A molecular probe with both chromogenic and fluorescent units for detecting serine proteases

Article abstract of DOI:10.3390/molecules26020482

A molecular probe with L-phenylalanine p-nitroanilide and L-lysin 4-methylcoumaryl-7-amide, in which these amino acid derivatives are connected through a succinic-acid spacer, was prepared. Trypsin and papain were detected by blue-fluorescence emission of generated 7-amino-4-methylcoumarin (AMC). α-Chymotrypsin and nattokinase were detected from both the blue-fluorescence emission of AMC and the UV absorbance of p-nitroaniline. In addition, different time courses of p-nitroaniline and AMC were observed between the reaction of P1 with α-chymotrypsin and that with nattokinase. In the case of nattokinase, both the fluorescence emission and UV absorbance slowly increased. In contrast, the increasing UV absorbance was saturated at the early stage of the reaction of the present probe with chymotrypsin, whereas the fluorescence emission continuously increased in the following stages.

Full text of DOI:10.3390/molecules26020482

molecules
Article
A Molecular Probe with Both Chromogenic and Fluorescent
Units for Detecting Serine Proteases
Kirara Ishida, Yushi Nakamura, Tetsuo Ohta and Yohei Oe *
Department of Biomedical Sciences and Informatics, Faculty of Life and Medical Sciences, Doshisha University,
Kyotanabe, Kyoto 610-0394, Japan; ctud1009@mail4.doshisha.ac.jp (K.I.); yusnakam@mail.doshisha.ac.jp (Y.N.);
tota@mail.doshisha.ac.jp (T.O.)
* Correspondence: yoe@mail.doshisha.ac.jp; Tel./Fax: +81-774-65-6505
Abstract: A molecular probe with L-phenylalanine p-nitroanilide and L-lysin 4-methylcoumaryl-7-
amide, in which these amino acid derivatives are connected through a succinic-acid spacer, was
prepared. Trypsin and papain were detected by blue-fluorescence emission of generated 7-amino-
4-methylcoumarin (AMC).
fluorescence emission of AMC and the UV absorbance of p-nitroaniline. In addition, different time
courses of p-nitroaniline and AMC were observed between the reaction of P1 with -chymotrypsin
α-Chymotrypsin and nattokinase were detected from both the blue-
α
and that with nattokinase. In the case of nattokinase, both the fluorescence emission and UV
absorbance slowly increased. In contrast, the increasing UV absorbance was saturated at the early
stage of the reaction of the present probe with chymotrypsin, whereas the fluorescence emission
continuously increased in the following stages.
Keywords: molecular fluorescence probe; chromogenic molecular probe; protease; analysis
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
1. Introduction
Citation: Ishida, K.; Nakamura, Y.;
Ohta, T.; Oe, Y. A Molecular Probe
with Both Chromogenic and Fluorescent
Units for Detecting Serine Proteases.
Molecules 2021, 26, 482. https://
doi.org/10.3390/molecules26020482
Proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins and
are implemented in the development of many pathological conditions [
protease assays include liquid chromatography, electrochemistry, mass spectrometry, and
fluorescence [ ]. A fluorescence assay is one of the most popular among them due to its
rapid responsibility and high sensitivity [ ]. As for a fluorescence assay, “turn-on” molecu-
1–3]. Methods for
4–9
4
lar probes are generally utilized. The fluorescence of fluorophores in these molecular probes
is initially quenched (“off” state), and the fluorescence turns on when the probes are reacted
Academic Editor: Sheshanath Bhosale
Received: 7 December 2020
Accepted: 15 January 2021
with serine proteases (“on” state). FRET [
nitrogen atom of fluorophores such as 7-amino-4-methylcoumarin (AMC) and the carboxy
group of amino acid residue [ ] have been utilized to achieve the “off” state. Being similar
to fluorescent molecular probes, chromogenic molecular probes, which sometimes use
p-nitroaniline as a chromogenic substrate, have also been reported [10 17]. In general,
4] and the amide-bond formations between the
Published: 18 January 2021
5
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
–
to detect the enzyme selectively, amino acid residues or peptides have been used. For
example, phenylalanine was used as an amino acid moiety in the “turn-on” probe to detect
α
-chymotrypsin, which is known to cleave the amide (or peptide) bond at the C-terminus
of tyrosine, tryptophan, and phenylalanine selectively [7,10]. Moreover, lysin was utilized
in a peptide FRET probe to detect trypsin activity [15 16]. These designed molecular probes
,
could measure the target enzymatic activities; however, untargeted enzymes sometimes
reacted with the molecular probe due to their poor substrate selectivity [18]. In this work,
we focused on such unexpected enzymatic activity in the reaction of proteases with the
“turn on” fluorescence and/or chromogenic molecular probes.
Our designed molecular probe, P1 hereafter, is shown schematically in Figure 1. Both
a chromogenic-probe and a fluorescence-probe are incorporated in P1. In P1, p-nitroaniline
is released when the corresponding amide bond in the phenylalanine unit is cleaved by
an enzyme that reacts with hydrophobic substrates like chymotrypsin. Moreover, AMC is
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 482. https://doi.org/10.3390/molecules26020482
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Molecules 2021, 26, 482
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released from the lysin unit by the reaction with an enzyme such as trypsin that reacts with
hydrophilic substrates. These two units are connected through the succinic acid moiety.
Installing the two molecular-probe units in one molecule brings the following advantage.
P1 has four possible reaction sites with proteases, which can generate six different fragment
probes with different sizes and hydrophilicities. It is therefore considered that various
timings to release the pigment and fluorescent moieties can be provided according to the
substrate selectivity of the enzymes. We hypothesized that the use of P1 would distinguish
the activity of proteases in more detail than a similar molecular probe with only one unit of
the two in the proposed probe. Hereafter, the synthesis of the new molecular probe with
the two different molecular-probe units is described, and its reactivity with several serine
proteases is demonstrated.
lysin unit
phenylalanine unit
Ph
H
N
O
O
H
N
N
N
O
O
H
H
O
O
O2N
NH2
possible reaction sites
P1
Ph
O
O
O
H
H
N
N
H2N
N
H
OH
N
O
O
O
O
H
O
O
O
1d
1e
1f
O2N
1a
O
NH2
NH2
Ph
O
O
O
H
N
H
N
OH
HO
N
H
N
H
O
O
O
1b
O2N
O2N
NH2
Ph
Ph
HO
H
O
N
H
N
NH2
N
H
N
H
O
O
O
O
1c
NH2
Figure 1. Designed molecular probe (P1) and plausible fragment probes 1a–f.
2. Results and Discussion
2.1. Synthesis of P1
As shown in Scheme 1, P1 can be synthesized by preparing p-nitrophenylanilide
(phenylalanine unit) and AMC anilide (lysin unit) and connecting both units to a
succinic acid spacer. Initially, anilide was prepared from commercially available L-
phenylalanine ( ) through the three steps (Scheme 1A). L-Phenylalanine is thus N-Boc
4
6
4
2
protected with (Boc)2O under basic conditions. The obtained N-Boc-phenylalanine is then
condensed along with p-nitroaniline in the presence of EDCI, Et3N, and DMAP to give
N-Boc-L-phenylalanine p-nitroanilide
3 (30% yield in two steps). The obtained N-Boc-L-

