Development of non-acidic 4-methylbenzenesulfonate-based aldose reductase inhibitors; Design, Synthesis, Biological evaluation and in-silicostudies.

Design and virtual screening of a set of non-acidic 4-methyl-4-phenyl-benzenesulfonate-based aldose reductase 2 inhibitors had been developed followed by chemical synthesis. Based on the results, the synthesized compounds 2, 4a,b, 7a-c, 9a-c, 10a-c, 11b,c and 14a-c inhibited the ALR2 enzymatic activity in a submicromolar range (99.29-417 nM) and among them, the derivatives 2, 9b, 10a and 14b were able to inhibit ALR2 by IC50 of 160.40, 165.20, 99.29 and 120.6 nM, respectively. Moreover, kinetic analyses using Lineweaver-Burk plot revealed that the most active candidate 10a inhibited ALR2 potently via a non-competitive mechanism. In vivo studies showed that 10 mg/kg of compound 10a significantly lowered blood glucose levels in alloxan-induced diabetic mice by 46.10 %. Moreover, compound 10a showed no toxicity up to a concentration of 50 mg/kg and had no adverse effects on liver and kidney functions. It significantly increased levels of GSH and SOD while decreasing MDA levels, thereby mitigating oxidative stress associated with diabetes and potentially attenuating diabetic complications. Furthermore, the binding mode of compound 10a was confirmed through MD simulation. Noteworthy, compounds 2 and 14b showed moderate antimicrobial activity against the two fungi Aspergillus fumigatus and Aspergillus niger. Finally, we report the thiazole derivative 10a as a new promising non-acidic aldose reductase inhibitor that may be beneficial in treating diabetic complications.

Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Branched alkyl of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates as unique cytochrome P450 1A1-activated antimitotic prodrugs: Biological evaluation and mechanism of bioactivation.

We recently discovered a new family of prodrugs deriving from phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) bioactivatable by cytochrome P450 1A1 (CYP1A1) into potent antimitotics referred to as phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs). PAIB-SOs display significant selectivity toward human breast cancer cells based on the N-dealkylation of PAIB-SOs into their corresponding PIB-SOs by CYP1A1. In this study, we have evaluated the molecular mechanism of the bioactivation of PAIB-SOs into PIB-SOs by branching the linear alkyl chain on the imidazolidin-2-one (IMZ) moiety of PAIB-SOs by branched alkyl groups such as isopropyl, isobutyl and sec-butyl. Our results show that PAIB-SOs bearing an isobutyl group on the IMZ moiety and either a methoxy, a chloro or a bromo group at positions 3, 3,5 or 3,4,5 on the aromatic ring B exhibit antiproliferative activity ranging from 0.13 to 6.9 µM and selectivity toward MCF7 and MDA-MB-468 mammary cancer cells comparatively to other cell lines tested. Moreover, the most potent and selective PAIB-SOs bearing an isobutyl group and either a 3,5-Cl (44), 3,5-Br (45) or a 3,4,5-OMe (46) on the IMZ moiety exhibit antiproliferative activity in the sub-micromolar range and high selectivity ratios toward mammary cancer cells. They stop the cell cycle of MCF7 cells in the G2/M phase and disrupt their cytoskeleton. Furthermore, our studies evidenced that PAIB-SOs bearing either an isopropyl, a sec-butyl or an isobutyl group are hydroxylated on the carbon atom adjacent to the IMZ (Cα-OH) but only PAIB-SOs bearing an isobutyl group are bioactivated into PIB-SOs. Finally, PAIB-SOs 45 and 46 exhibit low toxicity toward normal cells and chick embryos and are thus promising antimitotic prodrugs highly selective toward CYP1A1-expressing breast cancer cells.

Design, synthesis and biological activity of bis-sulfonyl-BODIPY probes for tumor cell imaging.

