Development of a fluorogenic small substrate for dipeptidyl peptidase-4

Article abstract of DOI:10.3762/bjoc.13.267

A series of aniline and m-phenylenediamine derivatives with electron-withdrawing 3,3,3-trifluoropropenyl substituents were synthesized as small and chemically stable fluorescent organic compounds. Their fluorescence performances were evaluated by converting 2,4-disubstituted aniline 1 to the non-fluorescent dipeptide analogue H-Gly-Pro-1 for the use as a fluorogenic substrate for dipeptidyl peptidase-4 (DPP-4). The progress of the enzymatic hydrolysis of H-Gly-Pro-1 with DPP-4 was monitored by fluorometric determination of 1 released into the reaction medium. The results suggest that 1 could be used as fluorophore in OFF-ON-type fluorogenic probes.

Full text of DOI:10.3762/bjoc.13.267

Development of a fluorogenic small substrate
for dipeptidyl peptidase-4
Futa Ogawa1, Masanori Takeda1, Kanae Miyanaga2, Keita Tani2, Ryuji Yamazawa1,
Kiyoshi Ito1, Atsushi Tarui1, Kazuyuki Sato1 and Masaaki Omote*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2017, 13, 2690–2697.
1Faculty of Pharmaceutical Sciences, Setsunan University, 45-1
Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan and 2Division of
Natural Sciences, Osaka Kyoiku University, Asahigaoka, Kashiwara,
Osaka 582-8582, Japan
doi:10.3762/bjoc.13.267
Received: 26 September 2017
Accepted: 29 November 2017
Published: 14 December 2017
Email:
Masaaki Omote* - omote@pharm.setsunan.ac.jp
This article is part of the Thematic Series "Organo-fluorine chemistry IV".
Guest Editor: D. O'Hagan
* Corresponding author
Keywords:
© 2017 Ogawa et al.; licensee Beilstein-Institut.
License and terms: see end of document.
dipeptidyl peptidase-4; fluorogenic substrate; fluorometry; small
fluorescent molecule
Abstract
A series of aniline and m-phenylenediamine derivatives with electron-withdrawing 3,3,3-trifluoropropenyl substituents were syn-
thesized as small and chemically stable fluorescent organic compounds. Their fluorescence performances were evaluated by
converting 2,4-disubstituted aniline 1 to the non-fluorescent dipeptide analogue H-Gly-Pro-1 for the use as a fluorogenic substrate
for dipeptidyl peptidase-4 (DPP-4). The progress of the enzymatic hydrolysis of H-Gly-Pro-1 with DPP-4 was monitored by fluoro-
metric determination of 1 released into the reaction medium. The results suggest that 1 could be used as fluorophore in OFF–ON-
type fluorogenic probes.
Introduction
Fluorescent organic probes have become indispensable for bio- enzymatic transformations. These fluorescent probes can act as
imaging or molecular imaging to visualize the presence of good sensors for enzymes but the fluorophore unit is usually
species such as particular enzymes and biologically important large because fused aromatic rings and an elongated π-conju-
inorganic ions. In biomedicine, these probes are used for the gated system are necessary to impart appropriate fluorescent
detection of a wide range of significant biomarkers and are properties to the probe [14]. The molecular size of the fluores-
essential for the diagnosis of critical diseases [1-3]. Selective cent probe is therefore large, which is sometimes inconvenient
and accurate determinations of specific enzymes, including in terms of membrane permeability, water solubility, and inhibi-
β-galactosidase [4-6], exoglycosidases [7], cyclooxygenases [8], tion of inherent interactions between the probe molecule and the
and others [9-13], have been achieved using the OFF–ON-type target enzyme. We have developed a new OFF–ON probe with
fluorogenic probes with fluorescence that can be turned on by a small fluorescent core unit. The same approach was recently
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used to produce a fluorophore for a push–pull system, which TFPE would be a reasonable approach to producing new fluo-
has a single benzene ring substituted by both electron-donating rescent molecules. However, there are few methods for synthe-
and electron-withdrawing groups [15-17]. We focused on the sizing TFPE-substituted compounds. In a previous study, we
3,3,3-trifluoropropenyl (TFPE) group, which is an electron- achieved the facile introduction of TFPE groups via Hiyama
withdrawing substituent that is free of oxygen atoms, which cross-coupling reactions of (E)-trimethyl-(3,3,3-trifluoroprop-1-
would form hydrogen bonds in protic solvents. We assumed enyl)silane and iodoanilines. This straightforward method is
that the use of the oxygen-free TFPE group would eliminate useful for TFPE introduction and a broad range of iodoanilines
hydrogen bonding with protic solvents, even water, to avoid can be used without protection of the amino group. We used
attenuation of the fluorescent characteristics of the fluorophore. this approach to synthesize several aniline derivatives contain-
In this paper, we report the synthesis of a new fluorophore with ing TFPE groups. The data in Table 1 show that Hiyama cross-
TFPE groups and its application as an OFF–ON-type fluoro- coupling reactions gave the desired TFPE-anilines and TFPE-
genic probe for determining dipeptidyl peptidase-4 (DPP-4) ac- phenylenediamines (1–4) in moderate to good yields. This
tivity.
