Concise synthesis of functionalized cyclobutene analogues for bioorthogonal tetrazine ligation

Article abstract of DOI:10.3390/molecules26020276

Novel bioorthogonal tools enable the development of new biomedical applications. Here we report the concise synthesis of a series of aryl-functionalized cyclobutene analogues using com-mercially available starting materials. Our study demonstrates that cyclobutene acts as a small, strained dienophile to generate stable substrates suitable for bioorthogonal tetrazine ligation.

Full text of DOI:10.3390/molecules26020276

molecules
Article
Concise Synthesis of Functionalized Cyclobutene Analogues
for Bioorthogonal Tetrazine Ligation
Jiayu Sun 1,†, Jie Li 1,†, Hongbao Sun 1,†, Chunling Li 2,3,* and Haoxing Wu 1,
*
1
Department of Radiology, West China Hospital, Sichuan University, Guo Xue Xiang 37,
Chengdu 610041, China; sjy080512@163.com (J.S.); lj_jievip@163.com (J.L.); hbsun07@sina.com (H.S.)
Department of Neurosurgery, Sichuan Provincial People’s Hospital,
2
University of Electronic Science and Technology of China, Chengdu 610072, China
Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu 610072, China
Correspondence: Lcl1206778081@163.com (C.L.); haoxingwu@scu.edu.cn (H.W.);
Tel.: +86-028-65261615 (H.W.)
3
*
†
These authors contributed equally to this work.
Abstract: Novel bioorthogonal tools enable the development of new biomedical applications. Here
we report the concise synthesis of a series of aryl-functionalized cyclobutene analogues using com-
mercially available starting materials. Our study demonstrates that cyclobutene acts as a small,
strained dienophile to generate stable substrates suitable for bioorthogonal tetrazine ligation.
Keywords: functionalized cyclobutene analogues; concise synthesis; tetrazine ligation; bioorthogo-
nal chemistry
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
1. Introduction
Citation: Sun, J.; Li, J.; Sun, H.; Li, C.;
Wu, H. Concise Synthesis of
Tetrazine has attracted increasing attention since the emergence of bioorthogonal
chemistry [
bioorthogonal reaction between tetrazine and dienophiles, an inverse electron demand
Diels–Alder (IEDDA) reaction [ ]. A wide range of dienophiles, each with different
advantages and limitations, have been developed as IEDDA substrates for various research
purposes. In particular, highly strained trans-cyclooctenes (TCO) can react with tetrazine
very rapidly, enabling a low concentration reaction in vivo in a short time [9–11]. The
reaction of allyl-substituted TCO with tetrazine leads to a cascade elimination that has
1–4]. Recent clinical trials have highlighted the biomedical usefulness of the
Functionalized Cyclobutene
Analogues for Bioorthogonal Tetrazine
Ligation. Molecules 2021, 26, 276.
https://doi.org/molecules26020276
5–8
Academic Editors: Pierre Audebert,
Jean-Cyrille Hierso and
Giuseppe Cirillo
been widely used in the design of prodrugs [12
with tetrazine to afford pyridazine as a single product, which can facilitate downstream
fluorogenic probe design [16 18]. Cyclopropene units have also emerged as alternative,
more compact tags that can react quickly with tetrazine and have proven useful in studies
of metabolic engineering [19 22]. The strained dienophiles norbornenes and norborna-
–15]. Bicyclooctynes (BCN) react rapidly
Received: 19 December 2020
Accepted: 5 January 2021
Published: 8 January 2021
–
–
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
dienes, although they react less quickly with tetrazine, serve as a handle for covalent
biomolecule labeling or offer a click-to-release feature that can be exploited for template-
promoted turnover amplification, allowing the detection of endogenous oncogenic mi-
croRNAs [23–27]. In addition, the vinyl ether group is small and easy to prepare and
can be decorated onto polymers and fluorophores under mild conditions [28–32]. There-
fore, the development of novel dienophiles with different features is expected to provide
straightforward access to new applications.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Cyclobutenes, as small and strained dienophiles, may be good mini-tags for labeling
proteins, since their compactness makes it unlikely that they will disturb the proteins’
physiological functions. In addition, cyclobutene is less volatile than cyclopropene, which
makes the synthesis of mini-tags easier. Despite the advantages of cyclobutene, only the
synthesis and bioorthogonal properties of alkyl-substituted cyclobutene analogues have
been investigated so far [33].
Molecules 2021, 26, 276. https://doi.org/10.3390/molecules26020276
https://www.mdpi.com/journal/molecules

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In this study, we report the concise synthesis of a series of aryl-substituted cyclobutene
derivatives from commercially available 1,3-cyclobutanediol derivatives. We also system-
atically investigated the stability of the derivatives and their kinetics in bioorthogonal
reactions.
2. Results and Discussion
We used 1,3-cyclobutanediol (1) as the starting material for the synthesis of the desired
cyclobutene derivatives, and we assumed that the two hydroxyl groups would be directly
converted into a double bond or functional groups upon reaction under appropriate condi-
tions. Treating 1,3-cyclobutanediol with p-toluenesulfonyl chloride (TsCl) and triethylamine
(TEA) yielded the mono-toluene-4-sulfonate ester
purification, the sulfonate ester reacted with a series of p-substituted thiophenols (3a
forming the corresponding thioethers 4a 4d in up to 86% yield. Subsequent tosylation
and elimination generated four novel cyclobutene derivatives (6a 6d) bearing a phenyl
2
in moderate yield. After flash column
–3d),
–
–
sulfide moiety useful for further functionalization (Scheme 1A). Phenolic cyclobutene
derivatives were prepared by slightly modifying the above mentioned steps and by using
the commercially available 3-(benzyloxy)cyclobutyl-4-methylbenzenesulfonate (7). We
followed a three-step process (Scheme 1B) to prepare two cyclobutene analogues (11a and
11b) in overall yield of 22–23.5%. None of the intermediates or final products were volatile,
and all were stored for months at −20 ◦C.
Scheme 1. Synthesis of the cyclobutene derivatives. (
A) Synthesis of phenylthio cyclobutene deriva-
tives 6a 6d; ( ) Synthesis of phenolic cyclobutene derivatives 11a and 11b. (i) TsCl, Et3N, DCM, r.t.,
–
B
9 h; (ii) tBuOK, tBuOH, 80 ◦C, 7 h, 32–86%; (iii) TsCl, Et3N, DCM, r.t., overnight, 55–79%; (iv) TFA,
◦
DCM, r.t., 1 h; (v) tBuOK, DMSO, r.t., 1 h, 46–89%; (vi) Cs2CO3, DMF, 80 C, 8 h, 48–70%; (vii) Pd/C,
EtOH, H2, r.t., overnight; (viii) Et3N, TsCl, DCM, r.t., 20 h, 70–86% for two steps; (ix) tBuOK, DMSO,
r.t., 1 h, 45–57%. TsCl: 4-tosyl chloride; TFA: trifluoroacetic acid.
The synthesized cyclobutene derivatives were then incubated in phosphate-buffered
saline (PBS) with 50% N,N-dimethylformamide (DMF) as co-solvent at 37 ◦C, and their
stability was monitored over time using liquid chromatography–mass spectrometry. All
cyclobutenes were relatively stable under these buffer conditions, irrespective of the phenyl
substituent. After incubation for 12 h, 90% of the original analogues were still present,
except for compound 11a (87%) (Figure 1A). There was slight degradation for all ana-
logues, with 79.4–92.7% of the original material remaining at 24 h. These aryl-substituted
cyclobutenes are slightly less stable than some other known dienophiles, such as TCO [
cyclopropene units [19 34], and norbornene derivatives [23 26]. However, they are facilely
prepared, so they could be a choice in the toolbox for preliminary studies.
9],
,
,