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phenylalanine p-nitroanilide (3) was then treated with trifluoroacetic acid (TFA) in CH2Cl2
to remove the Boc protecting group and afford the desired anilide (4) at 87% yield.
(A) Preparation of 4
Ph
Ph
Ph
HO
1. (Boc)2O, NaOH
in H2O/1,4-dioxane
TFA
in CH2Cl2
H
H
N
N
NH2
NHBoc
NH2
2. EDCI, Et3N, DMAP
O
O
O
O2N
p-nitroaniline
in CH2Cl2
O2N
3
4
2
(B) Preparation of 6
O
O
1. DCCin DMF
NHFmoc
NH2
HO
N
O
O
2. Piperidine
in DMF
H
O
O
NH2
AMC
6
NHBoc
NHBoc
5
(C) Prepraration of P1
Ph
O
O
H
H
1. Succinic anhydride
Et3N in pyridine
N
N
N
N
O
O
4
H
O
H
O
2. 6, DIC, HOBt
in DMF
H2N
NH2
TFA
3. TFA in CH2Cl2
P1
Scheme 1. Synthesis of P1.
Next, the lysin unit was prepared (Scheme 1B). Thus,
ε
-Boc-α-Fmoc-L-lysin (5) was
reacted with AMC [19] in the presence of DCC in DMF to give the corresponding adduct in
44% yield, which was then treated with piperidine in DMF to afford lysin unit at a yield
of 75%. To synthesize the probe P1, both prepared units and were connected through a
succinic acid spacer (Scheme 1C). Thus, the anilide was reacted with succinic anhydride
in the presence of Et3N in pyridine to afford the corresponding adduct, which was then
reacted with unit in the presence of DIC and HOBt in DMF to give N-Boc-protected probe.
6
4
6
4
6
Finally, the product was treated with TFA in CH2Cl2 at ambient temperature to provide P1
as a TFA adduct.
2.2. UV and Fluorescence Spectra of P1
Once the desired probe P1 was obtained, UV-vis absorbance and fluorescence proper-
ties of p-nitroaniline, AMC, and P1 were investigated (Figure 2). As shown in Figure 2A
both absorptions derived from p-nitroaniline (at 384 nm) and AMC (at 341 nm) are shifted,
and of P1 appeared at 320 nm. It is considered that these blue shifts are caused by the
,
λ
max
formation of amide bonds by chromophores in p-nitroaniline and AMC with phenylalanine
and lysin, respectively. The solution of P1 for these spectra was colorless; therefore, it is
expected that the release of p-nitroaniline by the reaction of P1 with proteases could also
be visually confirmed by the color change of the solution. As shown in Figure 2B, the
fluorescence-emission spectra of p-nitroaniline, AMC, and P1 excited at 360 nm reveal that
the fluorescence of AMC moiety in P1 is highly suppressed. To clarify the reason for this
effective suppression of fluorescence emission, several AMC derivatives were prepared,
and their fluorescence properties were investigated. Fluorescence emission spectra excited
at 300 nm are shown in Figure 2C. The maximum emission of AMC itself appears at a
wavelength of 441 nm. In contrast, AMCs connected with lysin (lys-AMC 7) or lysin

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derivatives (
6
and
8) show maximum fluorescence emission at wavelengths of 387, 390, and
390 nm, respectively. These blue shifts of the emission peaks are caused by the formation
of amide bonds between the amino group of AMC and the carboxylic group of lysin. It is
noteworthy that almost no fluorescence emission was observed with P1 excited at 300 nm.
It is considered that this quenching of fluorescence emission was caused by absorption of
fluorescence energy by p-nitroanilide. Fluorescence emission of P1 was therefore effectively
suppressed by both the amide-bond formation and the FRET effect.
Figure 2. UV-vis and fluorescence emission spectra of p-nitroaniline, AMC, P1 and AMC anilides.
2.3. Reaction of P1 with Proteases
The reaction of P1 with chymotrypsin and trypsin was preliminarily investigated.
P1 was reacted with α-chymotrypsin (4.8 U/mL) in pH 8.0 Tris-HCl buffer solution. A
test solution (with α-chymotrypsin) and a blank solution (without α-chymotrypsin) were
prepared, and the UV-vis and fluorescence emission spectra of both solutions were recorded
immediately after the preparation and after 5 min. According to the spectra, absorbance
at 320 nm derived from P1 slightly decreased after 5 min, while absorbance at 384 nm
derived from p-nitroaniline was increased (Figure 3(A-1)). The reaction solution turned
slightly yellow, indicating that p-nitroaniline was released into the reaction solution. On
the contrary, the UV-vis spectra and color of the blank solution did not change. As for both
the fluorescence-emission spectra of the test solution immediately after the preparation
and after 5 min, the fluorescent intensities at 441 nm apparently increased, suggesting that
AMC was apparently released under the described reaction conditions despite the fact
that
amino acid residue selectively (Figure 3(A-2)). It is considered that the amide bond at the
C-terminus of lysin in P1 was recognized as a reaction site by -chymotrypsin due to the
α-chymotrypsin is known to cleave the amide bond at the C-terminus of hydrophobic
α