method has the advantage that complete substitution of diiodo-
and triiodoarenes can proceed to give disubstituted (1 and 3)
and trisubstituted (2 and 4) products in one synthetic operation.
Results and Discussion
Synthesis of fluorescent compounds as
Next, the fluorescence profiles of these products were investi-
gated. The results are summarized in Figure 1 and Table 2. The
number of amino groups and TFPE groups introduced into com-
pounds 1–4 significantly affected the fluorescence profiles in
fluorophores
It has been reported that the introduction of a TFPE group
changes the properties of electron-rich aromatics and leads to a
good fluorophore structure. This suggests that substitution with
Table 1: Synthesis of TFPE-anilines 1–4 via Hiyama cross-coupling reactions of iodoanilines with (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane.a
Entry
1
ArI
Product
Yield (%)b
89
1
2
3
4
54
70
20
2
3
4
aReaction conditions: ArI (1.0 mmol), (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane (2 equiv), [PdCl(2-Me-allyl)]2 (10 mol %), CuF2 (2 equiv), 2,2'-
bipyridyl (2 equiv), DMF (6 mL). bIsolated yield.
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Figure 1: (a) UV–vis absorption and (b) fluorescence spectra of 1–4 in THF.
Table 2: UV–vis absorption and fluorescence data for 1–4 in THF.a
λabs,peak (nm) ε (M−1cm−1) λfl,peak (nm)
φb
1
2
3
4
359
388
373
385
4371
5763
7913
7004
436
460
433
455
0.31
0.30
0.25
0.03
aMeasurement conditions:1.0 × 10−5 M in THF, excitation at λ =
370 nm for 1 and 3, 400 nm for 2 and 4. bFluorescence quantum yield.
THF solution. TFPE-anilines 1 and 2 gave moderate fluores-
cence quantum yields of 0.31 and 0.30, respectively. In compar-
ison with these, the fluorescence quantum yields of TFPE-
phenylenediamine 3 was slightly lower, namely 0.25. The intro-
duction of one more TFPE group into 3, to give 4, further de-
creased the quantum yield to 0.03. In spectroscopic terms, the
fluorescence emission peaks of these compounds red-shifted
with increasing TFPE substitution. Additionally, a large Stokes
shift was observed for 1, which is important in view of the bio-
medical use of fluorescent compounds. Next, we changed the
solvent from THF to H2O/DMSO (9:1) as shown in Figure 2
and Table 3. The fluorescence quantum yield of a fluorophore
generally decreases significantly in an aqueous solvent because
of release of energy in the excited state of the fluorophore by
forming hydrogen bonds with water. However, in the cases of 1
and 3, the decreases in the fluorescence quantum yield were
Figure 2: Fluorescence spectra of 1 and 3 in H2O:DMSO (9:1).
