Survey of Dipeptidyl Peptidase III Inhibitors: From Small Molecules of Microbial or Synthetic Origin to Aprotinin

Dipeptidyl peptidase III (DPP III) was originally thought to be a housekeeping enzyme that contributes to intracellular peptide catabolism. More specific roles for this cytosolic metallopeptidase, in the renin-angiotensin system and oxidative stress regulation, were confirmed, or recognized, only recently. To prove indicated (patho)physiological functions of DPP III in cancer progression, cataract formation and endogenous pain modulation, or to reveal new ones, selective and potent inhibitors are needed. This review encompasses natural and synthetic compounds with experimentally proven inhibitory activity toward mammalian DPP III. Except for the polypeptide aprotinin, all others are small molecules and include flavonoids, coumarin and benzimidazole derivatives. Presented are current strategies for the discovery or development of DPP III inhibitors, and mechanisms of inhibitory actions. The most potent inhibitors yet reported (propioxatin A and B, Tyr-Phe- and Phe-Phe-NHOH, and JMV-390) are active in low nanomolar range and contain hydroxamic acid moiety. High inhibitory potential possesses oligopeptides from the hemorphin group, valorphin and tynorphin, which are poor substrates of DPP III. The crystal structure of human DPP III-tynorphin complex enabled the design of the transition-state peptidomimetics inhibitors, effective in low micromolar concentrations. A new direction in the field is the development of fluorescent inhibitor for monitoring DPP III activity.     

Synthesis and photophysical properties of a fluorescent TREN-type ligand incorporating the coumarin chromophore and its zinc complex

A new, UV-excited fluorescent Zn²⁺ indicator was synthesized and the spectral profile of its free and Zn²⁺ bound forms were studied. The fluorescent properties of this probe are due to the 7-amino-4-methylcoumarin fluorophore, which is conjugated with the tris(2-aminoethyl)amine (TREN) that functions as the zinc-chelating moiety. The compound exhibits a Zn²⁺ dissociation constant of 18.0 μM. The fluorescence spectra of the probe showed a clear shift in excitation wavelength maxima upon Zn²⁺ binding, indicating its potential use as ratiometric Zn²⁺ indicator.     

Observation of differential reactivity of cyclic amines in SN2 and SNAr displacement reactions in the course of synthesizing C-6, C-7 substituted quinolinecarbonitrile MEK1 kinase inhibitors

We have previously reported on a series of 4-anilino-6,7-dialkoxy-3-quinolinecarbonitriles as potent inhibitors of MEK1 kinase. Herein, we describe our synthetic efforts toward a series of 4-anilino-6-alkoxy-7-amino-3-quinolinecarbonitriles. In the course of this work, we were able to rapidly construct a library of 4-anilino-6-alkoxy-7-amino-3-quinolinecarbonitriles by simultaneous or sequential S_N2 (displacement) reactions on the C-6 chloroalkoxy moiety and S_NAr (addition/elimination) reactions at C-7 with nucleophilic amines.     

Asymmetric synthesis of 3-amino-2-hydroxy-4-phenylbutanoate

Asymmetric synthesis of 3-amino-2-hydroxy-4-phenylbutanoate, a key component of the natural product bestatin and HIV protease inhibitors of KNI-272 and R-87366, has been achieved from the stereoselective aldimine coupling reaction between 3-phenyl-2-aminopropanenitrile and (Z)-α-methoxy trimethylsilyl ketene acetal in the presence of Lewis acids.     

High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2′-Bipyridine Scaffold for Endogenous Zn2+ and Its Application to Living Cells

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn²⁺, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn²⁺ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (K_d = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn²⁺. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn²⁺ in living systems.     