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Figure 1.
(
A
) Stability of the cyclobutene derivatives 6a
–
6d
,
11a
,
and 11b during incubation in N,N-
◦
dimethylformamide/phosphate-buffered saline (1:1) at 37 C for 24 h. Absorption was monitored using high-performance
liquid chromatography. (
and 6a–6d, 11a, and 11b. Conditions: 0.5 mM tetrazine, 5 mM dienophile, DMF/H2O (1:1), 37 C.
B
) Kinetics of the inverse electron demand Diels–Alder (IEDDA) reactions between tetrazine 12
◦
Next, we reacted the six cyclobutene analogues 6a–6d, 11a, and 11b with di-pyridyl-
tetrazine under pseudo-first-order conditions (Figure 1B). The reactants were dissolved in
50% DMF in PBS, and the reaction at 37 ◦C was monitored using UV–vis spectroscopy to
track the disappearance of the characteristic absorption peak of tetrazine at 520 nm over
time. The IEDDA reaction between the cyclobutenes and tetrazine proceeded with rate
constants of 0.04–0.15 M−1 −1, similar to those of N-acylazetines with tetrazine [35
s ,36],
and faster than those of terminal alkenes with tetrazine [37]. Electron-donating substituents
on the phenyl ring of the cyclobutene analogues improved their reactivity, while a benzoic
acid group slightly reduced the reactivity of the double bond. In order to examine the
structure of the IEDDA product, the dihydropyridazine intermediate between 6a and 12
was further oxidized using 1.1 equivalent of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ), affording a single pyridazine product 14 as the major product in 82% overall yield.
The structure of 14 was confirmed by nuclear magnetic resonance (NMR) spectroscopy
(Figure 2) which indicated that almost no ring-opening or sideproduct formation had
occurred in the two-step reactions.
Figure 2.
(A) IEDDA-oxidation cascade reaction of cyclobutene 6a with dipyridinetetrazine 12.
(B) The 1H–NMR spectrum of compound 14.

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To examine the applicability of our cyclobutenes for biomolecular labeling, we first
synthesized a cyclobutene–N-hydroxysuccinimide (NHS) amide (17) using the cyclobutene
derivative 6d via a two-step process (Scheme 2). As a proof of concept, the cyclobutene–
NHS conjugate 17 was added to bovine serum albumin (BSA) to create a cyclobutene
“handle” for bioorthogonal labeling. The modified BSA was incubated with a tetrazine-Cy5
near-infrared probe (19, H-Tz-Cy5) for 4 h. Subsequent analysis with sodium dodecyl
sulfate–polyacrylamide gel electrophoresis showed that the cyclobutene-modified BSA was
fluorescent, but the control reactions lacking the conjugate 17 or the Cy5 probe were not
(Figure 3). A rough estimation of the yield of the labeling reaction on the model BSA protein
was calculated to be about 29.1%. These results suggest that cyclobutene derivatives can be
used to label proteins via tetrazine bioorthogonal reactions.
S
O
O
O
O
S
DSC, Et3N, THF
r.t., 4 h, 51%
Et3N, DCM
+
N
H
COOH
r.t., 3 h, 75%
NH2
3
6d
15
16
N
N
Cy5
N
N
O
O
O
S
19, H-Tz-Cy5
NH
O
N
3
PBS:DMSO = 9:1
pH = 8.2, r.t., 2 h
N2
PBS:DMSO = 1:1
37 , 4 h
O
°C
17
18
NH
H
N
NH
O
Cy5 =
Cy5
Me
N
N
Me
Me
Me
Me
20
Scheme 2. In vitro cyclobutene modification and bioorthogonal labeling of protein bovine serum
albumin (BSA).
Figure 3. In vitro labeling of bovine serum albumin (BSA) via cyclobutene–tetrazine bioorthogonal reaction. Upper panel:
fluorescent image of the same gel before Coomassie blue staining. Lower panel: Coomassie-blue-stained gel of BSA modified
through IEDDA reaction between conjugate 17 and the Cy5-containing fluorescent probe 19 (250
µM or 500 µM). Control
reactions contained only BSA or BSA with only the Cy5 probe (250 µM or 500 µM). “M”: protein marker.

