Design, synthesis, and use of novel photoaffinity probes in measuring the serum concentration of glycogen phosphorylase

Article abstract of DOI:10.3390/molecules24040798

A procedure to measure the serum concentration of glycogen phosphorylase during acute myocardial infarction is presented. This method was based on the synthesis of photoaffinity probes, and used the semiquantitative protein electrophoretic mobility shift technique. Three novel photoaffinity probes bearing different secondary tags were synthesized. Their potency was evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa). The inhibitory activity of probe 1 was only 100-fold less potent than the mother compound CP-320626. The photoaffinity labeling experiments were also performed, and a protein with molecular weight (MW) of about 90–100 kDa, which was consistent with the MW of GP, was clearly labeled by probe 1. A semiquantitative evaluation of the GP level in serum with probe 1 was also performed. The results showed that the protein band with a MW of about 90–100 kDa was tagged, and the concentration of the protein in serum was found to be between 25 and 50 ng/mL. Mass spectrometric analysis revealed that alpha-1,4 glucan phosphorylase (GPMM) was well-preserved in the bands.

Full text of DOI:10.3390/molecules24040798

molecules
Article
Design, Synthesis, and Use of Novel Photoaffinity
Probes in Measuring the Serum Concentration of
Glycogen Phosphorylase
1
1
1
2
1
Yuchao Zhang , Youde Wang , Zhiwei Yan , Chengjun Song , Guangxin Miao and
1
,
Liying Zhang *
1
Key Laboratory of Traditional Chinese Medicine Research and Development of Hebei Province,
Institute of Traditional Chinese Medicine, Chengde Medical University, Chengde 067000, China;
zyc9021@126.com (Y.Z.); wangyoude8686@126.com (Y.W.); cdyanzhiwei@163.com (Z.Y.);
mgx8088@163.com (G.M.)
Department of Human Anatomy, Chengde Medical University, Chengde 067000, China;
songchengjun@126.com
2
*
Correspondence: ly1981ly@cdmc.edu.cn; Tel.: +86-314-229-1000
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 30 December 2018; Accepted: 20 February 2019; Published: 22 February 2019
Abstract: A procedure to measure the serum concentration of glycogen phosphorylase during
acute myocardial infarction is presented. This method was based on the synthesis of photoaffinity
probes, and used the semiquantitative protein electrophoretic mobility shift technique. Three novel
photoaffinity probes bearing different secondary tags were synthesized. Their potency was
evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa).
The inhibitory activity of probe
The photoaffinity labeling experiments were also performed, and a protein with molecular weight
MW) of about 90–100 kDa, which was consistent with the MW of GP, was clearly labeled by probe
A semiquantitative evaluation of the GP level in serum with probe was also performed. The results
1 was only 100-fold less potent than the mother compound CP-320626.
(
1.
1
showed that the protein band with a MW of about 90–100 kDa was tagged, and the concentration
of the protein in serum was found to be between 25 and 50 ng/mL. Mass spectrometric analysis
revealed that alpha-1,4 glucan phosphorylase (GPMM) was well-preserved in the bands.
Keywords: CP-320626; glycogen phosphorylase; acute myocardial infarction; semiquantitative
protein electrophoretic mobility shift technique; photoaffinity probe
1
. Introduction
Early identification and confirmation of myocardial injury is essential for correct patient care
and disposition decision in the emergency department. In this respect, glycogen phosphorylase (GP)
has become an enzyme for early laboratory detection of ischemia based on its metabolic function [ ].
1
Three GP isoenzymes are present in human tissue. These are named according to the tissue of their
initial description: GPLL (liver), GPMM (muscle), and GPBB (brain). Heart muscle contains GPMM
and GPBB in high concentrations. During myocardial ischemia, activation of GP results in an increase
in glycogen degradation. Thus, GP is released from glycogen and then enters the bloodstream, which
is believed to occur via the T-tubulus system [
diagnosis of acute myocardial infarction (AMI) within 4 h after the onset of chest pain [
also considered a useful marker for the detection of perioperational myocardial ischemia and early
reperfusion [ ]. In addition, also it is considered to be a specific marker for detection of perioperative
myocardial injury in patients undergoing coronary artery bypass grafting [2].
2]. It is considered the most sensitive marker for the
3]. It is
4
Molecules 2019, 24, 798; doi:10.3390/molecules24040798
www.mdpi.com/journal/molecules

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Clinical detection of GP requires rapid and reliable test systems. Immunoassay methods and
chemical approaches have been developed to determine plasma GP concentrations during hypoxia and
ischemia injury in the heart [5,6]. Although these studies have been somewhat successful, they have
not yet reached definitive conclusions to allow the use of these approaches in routine determinations.
This is because most studies have only examined the measurement of GPBB, but not the efflux of
GPBB and GPMM. Continuous efforts for the development of methods that can accurately detect and
quantify the amounts of GP in plasma during acute myocardial infarction are becoming more and
more pressing.
Here we describe the development of a photoaffinity labeling method for the estimation of
GP. This method is based on the synthesis of photoaffinity probes and use of the semiquantitative
protein electrophoretic mobility shift technique [
7]. The probes were designed based on CP-320626
(
Figure 1), a high-affinity inhibitor of GP [ ]. X-ray crystallographic imaging of the complex of
8
CP-320626 with GP showed that the 5-chloro-1H-indole-2-carboxamide moiety had appropriate
interactions with GP at the dimer interface site. The 4-hydroxy-piperidyl moiety exhibited relatively
few Van der Waals interactions with the protein [9]. Therefore, chemical modification of CP-320626
was attempted by introducing a spacer arm in the hydroxy moiety to create a viable protein (GP)
scaffold. The benzophenone moiety was incorporated as the photochemically reactive group because
of its excellent chemical stability in solvents and easy preparation [10]. While a biotin tag is ideal
for affinity tagging purposes, its use is not optimal for higher throughput activity-based profiling
due to the time and labor involved in producing western blots. Additionally, many cells and tissues
contain endogenously biotinylated proteins that complicate analysis of biotinylated probe-based
western blots [11]. To circumvent this shortcoming, we synthesized a fluorophore-tagged probe
which possessed a dansyl group that exhibited relatively intense fluorescence in water [12]. For the
fluorescent probe, we added a biotin group to the structure of probe 1, creating a dual function probe
making it suitable for both affinity purification and fluorescence applications [13]. Moreover, in order
to avoid the steric hindrance caused by the reporter tag, we designed another tag-free probe that
1,
2
,
3
employed an azide handle for downstream conjugation of the reporter tag, via the click chemistry
reaction after proteome labeling [14].
Figure 1. Our design strategy for photoaffinity probes.
Copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a widely utilized, reliable, and
straightforward way for making covalent connections between building blocks containing various
functional groups. It has been used in organic synthesis, medicinal chemistry, surface and polymer
chemistry, and bioconjugation applications. In the present study, we designed and synthesized a series
of activity-based probes based on CP-320626 with a CuAAC reaction (Figure 2).