快速高分离度液相色谱/质谱方法筛选组织蛋白酶B的抑制剂

1引言组织蛋白酶B(CB)是溶酶体内半胱氨酸蛋白水解酶,属于木瓜蛋白酶家族,其特异性底物为Z-Arg-Arg-7-氨基-4-甲基香豆素[1],CB催化底物肽键断裂生成7-氨基-4-甲基香豆素。近年来的研究表明,CB在肿瘤的发展中起作用的一类关键酶[2],CB通过多种机制促进血管形成,从而促进肿瘤生长,同时降解基底膜主要成分以及激活胶原酶、尿激酶等其它蛋白酶发生协同作用,使肿瘤易侵袭、转移和扩散。在肿瘤组织中还发现CB与CB抑制剂平衡作用也参与肿瘤的发     

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O-GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group-transfer enzymes.     

Improved Spectral Coverage and Fluorescence Quenching in Donor–acceptor Systems Involving Indolo[3‐2‐b]carbazole and Boron‐dipyrromethene or Diketopyrrolopyrrole

A novel π‐conjugated triad and a polymer incorporating indolo[3,2‐b]‐carbazole (ICZ) and 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) were synthesized via a Sonogashira coupling. Compared to the parent BODIPY the absorption and fluorescence spectrum were for both compounds broader and redshifted. The redshift of the fluorescence and the decrease of the fluorescence quantum yield and decay time upon increasing solvent polarity were attributed to the formation of a partial charge‐transfer state. Upon excitation in the ICZ absorption band the ICZ fluorescence was quenched in both compounds mainly due to energy transfer to the BODIPY moiety. In a similar ICZ–π–DPP polymer (where DPP is diketopyrrolopyrrole), a smaller redshift of the absorption and fluorescence spectra compared to the parent DPP was observed. A less efficient quenching of the ICZ fluorescence in the ICZ–π–DPP polymer could be related to the unfavorable orientation of the transition dipoles of ICZ and DPP. The rate constant for energy transfer was for all compounds an order of magnitude smaller than predicted by Förster theory. While in a solid film of the triad a further redshift of the absorption maximum of nearly 100 nm was observed, no such shift was observed for the ICZ–π–BODIPY polymer.     

A Vinyl Sulfone‐Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells

Chemical probes are powerful tools for interrogating small molecule‐target interactions. With additional fluorescence Turn‐ON functionality, such probes might enable direct measurements of target engagement in live mammalian cells. DNS‐pE (and its terminal alkyne‐containing version DNS‐pE2) is the first small molecule that can selectively label endogenous 3‐phosphoglycerate dehydrogenase (PHGDH) from various mammalian cells. Endowed with an electrophilic vinyl sulfone moiety that possesses fluorescence‐quenching properties, DNS‐pE/DNS‐pE2 became highly fluorescent only upon irreversible covalent modification of PHGDH. With an inhibitory property (in vitro K_i=7.4 μm ) comparable to that of known PHGDH inhibitors, our probes thus offer a promising approach to simultaneously image endogenous PHGDH activities and study its target engagement in live‐cell settings.     

A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.     

Time-resolved fluorescence measurements using microlens array and area imaging devices

Time-resolved fluorescence (TRF) assay formats are frequently used technologies in high-throughput screening. In this article, we have characterised the novel Plate::Vision(2) 96-microlens array reader (Carl Zeiss Jena GmbH, Germany) and compared it to the novel LEADseeker Generation IV multimodality imaging system (LEADseeker Gen IV; Amersham Biosciences UK Ltd., UK) for applications in the TRF mode. In europium measurements using the TRF mode, the Plate::Vision displayed a limit of detection for europium of approximately 3 pM, which was comparable to two established TRF readers, the Discovery and the Victor V (both PerkinElmer Life Sciences Inc., USA). The LEADseeker's limit of detection only extended down to europium concentrations of approximately 10 pM in these experiments. For TRF resonance energy transfer (TR-FRET) experiments, a europium-biotin (Eu-biotin) conjugate was titrated with a streptavidin-allophycocyanin (SA-APC) conjugate. The Plate::Vision produced Z' values larger than 0.5 for the acceptor fluorophor emission with concentrations of Eu-biotin as low as 3 nM combined with 175 pM SA-APC. To achieve Z' values of at least 0.5 with the LEADseeker, concentrations of 10 nM Eu-biotin combined with SA-APC of at least 0.8 nM were required. In a drug screening application using TR-FRET, the energy transfer from a europium-labelled protein X (Eu-protein X) to a complex of biotinylated peptide Y with SA-APC was measured. Using the Plate::Vision, a Z' factor larger than 0.5 for the acceptor fluorophor emission was only obtained for a Eu-protein X concentration of at least 10 nM in combination with biotinylated peptide Y/SA-APC at saturating concentrations. Both the Plate::Vision and the LEADseeker show good quality results for applications in the TRF mode and enable an increased throughput based on their shortened measurement time in comparison to classic photomultiplier tube-based readers.     