Molecules 2021, 26, 276
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3. Experimental Section
3.1. Materials
All chemical reagents were purchased from commercial sources (Adamas-Beta, Shang-
hai, China; Energy Chemical, Shanghai, China; or Sigma-Aldrich, St. Louis, MO, USA)
and used without further purification. The silica gel plates were purchased from Yantai
Jiangyou Silica Gel Development (silica 200
×
200 mm, pH 6.2–6.8, glass-backed). All
reactions were monitored based on absorbance at 254 nm and liquid chromatography–mass
spectrometry (Agilent-Technologies Infinity Lab LC/MSD system, Agilent Technologies
Inc., Santa Clara, CA, USA). Products were purified by flash silica gel column chromatogra-
phy (SepaBeanTM, Santai Technologies, Changzhou, Jiangsu, China). H– and 13C–NMR
1
spectra were recorded on a Bruker (Billerica, MA, USA) NMR spectrometer (1H, 400 MHz;
13C, 101 MHz) and high-resolution mass spectrometry (Bruker, Billerica, MA, USA) was
performed on a Bruker Daltonics Data analysis 3.4 system. Reaction kinetics were de-
termined on a Quawell Scientific Q6000+ microvolume spectrophotometer. Protein gels
were analyzed for Cy5 fluorescence using the ChemiDoc™ MP imaging system (Bio-Rad
Laboratories Inc., Hercules, CA, USA).
3.2. Synthesis and Characterization
3.2.1. Synthesis of 3-Hydroxycyclobutyl 4-Methylbenzenesulfonate (2)
A solution of tosyl chloride (TsCl, 22 mg, 0.12 mmol, 0.7 equiv) in dichloromethane
(DCM, 0.5 mL) was added dropwise with stirring to a mixture of 1,3-cyclobutanediol (15 mg,
◦
0.17 mmol, 1.0 equiv) and TEA (47
µ
L, 0.34 mmol, 2.0 equiv) in DCM (0.3 mL) at 0 C under
Ar. The reaction mixture was stirred at room temperature for 9 h, followed by evapora-
tion in vacuo. The obtained residue was purified by silica gel column chromatography
(PE/EA = 2:1) to afford the desired analogue 2 in 38% yield as a light yellow oil.
3.2.2. Synthesis of Compounds 4a–4d
To synthesize 3-(p-tolylthio)cyclobutanol (4a), potassium tert-butoxide (265 mg, 2.35 mmol,
1.3 equiv) was added under intense stirring to a solution of 3-hydroxycyclobutyl-4-methyl
benzenesulfonate (439 mg, 1.81 mmol, 1.0 equiv) and 4-methylbenzenethiol (270 mg, 2.18 mmol,
◦
1.2 equiv) in tert-butanol (8 mL) at room temperature under Ar. The mixture was stirred at 80 C
for 7 h. After completion of the reaction monitored by TLC, the crude mixture was quenched
with water and extracted with ethyl acetate. The combined organic phases were washed with
brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue was purified by
silica gel column chromatography (PE/EA = 3:1) to afford 4a (186 mg, 86%) as a yellow solid.
1H–NMR (400 MHz, CDCl3)
δ
7.16 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 4.63–4.56 (m,
1H), 3.85–3.79 (m, 1H), 2.46–2.34 (m, 4H), 2.32 (s, 3H), 1.99 (s, 1H). 13C–NMR (101 MHz, CDCl3)
136.2, 132.9, 129.8, 129.7, 66.3, 39.6, 33.9, 21.1. HRMS [M+H]+ m/z calcd. for [C11H15OS]+
δ
195.0838, found 195.0837.
To synthesize 3-((4-methoxyphenyl)thio)cyclobutanol (4b), potassium tert-butoxide
(105 mg, 0.94 mmol, 1.3 equiv) was added under intense stirring to a solution of 3-
hydroxycyclobutyl-4-methylbenzenesulfonate (170.3 mg, 0.72 mmol, 1.0 equiv) and 4-
methoxybenzenethiol (121 mg, 0.86 mmol, 1.2 equiv) in tert-butanol (4 mL) at room temper-
ature under Ar. The mixture was stirred at 80 ◦C for 7 h. After completion of the reaction
monitored by TLC, the crude mixture was quenched with water and extracted with ethyl
acetate. The combined organic phases were washed with brine, dried over anhydrous
Na2SO4, and evaporated in vacuo. The residue was purified by silica gel column chro-
1
matography (PE/EA = 20:1) to afford 4b (81 mg, 77%) as a white solid. H–NMR (400 MHz,
CDCl3)
δ 7.31–7.26 (m, 2H), 6.87–6.81 (m, 2H), 4.58–4.52 (m, 1H), 3.79 (s, 3H), 3.77–3.69 (m,
1H), 2.34 (t, J = 6.4 Hz, 4H), 1.90 (s, 1H). 13C–NMR (101 MHz, CDCl3) δ 159.1, 133.2, 126.4,
114.7, 66.2, 55.4, 39.5, 35.2.
To synthesize methyl 4-((3-hydroxycyclobutyl)thio)benzoate (4c), potassium tert-
butoxide (304 mg, 2.70 mmol, 1.3 equiv) was added under intense stirring to a solution
of 3-hydroxycyclobutyl-4-methylbenzenesulfonate (504 mg, 2.08 mmol, 1.0 equiv) and