In recent years, BODIPY derivatives have become one of the research hotspots in the field of bioprobes, but most of them have the problems of poor hydrophilicity, low biocompatibility and no targeting. In this paper, novel ethylenediamine bridging bis-sulfonyl-BODIPY fluorescent probes were successfully designed and synthesized to solve these problems; What's more, the cytotoxicity analysis, cell imaging, in vivo imaging and apoptosis experiments were carried out. Ethylenediamine bridges and oxygen-rich sulfonyl groups made such probes had certain hydrophilicity, so they could be dissolved in dimethylsulfoxide and methanol. The IC50 value of compound 9 in HCT-116 cells was 93.12 ± 6.33 µM, and in HeLa cells was 89.09 ± 11.84 µM, which indicating that the probe had certain inhibitory effect on cancer cells. The excellent biocompatibility and potential tumor targeting properties of the compound were clearly observed in cell and mice imaging. This study is of great significance for the rational design of novel targeted BODIPY probes with good hydrophilicity and biocompatibility.

Development of succinimide-based inhibitors for the mitochondrial rhomboid protease PARL.

While the biochemistry of rhomboid proteases has been extensively studied since their discovery two decades ago, efforts to define the physiological roles of these enzymes are ongoing and would benefit from chemical probes that can be used to manipulate the functions of these proteins in their native settings. Here, we describe the use of activity-based protein profiling (ABPP) technology to conduct a targeted screen for small-molecule inhibitors of the mitochondrial rhomboid protease PARL, which plays a critical role in regulating mitophagy and cell death. We synthesized a series of succinimide-containing sulfonyl esters and sulfonamides and discovered that these compounds serve as inhibitors of PARL with the most potent sulfonamides having submicromolar affinity for the enzyme. A counterscreen against the bacterial rhomboid protease GlpG demonstrates that several of these compounds display selectivity for PARL over GlpG by as much as two orders of magnitude. Both the sulfonyl ester and sulfonamide scaffolds exhibit reversible binding and are able to engage PARL in mammalian cells. Collectively, our findings provide encouraging precedent for the development of PARL-selective inhibitors and establish N-[(arylsulfonyl)oxy]succinimides and N-arylsulfonylsuccinimides as new molecular scaffolds for inhibiting members of the rhomboid protease family.

Phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonates and phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamides as new antimicrotubule agents targeting the colchicine-binding site.

We recently designed and prepared new families of potent antimicrotubule agents designated as N-phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamides (PIB-SAs). Our previous structure-activity relationship studies (SAR) focused on the aromatic ring B of PIB-SOs and PIB-SAs leaving the impact of the phenylimidazolidin-2-one moiety (ring A) on the binding to the colchicine-binding site (C-BS) poorly studied. Therefore, the aim of the present study was to evaluate the effect of replacing the imidazolidin-2-one (IMZ) group by a pyrrolidin-2-one moiety. To that end, 15 new phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonate (PYB-SO) and 15 phenyl 4-(2-oxopyrrolidin-1-yl)benzenesulfonamide (PYB-SA) derivatives were designed, prepared, chemically characterised and biologically evaluated. PYB-SOs and PYB-SAs exhibit antiproliferative activity in the low nanomolar to low micromolar range (0.0087-8.6 µM and 0.056-21 µM, respectively) on human HT-1080, HT-29, M21 and MCF7 cancer cell lines. Moreover, they block cell cycle progression in G2/M phase. Immunofluorescence, tubulin affinity and tubulin polymerisation assays show that they cause microtubule depolymerisation by docking the C-BS. In addition, docking assays with the most potent derivatives show binding affinity toward the C-BS and they also exhibit weak or no toxicity toward chick embryos. Finally, physicochemical properties calculated using the SwissADME algorithm show that PYB-SOs and PYB-SAs are promising new families of antimicrotubule agents.

N-phenyl ureidobenzenesulfonates, a novel class of promising human dihydroorotate dehydrogenase inhibitors.

