Single-step fluorescent probes to detect decrotonylation activity of HDACs through intramolecular reactions

Article abstract of DOI:10.1016/j.ejmech.2020.113120

Lysine crotonylation plays vital roles in gene transcription and cellular metabolism. Nevertheless, methods for dissecting the molecular mechanisms of decrotonyaltion remains limited. So far, there is no single-step fluorescent method developed for enzymatic decrotonylation activity detection. The major difficulty is that the aliphatic crotonylated lysine doesn't allow π-conjugation to a fluorophore and decrotonylation can not modulate the electronic state directly. Herein, we have designed and synthesized two activity-based single-step fluorogenic probes KTcr-I and KTcr-II for detecting enzymatic decrotonylation activity. These two probes can be recognized by histone deacetylases and undergo intramolecular nucleophilic exchange reaction to generate fluorescence signal. Notably, peptide sequence-dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II with LGKcr peptide sequence preferentially reacted with HDAC3. Compared to other methods of studying enzymatic decrotonylation activity, our single-step fluorescent method has a number of advantages, such as facileness, high sensitivity, cheap facility and little material consumed. We envision that the probes developed in this study will provide useful tools to screen inhibitors which suppress the decrotonylation activity of HDACs. Such probes will be useful for further delineating the roles of decrotonylation enzyme and aid in biomarker identification and drug discovery.

Full text of DOI:10.1016/j.ejmech.2020.113120

European Journal of Medicinal Chemistry 212 (2021) 113120
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Single-step fluorescent probes to detect decrotonylation activity of
HDACs through intramolecular reactions
Yusheng Xie a, 1, Liu Yang
b
,
a
,
b
,
1
, Qingxin Chen b, Jie Zhang , Ling Feng a b,
a
,
a
,
b
,
Jian Lin Chen , Quan Hao , Liang Zhang , Hongyan Sun a
Department of Chemistry and COSDAF (Centre of Super-Diamond and Advanced Films), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong
Kong, China
Shenzhen Research Institute of City University of Hong Kong, Shenzhen, 518057, China
School of Science and Technology, The Open University of Hong Kong, Hong Kong Special Administrative Region
c
d
b,
e
, b, *
a
b
c
d
School of Biomedical Sciences, University of Hong Kong, Hong Kong, China
Department of Biomedical Science, College of Veterinary Medicine and Life Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong
e
Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 December 2019
Received in revised form
Lysine crotonylation plays vital roles in gene transcription and cellular metabolism. Nevertheless,
methods for dissecting the molecular mechanisms of decrotonyaltion remains limited. So far, there is no
single-step fluorescent method developed for enzymatic decrotonylation activity detection. The major
27 November 2020
difficulty is that the aliphatic crotonylated lysine doesn’t allow
p-conjugation to a fluorophore and
Accepted 29 November 2020
decrotonylation can not modulate the electronic state directly. Herein, we have designed and synthesized
two activity-based single-step fluorogenic probes KTcr-I and KTcr-II for detecting enzymatic decroto-
nylation activity. These two probes can be recognized by histone deacetylases and undergo intra-
molecular nucleophilic exchange reaction to generate fluorescence signal. Notably, peptide sequence-
dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II with
LGKcr peptide sequence preferentially reacted with HDAC3. Compared to other methods of studying
enzymatic decrotonylation activity, our single-step fluorescent method has a number of advantages, such
as facileness, high sensitivity, cheap facility and little material consumed. We envision that the probes
developed in this study will provide useful tools to screen inhibitors which suppress the decrotonylation
activity of HDACs. Such probes will be useful for further delineating the roles of decrotonylation enzyme
and aid in biomarker identification and drug discovery.
Available online 30 December 2020
Keywords:
Single-step
Fluorescent probe
Decrotonylation
Histone deacetylases
Sirtuin
Intramolecular reaction
©
2020 Elsevier Masson SAS. All rights reserved.
1
. Introduction
affect human health. Histone crotonylation specifically labels the
potential enhancers and active gene promoters and stimulates
transcription in mammalian cell genomes [2e4]. It has also been
reported that crotonylation in histone affects kidney injury and is
associated with cellular metabolism [5]. The enzymes responsible
for removal of crotonylation are termed as “erasers” of Kcr.
Increasing evidences have indicated that histone PTMs (post-
translational modifications) are involved in various biological pro-
cesses such as cell differentiation, gene transcription and organ-
ismal development [1]. Lysine crotonylation (Kcr), an evolutionarily
conserved PTM, was first identified in histone proteins in 2011 [2].