Molecules 2021, 26, 276
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methyl 4-mercaptobenzoate (420 mg, 2.50 mmol, 1.2 equiv) in tert-butanol (8 mL) at room
temperature under Ar. The mixture was stirred at 80 ◦C for 7 h. After completion of the
reaction monitored by TLC, the crude mixture was quenched with water and extracted
with ethyl acetate. The combined organic phases were washed with brine, dried over anhy-
drous Na2SO4, and evaporated in vacuo. The residue was purified by silica gel column
1
chromatography (PE/EA = 2:1) to afford 4c (200 mg, 48%) as a white solid. H–NMR (400
MHz, CDCl3)
δ 7.91 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H), 4.75–4.57 (m, 1H), 3.98–3.92
(m, 1H), 3.89 (s, 3H), 2.60–2.48 (m, 2H), 2.43–2.37 (m, 2H), 2.13 (s, 1H). 13C–NMR (101 MHz,
CDCl3) δ
167.0, 144.5, 130.1, 126.7, 126.0, 66.1, 52.2, 52.1, 39.4, 31.9. HRMS [M+Na]+ m/z
calcd. for [C12H14NaO3S]+ 261.0556, found 261.0563.
To synthesize tert-butyl (4-((3-hydroxycyclobutyl)thio)phenyl)carbamate (4d), potas-
sium tert-butoxide (200 mg, 1.78 mmol, 1.3 equiv) was added under intense stirring to a
solution of 3-hydroxycyclobutyl-4-methylbenzenesulfonate (331 mg, 1.36 mmol, 1.0 equiv)
and methyl tert-butyl (4-mercaptophenyl)carbamate (370 mg, 1.64 mmol, 1.2◦equiv) in
tert-butanol (8 mL) at room temperature under Ar. The mixture was stirred at 80 C for 7 h.
After completion of the reaction monitored by TLC, the crude mixture was quenched with
water and extracted with ethyl acetate. The combined organic phases were washed with
brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue was purified
by silica gel column chromatography (PE/EA = 2:1) to afford 4d (146 mg, 32%) as a brown
1
solid. H–NMR (400 MHz, CDCl3)
δ 7.29 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H), 6.48 (s,
1H), 4.63–4.49 (m, 1H), 3.82–3.72 (m, 1H), 2.36 (dd, J = 12.8, 6.4 Hz, 4H), 1.86 (s, 1H), 1.51 (s,
9H). 13C–NMR (101 MHz, CDCl3)
δ 152.7, 137.2, 131.4, 129.7, 119.2, 80.8, 66.2, 39.5, 34.5,
28.4. HRMS [M-H]− m/z calcd. for [C15H20NO3S]− 294.1169, found 294.1167.
3.2.3. Synthesis of Compounds 5a–5d
To synthesize 3-(p-Tolylthio)cyclobutyl-4-methylbenzenesulfonate (5a), a solution of TsCl
(365 mg, 1.91 mmol, 2.0 equiv) in DCM (5 mL) was added dropwise under stirring to a mixture
of 3-(p-tolylthio)cyclobutanol (186 mg, 0.96 mmol, 1.0 equiv), TEA (400
µ
L, 2.87 mmol, 3 equiv),
◦
and 4-dimethylaminopyridine (DMAP) (23 mg, 0.19 mmol, 0.2 equiv) in DCM (5 mL) at 0 C
under Ar. The resulting mixture was stirred at room temperature overnight. After completion,
the reaction was quenched with water and extracted with ethyl acetate. The combined organic
phases were washed with brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The
residue was purified by silica gel column chromatography (PE/EA = 2:1) to afford 5a (261 mg,
78%) as a light yellow oil. 1H–NMR (400 MHz, CDCl3)
δ
7.76 (d, J = 8.3 Hz, 2H), 7.33 (d,
J = 8.0 Hz, 2H), 7.11 (q, J = 8.3 Hz, 4H), 5.03–4.96 (m, 1H), 3.84–3.73 (m, 1H), 2.70–2.56 (m, 2H),
2.45 (s, 3H), 2.36–2.26 (m, 5H). 13C–NMR (101 MHz, CDCl3)
δ 145.0, 136.9, 133.8, 131.7, 130.4,
130.0, 129.9, 128.0, 73.9, 37.2, 34.6, 21.8, 21.2. HRMS [M+Na]+ m/z calcd. for [C18H20NaO3S2]+
371.0746, found 371.0749.
To synthesize 3-((4-methoxyphenyl)thio)cyclobutyl 4-methylbenzenesulfonate (5b),
a solution of TsCl (90.7 mg, 0.48 mmol, 2.0 equiv) in DCM (2 mL) was added dropwise
under stirring to a mixture of 3-((4-methoxyphenyl)thio)cyclobutanol (50 mg, 0.24 mmol,
1.0 equiv), triethylamine (TEA, 100 µL, 0.71 mmol, 3 equiv), and 4-dimethylaminopyridine
(DMAP) (5.8 mg, 0.05 mmol, 0.2 equiv) in DCM (2 mL) at 0 ◦C under Ar. The resulting
mixture was stirred at room temperature overnight. After completion, the reaction was
quenched with water and extracted with ethyl acetate. The combined organic phases were
washed with brine, dried over anhydrous Na2SO4, and evaporated in vacuo. The residue
was purified by silica gel column chromatography (PE/EA = 5:1) to afford 5b (47.6 mg,
55%) as a white solid. 1H–NMR (400 MHz, CDCl3)
δ 7.75 (d, J = 8.3 Hz, 2H), 7.33 (d,
J = 8.1 Hz, 2H), 7.25 (d, J = 8.9 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 4.97–4.90 (m, 1H), 3.79 (s,
3H), 3.74–3.67 (m, 1H), 2.63–2.51 (m, 2H), 2.44 (s, 3H), 2.30–2.25 (m, 2H). 13C–NMR (101
MHz, CDCl3)
δ 159.5, 145.0, 133.9, 133.7, 130.0, 127.9, 125.2, 114.8, 73.9, 55.4, 37.0, 35.8, 21.7.
HRMS [M+Na]+ m/z calcd. for [C18H20NaO4S2]+ 387.0695, found 387.0700.
To synthesize methyl 4-((3-(tosyloxy)cyclobutyl)thio)benzoate (5c), a solution of TsCl
(318.8 mg, 1.67 mmol, 2.0 equiv) in DCM (3 mL) was added dropwise under stirring to a mix-