Table 3: Fluorescence data for 1–3 in H2O:DMSO (9:1).a
λfl,peak (nm)
φb
1
2
3
461
-c
0.27
-c
447
0.20
aMeasurement conditions: 1.0 × 10−5 M in H2O:DMSO (9:1), excita-
tion at λ = 330 nm for 1 and 3. bFluorescence quantum yield. c2 is
insoluble in H2O:DMSO (9:1).
small, namely 0.27 and 0.20, respectively. This is reasonable for Design of fluorogenic probes
1 and 3 because the oxygen-free TFPE group is less likely to Fluorogenic OFF–ON-type probes for enzymes should be de-
form hydrogen bonds, even in aqueous solution. Additionally, signed carefully to ensure that the fluorescence profiles before
the emission peak of 1 is red-shifted from 436 to 461 nm. On and after the enzymatic reaction are different, ideally, non-fluo-
the basis of the properties of 1–4, we chose 1 for subsequent ex- rescence and intensive fluorescence, respectively. On the basis
periments involving enzymatic reactions.
of the structural features of 1, we expected that its fluorescence
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Beilstein J. Org. Chem. 2017, 13, 2690–2697.
Synthesis of the fluorogenic substrate for
would disappear as a result of peptide derivatization of the
amino group, because electron donation by the amino group DPP-4
would be attenuated and the fluorescence push–pull system The synthesis of H-Gly-Pro-1 is shown in Scheme 2. In the syn-
would be destroyed. To confirm this, we modified 1 to provide thesis, N-protected glycine was condensed with proline methyl
a peptidic substrate for an enzyme. The serine protease DPP-4 ester under microwave irradiation. Subsequent hydrolysis of the
was used as the test enzyme because its substrate specificity is ester gave N-protected Gly-Pro-OH (6). Condensation of dipep-
clear: it hydrolyses the C-terminal of proline or alanine second tide 6 with 1 was achieved using the corresponding acid chlo-
to the N-terminal of the peptide [18]. Additionally, DPP-4 is a ride. Deprotection of the amino group was achieved by treat-
significant biomarker for the progress of diabetes, and several ment with hydrazine, without disturbing the peptide bond struc-
chromogenic or fluorogenic substrates for DPP-4 have been de- ture, to give H-Gly-Pro-1.
veloped [19-21]. We therefore evaluated 1 as a fluorogenic
probe for DPP-4 activity using the peptide analogue H-Gly-Pro- DPP-4 activity measurement
1 as a substrate for DPP-4. The experimental strategy is summa- The fluorescence spectra of 1 and H-Gly-Pro-1 were recorded
rized in Scheme 1; non-fluorescent H-Gly-Pro-1 is hydrolysed to confirm their fluorescence profiles; the spectra are shown in
specifically by DPP-4 to release 1, enabling fluorometric mea- Figure 3. As expected, H-Gly-Pro-1 was non-fluorescent
surements.
because of the modification of the amino group to an amide
Scheme 1: Use of 1 as fluorogenic probe for DPP-4 activity.
Scheme 2: Synthesis of fluorogenic probe H-Gly-Pro-1.
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group. The loss of fluorescence is the result of attenuation of the DPP-4, with release of 1 as a fluorophore. In comparison with
electron-donating effect of the amino group to the benzene ring. existing fluorogenic substrates containing rhodamine fluoro-
In particular, a large difference between the fluorescence emis- phore, non-fluorescent characteristic of H-Gly-Pro-1 have
sions of 1 and H-Gly-Pro-1 at around 460 nm was observed. negligible background on the fluorometry to permit easy and
H-Gly-Pro-1 can therefore be used as a fluorogenic substrate for clear identification of reaction progress. On the other hand,
DPP-4 to minimize spectral interference from the background H-Gly-Pro-1 needs some improvements in terms of the reaction
state.
rate for this enzymatic reaction.
Conclusion
We synthesized the small fluorescent compounds 1–4. Among
them, 1 showed a good fluorescence profile despite its small
size. The use of 1 as a probe was investigated using the deriva-
tive H-Gly-Pro-1 as a fluorogenic substrate for DPP-4. The
enzymatic activity of DPP-4 was confirmed by measuring the
fluorescence intensity of the reaction medium, in which fluores-
cent 1 accumulated as the reaction progressed. H-Gly-Pro-1
behaves as an OFF–ON-type fluorogenic substrate for DPP-4.
