The synthesis of dihydropyridines, valuable molecules with diverse therapeutic properties, using eco-friendly heterogeneous catalysts as a green alternative received significant consideration. By selecting appropriate precursors, these compounds can be readily modified to induce the desired properties in the target product. This review focused on synthesising diverse dihydropyridine derivatives in single-pot reactions using magnetic, silica, and zirconium-based heterogeneous catalytic systems. The monograph describes preparation techniques for various catalyst materials in detail. It covers facile and benign magnetic, silica, zirconium-based, and ionic liquid catalysts, exhibiting significant efficacy and consistently facilitating excellent yields in short reaction times and in a cost-effective way. Most of the designated protocols employ Hantzsch reactions involving substituted aldehydes, active methylene compounds, and ammonium acetate. These reactions presumably follow Knoevenagel condensation followed by Michael addition and intra-molecular cyclisation. The multicomponent one-pot protocols using green catalysts and solvents have admirably increased the product selectivity and yields while minimising the reaction time. These sustainable catalyst materials retain their viability for several cycles reducing the expenditure are eco-friendly. Synthesis of small heterocyclic organic molecules has advanced a long way with numerous applications in the academic and pharmaceutical industry, which are also valuable synthons for preparing elaborate bioactive molecules . The strThe importance of sustainable heterogeneous catalysts and fiscal benefits is renascent through effective fusion procedures, which are deemed crucial in advanced organic chemistry . RecentlNitrogen-based heterocyclic skeletons have prominence in synthetic organic chemistry because of their valuable biological applications . Among tI) and chalcone (II) intermediates and further underwent cyclisation to generate a 1,4-dihydropyridine product   Hantzsc. The pro product   data. Catalyst accommodation capacity and microwave array capacity are the two fundamental factors for the optimisation of the catalyst.Lately, catalytic compounds in synthetic processes have been a topic of concern due to their nature and adverse effects on the environment, drawing attention to eco-friendly alternatives. The emerging challenge was to modify the naturally available compounds for multiple uses without compromising their vitality. The construction of magnetic nanoparticles with promising catalytic activity offers many advantages compared to traditional inorganic catalysts. A huge surface area-to-volume ratio and a high atomic accommodation capacity are advantageous for these particles. These catalysts can be easily separated from the final product using external magnetic fields owing to their magnetic efficiency. Magnetic nanocatalysts have gained paramount importance in organic and industrial reactions because of their interactions with various reagents and environmentally amiable properties, such as anchoring various functional groups or organic moieties onto the hydroxyl projections of their surface area. Integrating transitional metals or other plant-extracted enzymes with Feet al. (3O4/GO/Au–Ag) nano-catalyst with Au–Ag particles clipped on Fe3O4 and graphene oxide. The nanocatalyst’s activity was utilised to synthesise dihydropyridine-spiro oxindole analogues (4). The condensation reaction was carried out between uracil-6-amine (1), substituted isatin derivatives (2), and various barbituric acids (3) underwater at room temperature. The nanocatalyst was significantly thermally stable up to 600°C, and the average particle size was about 15 nm. The use of 20 mg of the nanocatalyst gave superior results with high yields (81%–93%) for <5 h of reaction time  utilising iron(III) phosphate aiming for a solvent-free synthetic method (5) with benzyl acetoacetate (6) and ammonium acetate (7) under the influence of a catalyst (Fe3PO4). The reaction proceeds by activating the carbonyl group of the aldehyde advocated by Fe3PO4, which acts as a Lewis acid, at a minimal catalytic usage of 10mol% to obtain an appropriate amount of yield. The reaction initially included benzaldehyde, benzyl acetoacetate, and ammonium acetate at an optimal temperature of 70°C. The aldehydes with +I substitution groups like -NO2, -OCH3, -CH=CH2 were observed to serve a better yield of 80% within a reaction time of 50 min using a minimal amount of 5 mol% of the catalyst.Behbahani and coworkers  practisec method . They emet al. 3.9H2O and Co (NO3)2.6H2O in a molar ratio of 2:1 in 100 ml of water and sonication. A volume of 20 ml of 1 M NaOH solution was added to the resulting solution and subjected to sonication once again. The resulting black precipitate was gathered using an external magnetic field and dried in the oven. The second step consisted of the addition of tetraethylorthosilicate to obtain CoFe2O4@SiO2. In the third step, NH2-Pr was synthesised by reacting triethanolamine, triethylenetetramine, and 3-chloropropyltriethoxysilane in ethyl alcohol. Upon adding Co (NO3)2.6H2O to the above-obtained product, NH2-Pr-Co(II) formed was dispersed in dry toluene to which a solution of CoFe2Co4@SiO2 in dry toluene was added dropwise, resulting in the precipitate of desired catalyst CoFe2O4@SiO2-NH2-Co(II). The XRD values of the synthesised catalyst explored its polycrystalline structure with an average particle size of 20–90 nm, as suggested by TEM analysis. The VSM values indicate that the magnetic properties of the catalyst depended on the surface arrangement of the catalytic particles, which were stable up to 560°C as per thermogravimetric analysis (TGA) analysis. The catalyst (180 mg) facilitated a 96% yield in 2 h, in aqueous ethanol (1:1). Several dihydropyridine derivatives (11) have been synthesised in a single-pot mild reaction, including aldehydes (9), ethyl acetoacetate (10), and ammonium acetate (7), aiding CoFe2O4@SiO4-NH2-Co(II) as a catalyst owing to its active metallic nucleus, sustainability, and high-yielding capacity  using benzaldehyde (12), ethyl acetate (10), and ammonium acetate (7) as reagents. Dihydropyrimidinone (14) compounds were also reported using urea (13) instead of ammonium acetate (7) (Wua and coworkers  developetate (7) . The cat2O3/Cu@cellulose), which showed magnificent catalytic efficiency during the Hantzsch reaction. High yields of 1,4 dihydropyridine (19) and polyhydroquinoline (20) were observed upon employing γ-Fe2O3/Cu@cellulose for their preparation. They prepared a homogenous solution of cellulose in an aqueous mixture of sodium hydroxide and urea bearing a ratio of 81:7:21, whose temperature was dropped to −12°C using an ice bath incorporated with salt. An aqueous solution of iron chloride with Fe+3 and Fe+2 in the ratio of 1:2 was added to the above solution while continuously stirring for 6 h, resulting in γ-Fe2O3-implanted cellulose assisted by co-precipitation. The CuSO4.4H2O solution was added to the above mixture; owing to the oxidative nature of the γ-Fe2O3 nanoparticles, Cu+2 is protected from reduction. The mixture was stirred continuously for 2 h before washing and collecting the product. From TEM analysis, the obtained catalyst’s particle size was 25–30 nm, and the EXD data confirmed the presence of C, O, Cu, and Fe. It showed engaging activity with a high yield of around 92%–95% within a short reaction time of 15–20 min in a solvent-free environment  and polyhydroquinoline (24) derivatives (21), ethyl acetoacetate (17), malononitrile (22), and ammonium acetate (7) in equal quantities. The reaction is presumed to follow Knoevenagel condensation to form arylidene malononitrile. The intermediate further participates in Michael addition. The catalyst increased the yield to 96% and dropped the reaction time to 30 min in an aqueous medium. The catalyst material was isolated by applying an external magnetic field.Bodaghifard et al.  fabricativatives . The synet al.  of ammonia solution to obtain a black precipitate of Fe3O4 nanoparticles after continuously stirring the solution at 80°C for 2 h under the influence of the N2 environment. A percentage of 28% (wt%) of tetramethyl orthosilicate was added to a solution of Fe3O4 nanoparticles suspended in ethanol:water (80:20 wt%) mixture to gather Fe3O4@SiO2 nanoparticles. The above-formed nanoparticles were taken in toluene and refluxed with (3-chloropropyl)triethoxysilane to obtain Fe3O4@SiO2@Si-(CH2)3Cl nanoparticles, which were further added to a solution of melamine and K2CO3, dissolved in 70 ml of DMSO to affix melamine to the above formed NPs. A methanolic solution of picoline aldehyde was added to the NPs, followed by 30 ml of dichloromethane to the pre-dried NPs under MgSO4. A 24-mmol chlorosulphonic solution in 20 ml of dichloromethane was further added dropwise to form Fe3O4@SiO2@Si-(CH2)3@melamine-picolineimine@SO3H nanoparticles. Different spectral data obtained from FT-IR and XRD suggest that the crystallite size of the catalyst is 14.98 nm whereas SEM analysis advocates for the high interaction of the HSO3 group on the surface along with TEM data which proves the lamination of the core–shell. The EDS analysis helped elucidate the nanoparticles’ morphology with dominant levels of Fe, Si, O, N, S, and Cl. The VSM analysis evaluated its magnetic nature, and the material was stable at 600°C as suggested by TGA data. A series of polyhydroquinolines (26) were reported using aryl aldehydes (25), β-keto esters (17), dimedones (19), and ammonium acetate (7) in equal proportions confirming the catalytic potential of the magnetic nanoparticles  using 1-(2-hydroxy-5-(4-nitrophenyl)-diazenyl)-phenyl)-ethenone. The final catalytic conclusion involves the induction of copper to the MNPs using CuCl2 to achieve a Copper/Schiff–base complex immobilised on KIT-6-NH2 magnetic nanoparticles. The gathered compound was morphologically elucidated using various methods such as BET analysis, representing its particle size of 51.43°m2/g. The VSM is used to assess its magnetic nature. TEM images explored the spherical shape of the particles with a diameter of 33–40 nm along with aggregation and stacking of particles. XRD showed a highly crystalline cubic spinel structure, and the elemental analysis energy-dispersive X-ray analysis (EDX) revealed that the catalyst contains Fe, O, N, C, Cu, and Si. TGA analysis suggested stability up to 520°C. The catalytic activity of MNPs was tested on Hantszch’s single-pot reaction method to synthesise polyhydroquinolines (28) using aryl aldehyde (27), cyclic ketone (19), and ammonium acetate (7) in the presence of the above-prepared catalyst 4-SO3H nanoparticles were isolated using an external magnetic field. The prepared catalyst was evaluated using SEM and TEM imaging, representing the uniform distribution of nanoparticles with spherical structure. The catalyst was stable up to 700°C owing to the dendrimers of D-NH-(CH2)4-SO3H side chains. The prepared catalyst was used in the production of tetrahydrobenzo pyrans (31), 2-amino-3-cyano-4H-pyrans (32), and polyhydroquinolines (33). The former pyrans were prepared by refluxing a reaction mixture of aldehydes (29), malononitrile (22), and 1,3-cyclohexanedione (19) or ethyl acetoacetate (17) in 5 ml of ethanol in the presence of the above-prepared catalyst. The polyhydroquinolines were produced by refluxing a reaction mixture containing aldehydes (29), 1,3-cyclohexanedione (19), ethyl acetoacetate (17), and ammonium acetate (7) in the presence of the same catalyst. The reaction was assumed to proceed