Cyclometalated Complexes of Palladium(II) and Platinum(II): cis-Configured Homoleptic and Heteroleptic Compounds with Aromatic C&arcraise;N Ligands

The palladium(II) and platinum(II) bis-homoleptic complexes M(C&arcraise;N)(2), where C&arcraise;N is benzo[h]quinoline (H-bhq), 2-phenylpyridine (H-phpy), 2-(2'-benzothienyl)pyridine (H-bthpy), 2-(2'-thienyl)quinoline (H-thq), and 2-(2'-thienyl)pyridine (H-thpy), were prepared by metal exchange of the lithiated ligands C&arcraise;N with M(Et(2)S)(2)Cl(2). The palladium(II) bis-heteroleptic complexes, Pd(C&arcraise;N)(C'&arcraise;N'), were synthesized from Pd(C&arcraise;N)(2) bis-homoleptic complexes, which were converted by HCl into the dichloro-bridged compounds [Pd(C&arcraise;N)Cl](2). By addition of Et(2)S, the Pd(C&arcraise;N)(Et(2)S)Cl complexes were formed, which were allowed to react with Li(C'&arcraise;N'), yielding M(C&arcraise;N)(C'&arcraise;N') compounds. An alternative way for obtaining the bis-heteroleptic molecules is by ligand exchange, according to the equilibrium M(C&arcraise;N)(2) + M(C'&arcraise;N')(2) = 2M(C&arcraise;N)(C'&arcraise;N'). The crystal structures of Pt(bhq)(2) (1) and Pt(thq)(2) (3) present an important distortion of the square planar (SP-4) geometry toward a two-bladed helix. Bis-homoleptic and some bis-heteroleptic complexes of palladium(II) have also been synthesized. In both cases, the steric interactions between the two ligands cause again a helical distortion rather than yielding trans compounds. For cis-bis(benzo[h]quinoline)platinum(II) (1), in the crystal (monoclinic, space group P2(1)/n, a = 13.728(3) ?, b = 6.9537(15) ?, c = 19.701(5) ?, beta = 106.17(2) degrees, Z = 4, rho(calcd) = 2.028 g.cm(-)(3); diffractometer measurements, block-matrix least-squares refinement, R = 0.043, R(w) = 0.047) the average Pt-N and Pt-C distances are 2.151(6) and 1.988(7) ?, respectively. One benzo[h]quinoline ligand is significantly less planar than the other. For cis-bis[2-(2'-thienyl)quinoline]platinum(II) (3), in the crystal (trigonal, space group P3(2)21, a = b = 9.373(1) ?, c = 20.152(3) ?, Z = 3, rho(calcd) = 2.022 g.cm(-)(3); diffractometer measurements, full-matrix least-squares refinement, R = 0.010, R(w) = 0.010) the molecule has C(2) symmetry and is chiral. The Pt-N and Pt-C bond lengths are 2.156(2) and 1.984(3) ?, respectively. The quinoline moitey is not planar but bent about the fused bond by 6.8 degrees. The thiophene moiety is inclined to the best plane through the quinoline moiety by 24.4 degrees.     