Further studies of the synthesis of different TFPE-aniline deriv-
atives and fluorogenic substrates for other enzymes are under-
way.
Figure 3: Fluorescence spectra of 1 and H-Gly-Pro-1. Measurement
conditions: 1.0 × 10−5 M in H2O:DMSO (9:1), excitation at λ = 300 nm.
Experimental
General information
Next, we determined whether or not DPP-4 could hydrolyse All experiments were carried out under an argon atmosphere in
H-Gly-Pro-1. The hydrolysis experiments were conducted using flame-dried glassware using standard inert techniques for intro-
H-Gly-Pro-1 with DPP-4 concentrations of 0.07 and ducing reagents and solvents, unless otherwise noted. N,N-
7.00 μg/mL, and a control. The results are shown in Figure 4. Dimethylformamide (DMF) was distilled over calcium hydride
Enzymatic hydrolysis occurred when the DPP-4 concentration and stored in a bottle with activated molecular sieves (4 Å). All
was 7.00 μg/mL, as shown by the increased fluorescence inten- commercially available materials were used as received with-
sity as the reaction proceeded. In contrast, hydrolysis was not out further purification. 1H NMR, 13C NMR and 19F NMR
observed in the absence of DPP-4 (control). The reaction did spectra were measured on a JEOL ECZS 400S spectrometer
not proceed at a low concentration of DPP-4 (0.07 μg/mL). (1H: 400MHz, 13C: 100 MHz, 19F: 376 MHz). Chemical shifts
These results suggest that H-Gly-Pro-1 could be used as an of 1H NMR and 13C NMR are reported in parts per million
OFF–ON-type fluorogenic substrate for enzymatic reactions of from tetramethylsilane (TMS), used as an internal standard at
Figure 4: Fluorescence intensity changes of H-Gly-Pro-1 on addition of DPP-4.
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Beilstein J. Org. Chem. 2017, 13, 2690–2697.
0 ppm. Chemical shifts of 19F NMR are reported in parts per 89.59 (dd, J = 2.2, 6.5 Hz, 6F), 90.24 (dd, J = 2.2, 6.5 Hz, 3F);
million from trichlorofluoromethane (CFCl3), used as an MS m/z: M+ 375, 316, 266; HRMS m/z: M+ calcd for
internal standard at 0 ppm. All dates are reported as follows: C15H10F9N3, 375.0670; found, 375.0668.
chemical shifts, relative integration value, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet), and 4,6-Bis((E)-3,3,3-trifluoroprop-1-en-1-yl)-1,3-diaminoben-
coupling constants (Hz). High-resolution mass spectroscopy zene (3): The title product was purified by column chromatog-
(HRMS) experiments were performed with a double-focusing raphy and was obtained in 70% yield (183.7 mg). A light brown
mass spectrometer with EI. Melting points were measured on solid: mp 165–167 °C (recrystallized from AcOEt and hexane);
Yanaco melting point apparatus MP-500V without correction. 1H NMR (CDCl3) δ 3.95 (s, 4H), 6.00 (s, 1H), 6.01 (qd, J = 6.6,
Microwave reactions were performed in microwave tubes with 16.0 Hz, 2H), 7.09 (qd, J = 2.1, 16.0 Hz, 2H), 7.27 (s, 1H); 13C
clip lids using a Biotage Initiator microwave reactor.