through Knoevenagel condensation to produce 2-benzylidenemelanonitrile. The intermediate endures Michael with enolised dimedone to generate enamine intermediate. The enamine undergoes cyclisation and tautomerism to give the product a high yield (96%) within 20 min using 0.05 g of the catalyst  and stirred continuously for a day and a half to obtain NiFe2O4@SiO2 nanoparticles. A volume of 2 g of the prepared nanoparticles was suspended in 15 ml of chloroform to which 0.5 g of sulphonyl chloride was added dropwise and stirred while maintaining a temperature range ≥5°C. The resulting mixture was filtered and dried at room temperature to obtain a black precipitate of NiFe2O4@SiO2@SO3H nanoparticles. The gathered catalytic nanoparticles were elucidated using various techniques such as XRD analysis which describes its rhombic crystalline structure. The SEM analysis showed a 60–90-nm nanosize, and the material was stable up to 600°C. The gathered catalyst was subjected to trial to determine its efficiency for the preparation of 1,4-dihydropyridines (37 and 38) by employing a single-pot Hantzsch reaction mixture consisting of aromatic aldehyde (34), ethyl acetoacetate (17), or 4-hydroxycoumarin (36) and 28% aqueous ammonia 28% (35) in 2 ml of DM water . A single-pot system consisting of equimolar amounts of aromatic aldehyde (39), 1,3-cyclohexadiene (30), acetoacetanilide (40), and ammonium acetate (7) was subjected to stirring for 20 min at room temperature  and subjected to ultrasonication which was added with 22 mmol of 3-aminopropyltriethoxysilane while continuously stirring for 6 h at 60°C the resulting Fe3O4/SiO2/KCC-1/aminopropyl MNPs, which were washed and vacuum dried for another 2 h at 60°C. A volume of 1 g of the gathered product was added to a solution of 1 ml of N-ethyldiisopropylamine and 10 ml of dry THF while maintaining the temperature below 5°C and adding 1 g of triazine trichloride while continuously stirring for a whole day. Fe3O4/KCC-1/TCl2 nanoparticles were gathered and washed with hot toluene to isolate pure products. A volume of 1 g of the currently gathered nanoparticles along with 1 g of pyridine was dissolved in a solution of 1.3 ml of N-ethyl diisopropylamine and 10 ml of dry toluene while continuously stirring for 16 h at 80°C. Fe3O4/KCC-1/BPAT nanoparticles were separated from the reaction solution by an external magnet, washed with ethanol, and dried at 50°C. Represented by SEM and TEM data, the stoked nanoparticles had 20–30-nm particle size. The magnetic saturation was 55.1 and 42.4 emu/g following VSM data, and the material was stable up to 700°C. The catalytic efficiency of the prepared nanocomposite was assessed by a single-pot synthesis of 1,4-dihydropyridines (45). The equimolar mixture of primary amine (43), dimethyl acetylene dicarboxylate (44), and methyl (arylmethylide) pyruvate (42) was refluxed for 4 h in the presence of Fe3O4/KCC-1/BPAT triazine, a green catalyst with good chemo selectivity . The proC-1/BPAT . The sol+2 and Fe+3 with a molar ratio of 1:2 and further treated with chlorosulphonic acid. The functionalised nanoparticles were reacted with boric acid, adhered to silica to obtain Fe3O4/SiO2-H3BO4-HSO3 nanoparticles, and filtered and washed with ethanol to isolate pure material. XRD analysis suggests a perovskite crystalline structure and stability up to 350°C as represented by TGA data with a boric acid concentration of 1.2 mmol/g. The catalytic efficiency was assessed in the Hantzsch ester formation reaction to synthesise 1.4-dihydropyridine derivatives (47). The single-pot reaction involved equimolar amounts of aldehyde (46) and ammonium acetate (7) and a double equimolar quantity of β-keto esters (10) (Azizi and his colleagues  have fabers (10) . The reaet al.  and hydroquinolines (50) was observed within a short reaction time of 10 min  were synthesised in the presence of the prepared material as a catalyst. A good 80% yield was observed with minimal use of N-1.2% weight of m-SiO2 in the presence of ethyl alcohol. The sequential mechanism of Knoevenagel condensation followed by Michael addition and cyclisation was observed. +I substituents in the reactant heterocyclic compound aided better yield as high as 92% within 6 h  was treated with 3-mercaptopropyl (trimethoxyl) silica and toluene and refluxed for 24 h. The resulting 3-mercaptopropyl silica is then filtered and treated with 30% H2O2 solution and concentrated H2SO4 while constantly stirring for 20 h. The obtained material by filtration was dried and characterised. The catalyst was a porous powder with an amorphous structure of the particles as deemed by SEM and TEM imaging. The material retained its form up to 360°C. A [BMIM][PF6]-laminated (20%) silica-based sulphonic acid catalyst was found to be most efficient among other derivatives. The material showed excellent catalytic activity in synthesising 1,4-dihydropyridines (55), offering high yields of 95% within a reaction time of 15 min 2.6H2O as the cobalt source and refluxing it for 16 h, which was later washed with ethanol to obtain the pure product free from by-products. The homogenous dispersion of the Co metal onto the surface of the modified SBA-15 gave a mesoporous cavity with a volume of 0.534 cm3/g and a surface area of 238.47 m2/g, which was represented by X-ray mapping and BET analysis, respectively. The prepared catalyst was used to synthesise polyhydroquinolines using the Hantzsch reaction mixture and oxidation of sulphides. The polyhydroquinolines (57) were prepared by using a reaction mixture of dimedone (19), aldehyde (56), ammonium acetate (7), and ethyl acetoacetate (17) using 1 mmol each aided with 8 g of SBA-15@AMPD-Co nanocatalyst . Alternatively, 250 ml of 0.2 M AlCl3 solution was added to the colloidal solution with continuous stirring to which 1.2 M NaOH solution was introduced in a molar ratio of 1:2 with Al. The solid obtained is centrifuged to collect it and washed with hot DM water to eliminate unwanted Cl− molecules and dried at 100°C. The gathered product was ground to powder and subjected to calcination for 2 h at 300°C to achieve AZP. Furthermore, 1 g of AZP was dissolved in a 20-ml solution of SA in water to gain a mesoporous surface. The aqueous solution was continuously stirred for 6 h at 90°C to attain a solid and further dried at 120°C to achieve a SAxAZP composite. The catalyst was subjected to various elucidation techniques, including XRD, which exposed its crystalline structure with an H3-type surface area of 118 m2/g and a pore volume of 0.19 cm3/g with a particle size of 25–50 nm as represented by SEM analysis. TGA analysis showed that the catalyst was stable up to 600°C. Its catalytic activity was evaluated using the Hantzsch reaction to prepare 1,4-dihydropyridines (59), exercising single-pot synthesis with a reaction mixture of aryl aldehyde (58), acetoacetic esters (17), and ammonium acetate (7) in the presence of the above-obtained catalyst  using zeolite-anchored Zr-ZSM-5 as an illustrious catalyst. To tailor the material, ammonium ZSM-5 was initially calcinated using a muffled furnace set to 500°C under aerobic conditions subjected to a heating ramp rate of 5°C per minute for 4 h to acquire H-ZSM-5. A solution of zirconium nitrate in water was added dropwise into H-ZSM-5 while constantly stirring the solution to incorporate zirconium nitrate into H-ZSM-5. The generated catalyst was dried at 120°C for 12 h, and the dry product was recalcinated in a muffled furnace at 500°C. The gathered catalyst was subjected to XRD, SEM/EDS, and BET SA-PSD spectroscopic techniques, exploring its crystalline structure. The high surface area of 341 m2/g allows the zeolite catalyst to interact with the reagents increasing the active sites. The classic Hantzsch reaction was examined using the Zr-ZSM-5 Zeolite catalyst in a single-pot reaction. The process involved benzaldehyde (60), ethyl acetoacetate (17), and ammonium hydroxide (61) in obtaining 1,4-dihydropyridines , different aromatic aldehydes (64), ammonium acetate (7), and malononitrile (22) was used to produce polyhydroquinoline derivatives (66)  . The obtet al. , adopting a single-pot synthetic method using a reaction mixture consisting of equimolar amounts of aldehyde (67), malononitrile (22), diethyl acetylene dicarboxylate (44), and 3-chloro 4-fluoroaniline (68) under the influence of RuO2/ZrO2 in 10 ml of ethyl alcohol medium. The reaction mixture was continuously stirred, and the produced derivatives were filtered and washed with ethanol , 5,5-dimethyl-1,3-cyclohexanedione (19), acetoacetanilide (40), and ammonium acetate (7) in the presence of V2O5/ZrO2 nanoparticles using 5 ml of ethanol as solvent (71) were isolated through solvent extraction using ethyl acetate followed by evaporation. The reaction proceeded through Knoevenagel condensation along with Michael addition and cyclisation. The gathered product was recrystallised from ethanol to gain a pure product which was subjected to various spectroscopic analyses for characterisation. The catalyst was prepared by wet impregnation method by dissolving equal amounts of V2O5 and ZrO2 in 100–150 ml of DM water while continuously stirring at room temperature for 8 h. The slurry obtained from filtration was oven-dried for 5 h at 120°C–150°C followed by calcination at 450°C for another 5 h to gain V2O5/ZrO2. Its structure explored using various analytical techniques such as XRD analysis confirms its polycrystalline structure with a 7.6 nm diameter. The SEM imaging and TEM imaging display massive irregular structures due to the aggregation of zirconium and vanadium moieties. The surface area of the catalyst was 89.2219 m2/g with a mesoporous pore volume of 0.327 cm3/g and a pore size of 10.32 nm. The catalytic use gained high yields around 90%–96% with a short reaction time of ≤20 min while retaining its catalytic efficiency for five to six consecutive cycles. The exciting feature of this method is the easy access to pure products without chromatographic separation.Bhaskaruni et al.  have con solvent . The reaIonic liquids are a brilliant alternative for the vintage volatile solvents, made of ionic components such as anions or cations with covalent crystalline structures. The only difference between molten salts and ionic liquids is the melting point which is presumed to be <100°C for ionic liquids, making them stable and efficient solvents. The low-volatility, low-melting-point, and low-vapour pressure impart heterogeneous characteristics to these ionic liquids aiding their hassle-free separation. The ionic components of the liquids account for their intense polarity, which validates their physical and chemical properties. Because of high solubility and eminent anti-corrosion activity, ionic liquids are used as anti-corrosion agents for metals and alloys and as a significant solvent catalyst in various industrial and laboratory preparations. Followed by the ammonium-hinged ionic liquid invention, researchers designed different ionic liquids incorporating imidazolium, pyridinium, phosphonium, and tetra-ammonium components.In contrast to their ionic nature, ionic liquids are poor conductors of electricity and can dissolve polar and non-polar materials. Ionic liquids are differentiated depending on the functional groups of the side chains, which act as electron-exchangeable sites, where the electron-donating groups impart basic nature. In contrast, electron-withdrawing nature imparts an acidic nature to the liquids.4 was used to form 1,4-dihydropyridine derivatives (73) which showed high yielding capacity and sustained its catalytic ability even after running for five consequent cycles. The