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Figure 2. Chemical structures of probes 1–3.
2
. Results and Discussion
2
.1. Chemistry
Introduction of an azide moiety into the structure of CP-320626 is summarized in Scheme 1.
◦
Reaction of tetraethylene glycol (
gave tosyl ester in 60% yield. The hydroxyl group of
with dihydropyran catalyzed by PPTS to afford the THP ether
tert-butyl 4-hydroxypiperidine-1-carboxylate using sodium hydride as base produced the desired ether
derivative . Subsequent deprotection with 3 M HCl afforded in excellent yield, which in turn was
reacted with Boc-L-4-fluorophe in the presence of HATU and DIPEA to deliver piperidineamide
Removal of the N-Boc group from with 3 M HCl furnished intermediate 10, and was directly reacted
with 5-chloroindole-2-carboxylic acid in the presence of HATU and Et N, resulting in alcohol 11 in 69%
4
) with p-toluenesulfonyl chloride (TsCl) in CH Cl at 0 C
2
2
5
5
was protected in the usual manner,
(71% yield). Treatment of with
6
6
7
8
9.
9
3
yield. Treatment of 11 with methanesulfonyl chloride (MsCl) and Et N afforded mesylate 12, which
3
was converted to intermediate 13 via a nucleophilic substitution reaction with sodium azide.

Molecules 2019, 24, 798
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◦
Scheme 1. Reagents and conditions: (a) TsCl, Et N, CH Cl , 0 C, 60%; (b) DHP, PPTS, CH Cl ,
3
2
2
2
2
room temperature, 71%; (c) tert-Butyl 4-hydroxypiperidine-1-carboxylate, NaH, THF, room temperature,
◦
◦
5
5
0
1%; (d) 3 M HCl/MeOH, 0 C to room temperature;
€
Boc-L-4-Fluorophe, HATU, DIPEA, DMF, 45 C,
◦
1%; (f) 5-Chloroindole-2-carboxylic acid, HATU, DIPEA, DMF, 45 C, 73%; (g) MsCl, Et N, CH Cl ,
C to room temperature, 89%; and (h) NaN , DMF, 60 C, 70%.
3
2
2
◦
◦
3
Click chemistry was handled as shown in Scheme 2. The alkyne 16 bearing a benzophenone
photophore and a fluorescent tag was readily prepared in five steps starting from Lys(Boc)-OMe (14
and 4-benzoylbenzoic acid (15) [15]. The corresponding azide intermediate 13 and alkyne 16 were
dissolved in CH Cl -H O, followed by the addition of catalytic sodium ascorbate and CuSO4·5H O,
)
2
2
2
2
resulting in triazole probe 1 in 11% yield.

Molecules 2019, 24, 798
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O
O
H
N
O
O
O
NH Cl
3
O
O
N
a
ref.14
+
COOH
HN
NHBoc
S
O
O
14
15
16
N
N
O
N
O
O
O
O
NH
O
O
O
N
O
O
H
O
S
N
N
H
O
NH
Cl
F
N
1
Scheme 2. Reagents and conditions: (a) 13, CuSO4 5H O, sodium ascorbate, CH Cl -H O, room
·
2 2 2 2
temperature, 11%.
The synthesis of the final probe
dual-labeled alkyne 18 starting from N-Cbz-N -Boc-L-lysine 17
2
is shown in Scheme 3. It took seven steps to prepare the
0
[
15]. It was similar to probe
1
, where the
reaction of the aforementioned azide 13 with the dual-labeled alkyne 18 resulted in probe
2
in 12% yield.
O
O
O
O
O
H
N
S
N
H
N
H
O
H
O
H
H
NH
O
N
HN
OH
ref.14
O
N
O
HN
O
S
O
NHBoc
O
18
17
H
O
N
O
N
HN
N
N
O
O
O
HN
S
O
O
O
O
O
a
N
O
O
HN
H
N
N
N
O
H
S
Cl
F
H
H
NH
HN
2
O
Scheme 3. Reagents and conditions: (a) 13, CuSO4 5H O, sodium ascorbate, CH Cl -H O, room
·
2 2 2 2
temperature, 12%.

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The tag-free probe
3
was also prepared as shown in Scheme 4. According to the reported method,
synthesis of the corresponding chloroacetyl compound 19 was based on Lys(Boc)-OMe (14) as well as
-benzoylbenzoic acid (15) [15]. Then, probe was synthesized by the CuI-catalyzed Huisgen (2 + 3)
4
3
dipolar cycloaddition reaction (click chemistry), followed by a nucleophilic substitution reaction with
sodium azide.
Scheme 4. Reagents and conditions: (a) 13, CuSO4·5H O, sodium ascorbate, CH Cl -H O, room
2
2
2
2
◦
temperature, 58%; and (b) NaN , DMF, 60 C, 50%.
3
2
.2. Enzyme Assay and SAR Analysis
To understand the validity of the synthetic probes, the synthesized probes 1–3 were evaluated in
an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa). As described
previously, the activity of RMGPa was measured through detecting the release of phosphate from
glucose-1-phosphate in the direction of glycogen synthesis, based on the published method. Caffeine,
a known allosteric GP inhibitor that shares the same binding site with CP-320626, was used as a
positive control [16]. The bioassay results are summarized in Table 1. Probes
activity against RMGPa. As expected, the steric interference was crucial for the inhibitory effect.
In contrast, probe inhibited RMGPa with an IC50 value of 13.20 M, and was approximately 100-fold
2 and 3 had no inhibitory
1
µ
less potent than that of CP-320626. To our surprise, the incorporation of the azide moiety resulted
in a diminished activity in the assay. Due to the lack of a binding mode of chemical probes with
GP, it is impossible to estimate how the introduction of different functional elements affected the
three-dimensional conformation of GP. We only confirm that there were significant differences in the
regulation of the active state of the GP enzyme between probe 3, containing the azide group, and probe
1
without the azide group.
Table 1.
Inhibition of rabbit muscle glycogen phosphorylase a (RMGPa) by probes 1–3,
and intermediates.
Compound
RMGPa IC50 a (µM)
13.20 ± 5.87
Compound
RMGPa IC50 (µM)
1
2
3
11
0.43 ± 0.16
0.11 ± 0.02
b
NI
CP-320626
c
NI
Caffeine
101.23 ± 13.18
a
S.D. of three determinations. b NI means no inhibition. c Caffeine was used as
Each value represents the mean
the positive control.
±