NMR (CDCl3) δ 102.3, 111.4, 113.9 (q, J = 33.4 Hz), 123.8 (q,
J = 268.8 Hz), 128.6, 132.4 (q, J = 6.7 Hz), 147.5; 19F NMR
(CDCl3) δ 89.83 (dd, J = 2.2, 6.5 Hz, 3F), 90.47 (dd, J = 2.2,
6.5 Hz, 3F). MS m/z: M+ 296, 277, 257, 226, 187; HRMS m/z:
Typical procedure for the Hiyama cross-cou-
pling reaction
Analogous as described in [24]. In a glovebox purged with M+ calcd for C12H10F6N2, 296.2116; found, 296.0749.
argon gas, iodo aniline (1.0 mmol), (2-methylallyl)palladium(II)
(0.1 mmol), CuF2 (2.0 mmol), and 2,2’-bipyridyl (2.0 mmol) 2,4,6-Tris((E)-3,3,3-trifluoroprop-1-en-1-yl)-1,3-diamino-
were placed in a flask. To the flask were added anhydrous DMF benzene (4): The title product was purified by column chroma-
(6.0 mL) and (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane tography and was obtained in 20% yield (78 mg). A orange
(2.0 mmol), and mixture was stirred at 80 °C. After the reaction solid: mp 196 °C (recrystallized from AcOEt and hexane);
mixture was stirred for 4 h, it was poured into ice water. The 1H NMR (CDCl3) δ 4.12 (s, 4H), 6.04 (qd, J = 6.4, 15.9 Hz,
mixture was extracted with CH2Cl2, and the organic layer was 2H), 6.21 (qd, J = 6.1, 16.4 Hz, 1H), 7.02 (qd, J = 2.0, 16.4 Hz,
dried over anhydrous MgSO4. After the solid was filtered, the 1H), 7.11 (qd, J = 2.0, 15.9 Hz, 2H), 7.28 (s, 1H); 13C NMR
solvent was removed in vacuo, and the residue was purified by (CDCl3) δ 105.7, 111.1, 115.4 (q, J = 33.7 Hz), 122.6 (q, J =
silica gel column chromatography to give product.
270.3 Hz), 123.6 (q, J = 269.0 Hz), 124.9 (q, J = 33.7 Hz),
128.6, 131.0 (q, J = 6.7 Hz), 132.3 (q, J = 6.7 Hz), 144.5; 19F
2,4-Bis[(E)-3,3,3-trifluoroprop-1-enyl]aniline (1): The title NMR (CDCl3) δ 89.18 (dd, J = 1.5, 5.8 Hz, 3F), 90.40 (dd, J =
product was purified by column chromatography and was ob- 1.5, 6.5 Hz, 6F); MS m/z: M+ 390, 321; HRMS m/z: M+ calcd
tained in 89% yield (251 mg). A light yellow solid: mp for C15H11F9N3, 390.0779; found, 390.0783.
106–107 °C (recrystallized from AcOEt and hexane); 1H NMR
(CDCl3) δ 4.05 (s, 2H), 6.04 (qd, J = 6.6, 16.2 Hz, 1H), 6.19 Fluorogenic substrate synthesis
(qd, J = 6.5, 15.8 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 7.03 (qd, 1-[2-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)acetyl]-L-
J = 2.1, 16.2 Hz, 1H), 7.20 (qd, J = 2.1, 15.8 Hz, 1H), 7.30 (dd, proline methyl ester (5): N-Phtaloylglycine (4 mmol),
J = 1.8, 8.2 Hz, 1H), 7.36 (d, J = 1.8 Hz, 1H); 13C NMR 1-hydroxybenzotriazole (4.4 mmol) and N-ethyl-N’(3-dimethyl-
(CDCl3) δ 112.9 (q, J = 33.7 Hz), 116.8, 117.9 (q, J = 33.7 Hz), aminopropyl)carbodiimide hydrochloride (10 mmol) were
119.2, 123.3 (q, J = 269.4 Hz), 123.9 (q, J = 269.4 Hz), 124.4, placed in a microwave vial. To the microwave vial was added
127.9, 132.7 (q, J = 6.7 Hz), 136.8 (q, J = 6.7 Hz), 146.3; 19F anhydrous DMF (8 mL) and anhydrous N,N-diisopropylethyl-
NMR (CDCl3) δ 89.84 (dd, J = 2.2, 6.5 Hz, 3F), 90.47 (dd, J = amine (20 mmol), and the mixture was stirred at room tempera-
2.2, 6.5 Hz, 3F); MS m/z: M+ 281, 242, 211; HRMS m/z: M+ ture. After the reaction mixture was stirred for 5 min, to the
calcd for C12H9F6N, 281.0639; found, 281.0634.