catalytic activity is aided by the mesoporosity of the material and the acidic nature of the ionic liquid with a product yield that gained excellent yields around 93% within a short reaction time.Tan and his colleagues  have for77), namely, N-aryl poly-hydro quinoline-based drugs, which resulted in excellent yields as high as 91% within a minimum reaction time of 2 h using water as a solvent  via the Hantzsch reaction  which was added and further subjected to mechanical stirring for 24 h to obtain black nanoparticles, which were washed and dried at 80°C. The gathered nanoparticles were laminated with silica employing the sol–gel method. A volume of 1.2 ml of tetraethyl orthosilicate is introduced into a nanoparticle-dispersed solution of ethanol, water, and ammonia while constantly stirring for 12 h. The obtained ferrite@SiO2 nanoparticles were washed and dried. The dried nanoparticles were gathered and dispersed in water. Simultaneously, ionic liquid, [Sipmim]Cl, was also prepared by refluxing 4.8 ml of 0.06 mol 1-methylimidazole and 14.5 ml of 0.07 mol (3-chloropropyl) triethoxysilane. Later, 2 g of ferrite@SiO2 NPs and 25 ml of dry toluene were mixed in a beaker, and 1 g of [Sipmim]Cl was introduced dropwise into it. The obtained nanoparticles were washed and dried at 60°C for 2 h. The size catalyst nanoparticle was 7–11 nm from TEM analysis, and the catalyst’s magnetisation was assessed to be 13 emu/g from VSM data. Its catalytic efficiency was investigated on the three-component Hantzsch reaction involving benzaldehyde (78), ethyl acetoacetate (10), and ammonium acetate (7)  . A good 83) , and difference thermo analysis (DTA) data were used to characterise the material. The single-pot four-component reaction involved substituted aldehyde (80), malononitrile (22), acetylene dicarboxylates (82), and anilines (81) in equal 1-mmol quantities in solvent-free abode at 50°C. 3,6-Dioxaoctamethylenediamminium trifluoromethanesulfonate showed remarkable activity. The reaction was assumed to follow Knoevenagel condensation along with Michael addition and intramolecular cyclisation. The yield of 1,4-dihydropyridines was high, around 93%–96%, with minimal use of a catalyst (2–5%) within 30 min  . To prepn 30 min . The proet al. (2 was prepared by mixing solutions of mono-Ts-βCD and 2.5 ml of dimethylformamide (DMF) into a mixture of 1,4-bis(imidazol-1-yl)-butane and 2.5 ml of DMF while maintaining the temperature between 40°C and 60°C. The white precipitate obtained was filtered and washed with CCl4. To adhere the prepared ionic product onto silica gel, to the solution containing [βCD/Im](OTs)2 in 30 ml of DMF and NaOH, 3-chloro propyl trimethoxysilane was added slowly under continuous stirring and temperature maintained at 90°C in an N2 environment after 30 min. The product was filtered and added to a solution containing tetraethoxysilane and 1-butanol in 0.7 ml of water. The mixture was neutralised using 2 M HCl. The hydrogel was stirred continuously for 3 h at 80°C to obtain a final white precipitate filtered and washed with lukewarm water and acetone. The gathered catalyst was characterised by FTIR, SEM, and NMR using TMS as a standard. The authors established the catalytic efficiency of the di-ionic liquid on a single-pot Hantzsch condensation reaction between aromatic aldehyde (84), a β-dicarbonyl compound (19), and ammonium acetate (7) at a temperature of 90°C to obtain the product, hydroquinolines (85). The product was filtered and recrystallised in ethyl alcohol  at room temperature. It showed significant potential to perform ideally in both polar and nonpolar solvents, making it a desirable catalyst. Gd(OTf)3 acted efficiently as a mild Lewis acid catalyst for the four-component coupling reaction between benzaldehyde (86), an active methyl group (ethyl acetoacetate) (17), 5,5-dimethyl-1,3-cyclohexanedione (dimedone) (19), and ammonium acetate (7)  . After c90), which paves a contemporary pathway for the drugs against cancer cells employing a single-pot method aside from Ag(I) and organo-N-heterocyclic carbenes used as a catalyst. This new catalyst was a progeny from labile Ag(I)-NHC in ethanol; therefore, it accommodates acidic Ag(I) alongside a basic NHC. A 3%–5% metal to organic–complex ratio was maintained for a smooth reaction. A single-pot four-component reaction between isatin (88), malononitrile (22), cyclo ketones (89), and ammonium acetate (7) was utilised  and phenyl hydroxyquinoline derivatives (93) were synthesized by refluxing a mixture of aldehydes (91), β-dicarbonyls (10), and ammonium acetate (7) in an ethanolic medium; the catalyst facilitated high 97% yields with minimal loading of 0.02 g within a reaction time of 15 min (Dekamin and his team  have expf 15 min . The rea95) using a copper catalyst under microwave irradiation. Cupric (II) trifluoromethanesulphonate [Cu(OTf)2] was perceived to be a promising catalyst to obtain a high yield around 90% with a minimal amount of catalytic molar percentage, i.e., 2% at 100°C within 15 min of reaction time (94), ethyl acetoacetate (17), dimedone (65), and ammonium acetate (7) along with ethyl alcohol and Cu(OTf)2. The components were irradiated under microwave to achieve 1,4-dihydropyridines.Pasunooti and his colleagues  have expion time . They opet al. (98). This nanocatalyst was prepared using powdered nano-WO3, and dichloromethane took in a suction flash connected to a pressure dropping funnel and a gas inlet tube facilitating the conduction of HCl gas over the water adsorbent. Chlorosulphonic acid was introduced into it dropwise, which resulted in the evolution of HCl gas. After the HCl gas ceased, the reaction mixture was stirred continuously for 30 min. The unreacted dichloromethane was removed under reduced pressure to obtain a dark green powder, washed with ethanol, and dried for 6 h at 120°C. The FE-SEM images suggest that the particle size of the nanocatalyst is around 60–70 nm. A single-pot three-component reaction involving aromatic aldehydes (96), β-dicarbonyls (97), and ammonium acetate (7) in a solvent-free abode in the presence of n-WSA was considered  and polyhydro quinolines (101), employing H5BW12O40 as a novel catalyst. They implemented a single-pot Hantzsch reaction mechanism using substituted aldehydes (99), acetoacetic esters (17), and ammonium acetate (7) refluxed using borotungstic acid (H5BW12O40) as a catalyst. The negative charge of the catalyst aids in its activity as a Bronsted acid. The resultants were assessed using TLC in combination with n-hexane and ethyl acetate in the ratio 7:3. The reminiscent was washed using ethyl acetate, and the products were dried using Na2SO4. The electron-withdrawing group, such as NO2 at aldehyde, was observed to gain the highest yield of 98% within 45 min 3.18H2O and stirred vigorously to obtain a white precipitate of Al(OH)3, which was washed with distilled water and dried. A volume of 20 g of Al(OH)3 was dissolved in 100 ml of 1 M NaOH, and 3% (v/v%) polyethylene glycol was added. The pH of the mixture was adjusted to eight and subjected to vigorous stirring to obtain a solid separated by centrifugation. The solid was collected and washed using distilled water and calcinated at 800°C for 3 h to get nano-γ-Al2O3. To 1 g of nano-γ-Al2O3 dissolved in 10 ml of dichloromethane, 0.5 ml of TiCl2 was added dropwise. The resulting solution was vigorously stirred till precipitation. The residue separated by filtration was washed using CHCl3 and dried at room temperature. The SEM revealed its amorphous shape with 50 nm of particle size and a surface area of 75.6 m2/g through BET analysis. The material was found stable up to 400°C. The catalyst’s capacity was assessed in the preparation of hydroquinolines (103) in a single-pot reaction method using 1 mmol of aryl aldehyde (102), 2 mmol of 1,3-dicarbonyl ester (17), and 1.5 mmol of ammonium acetate (7) in the presence of 0.05 ml of TiCl2/Nano-γ-Al2O3 in a solvent-free abode  catalysed by Zn/MCM-41. The impregnation method was used to induce ZnNO3 onto MCM-41. The vintage Hantzsch reaction mixture consisting of aryl aldehyde (104), dimedone (19), and methyl-3-aminocrotonate (105) was employed in the presence of the prepared catalyst in a solvent-free environment. The reaction proceeded by activating the carbonyl groups through Zn+2 for Knoevenagel condensation, and the enamine intermediate participates in Michael addition. The acquired product was filtered and dried. High yields were achieved by using 3- and 4-fluorobenzaldehyde, salicylaldehyde, and 4-hydroxybenzaldehyde in combination with methyl 3-aminocrotonate and 5-methyl furfural, while the lowest yield was attained with 3-indole-carboxaldehyde  and 2,4,6-triarylpyridines (109) adopting anomeric-based oxidation. Chitosan was refluxed with vanadium pentoxide in water to form a black precipitate after 24 h, which was filtered and washed with hot DM water followed by drying it at 80°C. By FT-IR, XRD, SEM, EDX, ICP-MS, and TGA analyses, the material was characterised. XRD amorphous structure SEM and wavelength-dispersive X-ray analysis (WDX) confirmed the presence of V, N, O, and C atoms. Tg and DTG stability of the catalyst was at 225°C. The reaction mixture of aldehyde (107), β-ketoester (17), and ammonium acetate (7) in the presence of 5 mg of ChVO catalyst in a solvent-free abode at 80°C gave 1,4-dihydropyridines in good yields  while reducing the reaction time to 45 min. The retrieved catalyst retained its viability and catalytic potential for several consequent cycles.Safaiee and his coworkers  have cond yields . The obt3H metal-organic framework, in the synthesis of 1,4-dihydropyridines (56) using Hantzsch reaction. Initially, a reaction mixture of monosodium 2-sulfoterephthalic acid and CrO3 was dissolved in a solution of 0.53 ml of HCl and 20 ml of water and autoclaved at 180°C for 168 h. It was allowed to drop to room temperature and filtered. The product was washed with 400 ml of DM water and 100 ml of methanol. The green powder was dissolved in DMF, heated at 120°C for 24 h, and again added to a solution of aqueous methanol at the same temperature to achieve microcrystalline powder. The collected green powder was dissolved in a solution containing 0.08 M of HCl. The obtained product was treated with aqueous methanol for three consecutive cycles. Finally, the green powder was washed with water and vacuum dried at 120°C. It showed a crystalline structure with 1,497 m2/g of surface area, as XRD represented and BET values. The MIL-101-SO3H aggregation was explored by SEM analysis. EDAX showed that the catalyst contains 13.47 wt% of chromium and 3.47% of sulphur which has stability up to 500°C. The catalytic potential was assessed by administering 20% (wt%) of MIL-101-SO3H MOF in Hantzsch single pot reaction, including aldehyde (111), 1,3-dicarbonyl compound (97), and ammonium acetate (7) to produce 1,4-dihydropyridines  and hydroquinolines (115) using a single-pot system under the influence of magnetic nanoparticles infused with glutathione. The desired catalyst was prepared by adding ferric oxide to water and methyl alcohol solution and subjecting it to sonication. A volume of 0.4 g of glutathione was added, and the mixture was further sonicated for 2 h. The magnetic material was separated using an external magnetic field, and the resulting solution was filtered by washing it with water and methyl alcohol and oven-dried at 50°C–60°C. The adhesion of glutathione onto the magnetic particles was confirmed by various analytical methods such as FT-IR, XRD, TGA, TEM, VSM, EDX, and elemental analysis. The EXD images show the presence of Fe, O, N, and S. The