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2
.3. Labelling of Serum Proteomes with Probe 1
Based on the results of the potency in the enzyme assay, probe
1
was selected and used throughout
photolabeling studies (Figure 3). The serum proteomes prepared from rats with acute myocardial
infarction were incubated with probe at concentrations of 10, 3, and 1 mM, and 300, 100, and 30 nM,
1
respectively. The mixture was exposed to UV light for 30 min, and then was imaged using the
Tanon-6100 Chemiluminescent Imaging system. The label transfer analysis is shown in Figure 3.
Interestingly, using the new photoprobe 1, the protein band with a MW of about 90–100 kDa, which was
consistent with the MW of GP, was clearly labelled. These results demonstrated that the synthesized
probe 1 might be used to label the GP in serum.
Figure 3. Gel-based fluorescence image after labeling of serum proteomes with various concentrations
of probe 1. Samples were prepared by incubating 2.0 mg/mL proteomes at different conditions:
Lane 1—Marker; Lanes 2–7 with 10, 3, and 1 mM, and 300, 10 and 30 nM probe 1, and exposed to UV
light for 30 min.
2
.4. Semi-Quantitative Analysis of Serum-Free Glycogen Phosphorylase (GP) Levels with Probe 1
An attempt to semi-quantitatively analyze the serum-free GP level after an acute myocardial
infarction was explored. Serum blanks were added in increasing quantities of RMGPa (0, 12.5,
5, 50, and 100 ng/mL at a final concentration) in the presence of an excess of probe (10 M).
In the same way, probe was added to the serum in an acute myocardial infarction model in a
2
1
µ
1
rat. All mixtures were exposed to UV light for 30 min, and then were imaged using the Tanon-6100
Chemiluminescent Imaging system. The label results are presented in Figure 4. These data demonstrate
that the synthesized probe
1 efficiently labeled the protein bands with a MW of about 90–100 kDa,
which was consistent with the MW of GP. The standard curve method was applied to detect the
concentration of the protein’s MW consistent with the MW of GP (Figure 4, Lane 2). Figure 5 shows the
standard curve of the protein concentration. Corresponding to the standard curve, the concentration
of the protein’s MW consistent with the MW of GP was found to be between 25 and 50 ng/mL.
For a more detailed proteomic analysis, we then used the mass spectrometric analysis method to
detect the protein bands with a MW of about 90–100 kDa. Several proteins were identified, and
the results are summarized in Table 2. This analysis revealed that the GPMM was well-preserved.

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However, it was unavoidable that the gel had typical high-abundance proteins such as cytoskeletal
protein, deoxynucleotidyltransferase terminal, interacting protein, Meiosis 1-associated protein,
Cation-transporting ATPase, etc. Interestingly, a homologous protein, alpha-1,4 glucan phosphorylase,
appeared in the search results in Table 2. The detailed results of the LC-MS/MS analysis are in Figure 6.
The parent ion with the highest protein percent coverage (74%), alpha-1,4 glucan phosphorylase
(GPMM), was selected for fragmentation and MS/MS analysis. The origin of sequence ions from
the protein indicated that variable modifications of GPMM, such as the acetyl of protein N-term and
oxidation (M), was presented.
Figure 4. Gel-based fluorescence image of the semiquantitative analysis with 10
Samples were prepared by incubating 2.0 mg/mL proteomes at different conditions: Lane 1—Marker;
Lane 2—Serum in a rat acute myocardial infarction model treated with probe ; Lanes 3–7—Serum
µM of probe 1.
1
blanks with increasing quantities of RMGPa (0, 12.5, 25, 50, and 100 ng/mL at a final concentration)
treated with probe 1.

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Figure 5. Determination the content of glycogen phosphorylase (GP) by the standard curve method.
Table 2. Comparative analysis of protein groups identified by LC-MS/MS.
Sequence
Coverage [%]
Mol. Weight
[kDa]
Protein
Peptides
10
Score
Alpha-1,4 glucan phosphorylase, Glycogen
phosphorylase, muscle form
1
74
97.288
66.017
60.044
323.31
116.05
57.792
Keratin, type II cytoskeletal 1
13
9
22.4
17.9
Keratin, type II cytoskeletal 6A; Keratin, type
II cytoskeletal 6C; Keratin, type II cytos
Keratin, type I cytoskeletal 16
Keratin, type I cytoskeletal 9
7
5
16.7
9.3
9.8
15.5
18
51.267
62.129
59.51
43.524
37.094
33.026
30.372
17.781
7.2216
6.1512
Keratin, type I cytoskeletal 10; Keratin 10
Keratin, type II cytoskeletal 2 epidermal
Alpha-1,4 glucan phosphorylase
6
8
65.865
95.794
62.378
253.76
17
5
Keratin, type II cytoskeletal 5
8.6
0.4
ATP binding cassette subfamily A member 1
1
Deoxynucleotidyltransferase terminal
interacting protein 1
1
2.7
37.042
6.0048
Meiosis 1 associated protein
Cation-transporting ATPase
1
1
2.4
1.1
59.223
138.21
5.9054
5.7721

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Figure 6. LC-MS/MS analysis of the Alpha-1,4 glucan phosphorylase (Glycogen phosphorylase,
muscle form) labeled by probe 1.