N-phenyl ureidobenzenesulfonates (PUB-SOs) is a new class of promising anticancer agents inducing replication stresses and cell cycle arrest in S-phase. However, the pharmacological target of PUB-SOs was still unidentified. Consequently, the objective of the present study was to identify and confirm the pharmacological target of the prototypical PUB-SO named 2-ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) leading to the cell cycle arrest in S-phase. The antiproliferative and the cytotoxic activities of SFOM-0046 were characterized using the NCI-60 screening program and its fingerprint was analyzed by COMPARE algorithm. Then, human dihydroorotate dehydrogenase (hDHODH) colorimetric assay, uridine rescuing cell proliferation and molecular docking in the brequinar-binding site were performed. As a result, SFOM-0046 exhibited a mean antiproliferative activity of 3.5 µM in the NCI-60 screening program and evidenced that leukemia and colon cancer cell panels were more sensitive to SFOM-0046. COMPARE algorithm showed that the SFOM-0046 cytotoxic profile is equivalent to the ones of brequinar and dichloroallyl lawsone, two inhibitors of hDHODH. SFOM-0046 inhibited the hDHODH in the low nanomolar range (IC50 = 72 nM) and uridine rescued the cell proliferation of HT-29, HT-1080, M21 and MCF-7 cancer cell lines in the presence of SFOM-0046. Finally, molecular docking showed a binding pose of SFOM-0046 interacting with Met43 and Phe62 present in the brequinar-binding site. In conclusion, PUB-SOs and notably SFOM-0046 are new small molecules hDHODH inhibitors triggering replication stresses and S-phase arrest.

Structural modification of the aryl sulfonate ester of cjoc42 for enhanced gankyrin binding and anti-cancer activity.

Gankyrin is an oncogenic protein involved in various biological processes, such as cellular growth and proliferation. Its overexpression in certain cancers results in an increase of gankyrin-mediated protein-protein interactions (PPIs), leading to cancer proliferation. To date, only one small molecule (cjoc42) has been identified to bind gankyrin, which simultaneously inhibits its interaction with the 26S proteasome. Despite this advance, 2nd generation inhibitors are needed to improve gankyrin binding and cellular efficacy. To this end, an extensive SAR for the aryl sulfonate ester moiety of the cjoc42 scaffold was explored, and showed that substitutions at the 2-, 3-, and 4-positions manifested significant increases in gankyrin binding, resulting in the most potent binders of gankyrin to date. Subsequent cell-based assay evaluation of our derivatives demonstrated antiproliferative activity against pediatric liver cancer cell lines Hep3B and HepG2, which was not previously observed for cjoc42.

Investigation on the impact of three different quaternary methyl ammonium cartridges on the radiosynthetic yields of [18 F]fluoromethyl tosylate.

Our recent investigations for the radiosynthesis of [18 F]fluoromethyl tosylate have highlighted that choice of quaternary methyl ammonium (QMA) cartridge used during the radiosynthesis can significantly impact the radiochemical yields. Often the details of the QMA cartridge used in fluourine-18 syntheses are not fully described. However, our studies demonstrate that the type, the size, and nature (method by which it has been conditioned) of the QMA cartridge used during the radiosynthesis can make a significant impact in the labelling efficiency. This paper investigates the use of three QMA cartridges and demonstrates that radiochemical yield (decay corrected) of [18 F]fluoromethyl tosylate can increase from 46% to 60% by simply changing the QMA cartridge (and leaving all other reagents and labelling conditions exactly the same). These learnings may be applied to improve the radiochemical yields of a number of [18 F]-fluorinated tracers (and synthons), where the labelling step is base-sensitive to increase the radiochemical yield, thereby significantly benefiting the radiochemistry and nuclear medicine community. This paper also highlights the necessity of the radiochemistry community to ensure the details of QMA cartridges used in fluorine-18 chemistry are fully and accurately described, since this will improve the translation of radiochemical methods from one laboratory to another.

An integrin-targeting glutathione-activated zinc(II) phthalocyanine for dual targeted photodynamic therapy.