Recently, it has been proved that non-histone proteins can also
undergo crotonylation [3]. Aberrant crotonylation of histones could
Histone deacetylases (HDACs) are hydrolases that remove the
acetyl groups from lysine residues in histones and other non-
histone proteins [6]. HDACs are interesting therapeutic targets
due to their important biological roles. To date, 18 distinct HDAC
proteins have been identified in humans. Among them, Class I, II
and IV utilize Zn as a cofactor. whereas Class III, also termed as
*
Corresponding author. Department of Chemistry and COSDAF (Centre of Super-
þ
Diamond and Advanced Films), City University of Hong Kong, 83 Tat Chee Avenue,
Kowloon, Hong Kong, China.
sirtuins (Sirt1-7), shows a different mode of action by using NAD
as cofactor [6c]. Emerging evidence has demonstrated that HDACs
can catalyze the removal of various alternative acyl groups of lysine
E-mail address: hongysun@cityu.edu.hk (H. Sun).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2020.113120
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

Y. Xie, L. Yang, Q. Chen et al.
European Journal of Medicinal Chemistry 212 (2021) 113120
efficiently, such as succinyl, malonyl and crotonyl [7]. Until now,
several HDACs, including Sirt2 and HDAC3, have been identified as
standard solid-phase peptide synthesis with 52% overall yield [14].
Intermediate 4 was treated with TFA to give compound 8 with a
quantitative yield. 8 underwent coupling reaction with peptide
building block 7 in the presence of the activator of HOBt/HBTU to
yield 9, which was subjected to substitution with NBD-F to afford
the desired probe KTcr-II. The final compound KTcr-I and KTcr-II
were both confirmed by NMR and mass spectrometry.
“
eraser” of Kcr [2,5b,7b,8]. Despite these advancements, our un-
derstanding of enzymes responsible for lysine decrotonylation and
their inhibitor design remains limited.
To detect the decrotonylation activity of enzymes, several
methods have been developed, including approaches that use Kcr
antibody and HPLC [2,5b,7b,8]. However, antibody-based methods
highly depend on the quality of the antibody used. HPLC ap-
proaches often need laborious preparation work and multistep
protocols. Fluorescence detection for examining enzymatic activity
has the advantage of high sensitivity and facileness [9]. In partic-
ular, fluorescence-based methods are useful for inhibitor screening
because they can be performed in a high-throughput manner. Olsen
et al. synthesized a fluorogenic probe for detecting decrotonylation
activity of enzymes through two-step enzyme-coupled reactions
2.2. Fluorescence turn-on molecular mechanism studies
With the probes in hand, we first carried out the HPLC studies
to evaluate whether crotonylated lysine in the probe can be
recognized by sirtuins. KTcr-I was used as the representative
probe. Briefly, 40
m
M KTcr-I was incubated with Sirt2 and cofactor
þ
ꢀ
NAD in HEPES buffer (pH 8.0) at 37 C. After 3 h of reaction, the
enzymatic reaction was quenched by methanol and aliquot was
analyzed by HPLC. HPLC analysis showed the appearance of a new
peak after reaction (Peak 2, retention time: 22.5 min, Fig. 1A).
Furthermore, the eluted fraction collected from this peak showed
the expected mass value of the decrotonylated/exchanged product
(Fig. 1B). Nevertheless, no product was observed in control ex-
[
10]. However, this method needs an additional step of protease
digestion. In addition, the aminomethylcoumarin (AMC) fluo-
rophore needs to be conjugated to the neighbouring C-terminus of
the acetylated lysine. This might not be ideal for enzyme recogni-
tion because of flanking effect [7b,11].
Till date, no single-step fluorescent method has been developed
for enzymatic decrotonylation activity assay. It was noted that
Buccella et al. has developed a peptide-based probe to detect
decrotonylase activity, however, their detection method was based
on absorbance readout [12]. The biggest challenge is that aliphatic
þ
periments, including samples without addition of NAD or/and
enzyme (Fig. 1A and Fig. S1). These data indicated that 1) the
probe can be recognized by the known Kcr “eraser” sirtuin2, 2) the
intramolecular exchange reaction between O-NBD and amine
occurred.
crotonylated lysine does not allow p-conjugation to a fluorophore
and decrotonylation is not able to modulate the electronic state
directly [9b]. Previously, our group and others have developed
several one-step fluorescent approaches for deacetylation/deli-
poylation activity detection based on intramolecular reactions [13].
We found that acylated lysine coupled with a NBD-conjugated and
hydrophilic aminoethoxyl linker can be recognized by HDACs with
moderate to good efficiency [13d-e]. We continued to explore
whether the intramolecular reaction strategy can be applied to the
design of single-step fluorescent probes to detect decrotonylation
activity.
