Resultsdemonstrate that the HG2 ligand assumes a tridentate coordinationmode with an N2O donor set and an octahedral coordinationsphere completed with coordinated waters. The magnesium:HG2 complex exhibits significant solubility and cellular uptake.Human chronic latentmagnesium deficiency is estimated to impacta substantive portion of the world’s population. A number ofmagnesium compounds have been developed to combat this deficiency;however, none are ideal due to issues of solubility, absorption, sideeffects  and/or formulation. Here, we describe thepH-dependent synthesis, chemical characterization  spectroscopies, and electrospray mass spectrometry) andin vitro uptake (in a cell model of the large intestine (CaCo-2 cells))of a magnesium complex of the glycine dimer (HG Additionally, previousstudies have indicated the substantial uptake of short glycine peptidesin man, including HG2, providing evidence for the efficacioustreatment of deficiencies utilizing ligands of this type.18First synthesized in 1901 byFischer and Fourneau,lglycine 1; HG2—th2 chelate of magnesium (1) that buildsupon and finally completes the initial 1955–1957 work of Martellet al.19 Complete solution- and solid-state characterizationand in vitro uptake of 1 in a standard cell model ofthe large intestine (CaCo-2), is described.To this end, the work herein describesthe synthesis and full characterizationof an HG22.12 , MgCl2 , citric acid , D2O, and DMSO-d6 NMR solvents, ethanol,and potassium bromide (KBr) for FT-IR analysis were purchased throughSigma-Aldrich . DI water was obtained in-house.Magnesium uptake colorimetric assay kits were purchased from BioVision . Stock solutionsfor magnesium uptake assays were made in-house, with magnesium bisglycinate(MgBG) provided by Balchem Corp  and confirmedfor purity in-house by 1H NMR and magnesium triglycine(MgG3) provided in-house. CaCo-2 (HTB-37) cells and Dulbecco’smodified Eagle medium (30–2002) were purchased from ATCC . Penicillin streptomycin , fetal bovine serum(FBS), and trypsin/EDTA (0.25%) were purchased from Gibco . White clear-bottomed 96-well Armadillo assay plates  were purchased from ThermoFisher .Magnesium oxide  was purchased from Fisher Scientific .HG2.22O/MeOH/0.1% TFAat a flow rate of 0.2 mL/min over a 1.5 min time frame and evaluatedfrom 0–600 m/z. 1D and 2DNMR were conducted on a Bruker Avance III HD 400 MHz instrument. FT-IRwas carried out on a Nicolet infrared spectrophotometer. Thermogravimetricanalysis (TGA) was carried out on a TA Instrument Q500 from 20–800°C with sample weights of 5–10 mg. ICP was conducted byIntertek Pharmaceutical Services . Uptake of magnesiumin CaCo-2 cells was determined on a Molecular Devices FlexStation3 (Molecular Devices). Cellular uptake data was plotted using Prism8 graphing software.Electrospray ionization massspectrometry was carried out on a Shimadzu 8040 LC-MS/MS with samplesanalyzed utilizing a solvent system of H2.32  wasdissolved in DI H2O (∼20 mL) in a 50 mL round-bottomflask, with constant heatingat 90 °C and stirring. A separate solution of magnesium oxide was taken up in DI H2O (∼20mL), with an addition of citric acid ), constantly stirred and heated to 90 °C. The MgO/CA solutionwas added to the HG2 solution—upon addition, thecombined solution turned an opaque white and was observed as translucentwhite/clear after about 10 min and up until reaction completion. Thereaction was left to run for 1 h at 90 °C. The reaction was cooledto room temperature, centrifuged to pellet any remaining solid, andthe supernatant was filtered through a 40 μm filter. The pHof the solution was noted as 10.2. The solution was concentrated invacuo to approximately 3 mL, and the solid was precipitated with anhydrousethanol. Centrifugation was employed to pellet the solid, and theethanol was decanted off. The solid was triturated with diethyl etherad libitum and centrifuged. The ether was decanted off. To ensurethat no trace solvent remained, the solid was reconstituted in DIH2O (∼50 mL), flash-frozen, and dried in vacuo ona lyophilizer. The dried material was collected and massed to 1.28g. Yield was found to be 74% relative to magnesium and 82.6% purewith a 17.3% impurity attributed to magnesium citrate. 