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hydrophobicity of the AMC and/or the probe molecule itself. The release of AMC was
confirmed visibly under UV irradiation (365 nm).
Figure 3. UV-vis and fluorescence emission spectra (excited at 360 nm) of the test and blank samples with
(A) or trypsin (B).
α-chymotrypsin
The reaction of P1 with trypsin (4.8 U/mL) gave a different result from that with
chymotrypsin. P1 was treated with trypsin in PBS buffer at pH 7.4. In a similar manner to
the reaction of P1 with -chymotrypsin, a test solution (with trypsin) and a blank solution
α-
α
(without trypsin) were prepared, and the UV-vis and fluorescence emission spectra of both
solutions were recorded immediately after the preparation and after 5 min. The UV-vis
spectrum suggested that p-nitroaniline was not obtained either immediately after the prepa-
ration of the test solution or 5 min after the preparation (Figure 3(B-1)). The solution was
colorless even after 5 min, indicating that p-nitroaniline was not released into the reaction
solution. Moreover, absorbance at 341 nm hardly increased, indicating the generation of
AMC. The fluorescence-emission spectrum (excited at 360 nm) clarified the generation
of AMC by the apparent increase of fluorescent intensity at 441 nm (Figure 3(B-2)). Al-
though several molecules, including the lysin unit, are known as substrates for the trypsin,
α
-chymotrypsin also reacted with the lys-AMC unit of P1 in this case. In other words,
chymotrypsin is hardly distinguishable from trypsin by the present fluorescence results.
The phenylalanine unit supports to solve this issue. p-Nitroaniline was released when P1
was reacted with α-chymotrypsin, whereas no p-nitroaniline was obtained with trypsin.
Therefore, P1 can distinguish between α-chymotrypsin and trypsin by the combination of
UV adsorption with fluorescence.
P1 was then reacted with
α-chymotrypsin, trypsin, pepsin, proteinase K, elastase,
nattokinase, and papain (4.8 U/mL) for 5 min, and the amounts of obtained p-nitroaniline

Molecules 2021, 26, 482
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and AMC were calculated by using high-performance liquid chromatography (HPLC) and
fluorescence-emission spectra, respectively. Yields of the obtained p-nitroaniline and AMC
are shown in Figure 4A. Both α-chymotrypsin and nattokinase cleaved the amide bond at
the C-terminus of L-phenylalanine in P1 to give p-nitroaniline at yields of 37.2 and 3.5%,
respectively. This result is not surprising since it is known that nattokinase is a subtilisin
endopeptidase and selectively cleaves the C-terminus of hydrophobic amino acid residue
in general. Trypsin, elastase, papain, and pepsin, which is known to cleave the peptide
at the C-terminus of small aliphatic, basic, and acidic amino acid residues, respectively,
and, as expected, no p-nitroaniline was detected after 5 min. Furthermore, no p-nitroaniline
was detected in spite of the wide substrate scope of proteinase K (presumably due to the
unsuitable reaction conditions for this enzyme). On the contrary, both the expected and
unexpected results were observed by the fluorescence emission spectra. Thus, AMC was
released when P1 was reacted with
Trypsin and papain are known to cleave the peptide bond of basic amino acid residue;
therefore, AMC was released as expected. -Chymotrypsin and nattokinase, which usually
α-chymotrypsin, trypsin, nattokinase, and papain.
α
react with hydrophobic amino acid residue, also afforded AMC presumably due to the
hydrophobic property of P1. Only a little amount of AMC was obtained when elastase and
pepsin were used. These results can be understood in terms of the substrate selectivity of
these two enzymes. Fluorescent intensity was almost zero when proteinase K was used.
These overall observations suggest that the balance between the obtained p-nitroaniline
and AMC after 5 min can partially explain the selectivity of proteases. Thus, P1 can
classify the seven proteases tested in this work into three categories. α-Chymotrypsin and
nattokinase were classified into category I, which react with both phenylalanine and lysin
units. Trypsin and papain were classified into category II since these two proteases were
reacted only with the lysin unit. Proteases in category III, elastase, proteinase K and pepsin,
obtained only a little fluorescence emission. In addition, it is considered that category I
could be subdivided by the ratio of p-nitroaniline to AMC. The ratio is 1.15 in the case
of
suggested that nattokinase reacted with the lysin unit more selectively compared with
-chymotrypsin. Therefore, we considered that P1 is more useful for the categorization
α-chymotrypsin, whereas the ratio is only 0.18 in the case of nattokinase. This result
α
of proteases than the molecular probe with a single reporter unit, which classifies these
enzymes into two groups (reactive or unreactive). The time courses of the yield of p-
nitroaniline and fluorescence intensities are shown in Figure 4B–D. Fluorescence emissions
(excited at 360 nm) are continuously increased with proteases in category I and II in
20 min (Figure 4B). Category I proteases,
nitoroaniline (Figure 4C). With -chymotrypsin, p-nitroaniline was obtained rapidly and
almost saturated after 5 min. The time course of fluorescence-emission spectra (excited
at 300 nm; emission at 390 nm) with -chymotrypsin is shown in Figure 4D, where the
fluorescent intensity was smoothly increased until 5 min later. This time course is very
similar to that of p-nitroaniline, indicating the generation of fragment 1f (shown in Figure 1
by selective cleavage of the p-nitroanilide moiety at the initial stage of the reaction of P1 with
-chymotrypsin. Interestingly, the fluorescent intensity excited at 300 nm was decreased
after this (Figure 4D), suggesting that the fragment molecular probe 1f generated in situ
was reacted with -chymotrypsin. We considered that a more detailed classification of
α-chymotrypsin and nattokinase, obtained p-
α
α
)
α
α
proteases would be possible by combining the categorization described above and the
investigation of time courses of the enzymatic reactions by use of P1.