2.3. Absorbance and fluorescence analysis
Next, we performed detailed absorbance and fluorescence
analysis with KTcr-I. The absorbance spectra were recorded before
and after enzymatic reaction (Fig. 2A). At the beginning, an obvious
absorption peak at 380 nm, which corresponds to that of O-NBD
group, was observed with KTcr-I. After enzymatic reaction with
þ
Sirt2 and NAD , a new absorption peak was detected at 480 nm,
indicating the formation of N-NBD. Some absorbance at 380 nm
was observed after enzymatic reaction. This is due to incomplete
reaction of enzymatic decrotonylation, which is consistent with our
HPLC analysis. The results also agrees well with the reported
literature that the decrotonylation activity of Sirt2 is much lower
than its deacetylation activity [7b]. Subsequently, more detailed
fluorescence studies were carried out (Fig. 2B). Results showed that
2
. Results and discussion
2.1. Design and synthesis
þ
In this study, we designed and synthesized two single-step ac-
KTcr-I only, and KTcr-I in the presence of Sirt2 or NAD displayed
tivity-based fluorogenic probes (KTcr-I, lysine decrotonylation
turn-on probe I and KTcr-II, lysine decrotonylation turn-on probe II,
Scheme 1A) for enzymatic decrotonylation activity detection. The
mechanism is based on a spontaneous intramolecular exchange
reaction between non-fluorescent O-NBD and a free amine group
which can be released by enzyme with decrotonylation activity
negligible fluorescence (
peak was observed upon addition of Sirt2 and NAD . The fluores-
cence increase was determined to be around 5-fold.
l
ex ¼ 480 nm). However, a strong emission
þ
2.4. Time-dependent experiments
(
Scheme 1A). The yielded product N-NBD is highly fluorescent. In
Following that time-dependent enzymatic reaction with Sirt2
was conducted by fluorescence analysis. The fluorescence spectra
of the probe under enzymatic condition were periodically recorded
(Fig. 3A). Initially, the fluorescence intensity of the probe was weak
and increased progressively during incubation. It plateaued after
3 h. Those data together unambiguously confirmed that the reac-
tion was catalysed by enzymatic activity rather than environmental
factors.
addition, the difference between KTcr-I and KTcr-II is the peptide
sequence incorporated in KTcr-II due to the sequence-dependent
effect for Kcr recognition.
The synthetic route for KTcr-I was illustrated as scheme 1B. The
synthesis started with Boc-Lys-OH, which is readily available from
commercial vendors. Selective protection is then performed with
Fmoc at ε-amino group of lysine side chain, followed by coupling
with a flexible and hydrophilic aminoethoxyl linker. Further
deprotection of Fmoc was subsequently carried out to afford
compound 3 with 75% yield. The use of crotonic anhydride to
functionalize the free amino group generated intermediate 4. The
final step was accomplished by nucleophilic substitution of NBD-F
under basic condition to generate the probe KTcr-I. The approach to
obtain probe KTcr-II (Scheme 1B) is slightly different. An Ac-Leu-
Gly-OH (compound 7) building block was first synthesized by
2.5. Inhibition experiments
We next investigated the capability of probe KTcr-I to evaluate
the potency of Sirt2 inhibitors by exposing Sirt2 to a known in-
hibitor, tenovin-6. Increasing concentrations (0, 2, 4, 10, 20, 40, 80,
120, 160
m
M) of inhibitor was firstly added to Sirt2 (40 ng/
m
l) in
ꢀ
HEPES buffer and incubated at 37 C for 30 min. KTcr-I (10
mM) was
2

Y. Xie, L. Yang, Q. Chen et al.
European Journal of Medicinal Chemistry 212 (2021) 113120
Scheme 1. (A) Mechanism of our strategy to detect enzymatic decrotonylation by single-step fluorescent method. (B) Synthetic route of probe KTcr-I and KTcr-II.
then added and incubated for another 3 h. Subsequently, the
fluorescence of the reaction samples was measured respectively.
The percentage of the total activity of Sirt2 was calculated from the
fluorescence intensity of samples at 545 nm relative to the positive
control (without tenovin-6). The IC50 value for Sirt2 was deter-
2.6. Selectivity studies
We then moved on to study the response of the designed fluo-
rescent probes using a series of HDACs (Fig. 4), including Sirt1-3, 5,
6 and HDAC3. The sequence-dependent effect of sirtuins recogni-
tion toward various substrates prompted us to include KTcr-II,
which contains a peptide fragment (LGKcr, Scheme 1A) [7b,10]. Our
results indicated that Sirt1 and Sirt2 showed decrotonylation ac-
tivity, which are in agreement with the previous literature [7b].
mined to be 33.99 mM by calculating from the plot of enzyme ac-
tivity and the log concentration of tenvoin-6 (Fig. 3B). These data
indicated that probe KTcr-I can provide useful tool to screen po-
tential inhibitors of decrontonylation enzymes.