Molecules 2021, 26, 276
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ture of methyl 4-((3-hydroxycyclobutyl)thio)benzoate (199 mg, 0.84 mmol, 1.0 equiv), TEA
(350
µ
L, 2.5 mmol, 3 equiv), and 4-dimethylaminopyridine (DMAP) (20.4 mg, 0.16 mmol,
◦
0.2 equiv) in DCM (4 mL) at 0 C under Ar. The resulting mixture was stirred at room
temperature overnight. After completion, the reaction was quenched with water and
extracted with ethyl acetate. The combined organic phases were washed with brine, dried
over anhydrous Na2SO4, and evaporated in vacuo. The residue was purified by silica
gel column chromatography (PE/EA = 5:1) to afford 5c (260 mg, 79%) as a white solid.
1H–NMR (400 MHz, CDCl3)
δ
7.91 (d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.34 (d,
J = 8.0 Hz, 2H), 7.11 (d, J = 8.5 Hz, 2H), 5.09–5.02 (m, 1H), 3.98–3.93 (m, 1H), 3.89 (s, 3H),
2.85–2.69 (m, 2H), 2.45 (s, 3H), 2.40–2.33 (m, 2H). 13C–NMR (101 MHz, CDCl3)
δ 166.7, 145.1,
143.2, 133.6, 130.2, 130.0, 127.9, 127.2, 126.4, 73.4, 52.2, 37.0, 32.5, 21.8. HRMS [M+Na]+ m/z
calcd. for [C19H20NaO5S2]+ 415.0644, found 415.0652.
To synthesize 3-((4-((tert-butoxycarbonyl)amino)phenyl)thio)cyclobutyl4-methylbenzene-
sulfonate (5d), a solution of TsCl (155 mg, 0.81 mmol, 2.0 equiv) in DCM (2 mL) was added drop-
wise under stirring to a mixture of tert-butyl (4-((3-hydroxycyclobutyl)thio)phenyl)carbamate
(120 mg, 0.41 mmol, 1.0 equiv), TEA (170 µL, 1.22 mmol, 3 equiv), and 4-dimethylaminopyridine
(DMAP) (10 mg, 0.08 mmol, 0.2 equiv) in DCM (2 mL) at 0 ◦C under Ar. The resulting mixture
was stirred at room temperature overnight. After completion, the reaction was quenched with
water and extracted with ethyl acetate. The combined organic phases were washed with brine,
dried over anhydrous Na2SO4, and evaporated in vacuo. The residue was purified by silica
gel column chromatography (PE/EA = 5:1) to afford 5d (136 mg, 74%) as a light yellow oil.
1H–NMR (400 MHz, CDCl3)
δ
7.75 (d, J = 8.3 Hz, 2H), 7.31 (dd, J = 15.9, 8.4 Hz, 4H), 7.19 (d,
J = 8.6 Hz, 2H), 6.51 (s, 1H), 5.04–4.89 (m, 1H), 3.77–3.71 (m, 1H), 2.64–2.52 (m, 2H), 2.44 (s,
3H), 2.31–2.25 (m, 2H), 1.51 (s, 9H). 13C–NMR (101 MHz, CDCl3)
δ 152.7, 145.0, 137.8, 133.7,
132.2, 130.0, 128.3, 127.9, 119.2, 80.9, 73.9, 37.0, 35.1, 28.4, 21.8. HRMS [M-H]− m/z calcd. for
[C22H26NO5S2]− 448.1258, found 448.1256.
3.2.4. Synthesis of Compounds 6a–6d
To synthesize cyclobut-2-en-1-yl(p-tolyl)sulfane (6a), potassium tert-butoxide (56 mg,
0.5 mmol, 2.0 equiv) was dissolved in dry DMSO (1 mL) under Ar, forming a colorless
solution. Then, a solution of 3-(p-tolylthio)cyclobutyl-4-methylbenzenesulfonate (87 mg,
0.25 mmol, 1.0 equiv) in dry DMSO (l mL) was added slowly to the colorless solution, and
the resulting mixture was stirred at room temperature for 1 h. Water was then added slowly,
followed by ethyl acetate. The separated water phase was extracted with ethyl acetate
three times, and the combined organic phases were washed once with water. The final
organic phase was dried over anhydrous Na2SO4 and evaporated in vacuo. The residue
was purified by silica gel column chromatography (PE) to afford compound 6a (38 mg,
1
86%) as a light yellow oil. H–NMR (400 MHz, CDCl3):
δ 7.25 (d, J = 8.0 Hz, 2H), 7.09 (d,
J = 7.9 Hz, 2H), 6.10 (dd, J = 17.4, 2.5 Hz, 2H), 4.28 (d, J = 3.7 Hz, 1H), 3.03 (dd, J = 13.8,
3.9 Hz, 1H), 2.49 (d, J = 13.8 Hz, 1H), 2.31 (s, 3H). 13C–NMR (101 MHz, CDCl3):
δ 138.1,
137.5, 136.5, 132.4, 130.7, 129.7, 46.9, 40.1, 21.2. HRMS [M+H]+ m/z calcd. for [C11H13S]+
177.0732, found 177.0740.
To synthesize cyclobut-2-en-1-yl(4-methoxyphenyl)sulfane (6b), potassium tert-butoxide
(29.3 mg, 0.26 mmol, 2.0 equiv) was dissolved in dry DMSO (1 mL) under Ar, forming a colorless
solution. Then, a solution of 3-((4-methoxyphenyl)thio)cyclobutyl 4-methylbenzenesulfonate
(47.6 mg, 0.13 mmol, 1.0 equiv) in dry DMSO (l mL) was added slowly to the colorless solution,
and the resulting mixture was stirred at room temperature for 1 h. Water was then added
slowly, followed by ethyl acetate. The separated water phase was extracted with ethyl acetate
three times, and the combined organic phases were washed once with water. The final organic
phase was dried over anhydrous Na2SO4 and evaporated in vacuo. The residue was purified
by silica gel column chromatography (PE) to afford compound 6b (24.5 mg, 89%) as a light
yellow oil. 1H–NMR (400 MHz, CDCl3)
(dd, J = 14.4, 2.6 Hz, 2H), 4.19 (d, J = 3.9 Hz, 1H), 3.80 (s, 3H), 2.97 (dd, J = 13.8, 4.0 Hz, 1H), 2.46
δ 7.35 (d, J = 8.7 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.07