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3
. Materials and Methods
3
.1. Chemistry
All commercially available solvents and reagents were used without further purification.
Melting points were uncorrected. NMR experiments were performed on Bruker Avance III 400
MHz and Bruker Fourier 300 MHz (Bruker Corporation, Billerica, MA, USA). The spectra were
referenced internally according to residual solvent signals of TMS (
ion LCMS data were obtained at 303 K by a quadrupole mass spectrometer Agilent LC/MSD 1200
Series (Agilent technologies Inc, Palo Alto, CA, USA) using a 50 4.6 mm (5 m) ODS column.
Reversed-phase-HPLC experiments were performed by flash welchrom C18 column (150 20 mm)
δ = 0.00 ppm). Positive or negative
×
µ
×
chromatography (Agela Technologies, Tianjin, China). The NMR spectrum of compounds were shown
in the Supplementary Flies (Figures S1–S11).
3
.1.1. Ethyl 2-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-4-methylbenzenesulfonate (5)
Under an ice bath, TsCl (40 mL, 7.93 g, 0.042 mol) was slowly added in groups to a mixture of
tetraethylene glycol (9.0 mL, 0.05 mol) and Et N (18.05 mL, 0.13 mol) in anhydrous CH Cl (60 mL).
3
2
2
◦
The reaction mixture was stirred at 0 C overnight. Then, the reaction mixture was diluted with CH Cl
2
2
and washed with water, 1 M aqueous HCl, and saturated NaHCO and brine, and then dried and
3
evaporated over anhydrous Na SO . The residue was purified by column chromatography on silica
2
4
gel [EtOAc-petroleum ether (35–90%)] to give the product (8.77 g, 60%) of colorless oil. HPLC analysis
1
was as follows: 99.0%; H-NMR (CDCl , 400 MHz)
δ
7.80 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H),
3
4
3
.17 (t, J = 4.8 Hz, 1H), 3.72–3.68 (m, 4H), 3.66–3.62 (m, 4H), 3.61–3.58 (m, 6H), 2.64 (s, 2H), and 2.45 (s,
13
H); C-NMR (CDCl , 100 MHz)
δ 144.9, 132.9, 129.8, 128.0, 72.5, 70.7, 70.6, 70.4, 70.3, 69.3, 68.7, 61.7,
3
and 21.6.
3
.1.2. Ethyl 2-[2-(2-{2-[(tetrahydro-2H-pyran-2-yl)oxy]ethoxy}ethoxy)ethoxy]4-methylbenzenesulfonate (
A mixture of Pyridinium 4-toluenesulfonate (PPTS, 0.73 g, 2.90 mmol) and 3,4-2H-dihydropyran
3.93 mL, 43.11 mmol) in CH Cl (50 mL) was added to a solution of compound (10.0 g, 28.74 mmol)
in anhydrous CH Cl (50 mL). The reaction mixture was stirred at room temperature for 5 h. Then,
6)
(
5
2
2
2
2
the reaction mixture was diluted with CH Cl and washed with water and saturated brine, dried over
2
2
anhydrous Na SO , and evaporated. The residue was purified by column chromatography on silica
2
4
gel [EtOAc-petroleum ether (15–50%)] to give the product (8.83 g, 71%) of colorless oil. HPLC analysis
1
was as follows: 90.1%; H-NMR (CDCl , 400 MHz)
δ
7.80 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H),
3
4
(
.62 (t, J = 3.2 Hz, 1H), 4.18–4.15 (m, 2H), 3.87–3.83 (m, 1H), 3.72–3.47 (m, 15H), 2.45 (s, 3H), 1.86–1.78
m, 1H), 1.75–1.68 (m, 1H), and 1.63–1.49 (m, 4H); 13C-NMR (CDCl , 100 MHz)
δ
144.8, 133.0, 129.8,
3
1
28.0, 99.0, 70.7, 70.64, 70.58, 70.53, 69.2, 68.7, 66.6, 62.2, 30.6, 25.4, 21.6, and 19.5.
3
.1.3. Tert-butyl 4-{2-[2-(2-{2-[(tetrahydro-2H-pyran-2-yl)oxy]ethoxy}ethoxy)ethoxy]ethoxy}piperidine
-
1-carboxylate (7)
A mixture of tert-butyl 4-hydroxypiperidine-1-carboxylate (0.50 g, 2.49 mmol) and NaH (0.3 g,
◦
0%, 7.47 mmol) in THF (35.0 mL) was stirred at 0 C for 1 h. Then, compound 6 (1.10 g, 2.55 mmol)
6
was slowly added to the reaction, which was stirred at room temperature overnight. The mixture
was diluted with water (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic phase
was washed with brine, dried over anhydrous Na SO , and evaporated. The residue was purified by
2
4
column chromatography on silica gel [EtOAc-petroleum ether (30–70%)] to give the product (0.58 g,
1
5
1%) of colorless oil. HPLC analysis was as follows: 96.8%; H-NMR (400 MHz, CDCl )
δ
4.63 (t,
3
J = 3.2 Hz, 1H), 3.90–3.84 (m, 2H), 3.79–3.76 (m, 2H), 3.69–3.63 (m, 15H), 3.51–3.45 (m, 2H), 3.09–3.02
m, 2H), 1.85–1.81 (m, 3H), 1.76–1.68 (m, 1H), 1.63–1.49 (m, 6H), and 1.45 (s, 9H); 1 C-NMR (CDCl3,
3
(
1
00 MHz) δ 154.8, 98.9, 79.4, 75.1, 70.8, 70.7, 70.6, 70.5, 67.4, 66.6, 62.2, 31.0, 30.6, 28.4, 25.4, and 19.5.

