Development of a fluorogenic small substrate for dipeptidyl peptidase-4

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.


Introduction
Fluorescent organic probes have become indispensable for bioimaging or molecular imaging to visualize the presence of species such as particular enzymes and biologically important inorganic ions. In biomedicine, these probes are used for the detection of a wide range of significant biomarkers and are essential for the diagnosis of critical diseases [1][2][3]. Selective and accurate determinations of specific enzymes, including β-galactosidase [4][5][6], exoglycosidases [7], cyclooxygenases [8], and others [9][10][11][12][13], have been achieved using the OFF-ON-type fluorogenic probes with fluorescence that can be turned on by enzymatic transformations. These fluorescent probes can act as good sensors for enzymes but the fluorophore unit is usually large because fused aromatic rings and an elongated π-conjugated system are necessary to impart appropriate fluorescent properties to the probe [14]. The molecular size of the fluorescent probe is therefore large, which is sometimes inconvenient in terms of membrane permeability, water solubility, and inhibition of inherent interactions between the probe molecule and the target enzyme. We have developed a new OFF-ON probe with a small fluorescent core unit. The same approach was recently  used to produce a fluorophore for a push-pull system, which has a single benzene ring substituted by both electron-donating and electron-withdrawing groups [15][16][17]. We focused on the 3,3,3-trifluoropropenyl (TFPE) group, which is an electronwithdrawing substituent that is free of oxygen atoms, which would form hydrogen bonds in protic solvents. We assumed that the use of the oxygen-free TFPE group would eliminate hydrogen bonding with protic solvents, even water, to avoid attenuation of the fluorescent characteristics of the fluorophore.
In this paper, we report the synthesis of a new fluorophore with TFPE groups and its application as an OFF-ON-type fluorogenic probe for determining dipeptidyl peptidase-4 (DPP-4) activity.

Synthesis of fluorescent compounds as 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 TFPE would be a reasonable approach to producing new fluorescent molecules. However, there are few methods for synthesizing TFPE-substituted compounds. In a previous study, we achieved the facile introduction of TFPE groups via Hiyama cross-coupling reactions of (E)-trimethyl-(3,3,3-trifluoroprop-1enyl)silane and iodoanilines. This straightforward method is useful for TFPE introduction and a broad range of iodoanilines can be used without protection of the amino group. We used this approach to synthesize several aniline derivatives containing TFPE groups. The data in Table 1 show that Hiyama crosscoupling reactions gave the desired TFPE-anilines and TFPEphenylenediamines (1)(2)(3)(4) in moderate to good yields. This method has the advantage that complete substitution of diiodoand triiodoarenes can proceed to give disubstituted (1 and 3) and trisubstituted (2 and 4) products in one synthetic operation.
Next, the fluorescence profiles of these products were investigated. The results are summarized in Figure 1 and Table 2. The number of amino groups and TFPE groups introduced into compounds 1-4 significantly affected the fluorescence profiles in  THF solution. TFPE-anilines 1 and 2 gave moderate fluorescence quantum yields of 0.31 and 0.30, respectively. In comparison with these, the fluorescence quantum yields of TFPEphenylenediamine 3 was slightly lower, namely 0.25. The introduction of one more TFPE group into 3, to give 4, further decreased 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 biomedical use of fluorescent compounds. Next, we changed the solvent from THF to H 2 O/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 small, namely 0.27 and 0.20, respectively. This is reasonable for 1 and 3 because the oxygen-free TFPE group is less likely to form hydrogen bonds, even in aqueous solution. Additionally, the emission peak of 1 is red-shifted from 436 to 461 nm. On the basis of the properties of 1-4, we chose 1 for subsequent experiments involving enzymatic reactions.

Design of fluorogenic probes
Fluorogenic OFF-ON-type probes for enzymes should be designed carefully to ensure that the fluorescence profiles before and after the enzymatic reaction are different, ideally, non-fluorescence and intensive fluorescence, respectively. On the basis of the structural features of 1, we expected that its fluorescence would disappear as a result of peptide derivatization of the amino group, because electron donation by the amino group would be attenuated and the fluorescence push-pull system would be destroyed. To confirm this, we modified 1 to provide a peptidic substrate for an enzyme. The serine protease DPP-4 was used as the test enzyme because its substrate specificity is clear: it hydrolyses the C-terminal of proline or alanine second to the N-terminal of the peptide [18]. Additionally, DPP-4 is a significant biomarker for the progress of diabetes, and several chromogenic or fluorogenic substrates for DPP-4 have been developed [19][20][21]. We therefore evaluated 1 as a fluorogenic probe for DPP-4 activity using the peptide analogue H-Gly-Pro-1 as a substrate for DPP-4. The experimental strategy is summarized in Scheme 1; non-fluorescent H-Gly-Pro-1 is hydrolysed specifically by DPP-4 to release 1, enabling fluorometric measurements.

Synthesis of the fluorogenic substrate for DPP-4
The synthesis of H-Gly-Pro-1 is shown in Scheme 2. In the synthesis, N-protected glycine was condensed with proline methyl ester under microwave irradiation. Subsequent hydrolysis of the ester gave N-protected Gly-Pro-OH (6). Condensation of dipeptide 6 with 1 was achieved using the corresponding acid chloride. Deprotection of the amino group was achieved by treatment with hydrazine, without disturbing the peptide bond structure, to give H-Gly-Pro-1.

DPP-4 activity measurement
The fluorescence spectra of 1 and H-Gly-Pro-1 were recorded to confirm their fluorescence profiles; the spectra are shown in Figure 3. As expected, H-Gly-Pro-1 was non-fluorescent because of the modification of the amino group to an amide group. The loss of fluorescence is the result of attenuation of the electron-donating effect of the amino group to the benzene ring.
In particular, a large difference between the fluorescence emissions of 1 and H-Gly-Pro-1 at around 460 nm was observed.
H-Gly-Pro-1 can therefore be used as a fluorogenic substrate for DPP-4 to minimize spectral interference from the background state.

Experimental General information
All experiments were carried out under an argon atmosphere in flame-dried glassware using standard inert techniques for introducing reagents and solvents, unless otherwise noted. N,N-Dimethylformamide (DMF) was distilled over calcium hydride and stored in a bottle with activated molecular sieves (4 Å). All commercially available materials were used as received without further purification. 1 H NMR, 13  Typical procedure for the Hiyama cross-coupling reaction Analogous as described in [24]. In a glovebox purged with argon gas, iodo aniline (1.0 mmol), (2-methylallyl)palladium(II) (0.1 mmol), CuF 2 (2.0 mmol), and 2,2'-bipyridyl (2.0 mmol) were placed in a flask. To the flask were added anhydrous DMF (6.0 mL) and (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane (2.0 mmol), and mixture was stirred at 80 °C. After the reaction mixture was stirred for 4 h, it was poured into ice water. The mixture was extracted with CH 2 Cl 2 , and the organic layer was dried over anhydrous MgSO 4 . After the solid was filtered, the solvent was removed in vacuo, and the residue was purified by silica gel column chromatography to give product.