3

Y. Xie, L. Yang, Q. Chen et al.
European Journal of Medicinal Chemistry 212 (2021) 113120
Fig. 1. (A) HPLC analysis of the enzymatic reaction of KTcr-I (40
mM) with recombinant Sirt2 (100 ng/
ml). UV channel: 254 nm. The retention time of peak 1 and 2 was 25.3 and
22.5 min respectively. (B) ESI mass spectrum of the peak at 22.5 min in LC. The mass peak corresponds to the tandem decrotonylated and exchanged product.
Fig. 2. (A) Absorption spectra of KTcr-I (10
m
M, prepared from 5 mM stock in DMSO) before and after enzymatic reaction with Sirt2 (40 ng/
m
l) in HEPES buffer (20 mM, pH ¼ 8.0)
þ
ꢀ
with 200
h.
mM NAD at 37 C for 3 h. (B) Fluorescence spectra of KTcr-I (10
m
M, prepared from 5 mM stock in DMSO) with Sirt2 (40 ng/
ml) under various conditions (
l
ex ¼ 480 nm) for
3
showed preference of Sirt2 over other HDACs. It is noted that KTcr-
II showed mediocre solubility because of hydrophobic amino acid
residues. Nevertheless, the probe KTcr-II was recognized by HDAC3
preferably compared with other sirtuins. This is consistent with
Olsen’s work [10]. In addition, we tested the selectivity of KTcr-I
with other potential interferents, including butylamine and various
amino acids. 10
mM KTcr-I was treated with different species
(
100 M), and the fluorescent spectra of reaction mixtures were
m
recorded respectively (Fig. S2). The results indicated that all the
tested species, including lysine, histidine and cysteine, only
induced negligible fluorescence increment. The substantial fluo-
rescence enhancement caused only by Sirt2 unambiguously proved
that the turn-on fluorescence comes from the intramolecular
nucleophilic exchange reaction upon enzymatic decrotonylation
reaction. Those experiments together proved that the current panel
of probes (KTcr-I and KTcr-II) are suitable for decrotonylation ac-
tivity detection.
Fig. 3. (A) Time dependent study of KTcr-I (10
DMSO) incubated with Sirt2 (40 ng/
m
M, prepared from 5 mM stock in
m
l) in HEPES buffer (20 mM, pH ¼ 8.0) with 200
ex ¼ 480 nm). (B) Dose-response inhibition curve of Sirt2 decroto-
nylation activity by Tenovin-6 using KTcr-I (10 M). Values of Y axis correspond to the
mM
þ
ꢀ
NAD at 37 C (
l
m
average enzyme activity of three independent experiments.
Sirt5 and Sirt6 did not show any activity toward both probes
because Sirt5 was reported to recognize Ksuc and Kmal and Sirt6
can remove long-chain fatty acid group preferably [7a,15]. The ac-
tivity of Sirt3 was not observed, indicating that the crotonylated
lysine of probe KTcr-I and KTcr-II might not be a good substrate for
Sirt3. Importantly, peptide-sequence dependent effect of sirtuins’
decrotonylation activity was observed by careful comparison of the
enzymatic data of the two probes. Sirt2 is known to possesses a
large hydrophobic active pocket and it can accommodate many acyl
groups such as acetyl, propionyl, butyryl, octanoyl, myristoyl and
crotonyl [16]. The newly designed probes KTcr-I/KTcr-II showed
that one single crotonylated lysine in probes can be recognized by
Sirt2 with a moderate to high efficiency. Specifically, probe KTcr-I
3
. Conclusions
In conclusion, we have developed two single-step fluorescent
probes (KTcr-I and KTcr-II) to detect decrotonylation activity of
HDACs. The probes can be recognized by HDACs and undergo
intramolecular nucleophilic exchange reaction to generate fluo-
rescence signal. Notably, peptide sequence-dependent effect was
observed. KTcr-I can be recognized by Sirt2 more efficiently, while
KTcr-II with LGKcr short sequence preferentially reacts with
HDAC3. Compared with other methods such as two-step fluores-
cent method to detect enzymatic decrotonylation activity, our
single-step fluorescent method is more facile and sensitive, and it
allows real-time monitoring the decrotonylation activity. We
4