Molecules 2019, 24, 798
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3
.1.4. N-{(S)-1-[4-(2-{2-[2-(2-mesyloxyethoxy)ethoxy]ethoxy}ethoxy)piperidin-1-yl]-3-
(
4-fluorophenyl)-1-oxopropan-2-yl}-5-chloro-1H-indole-2-carboxamide (12)
Under an ice bath, methanesulfonyl chloride (0.02 mL, 0.25 mmol) was slowly added in groups to
a mixture of compound 11 (0.15 g, 0.24 mmol) and Et N (0.10 mL, 0.72 mmol) in anhydrous CH Cl
3
2
2
(25 mL). The reaction mixture was stirred at room temperature for 5 h. Then, the reaction mixture
was diluted with CH Cl and washed with water, 1 M aqueous HCl, and saturated NaHCO and
2
2
3
brine, and then dried and evaporated over anhydrous Na SO . The residue was purified by column
2
4
chromatography on silica gel [CH OH-CH Cl (0–2%)] to give the product (0.15 g, 89%) as a white
3
2
2
◦
1
solid. HPLC analysis was as follows: 100.0%; m.p. 130–132 C. H-NMR (400 MHz, CDCl )
δ
9.64
3
(d, J = 15.2 Hz, 1H), 7.58 (s, 1H), 7.52 (t, J = 6.8 Hz, 1H), 7.31 (t, J = 6.0 Hz, 1H), 7.21 (d, J = 6.8 Hz,
1
3
1
H), 7.16–7.14 (m, 2H), 6.95 (t, J = 6.8 Hz, 2H), 6.91–6.86 (m, 1H), 5.37–5.33 (m, 1H), 4.38–4.35 (m, 2H),
.84–3.74 (m, 3H), 3.66–3.59 (m, 14H), 3.49–3.25 (m, 2H), 3.15–3.05 (m, 5H), 1.83–1.50 (m, 3H), and
.34–1.29 (m, 1H); 13C-NMR (CDCl , 100 MHz)
169.2, 162.0 (d, J = 195.0 Hz), 160.5, 134.7, 131.8 (d,
J = 15.4 Hz), 131.5, 131.1, 131.0, 130.9, 128.6, 126.2, 125.0, 121.2, 115.5 (d, J = 3.4 Hz), 115.3 (d, J = 3.6 Hz),
δ
3
1
3
13.0 (d, J = 3.1 Hz), 73.5, 70.82, 70.76, 70.68, 70.5, 69.2, 69.0, 67.6, 50.0 (d, J = 7.7 Hz), 42.7, 39.3, 38.76,
8.47, 31.0 (d, J = 7.6 Hz), and 30.4 (d, J = 3.6 Hz); EIMS m/z 698.1 [M] .
+
3
.1.5. General Procedure for Synthesis of Compounds 8 and 10
Aqueous HCl (3 M, 2 mL) was slowly added to a solution of compound
7 or 9 (0.58 g, 1.26 mmol)
in methanol (15 mL) in an ice bath. The reaction mixture was stirred at room temperature for 6 h,
and then concentrated to give the crude target products
without further purification.
8 or 10, which were used for the next reaction
3
.1.6. General Procedure for Synthesis of Compounds 9 and 11
HATU (1.0 equiv., dissolved in 1.5 mL DMF) and DIPEA (3.0 equiv., dissolved in
.5 mL DMF) were added to a solution of N-(tert-Butoxycarbonyl)-4-fluoro-L-phenylalanine or
-chloroindole-2-carboxylicacid (1.0 equiv.) in anhydrous DMF (1.5 mL). The reaction mixture was
1
5
stirred at room temperature for 10 min. Then, compound
The mixture was stirred at 45 C for 5 h. After cooling to room temperature, the mixture was diluted
8 or 10 (1.0 equiv.) was added to the reaction.
◦
with water (30 mL) and extracted with EtOAc (30 mL
with brine (1 L 2), dried over anhydrous Na SO , and evaporated. The residue was purified by
reversed-phase chromatography (methanol-water (20–90%)) to give the product 9 or 11.
×
3). The combined organic phase was washed
×
2
4
(
S)-3-(4-Fluorophenyl)-1-{4-[2-(2-{2-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]ethoxy}ethoxy)ethoxy]piperidin-1
1
-yl}-2-(tert-butoxycarbonylamino)propan-1-one (
9
). HPLC analysis was as follows: 98.9%; H-NMR
(400 MHz, CDCl3)
δ 7.16–7.12 (m, 2H), 6.98–6.94 (m, 2H), 5.39 (t, J = 6.4 Hz, 1H), 4.84–4.79 (m, 1H),
3
.81–3.71 (m, 3H), 3.67–3.58 (m, 14H), 3.54–3.18 (m, 4H), 2.98–2.92 (m, 2H), 1.79–1.48 (m, 3H), 1.41 (d,
13
J = 3.6 Hz, 9H), and 1.35–1.26 (m, 1H); C-NMR (CDCl , 100 MHz)
δ 169.6, 161.9 (d, J = 194 Hz), 155.0,
3
1
32.3 (d, J = 18.4 Hz), 131.0 (d, J = 5.9 Hz), 130.9 (d, J = 6.0 Hz), 115.3 (d, J = 5.3 Hz), 115.1 (d, J = 5.2 Hz),
79.7, 73.7 (d, J = 5.2 Hz), 72.5, 70.81, 70.77, 70.65, 70.61, 70.3, 67.5, 61.7, 50.9, 42.6, 39.4, 39.1, 31.1(d,
J = 3.8 Hz), 30.4, and 28.3.
N-{(S)-1-[4-(2-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethoxy)piperidin-1-yl]-3-(4-fluorophenyl)-1-oxopropan-2
◦
-
yl}-5-chloro-1H-indole-2-carboxamide (11). HPLC analysis was as follows: 94.5%; m.p. 103–105 C.
1
H-NMR (400 MHz, CDCl3)
δ
11.70 (t, J = 8.4 Hz, 1H), 8.91–8.86 (m, 1H), 7.70 (s, 1H), 7.40 (d, J = 6.8 Hz
,
1
2
3
H), 7.34 (dd, J = 6.4, 4.4 Hz, 2H), 7.25 (s, 1H), 7.17 (dd, J = 6.8, 1.6 Hz, 1H), 7.05 (t, J = 6.8 Hz,
H), 5.15–5.10 (m, 1H), 4.55–4.49 (m, 1H), 3.98–3.79 (m, 1H), 3.73–3.65 (m, 1H), 3.52–3.48 (m, 14H),
.42–3.39 (m, 2H), 3.33–3.21 (m, 1H), 3.14–2.94 (m, 3H), 1.75–1.66 (m, 2H), and 1.39–1.19 (m, 2H);
1
3
C-NMR (CDCl , 100 MHz)
δ
169.4, 161.5 (d, J = 190.3 Hz), 160.6, 135.3, 134.4 (d, J = 7.5 Hz), 132.9,
3
131.7, 131.6, 128.5, 124.7, 124.0, 121.1, 115.3, 115.2, 114.3, 103.4, 74.6, 73.8, 72.8, 70.5, 70.3, 70.2, 67.2 (d,