catalytic activity and viability of the material were established using the Hantzsch reaction to synthesise 1,4-dihydropyridines (113), ethyl acetoacetate (17), ammonium acetate (7), and dimedone (19). A Knoevenagel condensation followed by Michael addition generates the dihydropyridine derivative. The method offers a 90% yield within 35 min with minimal use of 0.02 g of the prepared catalyst.Maleki et al.  have proyridines . The rea117) and hydroxyquinoline (118) derivatives in a single-pot system. A lysed mixture of 0.25 mmol of Pd, Ni, and Ru in dehydrated tetrahydrofuran in 0.25 mmol of octane thiol was subjected to ultrasonication till the solution turned black-brown, suggesting the formation of Pd–Ni–Ru nanoparticles which was vacuum dried. The gathered nanoparticles were combined with graphene oxide in an equal molar ratio and subjected to sonication to gain Pd-Ni-Ru@GO nanoparticles. The XRD values of the gathered nanoparticles describe their perovskite crystalline structure with 3.64 nm of diameter with a surface area of 136.2 cm2/g as represented by BET images. The catalytic performance of the prepared nanoparticles was assessed in preparing 1,4-dihydropyridines. The reaction mixture involved an equimolar amount of ammonium acetate (7), ethyl acetoacetate (17), and 1 mmol of aldehyde (116) suspended in 2 ml of DMF, plus 6 mg of Pd-Ni-Ru@GO nanoparticles as a catalyst, under 70°C for 45 min (Demirci and the team  investigr 45 min . After tThe review highlighted the eminence of mainly the heterogeneous catalyst materials in preparing different novel dihydropyridine derivatives. The monograph described preparation techniques for various catalyst materials in detail. It covered facile and benign magnetic, silicon- and zirconium-bolstered, and ionic liquid-based heterogeneous catalysts, which can coherently facilitate excellent yields in short reaction times in a cost-effective and eco-friendly way. The target products are valuable molecules proficient in curing long-term disorders and drug delivery to the target tissue. The catalyst materials and protocols considered in this review are easy to handle, nontoxic, and easily prepared. High-temperature and pressure resistance ranges make them ideal and sustainable catalysts for preparing heterocyclic compounds, dihydropyridine in particular. These innovative pathways tailored for the synthesis of dihydropyridine derivatives have paved fresh approaches for further research.


Thermus play vital roles in its transformation in geothermal fluids. In this study, two Thermus tengchongensis strains, named as 15Y and 15W, were isolated from arsenic-rich geothermal springs and found different arsenite oxidation behaviors with different oxidation strategies. Arsenite oxidation of both strains occurred at different growth stages, and two enzyme-catalyzed reaction kinetic models were observed. The arsenite oxidase of Thermus strain 15W performed better oxidation activity, exhibiting typical Michaelis–Menten kinetics. The kinetic parameter of arsenite oxidation in whole cell showed a Vmax of 18.48 μM min–1 and KM of 343 μM. Both of them possessed the arsenite oxidase-coding genes aioB and aioA. However, the expression of gene aioBA was constitutive in strain 15W, whereas it was induced by arsenite in strain 15Y. Furthermore, strain 15Y harbored an intact aio operon including the regulatory gene of the ArsR family, whereas a genetic inversion of an around 128-kbp fragment produced the inactivation of this regulator in strain 15W, leading to the constitutive expression of aioBA genes. This study provides a valuable insight into the adaption of thermophiles to extreme environments.Arsenic is a ubiquitous constituent in geothermal fluids. Thermophiles represented by  However, it is widely distributed in many natural and artificial ecosystems, including geothermal springs . Due to –1 . Over 10–1 . The bio–1 . As spec–1 .Microorganisms have evolved multiple strategies to adapt to As-contaminated environments, including oxidation, reduction, and methylation of As. The first known bacterial strain capable of oxidizing As[III] was isolated in 1919 ; since tThermus are typically of the thermophilic group and are distributed globally across hot spring environments (Thermus species have highly versatile carbon metabolic pathways and that they perform incomplete denitrification (NO3–→N2O) and metabolize sulfur , extremely high temperature (82.5∼91.8°C), and slightly alkaline pH (7.4∼10) , located in Rehai geothermal area, Tengchong region, Yunnan province, China. These geothermal fluid samples were characterized for total As concentrations (197.3∼321.1 μg l(7.4∼10) . Water s4⋅2H2O, 1 g MgSO4⋅7H2O, 1.11 g Na2HPO4, 0.5 g FeCl3, and 5 ml trace element solution, which included 0.5 ml l–1 concentrated sulfuric acid, 2.28 g l–1 MnSO4⋅H2O, 0.5 g l–1 ZnSO4⋅7H2O, 0.5 g l–1 H3BO3, 0.025 g l–1 CuSO4⋅5H2O, 0.025 g l–1 Na2MoO4⋅2H2O, and 0.045 g l–1 CoCl2⋅6H2O. Enrichments were cultivated for 5 days while shaking on a rotary shaker (120 rpm) at 60°C in aerobic conditions.The filtered membranes were cut into pieces and then inoculated in a 100-ml enrichment medium  with 1 mM As[III], which consisted of (per liter) 0.25 g tryptone, 0.25 g yeast extract, 30 mM sodium lactate, and 1 ml 100 × stock solution, as listed in the following (in 100 ml): 1 g citric acid, 3.8 g KCl, 3.6 g NaCl, 0.2 g CaSO–7 dilution were spread on growth medium containing 1 mM As[III]. The inoculated agar plates were incubated at 60°C, and colonies were picked. To obtain pure strains, isolates were repeatedly subcultured on TYL medium. The As[III]-oxidizing isolates were first screened out by the KMnO4 method in TYL liquid medium  and 100-μl aliquot cultures with 10d medium . The spe2 and Na3AsO4⋅12H2O.Colony morphology and pigmentation were determined on TYL agar plates at 60°C. Cell morphology was observed using a fluorescence microscope . The ranges of temperature, pH, and salinity were determined at different temperatures , pH , and salinities  using TYL medium. MICs of As were determined by growing the strains in TYL medium with different concentrations of NaAsO7 CFU were inoculated into fresh TYL medium (100 ml) containing 1 mM As[III]. For short-term response, when cells were inoculated into fresh medium, 1 mM As[III] was not added until the cultures were in the stationary phase. Bacterial suspensions (1 ml) were taken for measurements of optical density at OD 660 nm and As[III]/As(V) concentrations. To estimate the As[III] oxidation rate quantitatively, a kinetics study was conducted under aerobic conditions. For this purpose, strains grown at the late exponential phase without As[III] were harvested by centrifugation at 8,000 g for 20 min at 4°C. Cells were washed twice with PBS buffer (pH 7.2) and resuspended into 1 ml fresh medium to obtain 107 CFU ml–1. Oxidation started by adding 0.2, 0.4, 0.8, 1.6, 2, 3, 4, and 5 μl of As[III] stock solution (500 mM). Control was set without cells, and samples were collected with a 25-min interval for As species determination.Cells were grown to late exponential phase in TYL medium (100 ml) under 60°C. A series of batch experiments were designed to determine the ability of As[III] oxidation with long-term and short-term responses under aerobic conditions. For long-term response, suspension cultures (1 ml) containing about 10As speciation was determined by liquid chromatography–hydride generation–atomic fluorescence spectrometry  . ControlaioA gene sequences extracted from genome sequences. Phylogenetic trees inferred from sequences of 16S rRNA gene and putative AioA were constructed using MEGA7  following the manufacturer’s instructions with some modifications. Cells were treated with 250 μl lysozyme (20 mg l–1) before RNA extraction. The RNA samples were treated with PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa) for 2 min at 42°C to remove genomic DNA. RNA was checked on agarose (2%) gel electrophoresis, and the concentration was determined using a NanoDrop spectrophotometer  and diluted to equal concentrations. DNA-free RNA (1 μg) was used for cDNA synthesis according to the manufacturer’s instructions. Each reaction mixture (20 μl) consisted of 4 μl RNase-free H2O, 4 μl 5 × PrimeScript buffer, 1 μl RT Primer Mix, 1 μl PrimeScript Enzyme Mix, and 10 μl DNase-treated RNA sample. Reactions were incubated at 37°C for 15 min followed by heat inactivation 85°C for 15 s.To compare the recG, was used as an endogenous control to normalize the cycle threshold (Ct) value (aioA and aioB) and two internal genes (recG and 16S rRNA) were quantified. Melting curve analysis was performed to check the specificity of amplification. The relative expression was calculated according to the 2–△△Ct method  using qPCR kit . Each reaction mixture contained 10 μl TB Green Premix Ex Taq II, 0.8 μl forward and reverse specific primers, 0.4 μl ROX Reference Dye II, 6 μl RNase-free water, and 2 μl diluted template . The housekeeping gene, encoding ATP-dependent DNA helicase-t) value . Two As to As(V) as part of both long- and short-term responses . We firsVmax and KM constants for the Aio enzyme of 18.48 μM min–1 and 343 μM, respectively. Some As[III] oxidizers have lower KM values than Thermus strains (343 μM), e.g., Variovorax sp. MM-1 (17 μM), which indicates that they have a higher affinity for As[III]. However, strain 15Y showed the highest Vmax based on previous literature reports regarding kinetic parameters for As[III] oxidation . However, when initial As[III] concentrations were > 700 μM, oxidation activity increased with As[III] concentration, possibly due to the shortened response time of aio operon regulation. After more than 2 mM, cell metabolism was significantly inhibited by As[III] toxicity. Compared with strain 15W, strain 15Y exhibited a slower oxidation rate, up to 3 μM min–1. The different As[III] oxidation models between two strains suggested that they employed different As[III] oxidation strategies to cope with As[III] stress. In summary, cells of strain 15Y would make different responses depending on the As[III] concentration. However, the reason why strain 15Y exhibited a special As[III] oxidation model needs to be further studied.The kinetic rate constants of As[III] oxidation for two strains were determined based on the whole-cell system. This could truly reflect their own kinetic characteristics of As[III] oxidation. An examination of the reaction kinetics of As[III] oxidation indicated that the two strains exhibited different relationships between the initial As[III] concentration and reaction rates . Strain xidation . Further species . Strain aioBA, we monitored the dynamic expression level at various time points under 1 mM As[III] treatment using quantitative real-time PCR (RT-qPCR)  . The aio As[III] . The peaoxidized , which maioA in strain 15W (aioA and 16S rRNA gene (as a control), were amplified to produce the fragments with the predicted sizes after cDNA was synthesized. A band matching the fragment size of aioA was present in strain 15W before the cells were treated with 1 mM As[III]  was used as another approach to verify the apparent constitutive expression of rain 15W . RNA sam As[III] . By cont As[III] . This suaioA was found to be widely distributed among members of the genus Thermus. Although the AioA sequence similarity for strains 15Y and 15W was 98.7%, genome annotation results indicated that the aio gene clusters were different. Compared with strain 15Y, strain 15W lacks a complete ArsR family regulatory gene, due to this gene being truncated as determined by sequence alignment  in strain 15W   . DDAIs hn (DDAI)  and are n (DDAI) .aioB and arsR family members in strains 15Y and 15W, indicating that high conservation of this sequence is essential for the transcription of up- and downstream genes  and −10 (GATAGT). We also predicted a transcription start site  and a ribosome-binding site , motif2 (5′-GTGAGGG-3′), motif3 (5′-ATGAGGACTGGTA-3′), motif4 (5′-GGAAAGGATGC-3′), motif5 (5′-CAGGCTATACTTTA-3′). However, further studies are needed to verify these binding sites. These demonstrated that the integrity of the promoter might be necessary for aioBA transcription in T. tengchongensis.Intriguingly, 234 base pairs were identical between am genes . Consisting site . 5 putatThermus, a predominant inhabitant of geothermal fluids, is involved in multiple elemental biogeochemical cycles. To adapt to extreme conditions, such as high temperature and heavy metal stress, these bacteria have evolved unique metabolic and regulatory mechanisms. A chromosomal inversion in the ArsR family regulatory gene likely provided strain 15W with an evolutionary advantage for inhabiting the As-rich geothermal environment within a limited As concentration range. Similar genetic mutations have been found in other microbes, which are thought to be adaptations for survival, such as mutator phenotypes and adaptive mutations  can be found below: CY was responsible for the design, execution of the study, and wrote the original manuscript. PL carried out reviewing and editing manuscript and study supervision. CQ, ZK, and HW assisted sampling for experiments. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