Molecules 2021, 26, 482
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Figure 4. Quantitative analysis of the reaction of P1 with proteases.
Finally, several fragment molecular probes (1b ) were reacted with
–e
α
-chymotrypsin,
trypsin, and nattokinase. UV-vis and fluorescence-emission spectra of these reaction
mixtures are shown in Figure 5. Although very weak fluorescence emissions were observed
when 1e was used as a substrate for the enzymatic reactions, almost no enzymatic reactions
took place with fragment molecular probes 1b–d. These results suggested that P1 was a
more suitable substrate than these fragment probes for the proteases tested here and that
combination of the tests by using 1b–d independently cannot classify the proteases in the
same way as using P1.

Molecules 2021, 26, 482
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Figure 5. UV-vis and fluorescence emission spectra (excited at 360 nm) of the reaction mixture of fragment probe 1b–e with
proteases. Enzymes showed their absorbance maxima at 280–290 nm (see Supporting Information).
3. Materials and Methods
3.1. General Methods
1H and 13CNMR spectra were recorded with a Bruker Ascend 400 spectrometer
(Parque Tecnológico de AndalucíaC/Severo Ochoa, Málaga, Spain, 400 MHz for 1H and
100 MHz for 13C) using TMS ( = 0 ppm) as an internal standard for 1H NMR and CDCl3
δ
(δ = 77 ppm) or DMSO-d6 (δ
= 39.7 ppm) as that for 13C NMR spectroscopy (Parque
Tecnológico de AndalucíaC/Severo Ochoa, Málaga, Spain). High-resolution mass spectra
(FAB) were recorded by using a JEOL JMS-700 instrument (Musashino Akishima, Tokyo,
Japan)with m-nitrobenzyl alcohol as the matrix and PEG-200 as the calibration standard.
UV-vis spectra were recorded on a Shimadzu UV-2600 spectrometer in the wavelength
range of 200–600 nm (Tokyo, Japan). Fluorescence emission spectra were recorded on
a Shimadzu RF-5300 in the wavelength range of 300–600 nm (Tokyo, Japan). Excitation
and emission wavelengths were, respectively, 300 and 390 nm or 360 and 430 nm. All
fluorescence emission spectra were recorded bandwidths of 1.5 nm for excitation and
1.5 nm for emission. HPLC analysis was performed on a Shimadzu LC system (Tokyo,
Japan, pump: LC-20AD, column oven: CTO-20A, UV detector: SPD-20AV at 370 nm,
and degasser: DGU-20A3) with a Shimadzu VP-ODS column. The mobile phase was
MeOH/H2O (2/3 v/v), the flow rate was 0.75 mL/min, and the column temperature was
kept at 40 ◦C.
3.2. Synthesis of Substrates
3.2.1. Synthesis of N-(Boc)-L-phenylalanine p-nitroanilide (3)
To a 100 mL round-bottomed flask, dichloromethane (30 mL), N-(Boc)-L-phenylalanine
(1.325 g, 5.0 mmol), EDCI (1.525 g, 8.0 mmol), and Et3N (1.280 g, 12.5 mmol) were added.
Then, p-nitroaniline (0.693 g, 5.0 mmol) and DMAP (0.625 g, 5.0 mmol) were added to the
mixture with stirring. The reaction mixture was stirred at ambient temperature overnight.
The solvent was removed, and the residue was dissolved in ethyl acetate. The organic
phase was washed with 10% NaHCO3 aq. and dried with Na2SO4. After filtration, the