Molecules 2019, 24, 798
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J = 6.1 Hz), 60.7, 50.6 (d, J = 9.6 Hz), 43.1, 42.7, 37.0 (d, J = 8.8 Hz), 31.7 (d, J = 33.1 Hz), and 31.1 (d,
+
J = 41.3 Hz). EIMS m/z 620.1 [M] .
3
.1.7. General Procedure for Synthesis of Compounds 3 and 13
NaN (3.0 equiv.) was added to a solution of compound 12 or 20 (1.0 equiv.) in anhydrous DMF
3
◦
(
10 mL). The reaction mixture was stirred at 60 C overnight. Then, the reaction mixture was poured
into ice water and extracted with EtOAc (30 mL
×
3). The combined organic phase was washed with
brine and dried over anhydrous Na SO . The residue was purified by column chromatography on
2
4
silica gel [CH OH-CH Cl (0–2%)] to give the product (0.10 g, 70%) as a thick solid.
3
2
2
N-{(S)-1-[4-(2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethoxy)piperidin-1-yl]-3-(4-fluorophenyl)-1-oxopropan-2-yl}
◦
1
-
(
2
3
1
1
5-chloro-1H-indole-2-carboxamide (13). HPLC analysis was as follows: 96.7%; m.p. 93–95 C; H-NMR
400 MHz, d -DMSO) 11.71 (d, J = 6.8 Hz, 1H), 8.91–8.86 (m, 1H), 7.70 (s, 1H), 7.33 (t, J = 5.6 Hz,
H), 7.25(s, 1H), 7.17 (d, J =7.2 Hz, 1H), 7.05 (t, J = 6.8 Hz, 2H), 5.17–5.09 (m, 1H), 3.98–3.79 (m, 1H),
.73–3.65 (m, 1H), 3.59–3.48 (m, 15H), 3.41–3.34 (m, 2H), 3.25–2.94 (m, 4H), 1.78–1.65 (m, 2H), and
.39–1.20 (m, 2H); 13C-NMR (d -DMSO, 100 MHz)
169.3, 161.4 (d, J = 192.1 Hz), 160.6, 135.3, 134.4,
32.9, 131.7, 131.6, 128.5, 124.7, 124.0, 121.1, 115.3 (d, J = 2.5 Hz), 115.1 (d, J = 2.6 Hz), 114.3, 103.4 (d,
δ
6
δ
6
J = 3.9 Hz), 74.6, 73.8, 70.5, 70.3, 70.2, 70.1, 70.0, 67.2 (d, J = 6.4 Hz), 50.6 (d, J = 8.4 Hz), 50.5, 43.1, 42.7,
+
3
7.0 (d, J = 6.8 Hz), 31.7 (d, J = 33 Hz), and 31.1 (d, J = 40.1 Hz). EIMS m/z 644.8 [M] .
N-[(S)-1-(4-{2-[2-(2-{2-[4-({[(S)-2-(4-Benzoylbenzamido)-6-(2-azidoacetamido)hexanoyl]oxy}methyl)-1H-1,2,
3
-triazol-1-yl]ethoxy}ethoxy)ethoxy]ethoxy}piperidin-1-yl)-3-(4-fluorophenyl)-1-oxopropan-2-yl]-5-chloro-1H-
◦
1
indole-2-carboxamide (
3
). HPLC analysis was as follows: 98.8%; m.p. 113–115 C; H-NMR (400 MHz,
d6-DMSO)
.05 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 7.6 Hz, 2H), 7.71 (t, J = 6.8 Hz, 2H), 7.59 (t,
J = 7.6 Hz, 2H), 7.40 (d, J = 8.8 Hz, 1H), 7.34 (t, J = 6.8 Hz, 2H), 7.26 (s, 1H), 7.18 (d, J = 8.4 Hz, 1H),
.06 (t, J = 8.8 Hz, 2H), 5.21 (s, 2H), 5.14–5.07 (m, 1H), 4.58–4.51 (m, 2H), 4.49–4.43 (m, 1H), 4.00–3.97 (m,
H), 3.83–3.79 (m, 4H), 3.73–3.66 (m, 1H), 3.49–3.45 (m, 13H), 3.31–3.17 (m, 1H), 3.13–2.91 (m, 5H),
δ 11.74 (d, J = 9.6 Hz, 1H), 8.97–8.91 (m, 2H), 8.13 (d, J = 5.2 Hz, 1H), 8.10 (t, J = 5.6 Hz, 1H),
8
7
1
1
1
1
1
3
.83–1.65 (m, 4H), and 1.43–1.23 (m, 6H); C-NMR (CDCl , 100 MHz)
δ 195.9, 172.3, 169.4, 167.5, 166.5,
3
61.4 (d, J = 238.3 Hz), 160.6, 142.0, 139.9, 137.5, 137.1, 135.3, 134.5, 133.5, 132.9, 131.7, 131.6, 130.2,
29.9, 129.1, 128.5, 128.2, 125.6, 124.7, 124.0, 121.1, 115.3, 115.1, 114.3, 103.3, 74.6, 73.9, 70.5, 70.2 (d,
J = 6.8 Hz), 70.1 (d, J = 7.9 Hz), 70.0 (d, J = 6.4 Hz), 69.1, 67.2 (d, J = 6.9 Hz), 58.3, 53.3, 51.3, 50.6 (d,
J = 9.4 Hz), 49.9, 43.1, 42.7, 38.8, 36.9 (d, J = 8.8 Hz), 31.7 (d, J = 34.9 Hz), 31.1 (d, J = 42.8 Hz), 30.5, 29.0,
+
and 23.6. EIMS m/z 1121.0 [M] .
3
.1.8. General Procedure for Synthesis of Compounds 1, 2, and 20
CuSO4 5H O (0.19 equiv.) and Vc-Na (0.4 equiv.) were added to a mixture of compound 13
1.0 equiv.) and (S)-prop-2-ynyl 2-(4-benzoylbenzamido)-6-(2-chloroacetamido)hexanoate (1.2 equiv.)
in CH Cl /H O (4 mL, v:v = 1:1). The reaction mixture was stirred at room temperature overnight.
·
2
(
2
2
2
Then, the reaction mixture was diluted with EtOAc and washed with water, dried over anhydrous
Na SO , and evaporated. The residue was purified by HPLC to give the product as a white solid.
2
4
N-[(S)-1-(4-{2-[2-(2-{2-[4-({[(S)-2-(4-Benzoylbenzamido)-6-(2-chloro-acetylamino)hexanoyl]oxy}methyl)-1H-
1
1
,2,3-triazol-1-yl]ethoxy}ethoxy)ethoxy]ethoxy}piperidin-1-yl)-3-(4-fluorophenyl)-1-oxopropan-2-yl]-5-chloro-
◦
1
H-indole-2-carboxamide (20). HPLC analysis was as follows: 95.9%; m.p. 137–139 C; H-NMR
11.74 (d, J = 9.6 Hz, 1H), 8.97–8.91 (m, 2H), 8.22 (t, J = 5.2 Hz, 1H), 8.13 (d,
(400 MHz, d -DMSO)
δ
6
J = 5.6 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 7.2 Hz, 2H), 7.71 (t,
J = 7.6 Hz, 2H), 7.59 (t, J = 7.6 Hz, 2H), 7.39 (d, J = 8.8 Hz, 1H), 7.34 (t, J = 6.8 Hz, 2H), 7.26 (s, 1H),
7
4
.17 (d, J = 8.8 Hz, 1H), 7.06 (t, J = 8.8 Hz, 2H), 5.20 (s, 2H), 5.15–5.09 (m, 1H), 4.55–4.51 (m, 2H),
.47–4.42 (m, 1H), 4.02 (s, 2H), 4.00–3.96 (m, 1H), 3.82–3.78 (m, 2H), 3.73–3.65 (m, 1H), 3.49–3.43 (m,
13
13H), 3.30–3.21 (m, 1H), 3.10–2.93 (m, 5H), 1.84–1.65 (m, 4H), and 1.48–1.23 (m, 6H); C-NMR
(
d6-DMSO, 100 MHz) 195.9, 172.3, 169.4, 166.5, 166.2, 161.5 (d, J = 238.3 Hz), 160.6, 142.0, 139.9, 137.5,
δ