These cadaverine recognition sites also interact with amines with four and three amino groups . Glu7.36 of sTAAR365 cooperates with Asp3.32 and Thr7.42 to recognize spermine, whereas mTAAR9 recognizes spermidine through an additional aromatic residue, Tyr7.43. These results suggest a conserved mechanism whereby independently evolved TAAR receptors recognize amines with two, three, or four amino groups using the same recognition sites, at which sTAAR365 and mTAAR9 evolved distinct motifs. These motifs interact directly with the amino groups of the polyamines, a class of potent and ecologically important odorants, mediating olfactory signaling.Biogenic amines activate G-protein-coupled receptors (GPCRs) in the central nervous system in vertebrate animals. Several biogenic amines, when excreted, stimulate trace amine-associated receptors (TAARs), a group of GPCRs in the main olfactory epithelium, and elicit innate behaviors. How TAARs recognize amines with varying numbers of amino groups is largely unknown. We reasoned that a comparison between lamprey and mammalian olfactory TAARs, which are thought to have evolved independently but show convergent responses to polyamines, may reveal structural determinants of amine recognition. Here, we demonstrate that sea lamprey TAAR365 (sTAAR365) responds strongly to biogenic polyamines cadaverine, putrescine, and spermine, and shares a similar response profile as a mammalian TAAR (mTAAR9). Docking and site-directed mutagenesis analyses show that both sTAAR365 and mTAAR9 recognize the two amino groups of cadaverine with the conserved Asp Biogenic amines are a group of signaling molecules that activate G-protein-coupled receptors (GPCRs) and regulate a wide variety of neurophysiologic and behavioral functions. Recognition of amine neurotransmitters, which are often monoamines that activate the aminergic family of GPCRs in vertebrate central nervous systems, has been examined extensively , 2, 3. BOdorous polyamines are found in natural excretions , decomposed tissues, and food sources, and can elicit significant physiological changes and behavioral responses in various species examined , 14, 15.3.32; Ballesteros–Weinstein indexing) and a tryptophan residue in transmembrane helix VII (Trp7.40)  , 23. Mol binding . By cont3.32 to recognize polyamines. In this study, we identified a sea lamprey olfactory TAAR receptor (sTAAR365) that shows a strikingly similar response profile to cadaverine, putrescine, and spermine as does mTAAR9. Through a systematic comparison of these two distant receptors with convergent functions, we show that sTAAR365 and mTAAR9 both possess conserved Asp3.32 and Tyr6.51 residues that interact with the two amino groups in cadaverine. In addition, sTAAR365 uses an extra Thr7.42 that stabilizes the recognition of cadaverine, serving as part of the amine-binding motif. In sTAAR365, this motif uses an additional negatively charged residue Glu7.36 that cooperates with Asp3.32 and Thr7.42 to recognize the tetraamine spermine. Likewise, mTAAR9 recognizes the triamine spermidine through an aromatic residue, Tyr7.43. Thus, sTAAR365 and mTAAR9 recognize these polyamines through a novel motif located in the transmembrane α-helices VI and VII. Taken together, our results propose a mechanism that sTAAR365 and mTAAR9 converged on their polyamine recognition through distinct motifs in a conserved binding site.We hypothesized that vertebrate TAARs rely on residues that form a cation–pi interaction or a hydrogen bond with the amino groups in addition to the salt bridge formed by Aspolf-mediated cAMP signal transduction (B). Rat TAAR9 showed the maximum responses to triethylamine, cadaverine, spermidine, and spermine compared with the other mammalian species. Cat TAAR9 displayed similar activation properties (potency and efficacy) to those of mTAAR9. These mTAAR9 ligands induced concentration-dependent activities in cells expressing mouse, rat, and cat TAAR9s (C). In contrast, human, hamster, and rabbit TAAR9s showed minimal activities for this set of ligands.We first asked whether mammalian TAAR9 orthologs are broadly tuned to triethylamine, cadaverine, spermidine, and spermine, as has been shown in mTAAR9 . We examsduction , 24. In sduction B. Rat TAt TAAR9s C. In conD). Triethylamine induced robust responses by mTAAR7f and moderate activities for mTAAR5 and mTAAR8c (D). Other TAARs were not activated by cadaverine or spermidine. Based on these findings, we concluded that several mammalian TAAR9 orthologs detect polyamines. As the mouse is a model animal for olfactory studies, we focused the remainder of our studies on mouse TAAR9 to further characterize TAAR interactions with polyamines.We then sought to determine if other mouse olfactory TAARs are activated by the mTAAR9 ligands. Only mTAAR8c showed modest activity to 500 μM spermidine D. Trieth mTAAR8c D. Other A). Putrescine and spermine elicited a half maximal response (EC50) at concentrations of 56 μM and 28 μM, respectively, in cells expressing sTAAR365 . These are comparable to the potency of odorant receptor agonists in similar assays, ranging from 100 nM to 100 μM . This level of sensitivity rivals the olfactory responses observed through in vivo recording (max) of sTAAR365 response to cadaverine was comparable to that for putrescine, whereas spermine elicited a maximal response of only one-third as much, suggesting that spermine likely acts as a partial agonist for sTAAR365.Next, we questioned whether the sea lamprey TAAR repertoire contains members that are broadly tuned to biogenic amines and that share similar response profiles with mTAAR9. In a previous study, we reported that sTAAR348 responds to spermine when expressed in HEK293T cells but not to other structurally related biogenic amines . SequencFig. 2o 100 μM , 26, 27.ecording . MoreovesTaar365 and mTaar9 in olfactory sensory neurons (OSNs) with in situ hybridization. For adult male and female sea lamprey, the antisense sTaar365 labeled cells were sparsely distributed in lamellae along the rostral-caudal axis of the main olfactory epithelium, displaying tall cell bodies situated in the deeper epithelium and long dendrites coursing toward the epithelium surface . The homology model with the highest C-score was chosen as the final structure for molecular docking. Both the sTAAR365 and mTAAR9 models displayed a canonical GPCR structure with seven hydrophobic transmembrane α-helices and an eighth intracellular helix (H8) in the C-terminus.A previous study of zTAAR13c by Li 3.32 contacts both amino groups of cadaverine, one is docked 2.67 Å away from the highly conserved Asp3.32, forming a salt bridge and a hydrogen bond with the carboxyl group of Asp3.32 (A). The second amino group of cadaverine also forms a pi–cation interaction with Tyr6.51 and a hydrogen bond with Thr7.42 (A). Similar to cadaverine, putrescine was predicted to interact with Asp3.32, Tyr6.51, and Thr7.42 (B). Notably, the distance between the carboxyl group of Asp3.32 and its salt-bridged amino group of putrescine is predicted at 3.61 Å. The difference in the salt bridge distance predicted for cadaverine and putrescine likely explains the 1000-fold difference in their potency for sTAAR365.Using the homology models, we performed Induced Fit Docking (IFD) with Schrodinger Maestro 11.5 to predict the residues of sTAAR365 and mTAAR9 involved in polyamine binding. Several poses of ligand–receptor interactions were generated, and the top result was chosen according to docking scores and glide models. For sTAAR365, the highly conserved Asp Asp3.32 A. The se Thr7.42 A. Simila Thr7.42 B. Notabl3.32 and Thr7.42 are involved in spermine binding (C). Asp3.32 forms a salt bridge and a hydrogen bond with the two middle amino groups of spermine. The distance between the charged aspartate side chain and the further amino group of spermine is 4.22 Å. Meanwhile, an amino group at one end of spermine contacts the backbone of Thr7.42 with a hydrogen bond, while the amino group at the other end is anchored on the negatively charged residue Glu7.36, located in the extracellular vestibule of TM VII, through a salt bridge and a hydrogen bond. The distance between the carboxyl group of Glu7.36 and the terminal amino group of spermine is predicted at 4.33 Å. The salt bridges involved in spermine recognition are longer than the typical cutoff value for a salt bridge at 4 Å. These relatively weak ionic interactions may explain the partial activation of sTAAR365 by spermine.We then docked spermine, a polyamine with four amino groups, into sTAAR365 homology model to infer how TAARs may interact with additional amino groups. As expected, the cadaverine recognition sites Asp binding C. Asp3.33.32 and Tyr6.51 are the primary binding sites. The carboxyl group of Asp3.32 forms a salt bridge with one amino group of cadaverine at a distance of 4.41 Å (A) and a hydrogen bond with the second amino group of cadaverine. Likewise, Tyr6.51 is predicted to be part of the cadaverine-binding pocket, forming a hydrogen bond with the amino group of cadaverine (A). However, mTAAR9 differs from sTAAR365 by having Val7.42 instead of Thr7.42. The larger distance of the salt bridge and the lack of scaffold interaction with Thr7.42 may explain the much lower potency of cadaverine for mTAAR9 compared with sTAAR365. mTAAR9 exhibits robust responses to spermidine, a biosynthetic precursor of spermine. Results from docking spermidine (with three amino groups) into the mTAAR9 model suggested that an extra residue, Tyr7.43, cooperates with Asp3.32 and Tyr6.51 to form the binding pocket (B). Asp3.32 recognizes the middle amino group of spermidine by a salt bridge, at a distance of 2.87 Å, and a hydrogen bond. Moreover, a pi–cation interaction is also predicted between the Tyr6.51 residue and the middle amino group. For the amino groups at the ends of spermidine, Tyr7.43 recognizes one and forms a pi–cation interaction, whereas Asp3.32 recognizes the amino group at the other end and forms a hydrogen bond (B). Furthermore, triethylamine, which has a similar