Synthesis and anticholinesterase activity of some new fluorogenic analogues of organophosphorus nerve agents

Eighteen new fluorogenic analogues of organophosphorus nerve agents were synthesised and characterised. They included analogues of tabun, sarin, cyclosarin, soman, VX, and Russian VX, with the 7-oxy-4-methylcoumarin or 7-oxy-4-(trifluoromethyl)coumarin leaving group. These analogues inhibited acetylcholinesterase (AChE) effectively in vitro and therefore have potential as tools for the identification of novel organophosphatases in biological systems. Analogues of VX and Russian VX with the 7-amino-4-methylcoumarin group, although poor AChE inhibitors, may have utility for screening enzyme libraries for phosphoramidases capable of cleaving P-N bonds.     

Ratiometric fluorescence detection of pyrimidine/purine transversion by using a 2-amino-1,8-naphthyridine derivative.

A new class of abasic site-binding fluorescence ligands, Naph-NBD in which 7-nitrobenzo-2-oxa-1,3-diazole (NBD) is connected to 2-amino-7-methyl-1,8-naphthyridine (Naph) by a propylene linker, is presented for the ratiometric assay for SNPs typing. In solutions buffered to pH 7.0 (I = 0.11 M, at 5 degrees C), Naph-NBD is found to selectively recognize pyrimidine bases over purine bases opposite the abasic site in DNA duplexes (K11/M(-1): T, 8.1 x 10(6); C, 2.5 x 10(6): G, 0.33 x 10(6); A, 0.27 x 10(6)). The binding of Naph-NBD is accompanied by significant quenching of the fluorescence from the naphthyridine moiety (lambda max, 409 nm), while the emission from the NBD (lamda max, 544 nm) is relatively unaffected. Such a fluorescence response of Naph-NBD allows the emission ratio detection of pyrimidine/purine transversion.     

(1,3‐Diphenyl‐1H‐Pyrazol‐4‐yl)‐Methylamine Analogues as Inhibitors of Dipeptidyl Peptidases

A number of pyrazole compounds reported in literatures elicit anti‐hyperglycemic effects. By modifying the side chain of the heterocyclic skeleton, a new chemical class of DPP‐IV inhibitors structurally derived from the (pyrazol‐4‐yl)‐methylamine scaffold have been discovered and evaluated the biological activities of these inhibitors against DPP‐IV, DPP8, DPP‐II and FAP. The SAR studies showed the (1,3‐diphenyl‐1H‐pyrazol‐4‐yl)‐methylamines with 2,4‐dichloro substituents at the 3‐phenyl ring selectively preferred as DPP‐IV inhibitors, whereas with difluoro substituents at the 3‐phenyl ring selectively preferred as DPP8 inhibitors. The binding mode of representative compound 15h at the active site of DPP‐IV was predicted by computer model. In additional, 15h exhibited the ability to significantly decrease the glucose excursion in mice.     

Determination of dissolved oxygen in water based on its quenching effect on the fluorescent intensity of bis(2,2'-bipyridine)-5-amino-1,10-phenanthroline ruthenium complex

A water-soluble fluorescence dye, bis(2,2'-bipyridine)-5-amino-1,10-phenanthroline ruthenium complex (Ru(bpy)(2)(5-NH(2)-1,10-phen)), was synthesized and used as a fluorescence probe for detecting dissolved oxygen in water. The fluorescence intensity of the probe in different dissolved-oxygen concentrations was investigated. The sensitivity of the probe was evaluated in terms of the ratio I(N(2))/I(O(2)), where I(N(2)) and I(O(2)) correspond to the detected fluorescence intensity of nitrogen and oxygen-saturated solutions, respectively. The experimental results showed that the probe yielded a linear Stern-Volmer plot, and had a I(N(2))/I(O(2)) ratio of about 5.2. The detection limit, defined as three-times the standard deviation, was 8.6 × 10(-7) mol L(-1) after eleven determinations of nitrogen-saturated blank solutions. Additionally, the probe was pH-insensitive and ionic strength-independent with good characteristics of practicality and selectivity.     