Molecules 2021, 26, 482
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filtrate was evaporated to afford the crude product as a solid, which was then washed with
diethyl ether to give anilide 3 (0.651 g, 34% yield).
1
N-(Boc)-L-phenylalanine p-nitroanilide (
3
): H NMR (400 MHz, CDCl3)
δ
= 8.50 (brs,
1H, -NHCO2), 8.18 (d, J = 9.2 Hz, 2H, Ar-H), 7.57 (d, J = 9.2 Hz, 2H, Ar-H), 7.24–7.35 (m,
5H, -C6H5), 5.04 (brs, 1H, -NHC(O)-), 4.46–4.50 (m, 1H, -NHCH(Bn)C(O)), 3.12–3.22 (m,
2H, Ph-CH2-), 1.44 (s, 9H, -C(CH3)3) ppm. [20]
3.2.2. Synthesis of L-phenylalanine p-nitroanilide (4)
To a 100 mL round-bottomed flask anilide
3 (0.346 g, 0.9 mmol) and dichloromethane
(30 mL) were added. TFA (4 mL) was added to the solution, and the reaction mixture was
stirred at ambient temperature for 1 h. After the removal of volatiles, ethyl acetate was
added to the residue. The organic layer was neutralized with 10% Na2CO3 aq., washed
with brine and then dried with Na2SO4. After filtration, the filtrate was evaporated, and
the residue was dried in vacuo to afford L-phenylalanine p-nitroanilide (4) (0.223 g, 87%
yield).
1
L-phenylalanine p-nitroanilide (
4
): H NMR (400 MHz, CDCl3)
δ
= 9.91 (s, 1H, -CO2H),
8.23 (d, J = 9.2 Hz, 2H, Ar-H), 7.78 (d, J = 9.2 Hz, 2H, Ar-H), 7.24–7.37 (m, 5H, -C6H5), 3.79
(dd, J = 4.0 Hz, 9.2 Hz, 1H, Ph-CH2-), 3.38 (dd, J = 4.0 Hz, 14 Hz, 1H, Ph-CH2-), 2.83 (dd,
J = 9.6 Hz, 13.6 Hz, 1H, -NHCH(Bn)C(O)) ppm. [21]
3.2.3. Synthesis of α-Fmoc-ε-Boc-L-lysin 4-methylcoumaryl-7-amide
To a 100 mL round-bottomed flask, N-
α
-Fmoc-N-
ε
-t-Boc-L-lysin (3.097 g, 6.66 mmol),
◦
dichloromethane (40 mL), and DCC (1.3986 g, 6.66 mmol) at 0 C were added, and the
◦
reaction mixture was stirred at 0 C. After 1 h, AMC (1.1681 g, 6.66 mmol) was added to the
solution, and the solution was stirred at ambient temperature overnight. The precipitates
were filtered off, and the filtrate was dried under reduced pressure. The obtained solid was
washed with hexane to afford
α
-Fmoc-
ε
-Boc-L-lysin 4-methylcoumaryl-7-amide (1.833 g,
44% yield).
α
-Fmoc-ε δ = 10.48
-Boc-L-lysin 4-methylcoumaryl-7-amide: 1H NMR (400 MHz, DMSO)
(s, 1 H, NH), 7.89 (d, J = 7.6 Hz, 2 H, Ar-H), 7.78–7.72 (m, 5 H, Ar-H, NH), 7.50 (d, J = 8.8
Hz, 1 H, Ar-H), 7.42 (t, J = 4.9 Hz, 2 H, Ar-H), 7.32 (t, J = 4.9 Hz, 2 H, Ar-H), 6.78 (s, 1 H,
NH), 6.27 (s, 1 H, Ar-H), 4.29 (m, 2 H, -OCH2CH-), 4.26 (d, J = 12.8 Hz, 1 H, -OCH2CH-),
4.30 (s, 1 H, -NHC(O)CH-), 2.90 (s, 2 H, -C(O)NHCH2-), 2.40 (s, 3 H, -CH3), 1.74–1.15 (m, 13
H, -CHC3H6-, -C(CH3)3) ppm. [22]
3.2.4. Synthesis of ε-Boc-L-lysin 4-methylcoumaryl-7-amide (6)
To a 100 mL round-bottomed flask, α-Fmoc-ε-Boc-L-lysin 4-methylcoumaryl-7-amide
(1.818 g, 2.908 mmol), DMF (32 mL), and piperidine (8 mL) were added. The reaction
mixture was stirred at ambient temperature for 30 min. The volatiles were removed under
reduced pressure, and the residue was dissolved in ethyl acetate. The organic layer was
washed with 10% Na2CO3 aq. and dried with Na2SO4. After filtration, the filtrate was
evaporated to afford the crude product as a solid, which was washed with t-butyl methyl
ether to afford amide 6 (0.879 g, 75% yield).
1
ε
-Boc-L-lysin 4-methylcoumaryl-7-amide (
6
): H NMR (400 MHz, DMSO)
δ
= 7.83 (d,
J = 2.0 Hz, 1 H, Ar-H), 7.72 (d, J = 8.0 Hz, 1 H, Ar-H), 7.56 (d, J = 2.0 Hz, 8.8 Hz, 1 H, Ar-H),
6.77 (s, 1 H, NH), 6.26 (d, J = 1.2 Hz, 1 H, Ar-H), 2.89 (s, 2 H, -C(O)NHCH2-), 2.40 (d, J = 1.2
Hz, 3 H, -CH3), 1.73–1.28 (m, 15 H, -CHC3H6-, -C(CH3)3) ppm. [8]
3.2.5. Synthesis of N-Succinyl-L-phenylalanine p-nitroanilide
To a 50 mL round-bottomed flask, pyridine (15 mL), Et3N (0.1 mL, 0.7 mmol), anilide
4
(0.120 g, 0.7 mmol), and succinic anhydride (0.219 g, 2.19 mmol) were added. The reaction
mixture was stirred at ambient temperature for 2 h. The volatiles were removed under
reduced pressure, and the residue was dissolved in ethyl acetate. The organic layer was
washed with 10% acetic acid aq. and then dried with Na2SO4. After filtration, the filtrate