potency as cadaverine for mTAAR9, forms a single salt bridge with the carboxyl group of Asp3.32 at a distance of 2.81 Å (C).Docking cadaverine into mTAA9 homology model suggested that Aspf 4.41 Å A and a hdaverine A. Howeveg pocket B. Asp3.3gen bond B. Furthef 2.81 Å C.Figure 3.32 and Tyr6.51 that interact with two amino groups in diamines. In addition, sTAAR365 uses an extra Thr7.42 to stabilize recognition of cadaverine and putrescine. The diamine recognition motifs, in addition to an extra Glu7.36 in sTAAR365 or Tyr7.43 in mTAAR9, enable the selective recognition of spermine and spermidine, respectively.Taken together, these docking results suggest that sTAAR365 and mTAAR9 have the conserved Asp3.32 in polyamine recognition by sTAAR365 and mTAAR9, we replaced their Asp3.32 with alanine, asparagine, or glutamate. In sTAAR365, a charge-neutralizing mutation of Asp3.32 (D3.32A or D3.32N) eliminated activation by cadaverine, putrescine, and spermine (A). In contrast, the D3.32E mutant, with the most conservative exchange of Asp3.32 to Glu, was activated by cadaverine and putrescine with comparable efficacy as wild-type, albeit with drastically decreased potency .To confirm the role of Aspspermine A. In conectively . However3.32 in mTAAR9 for responses to spermidine. Activity was abolished for the D3.32A, D3.32N, and D3.32E mutants (C). Likewise, cadaverine- and triethylamine-induced activities were abolished for mTAAR9 mutants (C). These empirical findings are consistent with docking predictions that the highly conserved Asp3.32 is a critical determinant for activation of sTAAR365 and mTAAR9 by various amines.Similar results were observed for mutations of Asp mutants C. Likewi mutants C. These 6.51 residue of sTAAR365 was predicted to interact with cadaverine and putrescine. We substituted the Tyr6.51 residue with phenylalanine, alanine, or leucine and examined the effect on receptor activity. These substitutions resulted in a loss of the hydrogen-bond interaction or pi-cation, or both. The sTAAR365 Y6.51A and Y6.51L mutants drastically reduced receptor responses to cadaverine and putrescine (B). In contrast, cadaverine was able to activate the Y6.51F mutant but with a potency 350-fold lower than the wild-type sTAAR365  and likely helps stabilize the spermine-binding pocket. Indeed, all three sTAAR365 Tyr6.51 mutants were activated by spermine with reductions in potency, ranging from 5- to 20-fold . The Y6.51A and Y6.51F mutants almost completely lost the receptor activity to cadaverine and spermidine (D). Tyr6.51 was not predicted to be a triethylamine recognition site. However, this residue is situated 5 Å from triethylamine in the docking model, well within the range of van der Waals interactions. For triethylamine, Tyr6.51 mutants impaired the receptor activity (D). In conclusion, results from site-directed mutagenesis suggest that the Tyr6.51 residue either constitutes or stabilizes an amine-binding pocket for polyamines in sTAAR365 and mTAAR9.To examine whether the Tyrtrations D. The Y6ermidine D. Tyr6.5activity D. In con7.42 is involved in amine recognition by forming a potential hydrogen bond. We substituted the Thr7.42 residue with alanine, valine, methionine, or serine to examine the effect on receptor activity. As the most conservative exchange, T7.42S mutant retained a comparable maximum response to the wild-type when exposed to polyamines (A). However, the potency of cadaverine, putrescine, and spermine for the mutant decreased about 32-fold, 5-fold, and 2-fold, respectively . Furthermore, spermine induced no receptor activity in these two mutants . Finally, the T7.42M mutant, with a bulky side chain that blocks the binding site, lost receptor activity to all ligands tested .Our docking studies of sTAAR365 predicted that the polar residue Thrlyamines A. Howeveectively . In cont7.42, as opposed to a polar Thr7.42 in sTAAR365, we determined whether a V7.42T mutant enhances the potency of polyamines for mTAAR9. Unexpectedly, the mTAAR9 V7.42T mutant lost activity to cadaverine and spermidine and showed a drastic reduction in its response to triethylamine (B). Taken together, these results suggest that sTAAR365 utilizes a polar Thr7.42 residue to form a hydrogen bond that recognizes polyamines, which is a mechanism distinct from mammalian TAAR9.Since mTAAR9 possesses a hydrophobic Valhylamine B. Taken 7.36 residue in the extracellular vestibule forms a salt bridge with an amino group at the distal end of spermine. However, Glu7.36 was not predicted to interact directly with the docked cadaverine and putrescine. It could also form an interhelical salt bridge with the Arg2.64 residue to potentially modulate structural stability of sTAAR365 .Docking of spermine into sTAAR365 homology model suggested that the GlusTAAR365 . We reas7.36 and Arg2.64 was observed in mTAAR9 docking models  in mTAAR9 and examined their receptor activity. Triethylamine, cadaverine, and spermidine activated the E7.36D mutant with little loss of potency (C). In sharp contrast, the charge-neutralizing mutants E7.36A and E7.36Q completely abolished mTAAR9 activation (C). These results indicate that the interhelical salt bridge between Glu7.36 and Arg2.64 may play an important role in modulating the stability of amine recognition pocket or structural conformation of mTAAR9.Likewise, the interhelical salt bridge between Glug models . We gene potency C. In shativation C. These 7.43 residue was not predicted to be part of the polyamine recognition motif in sTAAR365 homology model, docking studies suggested that the hydroxyl group of Tyr7.43 interacts with Asp3.32 by a hydrogen bond that stabilizes the conformation of the essential amine recognition site . The Y7.43F and Y7.43L mutants retained minimal putrescine-induced receptor activity, whereas the remaining Tyr7.43 mutants completely abolished receptor activity. Likewise, all five mutants were inactive to spermine (B). Taken together, all experimental data from the Tyr7.43 mutants are consistent with the prediction that Asp3.32 is anchored in place by a hydrogen bond to the hydroxyl group of Tyr7.43, which stabilizes polyamine recognition of sTAAR365.Although the Tyrion site . We posi000-fold . In cont000-fold B. The Y7spermine B. Taken 7.43 residue was predicted to interact with an amino group at the distal end of spermidine through a pi–cation interaction. Functional testing with spermidine revealed that all four mTAA9 mutants of Tyr7.43  eliminated the activity of the receptor (D). Furthermore, these mutants did not show receptor responses to cadaverine or triethylamine (D). However, flow cytometry analysis showed that mutation of the mTAAR9 Tyr7.43 residue to Ala (Y7.43A), Phe (Y7.43F), and Ser (Y7.43S) had impaired cell-surface expression of the receptor  for Trp7.40. All three sTAAR365 mutants showed reduction in potency to cadaverine, with increases in EC50 values for about 56-fold, 32-fold, and 3000-fold, respectively . The W7.40G mutant lost receptor activity to putrescine and spermine . All experimental results suggest that Trp7.40 is critical for polyamine recognition by sTAAR365, potentially stabilizing a positively charged amino group via hydrophobic or aromatic/aromatic interactions. A close investigation of the docking model for cadaverine and spermine indicates that Trp7.40 contacts Tyr7.43 with a pi–pi stacking to stabilize Asp3.32 in the polyamine-binding pocket . The efficacy of cadaverine for these two mutants was comparable to wild-type mTAAR9, whereas the efficacy of spermidine was significantly decreased (B). For monoamine detection, similar results were observed for these two mutants with triethylamine activation, with an 11-fold and 36-fold decrease of EC50, respectively . Thus, all experiments performed with the Trp7.40 mutants showed that substituting Trp7.40 with amino acid residues having a less bulky aromatic side chain increases the potency of ligands at mTAAR9. It is conceivable that the less bulky aromatic residue facilitates access of ligands into the internal binding pocket and generates a more sensitive receptor for amine detection. In contrast, the W7.40A mutation might result in a loss of structural stability by replacing the aromatic side chain with a methyl side chain, which abrogates potential aromatic interactions with neighboring amino acids, or it may reduce receptor overall stability. Taken together, these observations suggest that the Trp7.40 residue of the aminergic DW motif plays different pharmacological roles in modulating amine response profiles in sTAAR365 and mTAAR9.In sharp contrast to sTAAR365 mutants, the mTAAR9 W7.40F and W7.40Y mutants displayed a marked increase in potency of cadaverine and spermidine, resulting in a shift of the concentration–response curves to lower concentrations by about one order of magnitude B. The efecreased B. For mo3.32 or a noncanonical amine-detection site Asp5.42 (or both) to recognize various monoamines or diamines  or stabilize recognition of polyamines with more than two amino groups (Glu7.36 and Tyr7.43 for both sTAAR365 and mTAAR9). Our models provide strong evidence for a steric and functional odotope theory that sTAAR365 and mTAAR9 utilize convergent structural bases with distinct residues to detect various biogenic polyamines. The convergent mechanism of polyamine recognition by TAARs reveals additional insights into amine detection by GPCRs.Olfactory TAARs enable vertebrates to detect volatile or soluble amines that are ubiquitous in their habitat. The structural determinants for TAARs that recognize amines with a wide range of amino groups have remained elusive, largely due to the lack of crystal structures for any olfactory receptor. In the present study, we examined the structural basis of sTAAR365 and mTAAR9 for detection of polyamines with up to four amino groups. The sea lamprey and jawed vertebrate TAARs are suggested to have evolved independently, forming distinct clades. Receptors from both clades recognize polyamines, providing a unique opportunity to characterize polyamine recognition sites across independently evolved vertebrate GPCRs. Previous studies have demonstrated that teleost TAARs utilize a canonical amine-detection site Aspdiamines . Howevere theory , we prop3.32 residue of the aminergic DW motif (Asp3.32 and Trp7.40) has been reported as a critical element for amine recognition by TAARs , most mammalian TAARs , and some teleost TAARs (zebrafish 34/112). A few mammalian TAARs, such as two mouse TAARs (mTAAR7a and mTAAR7d) and two rat TAARs (rTAAR7a and rTAAR7c), contain a noncanonical amine-detection site Glu3.32 on transmembrane III. Here, we also showed that Asp3.32, but not Trp7.40, forms a salt bridge with one amino group in polyamines, which is an essential element for polyamine recognition by both sTAAR365 and mTAAR9. The Asp3.32 residue is highly conserved among class A GPCRs, and a similar salt bridge between Asp3.32 and the ligand amino group has been described in crystal structures of aminergic receptors, including β1 adrenergic receptor, β2 adrenergic receptor, and H1 histamine receptor  at the same plane of Asp3.32 serves as a binding site for polyamines in sTAAR365. Likewise, a polar residue at this position (Tyr7.42) is predicted to be part of the amine recognition pocket in crystal structures of aminergic receptors. Indeed, phylogenetic and evolutionary analyses indicate a close relationship between lamprey TAARs and the aminergic serotonin (5-HT)-4 receptor, supporting the idea that lamprey TAARs and aminergic receptors may use a polar residue at 7.42 that selectively recognizes biogenic amines  showed minimal loss of