Y. Xie, L. Yang, Q. Chen et al.
European Journal of Medicinal Chemistry 212 (2021) 113120
1
by using the corresponding solvent peaks as reference ( H,
1
3
CD
CD
1
3
OD ¼ 3.31 ppm, CDCl
3
¼ 7.26 ppm, CD
3
CN ¼ 1.94 ppm; C,
3
OD ¼ 49.00 ppm, CDCl
3
¼ 77.16 ppm, CD
3
CN ¼ 1.32,
18.26 ppm). Multiplicities are indicated as follows: m (multiplet),
dd (doublet of doublets), br (broad singlet), q (quartet), t (triplet),
d (doublet), s (singlet). To obtain mass spectra, a PC Sciex API 150 EX
ESI-mass spectrometer was used.
pH value was measured with a HANNA HI 2211 pH/ORP meter.
To carry out analytical high-performance liquid chromatography
(
1
HPLC) analysis, a Waters 2489 UV/Visible Detector and Waters
525 Binary HPLC Pump were used. A reverse-phase Phenomenex
Luna® Omega 5
m
m Polar C18 100 Å 250 ꢁ 4.6 mm column was
utilized for separation analysis. Acetonitrile and water were used as
eluents with the flow rate of 1 ml/min. Fluorescence detection was
conducted using a FluoroMax-4 fluorescence photometer. Absorp-
tion analysis was performed with UV-VS shimadzu 1700. Sirt1/2/3/
5/6 in this study were recombinantly expressed and purified as
previously reported [17]. HDAC3/NCOR1 was purchased from Enzo
Lifescience.
4.2. Enzymatic reaction
The probe KTcr-I or KTcr-II was incubated with sirtuin and
þ
ꢀ
cofactor NAD for specified time at 37 C in 20 mM HEPES buffer
pH ¼ 8.0) with 1 mM MgCl2, 2.7 mM KCl and 150 mM NaCl.
(
4.3. Enzymatic reaction monitoring with LC-MS using probe KTcr-I
A total reaction volume of 50
After specific reaction time, the enzymatic reactions were
quenched by addition of methanol (250 l), vortexed and spun-
ml was used for enzymatic reaction.
m
down. Supernatant was collected and applied for reversed HPLC
analysis. Water and acetonitrile were used as the mobile phase with
a flow rate (1 ml/min). The newly generated peak was collected for
ESI-MS analysis directly.
4.4. Measurement of absorption and fluorescence spectra of probes
KTcr-I and KTcr-II
A total reaction volume of 50 mL was set for the reaction. When
the reaction was complete, absorption and fluorescence spectra
were measured. The absorbance spectra were collected between
Fig. 4. (A) Fluorescence detection of KTcr-I with different HDACs. (B) Fluorescence
3
00 and 550 nm. The fluorescence spectra were collected between
10 and 600 nm with excitation wavelength of 480 nm and slit
width of 5 nm.
detection of KTcr-II with different HDACs. Reaction condition: probe, 10
mM (10
mM,
5
prepared from 5 mM stock in DMSO); HDACs, 40 ng/
HEPES buffer (20 mM, pH ¼ 8.0).
ml;
l
ex ¼ 480 nm;
l
em ¼ 545 nm in
4
.5. 6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-((tert-
envision that the probes will provide useful tools for further
delineating the roles of decrotonylation enzymes in biology.
butoxycarbonyl)amino)hexanoic acid (1)
N, N-Diisopropylethylamine (DIEA, 517 mg, 4 mmol) was added
to the solution of Fmoc N-hydroxysuccinimide ester (607 mg,
4
. Experimental section
1.8 mmol) and Na-(tert-Butoxycarbonyl)-lysine (493 mg, 2 mmol)
4
.1. Materials and instruments
in anhydrous dichloromethane, and the reaction was performed
overnight at room temperature. After completion of the reaction
and solvent removal under vacuo, the residue was purified by flash
chromatography (EA/MeOH, 100/1) to afford the product 1 as a
Starting materials were obtained from commercial vendors and
used directly. The anhydrous solvents used were purchased from
J&K company or produced as common procedures. To monitor
organic reactions, thin layer chromatography (TLC) analysis by UV
or ninhydrin stain visualization were conducted using pre-coated
1
light-yellow liquid (413 mg, 49% yield). H NMR (DMSO, 400 MHz)
(ppm): 12.41 (s, 1H), 7.89 (d, J ¼ 8.0 Hz, 2H), 7.68 (d, J ¼ 8.0 Hz, 2H),
7.41 (t, J ¼ 8.0 Hz, 2H), 7.33 (t, J ¼ 8.0 Hz, 2H), 7.28 (t, J ¼ 4.0 Hz, 1H),
7.04 (d, J ¼ 8.0 Hz, 1H), 4.29 (d, J ¼ 8.0 Hz, 2H), 4.20 (t, J ¼ 8.0 Hz,
silica plates (Merck 60 F254 nm, 250
mm thickness). Compound
13
purification was performed by flash column chromatography using
silica gel (Merck 60 F254 nm, 70e200 mesh). 1H NMR, C NMR
spectra were analyzed with Bruker 300 MHz/400 MHz NMR
spectrometers. Data are reported with chemical shift values in ppm
1H), 3.84e3.79 (m, 1H), 2.97e2.93 (m, 2H), 1.66e1.37 (m, 15H).