microwave vial was added L-proline methyl ester hydro-
chloride (4.8 mmol) and the mixture was heated by microwave
2,4,6-Tris((E)-3,3,3-trifluoroprop-1-en-1-yl)aniline (2): The irradiation for 20 min at 120 °C. The resulting mixture was
title product was purified by column chromatography and was quenched with water and extracted with AcOEt. The AcOEt
obtained in 54% yield (203 mg). A light yellow solid: mp layer was washed with brine and dried over MgSO4. The sol-
119–120 °C (recrystallized from AcOEt and hexane); 1H NMR vent was removed in vacuo and the residue was purified by
(CDCl3) δ 4.20 (s, 2H), 6.11 (qd, J = 6.5, 16.0 Hz, 1H), 6.20 column chromatography to give 1-[2-(1,3-dihydro-1,3-dioxo-
(qd, J = 6.4, 16.0 Hz, 2H), 7.05 (qd, J = 2.1, 16.0 Hz, 1H), 7.21 2H-isoindol-2-yl)acetyl]-L-proline methyl ester (5) in 86%
(qd, J = 2.1, 16.0 Hz, 2H), 7.40 (s, 2H); 13C NMR (CDCl3) δ yield (1.09 g, 3.4 mmol). A white solid: mp 166–167 °C ;
114.1 (q, J = 34.0 Hz), 119.8 (q, J = 33.6 Hz), 121.1, 123.0 (q, 1H NMR (CDCl3) δ 1.90–2.36 (m, 4H), 3.60–3.83 (m, 5H),
J = 269.4 Hz), 124.3, 124.7 (q, J = 269.2 Hz), 128.7, 132.3 (q, 4.40 (d, J = 16.5 Hz, 1H), 4.51–4.61 (m, 2H), 7.70–7.73 (m,
J = 6.8 Hz), 136.2 (q, J = 6.7 Hz), 143.9; 19F NMR (CDCl3) δ 2H), 7.85–7.88 (m, 2H); 13C NMR (CDCl3) δ 24.8, 28.9, 39.7,
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Beilstein J. Org. Chem. 2017, 13, 2690–2697.
46.2, 52.3, 59.1, 123.5, 132.2, 134.0, 164.4, 167.8, 172.1; MS 0.036 mmol). A light yellow solid: mp 129–130 °C; 1H NMR
m/z: M+ 316, 284, 257, 188, 160, 128; HRMS m/z: M+ calcd for (DMSO-d6) δ 1.90–2.25 (m, 4H), 3.51–3.80 (m, 2H), 4.56 (d,
C16H16N2O5, 316.3086; found, 316.1051.
J = 8.3 Hz), 6.75–6.95 (m, 2H), 7.21–7.53 (m, 4H), 7.75 (d, J =
8.2 Hz), 8.20 (s, 1H); 13C NMR (DMSO-d6) δ 24.8, 26.7, 46.2,
(S)-N-(2,4-Bis((E)-3,3,3-trifluoroprop-1-en-1-yl)phenyl)-1- 60.6, 70.3, 116.2 (q, J = 33.6 Hz), 117.2 (q, J = 33.3 Hz), 124.3
(2-(1,3-dioxo-2H-isoindolin-2-yl)acetyl)pyrrolidine-2- (q, J = 269.0 Hz), 124.6 (q, J = 269.0 Hz), 126.6, 127.2, 128.7,
carboxamide (7): 5 (2 mmol) and lithium hydroxide (12 mmol) 130.4, 131.2, 134.1 (q, J = 7.0 Hz), 137.6 (q, J = 7.0 Hz), 138.4,
were placed in a flask. To the flask was added THF (4.8 mL), 171.4; 19F NMR (DMSO-d6) δ 80.80 (dd, J = 2.2, 7.2 Hz, 3F),
methanol (1.5 mL), water (1.5 mL), and the mixture was stirred 80.91 (dd, J = 2.2, 7.2 Hz, 3F); MS m/z: M+ 435, 281, 242, 174,
overnight at room temperature. The resulting solution was 155, 127; HRMS m/z: M+ calcd for C19H19F6N3O2, 435.3635;
concentrated in vacuo, the residue was diluted with aq HCl and, found, 435,1382.