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(d, J = 13.8 Hz, 1H). 13C–NMR (101 MHz, CDCl3)
δ
159.2, 138.3, 137.2, 134.1, 125.6, 114.4, 55.4,
47.9, 39.8. HRMS [M+H]+ m/z calcd. for [C11H13OS]+ 193.0682, found 193.0686.
To synthesize 4-(cyclobut-2-en-1-ylthio)benzoic acid (6c), potassium tert-butoxide
(11.45 mg, 0.10 mmol, 2.0 equiv) was dissolved in dry DMSO (0.5 mL) under Ar, forming
a colorless solution. Then, a solution of methyl 4-((3-(tosyloxy)cyclobutyl)thio)benzoate
(20 mg, 0.05 mmol, 1.0 equiv) in dry DMSO (0.5 mL) was added slowly to the colorless
solution, and the resulting mixture was stirred at room temperature for 1 h. Water was
then added slowly, followed by ethyl acetate. The separated water phase was extracted
with ethyl acetate three times, and the combined organic phases were washed once with
water. The final organic phase was dried over anhydrous Na2SO4 and evaporated in
vacuo. The residue was purified by silica gel column chromatography (PE/EA = 1:1) to
1
afford compound 6c (5.4 mg, 47%) as a white solid. H–NMR (400 MHz, DMSO)
δ 7.85
(d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 6.25 (d, J = 24.8 Hz, 2H), 4.57 (s, 1H), 3.19
(dd, J = 13.9, 3.8 Hz, 1H), 2.43 (d, J = 14.0 Hz, 1H). 13C–NMR (101 MHz, DMSO)
143.3, 138.3, 137.2, 129.8, 129.1, 126.2, 43.8, 39.6. HRMS [M-H] m/z calcd. for [C11H9O2S]
205.0329, found 205.0330.
δ
167.−0,
−
To synthesize 4-(cyclobut-2-en-1-ylthio)aniline (6d), 3-((4-((tert-butoxycarbonyl)amino)
phenyl) thio)cyclobutyl 4-methylbenzenesulfonate (22 mg, 0.049 mmol, 1.0 equiv) was
dissolved in DCM (0.7 mL) and trifluoroacetic acid was added (0.3 mL). The resulting
mixture was stirred at room temperature for 1 h. Upon completion monitored by TLC, the
reaction mixture was evaporated under reduced pressure. The residue 5d’ was used for
the next step without further purification. Potassium tert-butoxide (11 mg, 0.097 mmol,
2.0 equiv) was dissolved in dry DMSO (0.4 mL) under Ar, forming a colorless solution.
Then, a solution of methyl 3-((4-aminophenyl)thio)cyclobutyl 4-methylbenzenesulfonate
(17.1 mg, 0.049 mmol, 1.0 equiv) in dry DMSO (0.4 mL) was added slowly to the colorless
solution, and the resulting mixture was stirred at room temperature for 1 h. Water was
then added slowly, followed by ethyl acetate. The separated water phase was extracted
with ethyl acetate three times, and the combined organic phases were washed once with
water. The final organic phase was dried over anhydrous Na2SO4 and evaporated in vacuo.
The residue was purified by silica gel column chromatography (PE/EA = 5:1) to afford
1
compound 6d (4 mg, 46%) as a light yellow solid. H–NMR (400 MHz, CDCl3)
δ
7.25 (d,
J = 9.3 Hz, 3H), 6.61 (d, J = 8.5 Hz, 2H), 6.05 (dd, J = 10.9, 2.6 Hz, 2H), 4.13 (d, J = 3.9 Hz,
1H), 3.73 (s, 2H), 2.93 (dd, J = 13.8, 4.0 Hz, 1H), 2.44 (d, J = 13.7 Hz, 1H). 13C–NMR (101
MHz, CDCl3) δ
146.2, 138.5, 137.0, 134.8, 122.2, 115.4, 48.3, 39.6. HRMS [M+H]+ m/z calcd.
for [C10H12NS]+ 178.0685, found 178.0687.
3.2.5. Synthesis of Compounds 9a and 9b
A solution of 3-(benzyloxy)cyclobutyl-4-methylbenzenesulfonate (1.0 equiv), com-
pound 8a or 8b (2.0 equiv), and Cs2CO3 (2.0 equiv) was prepared in DMF, and the resulting
◦
mixture was stirred at 80 C for 8 h. Then the reaction was quenched with water and
extracted with ethyl acetate. The combined organic phases were washed with brine, dried
over anhydrous Na2SO4, and evaporated in vacuo. The residue was purified by silica gel
column chromatography (PE/EA = 40:1) to afford the desired product 9a or 9b.
For the synthesis of (3-(benzyloxy)cyclobutoxy)benzene (9a), 1.32 g of 8a was used,
giving 484 mg of compound 9a (48% yield) as a light yellow oil.
For the synthesis of methyl 3-(4-(3-(benzyloxy)cyclobutoxy)phenyl)-2-((tert-butoxycar
bonyl)amino)propanoate (9b), 261 mg of 8b was used, giving 250 mg of compound 9b (70%
yield) as a white solid.
3.2.6. Synthesis of Compounds 10a and 10b
To prepare 3-phenoxycyclobutyl-4-methylbenzenesulfonate (10a), compound 9a (469 mg,
1.84 mmol, 1.0 equiv) was dissolved in ethanol (10 mL), followed by the addition of Pd/C
(94 mg). The resulting solution was purged with hydrogen five times, and the mixture was
then hydrogenated with a H2 balloon overnight at room temperature. After completion of