1: 1H NMR : δ 3.76 , 3.38 . ESMS m/z: [Mg (G2) + H+]+ calcd for Mg(C4H7N2O3) 155.4; found 155, [1 + H+]1+ calcd for [MgC4H12N2O6 + H+]1+ 209.5; found 210 . (Mg(C4H7N2O3)(H2O)(OH)): By ICP calcd: Mg, 8.66%; N, 9.99%;found: Mg, 8.78%; N, 9.89%; calcd ratio Mg:N = 0.87, found ratio Mg:N= 0.89  and cultured in 14mL of DMEM  with a seeding density of 3.6 ×104 cells/cm2 (CaCo-2) in a T-75 cm2 culture flask and left to grow in an incubator at 37 °C and5% CO2. Cells were subcultured at 90% confluency, and subculturingoccurred in a minimum of five times before use in uptake assays. Whencultures reached 90% confluency, media was removed and 3 mL of trypsin/EDTAwas added; the trypsinized culture flask was placed back in the incubatorfor ∼10 min to detach cells. Once detached, cells were confirmedunder a microscope, the culture flask was rinsed with 6 mL of freshmedia to neutralize the trypsin/EDTA, and cells for uptake assayswere then counted utilizing a DeNovix CellDrop. Once counted, cellswere pelleted down via centrifugation at 125 g for 10 min, and thesupernatant was removed via pipetting. Cells were resuspended in 5mL of room temperature magnesium assay buffer for cellular uptakeassay.CaCo-2cells were cultured from liquid N2.52× 6 H2O), 1, MgBG, and MgG3. Thekit provided standard for linearity confirmation consists of a 150nm/μL stock; as such, MgCl2× 6H2O, utilized as an internal standard, was prepared atthis concentration containing 17.93 mM Mg2+. All sampleswere prepared to contain the same amount of Mg2+ so asto evaluate magnesium uptake in a relative fashion. DMEM was removedfrom the plated cells, and cells were subsequently washed three timeswith HBSS in 100 μL volume. All samples were administered at150 μL/well as triplicate independent dilutions. Cells weretreated for 1–2 h at 37 °C and 5% CO2. Afterincubating, the sample volume was removed from each well and the cellswere again washed three times with HBSS. Cells were lysed utilizing200 μL of kit assay buffer, the post-lysis volume was collected,and each sample was centrifuged at 14,000g for 10min. The resulting supernatants were replated in the same order in50 μL volume. Fifty microliters of kit-provided enzyme/buffer/developermix was added to each well with a multichannel micropipette, and theplate was allowed to incubate for 40 min at 37 °C. Some wellswere left blank for required background subtraction. The kit-providedstandard was diluted to 0, 3, 6, 9, 12, and 15 nmol/μl in DIH2O and administered and developed in the same volumesas the samples and was used only to determine kit linearity . Each well was analyzed for endpointvalue over nine full-plate scans with triplet scans/well/plate scan, and the reported value of each wellwas the average value of these scans after background subtraction.All samples were analyzed in triplicate. Data was collected at 40min. Raw data was reduced and plotted as absorbance against magnesiumconcentration of each well. All assays were repeated in triplicate—errorbars were shown in a graph .A colorimetric magnesium uptake assay kit for use witha 96-well plate was purchased from BioVision . Samplesolutions for use with the kit were prepared in house utilizing magnesium-/calcium-freeHank’s balanced salt Solution (HBSS). The samples tested weremagnesium chloride hexahydrate . Additionally, HG2 exhibits a broad signalat 2055 cm–1, which is attributed to the terminalamine (−NH3+),21 a signal not observed for 1. This implicates that boththe terminal amine and the terminal acid are in coordination. TheFT-IR spectra of HG2 and 1 are provided as The FT-IR of 3.21 from the same synthetic batch. Acquisitionof nitrogen values is complicated by the substantially hygroscopicnature of 1. ICP analysis elucidated the percent magnesium presentin the samples and thus provided compositional insight utilizing aMg:N ratio. The ICP provided values of Mg = 8.78% and N = 9.89% . The values are consistent with aspecies of composition Mg(G2)(H2O)(OH) × 5 H2O. The theoretical nitrogen and magnesiumvalues for this species are Mg = 8.66% and N = 9.99%. Utilizing theMg:N ratio, the experimental ratio of M:N = 0.89 and is consistentwith the theoretical value of Mg:N = 0.87.Duplicate independent ICP analyseswere conducted on 2 exhibited a single, gradual percent weight decrease onset at 220°C (inflection point observed at 270 °C), which is consistentwith the melting point of HG2 at 220 °C. TGA analysisof 1 indicated a gradual decline in percent weight onsetfrom 30 °C until just before 200 °C. The weight change accountsfor a loss of 21%, which is consistent with the loss of 2 waters  (23 thus suggesting thecore magnesium species Mg(G2)(H2O)(OH) whererapid acquisition of subsequent water molecules is likely.TGA of HGto 19%) 3.This r3.31 was provided inthe form of solution-state NMR analysis at equimolar concentration. 1H NMR of 1 was conducted relative to the HG2 ligand. The 1H NMR spectrum of HG2 exhibitedtwo proton signals with a combined integration of two (2) (not surewhat this 2 means); the observed signals were a sharp singlet at 3.84ppm (H1) and a doublet centered around 3.80 ppm . Additionally, the 1H NMR 1 exhibited a combined integration of four (4). These observationswere consistent with the hypothesized observations. Like the 1H NMR spectrum of HG2, spectral analysis of 1 exhibited two proton signals with a combined integrationof four (4). Unlike HG2, the observed proton signals of 1 were both singlets, and each singlet exhibited a substantialupfield shift: H1 = 3.38 ppm (Δppm = 0.46) and H2 = 3.76 (Δppm = 0.04) . Furthermore,spectral analysis of 1 revealed a quartet between 2.50–2.70ppm attributed to