extracted with CHCl3:EtOH (2:1). The organic layer was
Assay of dipeptidyl peptidase activity using
washed with brine and dried over MgSO4. The solvent was re-
moved in vacuo to give 6. To a solution of 6 (1 mmol) in an- H-Gly-Pro-1 as a substrate
hydrous CH2Cl2 was added oxalyl chloride (2 mmol) and an- Stenotrophomonas dipeptidyl aminopeptidase IV was prepared
hydrous DMF (1 drop) at 0 °C, and mixture was stirred at room from the E. coli DH5 containing pUC19-SDP4 as described pre-
temperature. After the reaction mixture was stirred for 3 h, it viously [22]. The enzyme activity was assayed using H-Gly-
was concentrated in vacuo. To the residue was added 1 Pro-1 as a substrate following the procedure described previ-
(0.3 mmol), anhydrous 1,4-dioxane (3 mL) and, anhydrous pyri- ously [23]. The reaction mixture (total 100 μL) contained 0.1 M
dine (0.45 mmol), and mixture was stirred at room temperature. Tris–HCl (pH 8.0), 10–50 μM substrate and 10–400 ng of the
After the reaction mixture was stirred for 2 h, it was quenched enzyme. The reaction was initiated by the addition of the en-
with sat. NaHCO3 and, extracted with CHCl3. The CHCl3 layer zyme solution. Following incubation at 37 °C for 5 min in a
was washed with water and dried over MgSO4. The solvent was 96-well plate inside a SH-9000 plate reader (Corona electric,
removed in vacuo and the residue was purified by column chro- Japan), the amount of 1 liberated was determined fluorometri-
matography to give (S)-N-(2,4-bis((E)-3,3,3-trifluoroprop-1-en- cally. The excitation and emission wavelengths used were
1-yl)phenyl)-1-(2-(1,3-dioxo-2H-isoindolin-2-yl)acetyl)pyrrol- 300 nm and 460 nm for 1, respectively. The reaction velocities
idine-2-carboxamide (7) in 31% yield (71.9 mg, 0.12 mmol). A (µmol/min/mg protein) were compared.
white solid: mp 181–182 °C; 1H NMR (CDCl3) δ 1.87–1.97 (m,
1H), 2.14–2.28 (m, 2H), 2.63–2.67 (m, 2H), 3.58–3.77 (m, 2H), Acknowledgements
4.51 (d, J = 3.9 Hz, 2H), 4.82 (d, J = 7.8 Hz, 1H), 6.11 (qd, J = We thank Helen McPherson, PhD, from Edanz Group (http://
6.4, 16.0 Hz, 2H), 7.08 (qd, J = 2.2, 16.0 Hz, 1H), 7.28 (qd, J = www.edanzediting.com/ac) for editing a draft of this manu-
2.1, 16.0 Hz, 1H), 7.44 (d, J = 6.6 Hz, 2H), 7.73–7.90 (m, 4H), script.
8.08 (d, J = 9.0 Hz, 1H), 9.46 (s, 1H); 13C NMR (CDCl3) δ
ORCID® iDs
Keita Tani - https://orcid.org/0000-0002-9449-8406
26.3, 29.7, 39.7, 46.9, 61.2, 115.8 (q, J = 33.9 Hz), 119.4 (q,
J = 34.0 Hz), 122.7 (q, J = 270.0 Hz), 122.8, 123.5 (q, J =
269.0 Hz), 123.8, 125.8, 126.4, 129.9, 132.0, 132.7 (q, J = 6.7
References
1. Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Chem. Rev. 2013, 113, 192–270.
Hz), 136.4, 137.6 (q, J = 6.8 Hz), 137.6, 167.2, 167.8, 168.5;
19F NMR (CDCl3) δ 89.32 (dd, J = 2.2, 6.6 Hz, 3F), 89.54 (dd,
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