Molecules 2021, 26, 276
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the reaction monitored by TLC, the catalyst was removed by filtration over celite, and the
filter cake was washed three times with ethyl acetate. The combined organic layers were
evaporated in vacuo, affording a crude mixture that was used in the next step without
purification. TEA (772 µL, 5.53 mmol, 3.0 equiv) and DMAP (45 mg, 0.37 mmol, 0.2 equiv)
were dissolved in DCM◦(4 mL) under Ar and added to the crude mixture. The resulting
mixture was cooled to 0 C, and a solution of TsCl (1.05 g, 5.53 mmol, 3.0 equiv) in DCM (6
mL) was added. The reaction mixture was stirred at room temperature for 20 h. Then the
reaction was quenched with water and extracted with ethyl acetate. The combined organic
phases were washed with brine, dried over anhydrous Na2SO4, and evaporated in vacuo.
The residue was purified by silica gel column chromatography (PE/EA = 5:1) to afford 10a
1
in 86% yield (508 mg) as a light yellow oil. H–NMR (400 MHz, CDCl3)
δ
7.79 (d, J = 8.2 Hz,
2H), 7.35 (d, J = 8.3 Hz, 2H), 7.25 (t, J = 8.0 Hz, 2H), 6.94 (t, J = 7.4 Hz, 1H), 6.71 (d, J = 7.9 Hz,
2H), 5.11–5.00 (m, 1H), 4.84–4.79 (m, 1H), 2.67–2.58 (m, 2H), 2.54–2.48 (m, 2H), 2.45 (s, 3H).
13C–NMR (101 MHz, CDCl3)
δ 157.2, 145.1, 133.7, 130.1, 129.7, 128.0, 121.3, 114.9, 73.2, 68.2,
37.8, 21.8. HRMS [M+Na]+ m/z calcd. for[C17H18NaO4S]+ 341.0818, found 341.0826.
Methyl 2-((tert-butoxycarbonyl)amino)-3-(4-(3-(tosyloxy)cyclobutoxy)phenyl)propanoate
(10b) was isolated in 70% yield from compound 9b and TsCl as a light red solid by a method
analogous to that described for the synthesis of 10a above.
3.2.7. Synthesis of Compounds 11a and 11b
To prepare cyclobut-2-en-1-yloxy)benzene (11a), potassium tert-butoxide (198 mg,
1.77 mmol, 2.0 equiv) was dissolved in dry DMSO (2 mL) under Ar, forming a colorless
solution. A solution of compound 10a (281 mg, 0.88 mmol, 1.0 equiv) in dry DMSO (2 mL)
was then added slowly to the colorless solution, and the reaction mixture was left stirring
at room temperature for 1 h. After completion of the reaction monitored by TLC, water was
added slowly, followed by ethyl acetate. The separated water phase was extracted with
ethyl acetate three times, and the combined organic layers were washed once with water.
The final organic phase was dried over anhydrous Na2SO4 and evaporated in vacuo. The
residue was purified by silica gel column chromatography (PE) to afford 11a (73.6 mg, 57%)
1
as a colorless oil. H–NMR (400 MHz, CDCl3)
δ
7.28 (t, J = 9.8 Hz, 2H), 6.95 (t, J = 7.4 Hz,
1H), 6.89 (d, J = 7.8 Hz, 2H), 6.31 (d, J = 33.7 Hz, 2H), 5.10 (d, J = 3.2 Hz, 1H), 3.00 (dd, J = 13.4,
3.5 Hz, 1H), 2.63 (dd, J = 13.4, 1.0 Hz, 1H). 13C–NMR (101 MHz, CDCl3)
δ 158.2, 139.3, 137.0,
129.7, 121.0, 115.0, 74.5, 39.7.
The compound 2-((tert-butoxycarbonyl)amino)-3-(4-(cyclobut-2-en-1-yloxy)phenyl)
propanoic acid (11b) was obtained in 45% yield as a white solid from compound 10b and
potassium tert-butoxide by a method analogous to that described for the synthesis of 11a
above. 1H–NMR (400 MHz, DMSO-d6)
δ 12.49 (s, 1H), 7.11 (d, J = 8.4 Hz, 2H), 6.98 (d,
J = 8.3 Hz, 1H), 6.77 (d, J = 8.5 Hz, 2H), 6.30 (d, J = 32.6 Hz, 2H), 5.02 (d, J = 2.8 Hz, 1H),
4.02–3.96 (m, 1H), 2.98–2.84 (m, 2H), 2.71 (dd, J = 13.6, 10.3 Hz, 1H), 2.48–2.35 (m, 2H), 1.28
(s, 8H). 13C–NMR (101 MHz, DMSO-d6)
δ 173.7, 156.2, 155.5, 138.9, 137.2, 130.2, 130.1, 114.4,
78.0, 73.8, 55.4, 40.4, 35.7, 28.2, 27.9. HRMS [M-H]− m/z calcd. for [C18H22NO5]− 332.1503,
found 332.1493.
3.2.8. Synthesis of 5-((4-(Cyclobut-2-en-1-Ylthio)Phenyl)Amino)-5-Oxopentanoic Acid (16
A mixture of compound 6d (5 mg, 0.028 mmol, 1.0 equiv), glutaric anhydride (3.5 mg,
0.031 mmol, 1.1 equiv), and TEA (10 L, 0.07 mmol, 2.5 equiv) was dissolved in DCM
)
µ
(0.5 mL) under Ar. The resulting mixture was stirred at room temperature for 3 h, and then
quenched with water and extracted with ethyl acetate. The combined organic phases were
washed with brine, dried over anhydrous Na2SO4, and evaporated in vacuo. Due to the
small scale of the reaction, the residue was purified by thin-layer chromatography to afford
1
6.2 mg (75%) of 16 as a white solid. H–NMR (400 MHz, DMSO-d6)
δ
9.96 (s, 1H), 7.56 (d,
J = 8.6 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 6.15 (dd, J = 15.0, 2.6 Hz, 2H), 4.30 (d, J = 3.6 Hz, 1H),
3.00 (dd, J = 13.8, 3.9 Hz, 1H), 2.35 (dd, J = 13.7, 6.2 Hz, 3H), 2.26 (t, J = 7.3 Hz, 2H), 1.80 (p,
J = 7.3 Hz, 2H). 13C–NMR (101 MHz, DMSO-d6):
δ 174.2, 170.8, 138.1, 137.4, 131.0, 128.3,