Molecules 2021, 26, 482
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was evaporated to give the crude product as a solid, which was washed with diethyl ether
to afford N-succinyl-L-phenylalanine p-nitroanilide (0.240 g, 89% yield). [23]
1
N-Succinyl-L-phenylalanine p-nitroanilide: H NMR (400 MHz, DMSO)
δ
= 10.66 (s,
1H, -NHC(O)-), 8.42 (d, J = 7.6 Hz, 1H, -NHCH(Bn)C(O)), 8.22 (d, J = 9.2 Hz, 2H, Ar-H),
7.84 (d, J = 9.2 Hz, 2H, Ar-H), 7.17–7.29 (m, 5H, -C6H5), 4.63–4.68 (m, 1H, -NHCH(Bn)C(O)),
3.05 (dd, J = 5.2 Hz, 13.6 Hz, 1H, Ph-CH2-), 2.88 (dd, J = 9.2 Hz, 13.6 Hz, 1H, Ph-CH2-),
2.326–2.383 (m, 4H, -C2H4CO2H) ppm.
3.2.6. Synthesis of Boc-Protected Probe Boc-P1
To a 200 mL round-bottomed flask, N-succinyl-L-phenylalanine p-nitroanilide (0.212 g,
0.57 mmol), dichloromethane (35 mL), HOBt (0.0827 g, 0.615 mmol), and DIC (0.0827 g,
0.659 mmol) were added. After stirring for 10 min at ambient temperature, amide
6
(0.2298 g, 0.57 mmol) was added. The reaction mixture was stirred at ambient temperature
for 70 min and thereby evaporated, and the obtained residue was dissolved in ethyl acetate.
The organic layer was washed with sat. NaOH aq., 1 M HCl aq. and then with sat. NaHCO3
aq. and dried with Na2SO4. After filtration, the filtrate was dried under reduced pressure
to afford the crude product as a solid, which was washed with t-butyl methyl ether to give
Boc-protected P1 (0.351 g, 80% yield).
Boc-P1 1H NMR (400 MHz, DMSO)
δ = 10.63 (s, 1 H, NH), 10.41 (d, J = 17.2 Hz, 1 H,
NH), 8.50 (s, 1 H, NH), 8.30 (s, 1 H, NH), 8.17 (dd, J = 24 Hz, 9.2 Hz, 2 H, Ar-H), 7.85 (t, J =
10.0 Hz, 2 H, Ar-H), 7.79 (d, J = 6.4 Hz, 1 H, Ar-H), 7.66 (d, J = 17.6 Hz,8.4 Hz, 1 H, Ar-H),
7.48 (t, J = 11 Hz, 1 H, Ar-H), 7.29–7.26 (m, 4 H, -C6H5), 7.27 (s, 1 H, -C6H5), 6.76 (s, 1 H,
NH), 6.26 (d, J = 5.2 Hz, 1 H, Ar-H), 4.64 (s, 1 H, Ph-CH2CH-), 4.31 (s, 1 H, -C(O)NHCH-),
3.16–3.07 (m, 1 H, Ph-CH2-), 2.87 (s, 3 H, -NHC(O) CH2-, Ph-CH2-), 2.37 (d, J = 12 Hz, 7 H,
-CH3, -C2H4C(O)-), 1.699–1.23 (m, 15 H, -CH3, -CHC3H6-, -C(CH3)3) ppm. HRMS (FAB,
m/z) calc. 551.2294, found 551.2310.
3.2.7. Synthesis of P1
To a 200 mL round-bottomed flask, Boc-P1 (0.3784 g, 0.49 mmol) and dichloromethane
(30 mL) were added. TFA (5 mL) was added to the solution, and the reaction mixture
was stirred at ambient temperature for 3 h. After removal of volatiles, the obtained crude
product was washed with t-butyl methyl ether and dried in vacuo to afford P1 as a TFA
adduct (0.105 g, 32% yield).
P1 1H NMR (400 MHz, DMSO)
δ = 10.57 (d, J = 8.0 Hz, 1 H, NH), 10.37 (d, J = 22 Hz,
1 H, NH), 8.46 (d, J = 8.4 Hz, 1 H, NH), 8.29 (d, J = 7.2 Hz, 1 H, NH), 8.37–8.164 (m, 2 H,
Ar-H), 7.87–7.79 (m, 3 H, Ar-H), 7.67 (t, J = 9.8 Hz, 1 H, Ar-H), 7.61 (s, 2 H, NH2), 7.47 (t, J =
8.0 Hz, 1 H, Ar-H), 7.29–7.26 (m, 4 H, -C6H5), 7.19 (s, 1 H, -C6H5), 6.26 (d, J = 1.2 Hz, 1 H,
Ar-H), 4.64 (s, 1 H, Ph-CH2CH-), 4.35 (s, 1 H, -NHC(O)CH-), 3.10 (dd, J = 14 Hz, 5.2 Hz, 1
H, Ph-CH2-), 2.87 (t, J = 12.6 Hz, 1 H, Ar-H), 2.76 (s, 2 H, -C(O)NHCH2-), 2.37 (d, J = 16 Hz,
7 H, -CH3, -C2H4C(O)-), 1.74–1.32 (m, 6 H, -CHC3H6-) ppm. 13C NMR
δ (CDCl3) = 18.4,
22.9, 27.0, 27.3, 30.9, 31.4, 37.6, 38.9, 49.2, 54.0, 55.7, 106.25, 112.7, 115.5, 115.8, 116.3, 119.5,
125.3, 126.2, 126.9, 128.6, 129.6, 138.0, 142.7, 145.4, 153.5, 154.0, 158.2, 158.6, 160.5, 171.8,
172.0, 172.4, 172.5 ppm. HRMS (FAB, m/z) calc. 551.2294, found 551.2310.
3.2.8. Synthesis of L-lysin 4-methylcoumaryl-7-amide (1d)
To a 200 mL round-bottomed flask, ε-Boc-L-lysine 4-methylcoumaryl-7-amide (6)
(0.137 g, 0.34 mmol) and dichloromethane (30 mL) were added. TFA (5 mL) was added to
the solution, and the reaction mixture was stirred at ambient temperature for 3 h. After
removal of volatiles, the obtained crude product was washed with t-butyl methyl ether
and dried in vacuo to afford Lys-AMC 1d (0.072 g, 70% yield)
Lys-AMC 1d 1H NMR (400 MHz, DMSO)
δ = 8.4 (brs, 2 H, NH2), 7.85–7.77 (m, 4 H,
NH2, Ar-H), 7.58 (dd, J = 8.8 Hz, 2.0 Hz, 1 H, Ar-H), 6.32 (d, J = 1.2 Hz, 1 H, Ar-H), 5.58 (d,
J = 8.0 Hz, 1 H, Ar-H), 4.09–4.06 (brm, 1 H, -NHC(O)CH-), 2.81–2.74 (brm, 2 H, -CH2NH2),