A zinc(II) phthalocyanine substituted with three 2,4-dinitrobenzenesulfonate (DNBS) groups and a cyclic arginine-glycine-aspartic acid (cRGDfK) moiety was prepared and characterized. With three strongly electron-withdrawing DNBS groups, this compound was fully quenched in terms of fluorescence emission and singlet oxygen generation in N,N-dimethylformamide and phosphate buffered saline due to the strong photoinduced electron transfer effect. In the presence of glutathione (GSH), which is the most abundant intracellular thiol particularly in tumor cells, the DNBS moieties were cleaved, thereby restoring these photoactivities and making the conjugate as a GSH-activated photosensitizer. Being a well-known integrin antagonist, the cyclic RGD peptide sequence could enhance the localization of the conjugate in integrin-upregulated tumor cells. As shown by confocal laser scanning microscopy and flow cytometry, the intracellular fluorescence intensity of the conjugate was significantly higher in the integrin-positive A549 and MDA-MB-231 cells than in the integrin-negative MCF-7 and HEK293 cells. The photocytotoxicity of the conjugate against MDA-MB-231 cells was also higher than that toward MCF-7 cells. The results suggest that this dual-functional photosensitizer is a promising candidate for targeted photodynamic therapy.

Microwave-assisted synthesis of 1-substituted-1H-benzimidazolium salts: Non-competitive inhibition of human carbonic anhydrase I and II.

A series of 1-substituted-1H-benzimidazolium p-toluenesulfonate salts were synthesized in good yields by the reaction of 1-substituted benzimidazole derivatives and p-toluenesulfonic acid under microwave irradiation. Two iodide salts were synthesized by the anion exchange reaction of the corresponding p-toluenesulfonate salt and NaI. All compounds were characterized by 1 H NMR, 13 C NMR, IR, LC-MS spectroscopic methods, and elemental analyses. The crystal structure of 1-methoxyethyl-1H-benzimidazolium p-toluenesulfonate 2d showed that cation and anion are interconnected by N-H···O and C-H···O hydrogen bonds. All compounds were examined as inhibitor of human carbonic anhydrase (hCA) I and II, and all of them inhibited hCA I and hCA II. Kinetic investigation results revealed that these compounds inhibit hCA I and hCA II in a non-competitive manner. The iodide salts had higher inhibitory activity than their corresponding p-toluenesulfonate salts.

A dual-responsive fluorescent probe for detection of fluoride ion and hydrazine based on test strips.

Hydrazine (N2H4) and fluoride ion (F-) are regarded as environmental pollutants and potential carcinogens. A dual-functional fluorescent probe (probe 1) was developed for both F- and N2H4 with high selectivity and sensitivity. 1 was based on nucleophilic aromatic substitution reaction for N2H4 detection and selective cleavage of 4-nitrobenzenesulphonyl group for the determination of F-. The limits of detection of probe for F- and N2H4 were 77.82 nM and 29.34 nM, respectively, which are far below the threshold limit value (TLV) of United States Environmental Protection Agency (EPA). The home-made test strips of 1 provided the positive tool for F- and gaseous N2H4 in different system. And the confocal fluorescence images indicated that 1 can quantitatively detect N2H4 in living PC12 cells. Promisingly, 1 has great prospects for N2H4 imaging and determining in living system.

Preparation, characterisation and biological evaluation of new N-phenyl amidobenzenesulfonates and N-phenyl ureidobenzenesulfonates inducing DNA double-strand breaks. Part 3. Modulation of ring A.