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119.5, 46.1, 39.4, 35.4, 33.1, 20.5. HRMS [M+Na]+ m/z calcd. for [C15H17NNaO3S]+ 314.0821,
found 314.0821.
3.2.9. Synthesis of 2,5-Dioxopyrrolidin-1-yl-5-((4-(Cyclobut-2-en-1-Ylthio)Phenyl)Amino)-
5-Oxopentanoate (17)
A mixture of compound 16 (6.2 mg, 0.021 mmol, 1.0 equiv), N,N-disuccinimidyl
carbonate (DSC) (5.4 mg, 0.021 mmol, 1.0 equiv), and TEA (4.5 µL, 0.032 mmol, 1.5 equiv)
was dissolved in tetrahydrofuran (THF) (0.5 mL) under Ar. The resulting mixture was
stirred at room temperature for 4 h. After reaction completion, the reaction was quenched
with water and extracted with ethyl acetate. The combined organic phases were washed
with brine, dried over anhydrous Na2SO4, and evaporated in vacuo. Similarly to analogue
16, the residue was purified by thin-layer chromatography to afford the desired product 17
1
as a white solid in 51% yield (4.2 mg). H–NMR (400 MHz, CDCl3)
δ
8.02 (s, 1H), 7.47 (d,
J = 8.5 Hz, 2H), 7.30 (d, J = 8.6 Hz, 2H), 6.09 (dd, J = 21.8, 2.5 Hz, 2H), 4.26 (d, J = 3.5 Hz,
1H), 3.02 (dd, J = 13.8, 3.6 Hz, 1H), 2.88 (s, 4H), 2.75–2.68 (m, 2H), 2.47 (dd, J = 12.9, 5.8 Hz,
3H), 2.23–2.12 (m, 2H). 13C–NMR (101 MHz, CDCl3)
δ 170.2, 169.7, 168.5, 138.0, 137.6,
136.9, 131.7, 130.9, 120.3, 47.0, 40.0, 35.5, 30.0, 25.8, 21.2. HRMS [M+Na]+ m/z calcd. for
[C19H20N2NaO5S]+ 411.0985, found 411.0989.
3.3. Stability of the Cyclobutene Derivatives 6a–6d, 11a, and 11b
To examine the stability of the synthesized analogues for potential applications in
bioorthogonal chemistry, dienophiles 6a–6d
mixture (1:1) at a final concentration of 0.5 mM at pH 7.4 and 37 C. Samples were monitored
,
11a, and 11b we◦re dissolved in a DMF/PBS
at 0, 12, and 24 h using high-performance liquid chromatography (3 µL injected per time
point). Absorbance was measured at 260 nm for 6b, 280 nm for 11a and 11b, 254 nm for 6a
and 6d, and 300 nm for 6c. The peak area at each time point was expressed as a percentage
of the area at 0 h.
3.4. Kinetics of the Reactions between Cyclobutene Derivatives and Tetrazine
Tetrazine 12 was dissolved in a DMF/H2O (1:1) mixture at a final concentration of
0.5 mM at 37 ◦C. Dienophiles 6a–6d
, 11a, and 11b were then added to the quartz cuvettes
with final concentration of 5 mM. A Quawell Q6000+ UV–Vis spectrophotometer was used
to monitor the disappearance of the characteristic absorption peak of tetrazine at 520 nm
at 10-min intervals for a duration of 6 h. The experimental data were processed using
GraphPad Prism 6.0.
3.5. IEDDA-Oxidation Cascade Reaction of Cyclobutene 6a with Dipyridinetetrazine 12
Compound 6a (5 mg, 0.028 mmol, 1.05 equiv) and dipyridine tetrazine 12 (6.38 mg,
0.027 mmol, 1.0 equiv) were dissolved in THF (0.3 mL) and H2O (0.1 mL). The resulting
◦
mixture was stirred at 60 C for 2 h. Upon completion monitored by HPLC, to the reaction
mixture was added DDQ (6.74 mg, 0.029 mmol, 1.1 equiv). The resulting mixture was
stirred at room temperature for 1.5 h. Upon completion monitored by HPLC, the reaction
was quenched with water and extracted with ethyl acetate. The combined organic phase
was washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was purified via silica column chromatography to afford the product
14 (8.5 mg, 82% yield) as a light red solid.
3.6. Protein Labeling
3.6.1. Protein Modification In Vitro
The cyclobutene derivative 17 (0.5
µL, 50 mM, 10.0 equiv) was mixed with BSA (5
µL,
0.5 mM in PBS) in sodium bicarbonate buffer (39
µL, pH 8.2) containing DMSO (4.5
µL).
The mixture was kept at room temperature for 2 h, then excess 17 was removed using a
Zeba desalting spin column (0.5 mL). The modified protein was recovered in PBS and its
concentration quantified at 19.2 µM based on Quawell Q6000+ UV–Vis absorption scanning.

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3.6.2. Protein Labeling In Vitro
Three samples were incubated at 37 ◦C for 4 h: the BSA-labeling reaction containing
H-Tz-Cy5 (2.5 L, 5 mM in DMSO) and cyclobutene-modified BSA (22.5 L, 19.2 M in
PBS), giving a final dye concentration of 250 M or 500 M; a negative-control reaction
containing H-Tz-Cy5 (2.5 L, 5 mM in DMSO) and unmodified BSA (22.5 L, 19.2 M in
PBS), again giving a final dye concentration of 250 M or 500 M; and a “blank” reaction
of only neat, unmodified BSA (25 L, 19.2 M in PBS). After incubation, all of the samples
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
were passed through a Zeba desalting spin column (0.5 mL) to remove excess dye. All
of the column eluates contained the same concentration of about 0.4 mg/mL, based on
Quawell Q6000+ spectrophotometry. An aliquot (20
L of 5X SDS–PAGE loading buffer and vortexed for 10 sec, and an aliquot (15
supernatant was loaded onto SDS–PAGE gels. The gels were imaged for fluorescence using
the ChemiDoc MP imaging system (Bio-Rad), then stained with Coomassie blue and
µL) of each sample was mixed with
2
µ
µL) of
™
imaged under visible light.
3.6.3. Rough Estimation Method of the Yield of the Labeling Reaction on the Model
BSA Protein
The second-order reaction rate constant of compound 6d and 3-phenyl-1,2,4,5-tetrazine
(H-Tz-Ph) was determined by a method similar to that described above for determining
reaction kinetics, giving k2 = 0.056 M−1s−1
.
The first-order reaction rate constant (k1) was determined according to the formula:
k1 = ck2
(1)
c is the concentration of the reactant, which is greatly excessive.
The half-life (t1/2) of the reaction was calculated with the following formula:
ln 2
k1
t1/2
=
(2)
The final dye concentration in the labeling reaction was 500
µM and the reaction time
was 4 h. The half-life (t1/2) was calculated according to the above formulas and it was
about 6.875 h. A rough estimation of the yield of the labeling reaction was about 29.1%,
which is determined according to the formula:
4 h
t1/2
Yield (4 h) =
× 50%
(3)
4. Conclusions
We developed concise synthesis of a series of novel aryl-substituted cyclobutene
derivatives. These small, strained cyclobutenes are stable enough to serve as small
dienophiles for bioorthogonal tetrazine ligations that proceed with good kinetics and
high yields. Yet, the aryl-substituted cyclobutenes might not be small enough for use as
mini-tags for bioorthogonal metabolic labeling applications and of relatively poor water
solubility. Our synthesis strategy provides a potential way to synthesize hydrophilic alky-
substituted cyclobutene. Based on these promising results, we expect that the synthesis
of additional aryl-substituted cyclobutene analogues will significantly enrich available
bioorthogonal tools and facilitate the development of new biomedical applications.
Author Contributions: H.W. and H.S. designed the experiments; J.S., J.L. and H.S. performed the
experiments and analyzed the data; J.S. and C.L. wrote the original draft preparation; H.W., J.L. and
H.S. reviewed and edited the manuscript. All authors have read and agreed to the published version
of the manuscript.

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Funding: This work was supported by the Key Research and Development Projects in Sichuan
Province (2020YFS0123); National Natural Science Foundation (21801178); and 1.3.5 project for
disciplines of excellence, West China Hospital, Sichuan University.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing not applicable.
Acknowledgments: We acknowledge the Analytical & Testing Center of Sichuan University for
NMR Tests.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not available.
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