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2.41 (d, J = 1.2 Hz, 3 H, -CH3), 1.90–1.80 (m, 1 H, -CHC3H6-), 1.73–1.68 (m, 1 H, -CHC3H6-),
1.62–1.40 (m, 2 H, -CHC3H6-), 1.30–1.02 (m, 2 H, -CHC3H6-) ppm.
3.2.9. Synthesis of AMC-lys(Boc)-suc
To a 50 mL round-bottomed flask, pyridine (15 mL), Et3N (0.1 mL, 0.7 mmol),
ε-Boc-L-
lysine 4-methylcoumaryl-7-amide ( ) (0.15 g, 0.37 mmol), and succinic anhydride (0.219 g,
6
2.19 mmol) were added. The reaction mixture was stirred at ambient temperature for 2 h.
The volatiles were removed under reduced pressure, and the residue was dissolved in
ethyl acetate. The organic layer was washed with 10% acetic acid aq. and then dried with
Na2SO4. After filtration, the filtrate was evaporated to give the crude product as a solid,
which was washed with diethyl ether to afford AMC-lys(Boc)-suc (0.024 g, 13% yield).
AMC-lys(Boc)-suc 1H NMR (400 MHz, DMSO)
δ = 10.4 (s, 1 H, NH), 8.23 (d, J = 7.2
Hz, 1 H, NH), 7.81 (s, 1 H, Ar-H), 7.71 (d, J = 8.8 Hz, 1 H, Ar-H), 7.55 (d, J = 9.6 Hz, 1 H,
Ar-H), 6.77–6.74 (m, 1 H, NH), 6.26 (d, J = 1.2 Hz, 1 H, Ar-H),5.56 (d, J = 8.4 Hz, 1 H, NH),
4.37–4.32 (brm, 1 H, -NHC(O)CH-), 2.91–2.86 (m, 2 H, -C(O)NHCH2-), 2.40 (s, 7 H, -CH3,
-C2H4C(O)-), 1.74–1.68 (m, 2 H, -CHC3H6-), 1.63–1.57 (m, 2 H, -CHC3H6-), 1.37–1.20 (m, 10
H, -CH3, -CHC3H6-, -C(CH3)3) ppm.
3.2.10. Synthesis of AMC-lys-suc (Fragment Probe 1e)
To a 50 mL round-bottomed flask, AMC-lys(Boc)-suc (0.044 g, 0.09 mmol) and
dichloromethane (10 mL) were added. TFA (2 mL) was added to the solution, and the
reaction mixture was stirred at ambient temperature for 3 h. After removal of volatiles, the
obtained crude product was washed with t-butyl methyl ether and dried in vacuo to afford
AMC-lys-suc 1e (0.036 g, 85% yield)
AMC-lys-suc 1e 1H NMR (400 MHz, DMSO)
δ = 10.4 (s, 1 H, NH), 8.24 (d, J = 8.0
Hz, 1 H, NH), 7.79 (d, J = 2.0 Hz, 1 H, NH), 7.73 (d, J = 8.4 Hz, 2 H, Ar-H), 7.51 (d, J = 8.8
Hz,2.4 Hz, 1 H, Ar-H), 6.28 (s, 1 H, Ar-H), 5.57 (d, J = 8.0 Hz, 1 H, NH), 4.43–4.37 (brm, 1 H,
-NHC(O)CH-), 2.77 (t, J = 7.8 Hz, 2 H, -CH2NH2), 2.43–2.38 (m, 7 H, -CH3, -C2H4C(O)-),
1.74–1.68 (m, 2 H, -CHC3H6-), 1.64–1.50 (m, 3 H, -CHC3H6-) 1.37–1.05 (m, 1 H, -CHC3H6-)
ppm.
3.3. Preparation of Solutions
3.3.1. pH 8.0 Tris-HCl Buffer Solution
2-Amino-2-hydroxymethyl-1,3-propanediol (1.938 g) was dissolved in 100 mL of
distilled water. The pH of the solution was adjusted to 8.0 by the careful addition of 1 M
HCl aq. The final volume of the solution was brought to 200 mL with distilled water.
3.3.2. pH 7.4 PBS Buffer Solution
Potassium dihydrogen phosphate (0.06 g, 0.6 mmol), potassium chloride (0.06 g,
0.81 mmol), disodium hydrogen phosphate 12-water (0.87 g, 2.4 mmol), and sodium
chloride (2.4 g, 41.1 mmol) were dissolved in distilled water. After confirming the pH
of the solution was 7.4, the final volume of the solution was brought to 300 mL with
distilled water.
3.3.3. pH 2.2 Glycine-HCl Buffer Solution
Glycine (1.5 g, 20 mmol) was dissolved in 100 mL of distilled water. Sodium chloride
(1.18 g, 20 mmol) was then added to the solution; the pH of the solution was adjusted
to 2.2 with 1 M HCl aq. The final volume of the solution was brought to 200 mL with
distilled water.
3.3.4. 0.59 mM P1 Solution
P1 (0.0046 g, 5.9
µmol) was dissolved in DMSO (0.8 mL) and methanol (2 mL). The
final volume of the solution was brought to 10 mL with distilled water.