N-Phenyl ureidobenzenesulfonates (PUB-SOs) are a new class of anticancer agents blocking the cell cycle progression in S-phase, inducing replicative stress and DNA double-strand breaks (DSBs). In this study, we evaluate the effect of modifying the nature and the position of different substituents on ring A of PUB-SOs on the antiproliferative activity, pharmacological activity as well as on calculated physicochemical, pharmacokinetics and drug-likeness properties. Modification of the urea group by an amide group led to new PUB-SO analogs designated as N-phenyl amidobenzenesulfonates (PAB-SOs). The 2-chloroethyl moiety on ring A was also substituted by different alkyl, cycloalkyl and chloroalkyl groups. The new PAB-SOs and PUB-SOs blocking the cell cycle progression in S-phase exhibit antiproliferative activity in the submicromolar to low micromolar range (0.14-27 µM) on four human cancer cell lines, namely HT-1080, HT-29, M21 and MCF7. Moreover, selected PUB-SO and PAB-SO derivatives induced the phosphorylation of H2AX in M21 cells and do not exhibit or only slightly alkylating activity as confirmed by the 4-(4-nitrobenzyl)pyridine (NBP) assay. Finally, our results show that structure modifications weakly affect the calculated physicochemical, pharmacokinetics and drug-likeness properties of PAB-SOs and PUB-SOs. Therefore, PAB-SOs and PUB-SOs are promising anticancer agents inducing replicative stress and DNA damage via a mechanism of action unrelated to DNA alkylation.

Synthesis, biological evaluation and computational studies of novel iminothiazolidinone benzenesulfonamides as potent carbonic anhydrase II and IX inhibitors.

A series of iminothiazolidinone-sulfonamide hybrids (2a-k) was synthesized by heterocyclization of sulfanilamide thioureas with methyl bromoacetate and characterized by spectroscopic techniques, mass and elemental analysis. The synthesized derivatives were screened against four relevant human (h) isoforms of carbonic anydrases (CAs, EC 4.2.1.1) I, II, IV and IX. These enzymes are involved in a variety of diseases, including glaucoma, retinitis pigmentosa, epilepsy, arthritis, and tumors. Derivatives 2a-2k exhibited the best inhibitory activity against the cytosolyc hCA II (KIs are reaching the sub-nanomolar range, 0.41-37.8 nM) and against the tumor-associated isoform hCA IX (KIs are spanning between 24.3 and 368.3 nM). The binding mode of the reported iminothiazolidinone benzenesulfonamides within hCA II and IX catalytic clefts was investigated by docking studies.

1-(Benzenesulfonyl)-1,5-dihydro-4,1-benzoxazepine as a new scaffold for the design of antitumor compounds.

AIM: Bozepinib is a potent and selective anticancer compound which chemical structure is made up of a benzofused seven-membered ring and a purine moiety. We previously demonstrated that the purine fragment does not exert antiproliferative effect per se. METHODOLOGY: A series of 1-(benzenesulfonyl)-4,1-benzoxazepine derivatives were synthesized in order to study the influence of the benzofused seven-membered ring in the biological activity of bozepinib by means of antiproliferative, cell cycle and apoptosis studies. RESULTS & CONCLUSION: Our results show that the methyleneoxy enamine sulfonyl function is essential in the antitumor activity of the structures and thus, it is a scaffold suitable for further modification with a view to obtain more potent antitumor compounds.

A small-molecule screen identifies the antitrypanosomal agent suramin and analogues NF023 and NF449 as inhibitors of STAT5a/b.

The transcription factor STAT5a is a potential target for tumor therapy. We present a fluorescence polarization-based, high-throughput screen of chemical libraries containing natural products and known bioactive molecules, for the identification of small-molecule inhibitors of the STAT5a SH2 domain. This screen identified suramin, a drug used to treat African trypanosomiasis, and its analogues NF023 and NF449 as inhibitors of the SH2 domains of STAT5a/b. Our data extend the known in vitro targets of suramin and analogues to include members of the STAT protein family.

Activation of Phenyl 4-(2-Oxo-3-alkylimidazolidin-1-yl)benzenesulfonates Prodrugs by CYP1A1 as New Antimitotics Targeting Breast Cancer Cells.

Prodrug-mediated utilization of the cytochrome P450 (CYP) 1A1 to obtain the selective release of potent anticancer products within cancer tissues is a promising approach in chemotherapy. We herein report the rationale, preparation, biological evaluation, and mechanism of action of phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) that are antimicrotubule prodrugs activated by CYP1A1. Although PAIB-SOs are inert in most cells tested, they are highly cytocidal toward several human breast cancer cells, including hormone-independent and chemoresistant types. PAIB-SOs are N-dealkylated into cytotoxic phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) in CYP1A1-positive cancer cells, both in vitro and in vivo. In conclusion, PAIB-SOs are novel chemotherapeutic prodrugs with no equivalent among current antineoplastics and whose selective action toward breast cancer is tailored to the characteristic pattern of CYP1A1 expression observed in a large percentage of human breast tumors.