Molecules 2019, 24, 798
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1
1
37.1, 135.3, 134.5, 133.5, 132.9, 131.7, 131.6, 130.2, 129.9, 129.1, 128.5, 128.2, 125.6, 124.7, 124.0, 121.1,
15.4, 115.1, 114.3, 103.3, 74.6, 73.9, 70.5, 70.2 (d, J = 6.1 Hz), 70.0 (d, J = 3.4 Hz), 69.9 (d, J = 1.7 Hz), 69.1,
6
7.1 (d, J =7.4 Hz), 58.2, 53.3, 50.7, 49.9, 43.1, 42.7, 39.1, 36.9 (d, J = 8.1 Hz), 31.7 (d, J = 36.1 Hz), 31.1(d,
+
J = 43.4 Hz), 30.4, 28.9, and 23.5. EIMS m/z 1114.0 [M] .
N-[(S)-1-(4-{2-[2-(2-{2-[4-({[(S)-2-(4-Benzoylbenzamido)-6-(dansylamide)hexanoyl]oxy}methyl)-1H-1,2,3
-
triazol-1-yl]ethoxy}ethoxy)ethoxy]ethoxy}piperidin-1-yl)-3-(4-fluorophenyl)-1-oxopropan-2-yl]-5-chloro-1H-
◦
1
indole-2-carboxamide (
CD OD) 8.73 (d, J = 10.0 Hz, 1H), 8.57 (d, J = 10.8 Hz, 1H), 8.41 (d, J = 11.2 Hz, 1H), 8.23 (d, J = 9.6 Hz,
H), 8.10 (d, J = 12.0 Hz, 1H), 8.04–8.01 (m, 2H), 7.88–7.82 (m, 4H), 7.71 (t, J = 11.2 Hz, 1H), 7.62–7.55 (m,
1). HPLC analysis was as follows: 100.0%; m.p. 151–153 C; H-NMR (400 MHz,
δ
3
1
5
3
2
H), 7.44 (d, J = 11.6 Hz, 1H), 7.33–7.20 (m, 4H), 7.13 (s, 1H), 7.02 (t, J = 11.2 Hz, 2H), 5.38–5.33 (m,
H), 4.81–4.76 (m, 1H), 4.62–4.58 (m, 3H), 3.93–3.81 (m, 4H), 3.63–3.59 (m, 13H), 3.24–3.07 (m, 3H),
.99–2.92 (m, 8H), 1.90–1.72 (m, 4H), and 1.61–1.40 (m, 6H); 13C-NMR (d -DMSO, 100 MHz)
δ 195.9,
6
172.2, 169.3, 166.5, 162.7, 160.6, 158.7 (d, J = 30 Hz), 151.8, 142.0, 139.9, 137.4, 137.1, 136.6, 135.3, 133.5,
132.9, 131.7, 131.6, 130.2, 129.9, 129.8, 129.5, 129.4, 129.1, 128.6, 128.5, 128.2, 128.1, 125.6, 124.7, 124.0,
121.1, 119.6, 115.5, 115.3, 115.1, 114.3, 103.4, 74.6, 73.8, 70.4, 70.2 (d, J = 5.8 Hz), 70.1 (d, J = 8.0 Hz),
7
2
0.0 (d, J = 5.1 Hz), 69.1, 67.1 (d, J = 7.0 Hz), 58.2, 53.3, 49.9, 49.0, 45.5, 42.6, 30.2, 29.5, 29.2, 27.0, and
3.2. EIMS m/z 1271.0 [M] .
+
N-[(S)-1-(4-{2-[2-(2-{2-[4-({[(S)-2-[(S)-2-(4-Benzoylbenzamido)-6-(dansylamide)hexanamido]-6-(5-{(3aR,4S,
aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-1-yl}pentanamido)hexanoyl]oxy}methyl)-1H-1,2,3-triazol-1-
yl]ethoxy}ethoxy)ethoxy]ethoxy}piperidin-1-yl)-3-(4-fluorophenyl)-1-oxopropan-2-yl]-5-chloro-1H-indole-2-
6
◦
2). HPLC analysis was as follows: 99.4%; m.p. 159–161 C; H-NMR (400 MHz, d -DMSO)
6
1
carboxamide (
11.74 (d, J = 10.0 Hz, 1H), 8.98–8.92 (m, 1H), 8.58 (d, J = 7.6 Hz, 1H), 8.44 (d, J = 8.8 Hz, 1H), 8.34 (d,
J = 7.2 Hz, 1H), 8.30 (d, J = 8.8 Hz, 1H), 8.11–8.09 (m, 2H), 8.02 (d, J = 8.0 Hz, 2H), 7.91 (t, J = 5.6 Hz,
δ
1
1
H), 7.81–7.69 (m, 7H), 7.63–7.54 (m, 4H), 7.39 (dd, J = 8.8, 1.6 Hz, 1H), 7.34 (t, J = 6.4 Hz, 2H), 7.26 (s,
H), 7.23 (d, J = 7.6 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 7.06 (t, J = 8.8 Hz, 2H), 6.43 (br s, 1H), 6.37 (br
s, 1H), 5.18–5.11 (m, 3H), 4.53–4.49 (m, 2H), 4.45–4.40 (m, 1H), 4.28 (t, J = 6.4 Hz, 1H), 4.24–4.21 (m,
H), 4.11–4.08 (m, 1H), 4.00–3.97 (m, 1H), 3.82–3.77 (m, 2H), 3.72–3.64 (m, 1H), 3.50–3.44 (m, 13H),
.30–3.18 (m, 1H), 3.13–2.90 (m, 6H), 2.81 (s, 6H), 2.78–2.76 (m, 3H), 2.56 (d, J = 12.4 Hz, 1H), 2.03 (t,
1
3
13
J = 7.2 Hz, 2H), 1.73–1.56 (m, 6H), and 1.48–1.24 (m, 16H); C-NMR (d -DMSO, 100 MHz)
δ
195.9,
6
1
1
1
72.5, 172.3, 172.2, 169.4, 166.2, 163.2, 161.5 (d, J = 241.1 Hz), 160.6, 151.8, 141.9, 139.7, 137.9, 137.2,
36.6, 135.3, 135.1, 134.4, 133.5, 132.9, 131.7, 131.6, 130.2, 129.8, 129.6, 129.5, 129.1, 128.6, 128.5, 128.2,
28.1, 125.5, 124.7, 124.0, 121.1, 119.6, 115.5, 115.3, 115.1, 114.3, 103.4, 74.6, 73.9, 70.5, 70.2 (d, J = 6.2 Hz),
7
4
2
0.1 (d, J = 3.4 Hz), 70.0 (d, J = 1.7 Hz), 69.1, 67.1 (d, J = 7.1 Hz), 61.5, 59.7, 58.1, 55.9, 53.6, 52.4, 50.5,
9.9, 45.5, 43.1, 42.9, 38.6, 35.7, 31.9, 31.4 (d, J = 6.2 Hz), 30.9 (d, J = 10.9 Hz), 29.6, 29.2, 28.7, 28.5,25.8,
+
3.3, and 23.2. EIMS m/z 1625.0 [M] .
3
.2. Enzyme Kinetics
The inhibitory activity of the test compounds against RMGPa was monitored using a microplate
reader (BIO-RAD, Bio-Rad Laboratories, Hercules, CA, USA), based on the published method [16].
In brief, RMGPa activity was measured in the direction of glycogen synthesis by the release of
phosphate from glucose-1-phosphate. Each test compound was dissolved in DMSO and diluted
at different concentrations for IC50 determination. The enzyme was added to 100
µL of buffer
containing 50 mM Hepes (pH = 7.2), 100 mM KCl, 2.5 mM MgCl , 0.5 mM glucose-1-phosphate,
2
1
mg/mL glycogen, and the test compound in 96-well microplates (Costar, Corning Incorporated,
Corning, NY, USA). After the addition of 150
µ
L of 1 M HCl containing 10 mg/mL ammonium
◦
molybdate and 0.38 mg/mL malachite green, reactions were run at 22 C for 25 min, and then the
phosphate absorbance was measured at 655 nm. The IC50 values were estimated by fitting the inhibition
data to a dose-dependent curve using a logistic derivative equation.