potency to polyamines. Moreover, we observed that the maximal efficacy of spermine on the T7.42S mutant differs from that on the wild-type. The greater Emax value is likely imparted by the ability of GPCRs coupling to G proteins and the formation of agonist-induced active conformation. These findings encouraged us to examine the role of the hydrophobic Val7.42 in mTAAR9. In contrast to the requirement for a polar residue at this position in sTAAR365, swapping Thr7.42 for Val7.42 in mTAAR9 failed to increase the potency of mTAAR9 ligands and impaired receptor activation, suggesting that the residue at position 7.42 plays disparate roles in sTAAR365 and mTAAR9. Thr7.42 is involved in polyamine recognition by sTAAR365. In contrast, mTAAR9 has evolved a hydrophobic Val7.42 that does not directly contribute to polyamine recognition but plays an important role in maintaining the structural stability of the receptor, conceivably due to effects of hydrophobic residues that point toward the lipid–protein interface.Our docking and mutagenesis data indicate that a distinctive polar residue , serving as a cryptic pocket for mAChR ligands . While the more drastic substitutions, E7.63Q and E7.63A, either reduced potency of cadaverine or putrescine for sTAAR365, or radically diminished the magnitude of activation of mTAAR9. In contrast to cadaverine/putrescine, all three sTAAR365 mutants showed a significant loss of potency to spermine, conceivably because spermine has a much longer carbon chain and Glu7.36 serves as an extracellular vestibular site to stabilize the terminal amino group of spermine.Aside from the abovementioned key residues in the upper region of transmembrane domains, an extra negatively charged residue, Glu ligands . More inmax > 0.5 of WT sTAAR365) gave Hill slopes >1.2. The pharmacological mechanism underlying this phenomenon remains to be examined. This may be due to the much lower potency and attendant artifacts of the high concentrations. However, there could be mutation-induced loss of major interaction, which then requires two separate ligands to contact the multiple sites needed to activate the receptor.We noticed that compared with the wild-type, some sTAAR365 mutants (such as D3.32E with Hill slope 1.44) show steeper slopes in their concentration–response curves for cadaverine. Overall, three mutant receptors with good responses  and low (10−9 M) concentrations  and divergent  motifs for recognition of polyamine odorants  were approved by the Michigan State University Institutional Animal Use and Care Committee (03/14-054-00 and 02/17-031-00). Sea lamprey used for in situ hybridization was euthanized with 3-aminobenzoic acid ethyl ester  followed by dissection of the olfactory organ.All procedures involving sea lamprey . Mice used for Amine compounds of the highest purity available were purchased from Sigma-Aldrich. All chemicals were dissolved in dimethyl sulfoxide  at a final concentration of 200 mM and stored at −20 °C.2 and grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 1× Antibiotic-Antimycotic (Gibco) Hana3A cells derived from HEK293 used for all mTAAR9 experiments were maintained at 37 °C with 5% CO2 and grown in DMEM supplemented with 10% fetal bovine serum and 1× Antibiotic-Antimycotic.HEK293T cells used for all sTAAR365 experiments were maintained at 37 °C with 5% COAll mammalian TAAR genes were cloned from genomic DNA inserted into a modified pcDNA3.1- (Invitrogen) vector containing a Rho-tag (the first 20 amino acids of bovine rhodopsin) as described previously  6). The. The6). Site-directed mutations of sTAAR365 and mTAAR9 were introduced following the protocol of Quik Change site-directed mutagenesis kit (Agilent Technologies). In brief, PCR primers were designed by Agilent QuikChange Primer Design  and S4 a4 cells per well with 50 μl DMEM medium with 10% FBS and incubated for 24 h at 37 °C with 5% CO2. Cells in each well were transfected with 10 ng CRE-Luc, 10 ng pRL-SV40, 10 ng olfactory mRTPs, and 50 ng mTAAR9 or mutants by Lipofectamine 2000 (Invitrogen), and then incubated for 18 h at 37 °C with 5% CO2. Subsequently, the media was aspirated and replaced with 50 μl fresh CD293 media (with 1% glutamine) and incubated for 30 min at 37 °C with 5% CO2. Then, cells were stimulated with serial dilutions of compounds (diluted in CD293 media) and incubated for 4 h at 37 °C with 5% CO2. The firefly luciferase and renilla luciferase activity was measured with a BioTek microplate reader following manufacturer’s instructions.Hana3A cells were seeded in poly-D-lysine pre-coated 96-well plates at a density of 1× 102 and grown in DMEM supplemented with 10% FBS with 1× Antibiotic-Antimycotic. The cAMP production assay was performed in 384-well plates as described in LANCE Ultra cAMP Kit manual (PerkinElmer) to characterize the cAMP production induced in HEK293T cells expressing sTAAR365 and its mutants. Briefly, HEK293T cells were seeded in a 100 mm dish with 3 × 106 cells in 10 ml DMEM medium with 10% FBS and incubated for 24 h at 37 °C with 5% CO2. Cells were then transfected with 5 μg pGL4.29, 1 μg pCI-mRTPs, 1 μg pCI-Gαolf, and 1 μg sTAAR365 plasmid or sTAAR365 mutants and incubated at 37 °C with 5% CO2 for 24 h. Transfected cells were detached with 2 ml Versene (Gibco) and transferred to 384-well plates at 5 μl (2000 cells) per well. Then, 5 μl of the 2× compound serial dilutions was added to each well and incubated for 30 min at room temperature. Afterward, 5 μl 4× Eu-cAMP tracer working solution and 5 μl 4× ULight-anti-cAMP working solution were added to each well and incubated for 1 h at room temperature. Plates were read in the Synergy Neo multimode microplate reader for TR-FRET emissions at 620 nm  and 665 nm . The ratio of 665/620 allows normalization for the well-to-well variability and interference due to assay components.HEK293T cells were maintained at 37 °C with 5% COsTaar365 anti-sense probes were designed against the 372 bp nucleotide sequences of sTAAR365 open reading frame. Amplified fragments were cloned into the pGEM-T vector (Promega) for sequence verification. Plasmids were linearized using restriction enzyme NcoI (anti-sense probe) or SpeI (sense probe) and used for synthesis of digoxigenin (DIG)-labeled RNA probes with DIG RNA labeling kit (SP6/T7) (Roche). mTAAR9 anti-sense probe was designed against the entire coding region of mTAAR9 (1044 bp). Amplified fragments were cloned into the TOPO TA cloning vector (Invitrogen) for sequence verification. Plasmids were linearized using restriction enzyme NotI and used for synthesis of digoxigenin-labeled RNA probes with DIG RNA labeling kit (SP6/T7).In situ hybridization was conducted following previously described methods by Chung-Davidson and colleagues. Briefly (sTaar365 and mTaar9 sense probe) were conducted were conducted simultaneously. Briefly , 20 μm fHomology models of sTAAR365 and mTAAR9 were generated using GPCR-I-TASSER based on the crystal structure of nine homologous templates  . Rank ofhttps://pubchem.ncbi.nlm.nih.gov) and prepared by LigPrep in Maestro , and 75 ng sTAAR365 plasmid or sTAAR365 mutants and incubated at 37 °C with 5% CO2 for 24 h. The null plasmid, pGL4.29, pCI-mRTPs, and pEGFP-N1 were cotransfected as a negative control. Subsequently, 100 μl of 37% formaldehyde was added to each well and incubated for 15 min at room temperature and then washed with 500 μl of PBS three times (5 min each). Cells were permeabilized or not permeabilized with 0.5% Triton X-100 for 10 min at room temperature to label the whole cell or only cell membrane, respectively. Then, cells were washed with 500 μl of PBS three times (5 min each). Permeabilized cells were treated with 500 μl blocking buffer (5% BSA and 0.3% Triton X-100 in PBS) for 1 h at room temperature. Nonpermeabilized cells were treated with 500 μl blocking buffer (5% BSA in PBS) for 1 h at room temperature. Then, the blocking buffer was removed and 300 μl mouse monoclonal anti-rhodopsin antibody  was added to each well and incubated at 4 °C overnight. The antibody solution was aspirated and washed five times for 5 min each with 500 μl of PBS. Cells were incubated with 300 μl red-fluorescent Alexa Fluor 594 goat anti-mouse IgG  for 1 h at room temperature. Cells were washed three times for 5 min each with 500 μl of PBS. Cells were counterstained with 300 μl DAPI  and incubated in the dark for 5 min. Cells were washed three times for 5 min each with 500 μl of PBS. Images were acquired at 400 × magnification under DMI8 Thunder (Leica) with DAPI filter, GFP filter, and Texas Red filter . The mean value of the red fluorescent signal was quantified with LAS X software (Leica).HEK293T cells were seeded in collagen I coated 24-well glass bottom plates at a density of 5 × 105 cells per well with 2 ml DMEM medium with 10% FBS and incubated for 24 h at 37 °C with 5% CO2. Cells were then transfected with 0.4 μg pCI-mRTPs, 0.3 μg pEGFP-N1, and 2 μg wild-type receptor plasmid (mTAAR9 or sTAAR365) or mutant receptor plasmid, and incubated at 37 °C with 5% CO2 for 24 h. The null pCI plasmid, pCI-mRTPs, and pEGFP-N1 were cotransfected as a negative control. Subsequently, transfected cells were dissociated with CellstripperTM (Corning) and transferred into 5 ml tubes for antibody incubation. 100 μl mouse monoclonal anti-rhodopsin antibody  was added to each tube and incubated at 4 °C for 45 min. Cells were washed twice by adding 2 ml staining buffer and centrifuged at 200 × g for 3 min at 4 °C. In the next step, 100 μl phycoerythrin-conjugated donkey anti-mouse IgG  was added and incubated at 4 °C for 30 min. Cells were washed twice by adding 2 ml staining buffer and then centrifuged at 200 × g for 3 min at 4 °C. Cell pellets were resuspended in 500 μl staining buffer for flow cytometry analysis .Hana3A or HEK293T cells were seeded in 6-well plates at a density of 3 × 10The data that support the findings of this study are available from the corresponding author upon reasonable request. All other data related to receptor expression and activity are contained in the article and supplementary material.