Crystal structures and solution properties of discrete complexes composed of saddle-distorted molybdenum(v)-dodecaphenylporphyrins and keggin-type heteropolyoxometalates linked by direct coordination

Reactions of a saddle-distorted Mo(V)-porphyrin complex, [Mo(DPP)(O)(H(2)O)]ClO(4) (1·ClO(4); DPP(2-) = dodecaphenylporphyrin dianion), with tetra-n-butylammonium (TBA) salts of Keggin-type heteropolyoxomatalates (POMs), α-[XW(12)O(40)](n-) (X = P, n = 3, 2; X = Si, n = 4, 3; X = B, n = 5; 4), in ethyl acetate/acetonitrile gave 2:1 complexes formulated as [{Mo(DPP)(O)}(2)(HPW(12)O(40))] (5), [{Mo(DPP)(O)}(2)(H(2)SiW(12)O(40))] (6), and [(n-butyl)(4)N](2)[{Mo(DPP)(O)}(2)(HBW(12)O(40))] (7) under mild reaction conditions. The crystal structures of the complexes were determined by X-ray crystallography. In these three complexes, named Porphyrin Hamburgers, the POM binds to two Mo(V) centers of porphyrin units directly via coordination of two terminal oxo groups. In spite of the similarity of those POM's structures, those Porphyrin Hamburgers exhibit different coordination bond angles between POM and the Mo(V) center in the porphyrin: 5 and 7 show two different coordination bond angles in one molecule in contrast to 6, which exhibits only one coordination bond angle. The Porphyrin Hamburgers involve protonation of the POM moieties to adjust the charge balance, as confirmed by spectroscopic titration with bases. In the crystals, the Porphyrin Hamburgers form two-dimensional (2D) sheets in the ac plane based on π-π interactions among peripheral phenyl substituents. Stacking of the 2D sheets toward the b axis constructs a 3D layered structure involving channels running into the crystallographic [1 0 0] and [0 0 1] directions in the crystal to include solvent molecules of crystallization for 5-7, and also counter cations for 7. Three complexes were revealed to be stable enough to maintain their structures even in solutions to show molecular ion peaks in the MALDI-TOF-MS measurements. They also exhibited different electron paramagnetic resonance (EPR) signals because of the Mo(V) (S = 1/2, I = 0) centers, reflecting the difference in the crystal structures. In addition, these complexes showed reversible multistep redox processes as observed in their cyclic voltammograms in benzonitrile to demonstrate high stability throughout the redox reactions in solution.     

Near‐Infrared Fluorescent Probe with New Recognition Moiety for Specific Detection of Tyrosinase Activity: Design,Synthesis, and Application in Living Cells and Zebrafish

Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4‐hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase‐recognition 3‐hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed. Using the recognition moiety, we develop a near‐infrared fluorescence probe for tyrosinase activity, which effectively eliminates the interference from ROS. The high specificity of the probe was demonstrated by imaging and detecting endogenous tyrosinase activity in live cells and zebrafish and further validated by an enzyme‐linked immunosorbent assay. The probe is expected to be useful for the accurate detection of tyrosinase in complex biosystems.     

Application of 2-(3,5,6-trifluoro-2-hydroxy-4-methoxyphenyl)benzoxazole and -benzothiazole to fluorescent probes sensing pH and metal cations.

2-(3,5,6-Trifluoro-2-hydroxy-4-methoxyphenyl)benzoxazole (3) and benzothiazole analogue (4) are prepared by the two-step procedures from the corresponding 2-(pentafluorophenyl)benzazoles. Benzoxazole 3 is applicable to a fluorescent probe sensing magnesium cation, and 4 is suitable for sensing zinc cation. Both fluorophores 3 and 4 are sensitive to the pH change at pH 7-8, resulting in large fluorescence enhancement under basic conditions. Their high sensitivity to pH and selectivity in metal cations are ascribed to the high acidity of the fluorophenol moiety.     

A Molecular Probe with Both Chromogenic and Fluorescent Units for Detecting Serine Proteases

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.