Molecules 2019, 24, 798
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3
.3. Production of Acute Myocardial Infarction
Male Sprague–Dawley rats weighing 250–350 g were intraperitoneally anesthetized with sodium
phenobarbital (10 L/g). The rats were intubated and ventilated with a volume-cycled small-animal
µ
ventilator (Chengdu Taimeng Science And Technology Co. Ltd., Chengdu, China). An anterior
thoracotomy was performed to open the pericardium. The heart was then rapidly exteriorized, and a
6
-0 silk suture was tightened around the proximal left anterior descending coronary artery (before the
first branch of diagonal artery). Positive end-expiratory pressure was applied to fully inflate the lungs.
The muscle layer and skin were closed separately after plasmid injection, and the animals were allowed
to recover. The experimental protocol was approved by the Chengde Medical University committee
on animal experiments.
3
.4. Photoaffinity Labeling Experiment
Blood samples prepared from rats with acute myocardial infarction were drawn from the
abdominal aorta in rats after coronary ligation using a 23-gauge needle. The serum blank was obtained
◦
from male rats without surgery. The blood sample was stored at 4 C for 30 min, and then centrifuged
◦
◦
at 3000 rev./min for 15 min at 4 C. Serum was frozen at
−
80 C until assay. The protein concentration
of the suspension was measured according to the Bradford method. The serum proteomes were
diluted to 2.0 mg/mL with 50 mM Tris
by incubating proteomes with the probe (dissolved in DMSO) at 4 C for 8 h, and then exposed
to UV 365 nm (220 v, 6 W, 365 nm) at a distance of 3 cm. The reaction mixture was centrifuged at
·
HCl buffer (pH 7.4). The labeling reaction was initiated
◦
4
8,000
×
g for 10 min, then the supernatant was removed and the precipitate was resuspended in lysis
buffer (urea 480 mg/L, chaps 40 mg/L, Tris-base 4.8 mg/L, DTT 10 mg/L, Ampholate 50 mL/L, and
bromophenol blue 0.002%) at 4 C for 1 h, and then centrifuged at 18,000
was dialyzed against 50 mM Tris
test probe, the homogenate photolabeled with the probe was subjected to SDS-PAGE electrophoresis,
and then transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with
◦
×
g for 2 h. The supernatant
·
HCl buffer (pH 7.4). To detect the proteins photo-cross-linked by
1
0
(
% BSA in PBS with 0.05% Tween 20 at room temperature for 1 h, washed twice using PBS with
.05% Tween 20 for 5 min, and then imaged using the Tanon-6100 Chemiluminescent Imaging system
Tanon Science and technology Co., Ltd., Shanghai, China). The band densities were calculated with
Quantity One software 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The serum in the rat
acute myocardial infarction model treated with probe 1 (Figure 4, lane 2) was excised in gel digested
with trypsin, and subjected to LC-MS/MS. The data were recorded on an LTQ-Orbitrap Fusion mass
spectrometer (ThermoFisher Scientific Inc., Waltham, MA, USA) coupled to an Easy-nLC 1000 LC
System (Thermo Fischer Scientic). Label-free MS analysis was performed by Thermo Q-Exactive mass
spectrometry (ThermoFisher Scientific Inc., Waltham, MA, USA), and the MS raw data were processed
using MaxQuant 1.5.2.8 software with the Uniprot-Orytolagus cuniculus database.
4
. Conclusions
In summary, three novel activity-based probes were designed, synthesized, and applied in the
semiquantitative analysis of serum-free-GP levels after an acute myocardial infarction. The synthesized
probes were evaluated for their potency in an enzyme inhibition assay against RMGPa. The results
showed that the probe 1 exhibited inhibitory activity against RMGPa with an IC50 value of 13.20 µM,
which was approximately 100-fold less potent than that of CP-320626. Photoaffinity labeling studies
were also performed, and the protein with a MW of about 90–100 kDa was efficiently and specifically
labeled by probe
the GP level in serum with probe
0–100 kDa was detected. Mass spectrometric analysis revealed that the GPMM was well-preserved in
1. This was consistent with the MW of GP. The attempt to semi-quantitatively evaluate
1
was also performed, and a protein band with a MW of about
9
the bands. For semiquantitative data analysis, the concentration of the GPMM protein was found to be
between 25 and 50 ng/mL.

Molecules 2019, 24, 798
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Supplementary Materials: Supplementary materials associated with this article are available online.
Figure S1–S11: Data and NMR spectra of the prepared compounds.
Author Contributions: Conceived and designed the study: L.Z. Performed the experiments: Y.Z., Y.W., Z.Y., C.S.
and G.M. Analyzed the data: Y.Z., Y.W. and Z.Y. Contributed reagents/materials/analysis tools: Y.Z., Y.W., Z.Y.
Wrote the manuscript: L.Z.
Funding: This research was supported by the National Natural Science Foundation of China (No. 81473101
and 81001401), the Natural Science Foundation of Hebei Province (No. H2017406049), and the Key Programs of
Education Department of Hebei Province (No. ZD2018080).
Conflicts of Interest: The authors declare no conflict of interest.
References
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[
PubMed]
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4.
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Sample Availability: Samples of the compounds are available from the authors.
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