C
13
NMR (DMSO,100 MHz) (ppm): 174.7, 156.5,156.1, 144.4, 141.2, 128.1,
127.5, 125.6, 120.6, 78.4, 65.6, 60.2, 47.2, 31.2, 30.9, 29.4, 28.7, 23.4.
-
ESI-MS calcd for [M ꢂ H] 467.23; Found 467.60.
5

Y. Xie, L. Yang, Q. Chen et al.
European Journal of Medicinal Chemistry 212 (2021) 113120
4
.6. (9H-fluoren-9-yl)methyl tert-butyl (6-((2-(2-hydroxyethoxy)
(t, J ¼ 4.0 Hz, 2H), 1.83 (d, J ¼ 8.0 Hz, 3H), 1.74e1.40 (m, 15H). 13C
ethyl)amino)-6-oxohexane-1,5-diyl)dicarbamate (2)
3
NMR (CD OD, 100 MHz) (ppm): 175.5, 168.7, 157.8, 155.9, 146.9,
1
45.6, 140.7, 136.2, 131.0, 126.2, 106.8, 80.6, 71.8, 71.0, 69.9, 56.2,
0
þ
N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochlo-
40.4, 40.0, 33.2, 30.1, 28.8, 24.3, 17.8. HRMS calcd for [MþNa]
ride (230 mg, 1.2 mmol) and N-Hydroxysuccinimide (127 mg,
587.2441; Found 587.2428.
1.1 mmol) were added to a solution of 1 (413 mg, 0.88 mmol) in
anhydrous dichloromethane (2 ml), and the reaction was per-
4.10. 2-amino-4-methylpentanoic acid (5)
formed for 2 h at room temperature. N, N-Diisopropylethylamine
(
1
336 mg, 2.6 mmol) and 2-(2-Aminoethoxy)ethanol (116 mg,
.1 mmol) were then added, and the mixture was reacted overnight
A solution of Fmoc-Leu-OH (3 g, 8.5 mmol) in 20% piperidine in
DMF was reacted at room temperature for 1 h and monitored with
TLC. After reaction finish, cold ethyl ether was added to wash the
furtherly. The solvent was removed under vacuo, the residue was
purified by flash chromatography (DCM/MeOH, 50/1) to afford the
reaction residues and obtained the pure product 5 (1.1 g, 100%
1
1
product 2 as a colorless liquid (254 mg, 52% yield). H NMR (CD
3
OD,
yield). H NMR (CD
3
OD, 400 MHz)
d
(ppm): 3.54e3.51 (m, 1H),
4
00 MHz) (ppm): 7.78 (d, J ¼ 8.0 Hz, 2H), 7.63 (d, J ¼ 8.0 Hz, 2H),
1.79e1.75 (m, 2H), 1.65e1.58 (q, J ¼ 8.0 Hz, 1H), 0.98 (t, J ¼ 8.0 Hz,
7
4
3
.38 (t, J ¼ 8.0 Hz, 2H), 7.30 (t, J ¼ 8.0 Hz, 2H), 4.33 (d, J ¼ 8.0 Hz, 2H),
6H). ESI-MS calcd for [MþNa]þ 154.09; Found 154.3.
.17 (t, J ¼ 8.0 Hz, 1H), 3.99e3.98 (m, 1H), 3.64 (t, J ¼ 4.0 Hz, 2H),
.55e3.47 (m, 4H), 3.37 (t, J ¼ 4.0 Hz, 2H), 3.12e3.08 (m, 2H),
4.11. 2-acetamido-4-methylpentanoic acid (6)
13
3
1.73e1.42 (m, 15H). C NMR (CD OD, 100 MHz) (ppm): 175.4,159.0,
157.9, 145.4, 142.7, 128.8, 128.2, 126.2, 121.0, 80.7, 73.5, 70.6, 67.7,
2
To a solution of Leu-OH (5, 1.1 g, 8.5 mmol) in 70% THF/H O
6
2.3, 56.2, 41.5, 40.4, 33.2, 30.9, 30.6, 28.8, 24.1. ESI-MS calcd for
(80 ml) was added acetic anhydride dropwise and slowly in ice-
batch. The mixtures were monitored with TLC. After reaction fin-
þ
[MþNa] 578.29; Found 578.6.