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Sulfolobus solfataricus (S. solfataricus), an extremophile, are active even at high temperatures. The single-stranded DNA (ssDNA) binding protein of S. solfataricus (SsoSSB) is overexpressed to protect ssDNA during DNA metabolism. Although SsoSSB has the potential to be applied in various areas, its structural and ssDNA binding properties at high temperatures have not been studied. We present the solution structure, backbone dynamics, and ssDNA binding properties of SsoSSB at 50 °C. The overall structure is consistent with the structures previously studied at room temperature. However, the loop between the first two β sheets, which is flexible and is expected to undergo conformational change upon ssDNA binding, shows a difference from the ssDNA bound structure. The ssDNA binding ability was maintained at high temperature, but different interactions were observed depending on the temperature. Backbone dynamics at high temperature showed that the rigidity of the structured region was well maintained. The investigation of an N-terminal deletion mutant revealed that it is important for maintaining thermostability, structure, and ssDNA binding ability. The structural and dynamic properties of SsoSSB observed at high temperature can provide information on the behavior of proteins in thermophiles at the molecular level and guide the development of new experimental techniques.Proteins from  Sulfolobus is one of the well-known hyperthermophilic archaebacterial genera [Sulfolobus can survive at extremely low pH or high temperature [Sulfolobus solfataricus (S. solfataricus) is adapted to high temperature via lipid composition changes [S. solfataricus are widely used in biological experiments and industrial applications that require low pH or high-temperature conditions [S. solfataricus have been applied as industrial biocatalysts [l genera . Unlike perature ,2,3,4,5. changes , protect changes , and exp changes . Becauseatalysts .S. solfataricus are also used in biotechnological applications under harsh conditions. SSBs are proteins that bind to ssDNA non-sequence specifically. During the DNA replication or repair process, they prevent ssDNAs released by a helicase from returning to double-stranded DNAs (dsDNAs), and thereby increase the DNA polymerase activity. Thus, SSBs are essential for all living organisms to preserve their genomes [S. solfataricus, that inhabit extremely hot environments, overexpress SSBs to protect single-stranded nucleic acids from severe conditions and retain their genes [Single-stranded DNA (ssDNA) binding proteins (SSBs) of  genomes ,11. At h genomes . Therefoir genes .S. solfataricus SSB (SsoSSB) consists of 148 amino acids. The unbound structure determined using X-ray crystallography (PDB ID: 1O7I [12 and L45 loops [Escherichia coli SSB [ID: 1O7I ) showed 45 loops ,17. Huma45 loops , Eschericoli SSB , and humcoli SSB  are examcoli SSB ,14,15. Acoli SSB ,4,5).Here, we determined the structure of SsoSSB at high temperature (50 °C) using NMR spectroscopy. SsoSSB was shown to maintain a well-conserved OB-fold structure even at this high temperature, and its thermostability was measured using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. The ssDNA binding activity and backbone dynamics of the protein were also investigated at high temperature using NMR spectroscopy. The spin relaxation experiments revealed that the protein surprisingly retained a highly rigid structure even at high temperature. Moreover, the protein was still able to interact with ssDNA at elevated temperatures. To determine the role of the N-terminal region in the thermostability and DNA binding of the protein, we analyzed the properties of an N-terminal deletion mutant using DSC, CD spectroscopy, isothermal titration calorimetry (ITC), and NMR. Our findings provide important understanding of thermophilic SsoSSB near its physiological conditions and fundamental insights into its potential for biotechnological applications in high-temperature conditions.SSBs have highly conserved sequences ranging from mesophiles to hyperthermophiles . Unlike m) of SsoSSB1−114 was measured at 84.16 °C (2 dmol−1. This suggests that the secondary structure became destabilized but not completely denatured [1–114 refolded after heating (1H-15N heteronuclear single-quantum coherence (HSQC) spectra [To compare the thermostability of SsoSSB with other thermophilic SSBs, we performed DSC. From the DSC data, the melting temperature  and presented 2D signment . To obtalgorithm  generatelgorithm  and Xplolgorithm  operatio10 helical structure. For the structure calculation at room temperature, a similar proportion of medium-range restraints  was used [We obtained 893 distance constraints from the NOESY data and 193 angle constraints from TALOS-N. In the previous study of the NMR structure at room temperature, 2294 intramolecular constraints were used for protein structure calculation . It is kwas used . The num10 helix .The solution structure of the protein was calculated with Xplor-NIH in the PONDEROSA-C/S software package , startin10 helix . Unstruc1–114 consists of nine β strands and three 310 helices [10 helices, and the DNA-bound NMR structure at room temperature (25 °C) consisted of five β strands and two 310 helixes  was calculated as 1.406 Å, and that in the solution structure at room temperature bound to ssDNA (PDB ID: 2MNA) was 1.954 Å.It was already known that β strands β1, β4, and β5 are broken by residues 26, 72–73, and 89, respectively, which differs from the general OB-fold domain . Seconda helices . The pre helixes . Most β 12 loop in the high-temperature structure of SsoSSB highly deviates from the other two SsoSSB structures. Significant structure fluctuations in L12 were also observed in the 20 ensemble structures , spin-spin relaxation (R2), and 1H-15N heteronuclear Overhauser effect (hetNOE) experiments were performed at 25 °C and 50 °C. The average R1 at 25 °C was 1.078 ± 0.067 Hz. Most residues had R1 values within 2 standard deviations of the average. At 50 °C, the average R1 value (1.928 ± 0.141 Hz) was higher than that at 25 °C, and we found more deviations. This implies that the overall motion increases at 50 °C. Residues N34, G35, and V36 in loop L12; residues S97 and E98 located in the loop between β5’ and H3; residue N110 at the C-terminus had R1 values > 2 standard deviations below the average  values calculated from R2/R1 were 12.657 ns and 5.012 ns at 25 °C and 50 °C, respectively [To understand how the protein’s backbone dynamics change with temperature, spin-lattice relaxation  was prepared. DSC was used to measure the Tm value of SsoSSB12–114 as 53.12 °C  ss show53.12 °C a. This w cooling b. Unlikend 60 °C . This isat 25 °C . The NMRdeletion . 12–114 could still interact with ssDNA. There were some chemical shift changes observed due to the added ssDNA (d) and stoichiometry (n) of SsoSSB1–114 in the presence of dA(15) were 1.75 μM and 1.009 (12–114 and dA(15) to determine thermodynamic parameters . While the protein contains a well-conserved OB-fold domain and its structural aspects were already studied at room temperature ,15, the It is usually considered that a sufficient number of NOE  are required for the reliable protein NMR structure calculation . However12 were revealed  and lower R2 (elevated T2) values [1 increased and R2 decreased compared to the values at 25 °C  values . At 50 °at 25 °C a,b. This average a. This mr weight . Remarkam to decrease ~30 °C. The absence of the N-terminus resulted in partial destabilization of the protein  and transformed into BL21(DE3) cells. We cultivated cells for more than 12 h in 10 mL LB medium (25 g/L) with ampicillin  at 37 °C. Into 1 L of LB medium containing ampicillin, 15 mL of overnight cultured cells was poured. Cells were grown at 37 °C until the optical density at 600 nm reached 0.5–0.6, and then isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 0.5 mM. Cells were incubated for an additional 14–18 h at 18 °C. Cells were centrifuged for 15 min at 7500 rpm at 4 °C. For separating endogenous nucleic acids from protein, we used a high salt binding buffer  and a high salt wash buffer . Cells were resuspended and sonicated in the high salt binding buffer. The sample was centrifuged for 15 min at 13,000 rpm, 4 °C, and the supernatant put into an Ni-NTA column . The high salt wash buffer and an elution buffer  were used sequentially to purify the proteins. The proteins were further purified by gel filtration chromatography using a Hi-Load 16/600 75 pg column  with buffer A ethanesulfonic acid (pH 6.5). For expression of 15N- and 13C-labeled protein, cells were grown in M9 minimum media that included 15NH4Cl and 13C-D-Glucose  as nitrogen and carbon sources. The composition of M9 minimum media was 870 mL of distilled water, 1 g of 15NH4Cl, 100 mL of M9 10X salt, 20 mL of 10% glucose (13C-labeled) solution, 2 mL of 1 M MgSO4 solution, 0.3 mL of 1 M CaCl2 solution, 0.33 mL of vitamin solution, and 10 mL of trace metal solution.SsoSSB15N- and 13C-labeled SsoSSB1–114 sample was dissolved to a final protein concentration of 0.5–1.2 mM with 10% D2O in buffer A. A Bruker 900 MHz NMR spectrometer equipped with a cryogenic triple-resonance probe at the Korea Basic Science Institute , Bruker AVANCE Neo 600 MHz spectrometers at GIST Central Research Facilities with a cryogenic triple-resonance probe , and an Agilent DD2 700 MHz NMR spectrometer at Gyeongsang National University  were used to collect NMR spectra. Backbone and sidechain assignments were performed in previous studies [15N- and 13C-edited NOESY-HSQC were collected at 50 °C with 150 ms and 300 ms mixing times for structure calculation. In CSP experiments, ssDNA composed of 15 adenines (dA(15)) was added at molar ratios ranging from 0:1 to 2:1 to 15N-labeled SsoSSB1–114. Average CSP values (Δδavg) were calculated using the following equationThe  studies ,66. 15N-1–114 at 50 °C was calculated using Xplore-NIH-based computations in the PONDEROSA-C/S package [http://www.pymol.org, accessed on 8 March 2022) was used to create the protein structural diagrams and align the protein structures. The NOE constraints and final coordinates were deposited in the RCSB PDB under the accession number 7WCG (BMRB ID: 50523).The 3D structure of SsoSSB package , and NOE package . Followi package  was used package  was used1 and R2 of 15N, and 1H-15N hetNOE data, were recorded on the Bruker AVANCE Neo 600 MHz spectrometers at GIST Central Research Facilities with cryogenic triple-resonance probes . Pseudo-3D NMR spectra were collected with relaxation delays of 20, 60, 100, 200, 400, 600, 800, 1000, 1200, and 1600 ms at 25 °C and 50 °C for the 15N R1 measurements, and with relaxation delays of 16.96, 33.92, 67.84, 101.76, 135.68, 203.52, 271.36, 339.2, 407.04, and 547.72 ms at 25 °C and 50 °C for the 15N R2 measurements. POKY was used to extract the relaxation rate constants by fitting the decay of peak height as a function of the relaxation delay to a single exponential function [1H-15N HSQC spectra were acquired with and without an initial proton saturation of 2.5 s at 25 °C and 50 °C. hetNOE values were obtained from the ratios of peak heights between pairs of spectra, calculated with a POKY script [c) was calculated by this equation [N is the resonance frequency of 15N in Hz.Rfunction . For theY script . For morms of SsoSSB1–114, SsoSSB12–114, and hRPA70A were measured by DSC using a NanoDSC system . The protein samples were prepared at concentrations of 5 mg/mL in buffer A. The thermograms were recorded as the temperature was increased at a rate of 1 °C/min from 50 °C to 110 °C (SsoSSB1–114) or 20 °C to 80 °C (SsoSSB12–114 and hRPA70A). The pressure was kept constant at 3 atm to prevent evaporation of the solvent. Individual component peaks were resolved from the complex profiles after polynomial baseline correction, and the two-state scaled curve fittings were performed by the NanoAnalyze software .The T4, pH 6.5) and placed in a cuvette with a 0.2 mm path length. CD spectra were measured from 190 to 250 nm at 0.5 nm intervals at 20 °C. A 50 μM protein sample was dissolved in buffer A and placed in a cuvette with a 1 mm path length. CD spectra were measured from 210 to 250 nm at 0.5 nm intervals. The temperature was increased from 20 to 80 °C in 2 °C increments. After heating, the temperatures were decreased from 80 °C to 20 °C in 5 °C decrements. Every measurement was performed after waiting for 1 min between temperature changes. Temperature-dependent ellipticity changes at 228 nm were observed to monitor the heat denaturation of the protein. θ was calculated as described in previous papers [The secondary structure of SsoSSB at various temperatures was assessed by far-UV CD experiments using a J-815 spectropolarimeter . It was measured under two different conditions. A 100 μM protein sample was dissolved in buffer B  and stoichiometry (n) were calculated by fitting to the independent model in the NanoAnalyze software .ITC experiments were carried out in buffer A with a Nano-ITC SV instrument . Twenty-four aliquots of 10 μL of 500 μM dA(15) were titrated at 25 °C into 50 μM of SsoSSB