Phenylbenzenesulfonates and -sulfonamides as 17ß-hydroxysteroid dehydrogenase type 2 inhibitors: Synthesis and SAR-analysis.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) converts the potent estrogen estradiol into the weakly active keto form estrone. Because of its expression in bone, inhibition of 17ß-HSD2 provides an attractive strategy for the treatment of osteoporosis, a condition that is often caused by a decrease of the active sex steroids. Currently, there are no drugs on the market targeting 17ß-HSD2, but in multiple studies, synthesis and biological evaluation of promising 17ß-HSD2 inhibitors have been reported. Our previous work led to the identification of phenylbenzenesulfonamides and -sulfonates as new 17ß-HSD2 inhibitors by ligand-based pharmacophore modeling and virtual screening. In this study, new molecules representing this scaffold were synthesized and tested in vitro for their 17ß-HSD2 activity to derive more profound structure-activity relationship rules.

Tos-Nos-Mos: Synthesis of different aryl sulfonate precursors for the radiosynthesis of the alpha7 nicotinic acetylcholine receptor radioligand [(18)F]NS14490.

Radiopharmacological investigations of [(18)F]NS14490 have proven that this radiotracer could be a potential PET radiotracer for imaging of alpha7 nicotinic acetylcholine receptor particularly with regard to vulnerable plaques of diseased vessels. For further optimisation of the previously automated one-pot radiosynthesis of [(18)F]NS14490 using a tosylate precursor, precursors with other leaving groups (nosylate and mosylate) were synthesized and compared with the tosylate with respect to their reactivities towards [(18)F]fluoride. The use of these different precursors resulted in comparable labelling yields of [(18)F]NS14490. A novel mosylate precursor was synthesized and evaluated, which has revealed a higher stability during a storage period of five months compared to the corresponding tosylate and nosylate.

Reduction of Synthetic Ubiquinone QT Catalyzed by Bovine Mitochondrial Complex I Is Decoupled from Proton Translocation.

We previously succeeded in site-specific chemical modifications of the inner part of the quinone binding pocket of bovine mitochondrial complex I through ligand-directed tosylate (LDT) chemistry using specific inhibitors as high-affinity ligands for the enzyme [Masuya, T., et al. (2014) Biochemistry 53, 2304-2317, 7816-7823]. To investigate whether a short-chain ubiquinone, in place of these specific inhibitors, serves as a ligand for LDT chemistry, we herein synthesized a LDT reagent QT possessing ubiquinone scaffold and performed LDT chemistry with bovine heart submitochondrial particles (SMP). Detailed proteomic analyses revealed that QT properly guides the tosylate group into the quinone binding pocket and transfers a terminal alkyne to nucleophilic amino acids His150 and Asp160 in the 49 kDa subunit. This result clearly indicates that QT occupies the inner part of the quinone binding pocket. Nevertheless, we noted that QT is a unique electron acceptor from complex I distinct from typical short-chain ubiquinones such as ubiquinone-1 (Q1) for several reasons; for example, QT reduction in NADH-QT oxidoreduction was almost completely insensitive to quinone-site inhibitors (such as bullatacin and piericidin A), and this reaction did not produce a membrane potential. On the basis of detailed comparisons of the electron transfer features between QT and typical short-chain quinones, we conclude that QT may accept electrons from an N2 cluster at a position different from that of typical short-chain quinones because of its unique side-chain structure; accordingly, QT reduction is unable to induce putative structural changes inside the quinone binding pocket, which are critical for driving proton translocation. Thus, QT is the first ubiquinone analogue, to the best of our knowledge, the catalytic reduction of which is decoupled from proton translocation through the membrane domain. Implications for mechanistic studies on QT are also discussed.