ish, the volatile was removed to obtain the pure product 6 in a
1
4.7. tert-butyl (6-amino-1-((2-(2-hydroxyethoxy)ethyl)amino)-1-
quantitative yield. H NMR (DMSO, 400 MHz)
d
(ppm): 12.48 (br,
oxohexan-2-yl)carbamate (3)
1H), 8.09 (d, J ¼ 8.0 Hz, 1H). 4.21e4.16 (m, 1H), 1.83 (s, 3H),
13
1
.67e1.57 (m, 1H), 1.50e1.47 (m, 2H), 0.89e0.83 (m, 6H). C NMR
A solution of 2 (254 mg, 0.46 mmol) in piperidine/DCM (1:1,
ml) was reacted at room temperature and monitored with TLC.
After completion of the reaction, cold ethyl ether was added to
(DMSO, 100 MHz) (ppm): 174.3, 169.2, 50.1, 24.3, 22.8, 22.3, 21.3.
ESI-MS calcd for [M ꢂ H] 172.11; Found 172.10.
-
1
wash the reaction residues and obtained the product 3 as a light
4.12. 2-(2-acetamido-4-methylpentanamido)acetic acid (7)
1
yellow oil (114 mg, 75% yield). H NMR (CD
3
OD, 400 MHz)
d
(ppm):
4
3
9
.02e3.94 (m, 1H), 3.63 (t, J ¼ 4.0 Hz, 2H), 3.50 (t, J ¼ 4.0 Hz,4H),
It was synthesized on a 0.5 mmol scale Fmoc-Gly-Wang resin
(capacity, 0.41 mmol/g). To the swelled and pre-deprotected Gly-
Wang resin in DMF were added 2-(1H-benzotriazole-1-yl)-1,1,3,3-
.38e3.32 (m, 2H), 2.91 (t, J ¼ 4.0 Hz, 2H), 1.71e1.53 (m, 6H), 1.40 (s,
H). ESI-MS calcd for [MþH]þ 334.23; Found 334.6.
tetramethyluroniumhexafluorophosphate
(HBTU,
948
mg,
4.8. (E)-tert-butyl (6-(but-2-enamido)-1-((2-(2-hydroxyethoxy)
2.5 mmol), 1-Hydroxybenzotriazole hydrate (HOBt, 338 mg,
ethyl)amino)-1-oxohexan-2-yl)carbamate (4)
2.5 mmol), 6 (433 mg, 2.5 mmol) and N, N-Diisopropylethylamine
(DIEA, 646 mg, 5 mmol). The mixtures were reacted for 2 h, fol-
3
(83 mg, 0.25 mmol) was dissolved in DMF/acetonitrile mixture
lowed by cleavage by trifluoroacetic acid, precipitation in ethyl
ether to obtain the crude peptide. With further purification by
(
1:3, 1.2 ml) in ice-bath. To this solution was added crotonic an-
hydride (50 mg, 0.32 mmol) slowly. After completion of the reac-
tion and removal of the solvents, the crude product was purified
preparative HPLC, the pure product 7 was obtained (60 mg, 52%
1
yield). H NMR (CD
3
OD, 400 MHz)
d
(ppm): 4.44e4.41 (m, 1H),
with column chromatography (DCM/MeOH, 100/1e30/1) to yield 4
3.95e3.82 (m, 2H), 1.98 (s, 3H), 1.70e1.65 (m, 1H), 1.63e1.53 (m,
1
-
(
(
(
49 mg, 49% yield). H NMR (CD
3
OD, 400 MHz)
d
(ppm): 6.80e6.70
2H), 0.96e0.91 (m, 6H). ESI-MS calcd for [M ꢂ H] 229.13; Found
m, J
m,1H), 3.65 (t, J ¼ 4.0 Hz, 2H), 3.52 (t, J ¼ 4.0 Hz, 4H), 3.42e3.35 (m,
H), 3.21 (t, J ¼ 4.0 Hz, 2H), 1.83 (d, J ¼ 4.0 Hz, 3H), 1.76e1.43 (m,
1
¼ 4.0 Hz, J
2
¼ 16.0 Hz, 1H), 5.91 (d, J ¼ 16.0 Hz, 1H), 4.02e3.94
229.2.
