines and 2H-
Azirines. Chem. Commun. 2011, 47, 7848−7850. (e) Jiang, Y.; Park,
C.-M. A Catalyst-Controlled Selective Synthesis of Pyridines and
Pyrroles. Chem. Sci. 2014, 5, 2347−2351. (f) Jiang, Y.; Park, C.-M.;
Loh, T.-P. Transition-Metal-Free Synthesis of Substituted Pyridines
via Ring Expansion of 2-Allyl-2H-azirines. Org. Lett. 2014, 16, 3432−
3435.
(15) (a) Okamoto, K.; Watanabe, M.; Mashida, A.; Miki, K.; Ohe, K.
Rhodium-Catalyzed Reaction of 2H-Azirines with Carbonyl-ene-yne
Compounds Giving 1-Furyl-2-aza-1,3-dienes. Synlett 2013, 24, 1541−
1544. (b) Xuan, J.; Xia, X.-D.; Zeng, T.-T.; Feng, Z.-J.; Chen, J.-R.;
Lu, L.-Q.; Xiao, W.-J. Visible-Light-Induced Formal [3 + 2]
Cycloaddition for Pyrrole Synthesis under Metal-Free Conditions.
Angew. Chem., Int. Ed. 2014, 53, 5653−5656.
(16) (a) Ding, H.-L.; Wang, Z.-B.; Bai, S.-L.; Lu, P.; Wang, Y.-G. Rh-
Catalyzed Conversion of 3-Diazoindolin-2-imines to 5H-Pyrazino-
[2,3-b]indoles with Photoluminescent Properties. Org. Lett. 2017, 19,
6514−6517. (b) Novikov, M. S.; Rostovskii, N. V.; Koronatov, A. N.;
Zavyalov, K. V.; Zubakin, G. V.; Khlebnikov, A. F.; Starova, G. L.
Synthesis of 1,2-Dihydropyrimidine-2-carboxylates via Regioselective
Addition of Rhodium(II) Carbenoids to 2H-Azirine-2-carbaldimines.
J. Org. Chem. 2017, 82, 13396−13404.
(17) Ge, Y.; Sun, W.; Pei, B.; Ding, J.; Jiang, Y.; Loh, T.-P. Hoveyda-
Grubbs II Catalyst: A Useful Catalyst for One-Pot Visible-Light-
Promoted Ring Contraction and Olefin Metathesis Reactions. Org.
Lett. 2018, 20, 2774−2777.
(18) (a) Suryakiran, N.; Prabhakar, P.; Srikanth Reddy, T.; Chinni
Mahesh, K.; Rajesh, K.; Venkateswarlu, Y. Chemoselective Mono
Halogenation of β-Keto-Sulfones Using Potassium Halide and
Hydrogen Peroxide; Synthesis of Halomethyl Sulfones and
Dihalomethyl Sulfones. Tetrahedron Lett. 2007, 48, 877−881.
(b) Powers, L. J.; Fogt, S. W.; Ariyan, Z. S.; Rippin, D. J.; Heilman,
R. D.; Matthews, R. J. Effect of Structural Change on Acute Toxicity
and Antiinflammatory Activity in A Series of Imidazothiazoles and
Thiazolobenzimidazoles. J. Med. Chem. 1981, 24, 604−609.
F
https://dx.doi.org/10.1021/acs.orglett.0c00415
Org. Lett. XXXX, XXX, XXX−XXX