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3.3.5. 0.59 mM p-Nitroaniline and AMC Solution
p-Nitroaniline (0.0008 g, 5.9 mol) or AMC (0.001 g, 5.9
µ
µmol) was dissolved in DMSO
(0.8 mL) and methanol (2 mL). The final volume of the solution was brought to 10 mL with
distilled water.
3.3.6. 0.59 mM Solution of Fragment Probe 1b–e
Fragment probe 1b (0.0018 g, 5.9
DMSO (0.8 mL) and methanol (2 mL). The final volume of the solution was brought to
10 mL with distilled water. 1d (0.0017 g, 4.4 mol) was dissolved in DMSO (0.6 mL) and
methanol (1.5 mL). The final volume of the solution was brought to 10 mL with distilled
water. 1e (0.0012 g, 42.95 mol) was dissolved in DMSO (0.4 mL) and methanol (1 mL).
µmol) or 1c (0.0017 g, 5.9 µmol) was dissolved in
µ
µ
The final volume of the solution was brought to 5 mL with distilled water.
3.3.7. 36 µM P1 in PBS Buffer Solution
P1 (0.0016 g, 2.0 µmol) was dissolved in DMF (0.8 mL), and then the volume of the
solution was brought to 20 mL with pH 7.4 PBS buffer solution. The obtained solution
(3.6 mL) was diluted to a pH 7.4 PBS buffer solution, and the final volume of the solution
was brought to 10 mL with the buffer solution.
3.3.8. 36 µM Solution of Fragment Probe 1b and 1e in PBS
Fragment probe 1b (0.0040 g, 0.01 mmol) was dissolved in DMF (0.4 mL), and the
volume of the solution was brought to 20 mL with pH 7.4 PBS buffer solution. The obtained
solution (0.36 mL) was diluted to a pH 7.4 PBS buffer solution, and the final volume of the
solution was brought to 10 mL with pH 7.4 PBS buffer solution. 1e (0.0030 g, 0.01 mmol)
was independently dissolved in DMF (0.2 mL), and the volume of the solution was brought
to 5.0 mL with pH 7.4 PBS buffer solution. The obtained solution (0.36 mL) was diluted to
a pH 7.4 PBS buffer solution, and the final volume of the solution was brought to 10 mL
with the buffer solution.
3.3.9. Solution of α-Chymotrypsin, Pepsin, Elastase, Proteinase K, and Papain in HCl
α
-chymotrypsin (0.01 g), pepsin (0.0012 g), elastase (0.0023 g), proteinase K (0.0062 g)
or papain (0.1 g) was dissolved in 1.0 mM HCl aq. (1 mL). Nattokinase (0.208 g) was
dissolved in 1.0 mM HCl aq. (0.25 mL). These solutions were stored in an ice bath before
use.
3.3.10. Solution of Trypsin in PBS Buffer
Trypsin (0.01 g) was dissolved in pH 7.4 PBS buffer solution (10 mL). The obtained
solution (0.075 mL) was diluted to a pH 7.4 PBS buffer solution, and the final volume of
the solution was brought to 10 mL with the buffer solution.
3.4. Reaction of Molecular Probes with Proteases
The reaction of P1 with proteases was monitored by using both a test solution (with
protease) and a blank solution (without protease) for control experiments. The reactions
were performed at ambient temperature.
3.4.1. Reaction of P1 with α-Chymotrypsin
The 0.59 mM P1 solution (0.13 mL) was diluted to pH 8.0 Tris-HCl buffer (3 mL). To the
solution was added CaCl2 aq. (0.04 mL), the α-chymotrypsin solution above (0.05 mL) for
the test sample or 1 mM HCl aq. (0.05 mL) for the blank sample. After 5 min, UV-vis and
fluorescent emission spectra of both the test sample and the blank sample were recorded.
3.4.2. Reaction of P1 with Trypsin
The 36
µ
M
P1 solution (2.0 mL) was mixed with pH 7.4 PBS buffer solution (1 mL) to
Probe 2 solution (2 mL), trypsin solution (above
obtain the blank sample. To the 36
µM

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1 mL) was added to obtain the test sample. After 5 min, UV-vis and fluorescent emission
spectra of both the test sample and the blank sample were recorded.
3.4.3. Reaction of P1 with Proteases
Preparation of Test Solutions
Test solutions with α-chymotrypsin, proteinase K, elastase, papain, and nattokinase:
The P1 solution (0.13 mL) was mixed with pH 8.0 Tris-HCl buffer solution (3 mL) and
CaCl2 aq. (0.04 mL). The protease solution (0.05 mL) was added to the solution to obtain
the test sample.
Test solutions with trypsin: The P1 solution (2.0 mL) was mixed with the trypsin
solution (1 mL) to obtain the test sample.
Test solutions with pepsin: The P1 solution (0.13 mL) was mixed with a pH 2.2 glycine-
HCl buffer solution (3 mL) and CaCl2 aq. (0.04 mL). The pepsin solution (0.05 mL) was
added to the obtained solution to obtain the test sample.
Determination of p-Nitroaniline Concentration after 5 min
Five minutes after the test sample was prepared, some of it was injected into the HPLC
machine. The concentration of p-nitroaniline was calculated from the obtained peak area
(Rt = 3.8 min) and the calibration curves (Supplementary Materials).
Time Course of p-Nitroaniline Concentration
After the test sample was prepared, some of it was injected into the HPLC spectrometer
after 0.5, 1, 3, 5, 7, 9, and 11 min. Concentrations of p-nitroaniline at each time were
calculated from the obtained peak area (Rt = 3.8 min) and the calibration curves.
Determination of AMC Concentration
Five minutes after the test sample was prepared, its fluorescence emission spectrum
was recorded. The concentration of AMC was calculated from the obtained fluorescent
intensity and the calibration curves.
Time Course of AMC Concentration
After the test sample was prepared, its fluorescent intensity (ex: 360 nm; em: 441 nm)
was measured every 0.8 s. The obtained fluorescent intensity was plotted to obtain time-
course curves.
3.4.4. Reaction of P1 Fragment Probes
The enzymatic reactions were performed under the conditions described above by
using the 1b–e solutions instead of the P1 solution.
4. Conclusions
A new chromogenic and fluorescent molecular probe (P1) for detecting proteases
was designed and synthesized. P1 reported the presence of
α-chymotrypsin, trypsin,
papain, and nattokinase by releasing p-nitroaniline and/or AMC. The type of protease
can be distinguished by the differences in the balances of the obtained p-nitroaniline and
AMC. In addition, the time courses of the reactions could report the difference in substrate
selectivity between
α-chymotrypsin and nattokinase. Further studies now underway
include synthesizing other probes bearing various amino acid moieties and categorizing
proteases by using these molecular probes.
Supplementary Materials: The following are available online, Figure S1–S4: calibration curves of
p-nitroaniline and AMC, Figure S5–S14: NMR and MS spectra of substrates, Figure S15: UV-vis
spectra of proteases.

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Author Contributions: Conceptualization, Y.O., T.O. and Y.N.; methodology, Y.O, T.O. and Y.N.;
validation, K.I. and Y.O.; formal analysis, K.I.; investigation, K.I.; data curation, K.I.; writing—original
draft preparation, K.I. and Y.O.; writing—review and editing, K.I. and Y.O.; supervision, T.O. and
Y.O. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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