2
4.13. 2-amino-6-(but-2-enamido)-N-(2-(2-hydroxyethoxy)ethyl)
hexanamide (8)
13
15H). C NMR (CD
3
OD, 100 MHz) (ppm): 175.3, 168.6, 157.8, 140.6,
1
1
26.2, 80.6, 73.5, 70.5, 62.3, 56.1, 40.4, 40,1, 33.2, 30.2, 28.8, 24.3,
7.9. ESI-MS calcd for [MþNa]þ 424.25; Found 424.5.
A solution of 4 (70 mg, 0.17 mmol) in trifluoroacetic acid/DCM
(1:1) was reacted at room temperature and monitored with TLC.
4.9. (E)-tert-butyl (6-(but-2-enamido)-1-((2-(2-((7-nitrobenzo[c]
After reaction finish, cold ethyl ether was added to wash the re-
[
1,2,5]oxadiazol-4-yl)oxy)ethoxy)ethyl)amino)-1-oxohexan-2-yl)
action residues two times and the pure product 8 was obtained in
1
carbamate (KTcr-I)
quantitative yield. H NMR (CD
3
OD, 400 MHz)
d
(ppm): 6.81e6.72
(
m, J
1
¼ 8.0 Hz, J ¼ 16.0 Hz, 1H), 5.92 (d, J ¼ 16.0 Hz, 1H), 4.52e4.50
2
NBD-F (29 mg, 0.16 mmol) and N, N-Diisopropylethylamine
(m, 1H), 3.85e3.75 (m, 2H), 3.68e3.52 (m, 4H), 3.46e3.37 (m, 2H),
3.23 (t, J ¼ 8.0 Hz, 2H), 1.83 (m, J ¼ 8.0 Hz, 5H), 1.59e1.52 (m, 2H),
1.44e1.37 (m, 2H). ESI-MS calcd for [MþH]þ 302.20; Found 302.60.
(
0
40 mg, 0.31 mmol) were added to a solution of 4 (49 mg,
.12 mmol) in anhydrous DCM/DMF (3:1). The mixture was stirred
overnight at room temperature. After completion of the reaction
and solvent removal under reduced vacuo, the residue was purified
by flash column chromatography (DCM/MeOH, 50/1e20/1), fol-
4.14. 2-(2-(2-acetamido-4-methylpentanamido)acetamido)-6-((E)-
but-2-enamido)-N-(2-(2-hydroxyethoxy)ethyl)hexanamide (9)
lowed by preparative TLC, to afford the product KTcr-I as a yellow
1
solid (16 mg, 24% yield). H NMR (CD
(
3
OD, 400 MHz)
d
(ppm): 8.64
¼ 8.0 Hz,
1-Hydroxybenzotriazole hydrate (HOBt, 18 mg, 0.13 mmol) and
N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride
(EDCI, 25 mg, 0.13 mmol) were added to a solution of compound 7
(22 mg, 0.1 mmol) in anhydrous DMF (0.5 ml), and the mixture was
0
d, J ¼ 8.0 Hz, 1H), 6.99 (d, J ¼ 8.0 Hz, 1H), 6.77e6.67 (m, J
1
J
2
¼ 16.0 Hz, 1H), 5.88 (d, J ¼ 16.0 Hz, 1H), 4.57 (t, J ¼ 4.0 Hz, 2H),
3
.98e3.94 (m, 3H), 3.65 (t, J ¼ 4.0 Hz, 2H), 3.49e3.33 (m, 2H), 3.17
6

Y. Xie, L. Yang, Q. Chen et al.
European Journal of Medicinal Chemistry 212 (2021) 113120
stirred for 1.5 h at room temperature. After activation, 8 (33 mg,
Appendix A. Supplementary data
0
.11 mmol) and N, N-Diisopropylethylamine (DIEA, 39 mg,
0
.3 mmol) were then added, and the residues were stirred over-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113120.
night furtherly. After completion of the reaction and solvent
removal under vacuo, the residue was purified by flash chroma-
tography (DCM/MeOH, 30/1e10/1) to afford the product 9 (27 mg,
5
3% yield). 1H NMR (CD
¼16.0 Hz, J ¼ 4.0 Hz, 1H), 6.03 (d, J ¼ 16.0 Hz, 1H), 4.39e4.30 (m,
3
OD, 400 MHz) d (ppm): 6.87e6.78 (m,
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[
þ
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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This work was supported by the Science Technology and Inno-
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vation
Committee
of
Shenzhen
Municipality
297.
(
JCYJ20180507181654823, JCYJ20170413141047772), the National
[
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Natural Science Foundation of China (21778044) and Sichuan Science
and Technology Program (2018JY0360).
7