magnesium citrate of a 1:1 magnesium:citrate compositionwhen compared to a magnesium citrate standard, which is a result ofcitric acid utilization during synthesis . Full spectra 1H NMR overlay indicating solvent calibrationis provided as Figure S4.Furthersupport for the coordination mode of 1) implicates the participationof this moiety in coordination.20 Additionally,coalescing of the H2 proton signal for 1 relativeto HG2 suggests coordination to the carboxylic acid thatresults in an even distribution of electron density and subsequentlack of observed splitting. Furthermore, the observation of upfieldproton shifting coincides with generalized magnesium coordinationand may be observed for the α-proton adjacent to the point ofcoordination and as far-reaching as the γ-proton.27 Conserved integration of the HG2 proton signals is consistentwith no formation of new ligand-based product and is consistent withthe 1:1 stoichiometric yield of the previously discussed synthesis.As previously reportedby Case et al. when analyzing a triglycinechelate of magnesium, the more substantial upfield shift of the protonnearest the terminal amine  and two signals between160–180 ppm attributed to the sp2-hybridized R–CO–Rmoieties   and C2 (Δppm = 8) signalsto 43.2 ppm and 175 ppm, respectively. These observations are consistentwith those reported by Drevenṧek et al.24 and Chang et al.27 in theirstudies of magnesium testosterone and magnesium ofloxacin/levofloxacin,respectively and are consistent with magnesium coordination in regionsadjacent to these moieties. Additionally, in agreement with 1H NMR analysis, 13C NMR analysis of 1 exhibitedfour signals attributed to magnesium citrate , and no new ligand-based carbon signals were observed, furthersupporting the formation of 1 as a 1:1 complex.6.4 ppm) 5. 13Can1H/13C heteronuclear single quantumcoherence (HSQC), which shows correlations between a defined protonand the carbon a single bond distance away, and heteronuclear multiple-bondcorrelation (HMBC), which confirms the interaction defined protonand corresponding carbons over multiple bond lengths, confirmed allproton and carbon signal assignments. The HSQC spectrum of HG2   7. Given 1H/13C NMRprovides insight into the overall coordination mode of the HG2 ligand and indicates that the ligand acts as a tridentatechelate that coordinates via an N2O donor set. This coordinationis achieved via the terminal amine, the backbone amide, and the terminalcarboxylic acid. These results are consistent with magnesium chelateligands assuming higher-order coordination modes that favor entropicallystable complexes,30 as well as previous studies that indicate the tendencyof ligands such as HG2 and triglycine to act as tri- andtetradentate magnesium chelates.19 Lastly, allNMR analyses of 1 exhibit minimum amounts of magnesiumcitrate, confirming an 82.6% purity of 1.A combination of both 1D and 2D 3.42 ligand acts as a tridentatemagnesium chelate and assumes an N2O donor set to forman entropically-favored complex. Additionally, the six-coordinate,octahedral coordination sphere is completed by two waters and onehydroxide. Confirmation of the presence of a hydroxide anion was providedby a series of potentiometric experiments evaluating the conductivityof 1 relative to potassium chloride .Solutions of both KCl and 1 were prepared at concentrationsof 1, 10, and 100 mM, respectively. At 100 mM, data indicated thatKCl has a conductivity of 13.11mS and 1 has a conductivityof 5.08 mS (approximately 2.5× less than that of KCl). Thesedata indicate that 1 acts as a 1:1 electrolyte with onlypartial dissociation, which is consistent with the previously reportedMgG3.20 Conductivity valuesfor both KCl and 1 trend linearly at the given concentrationrange with R2KCl = 1.0000 and R21 = 0.9995.The combined solution- and solid-state data isconsistent with the structure as shown in 3.51 was determined via triplicate independentanalyses and compared to other reported magnesium chelate complexes.Analysis indicated that 1 has a water solubility of approximately39 g/100 mL. While more soluble than MgO, 1 is less solublethan MgCl2 and about one fifth as soluble as MgG3, which is previously reported by Case et al.  cell line relative to selectpreviously reported magnesium chelate complexes and MgCl2 . Further in vivo testingis required to confirm the observed in vitro trends.Both solution- and solid-state methodsconfirm the successful synthesisof a 1:1 magnesium-HG


Plasmodium life cycle, surface-associated proteins establish key interactions with the host and play critical roles in parasite survival. The 6-cysteine (6-cys) protein family is one of the most abundant surface antigens and expressed throughout the Plasmodium falciparum life cycle. This protein family is conserved across Plasmodium species and plays critical roles in parasite transmission, evasion of the host immune response and host cell invasion. Several 6-cys proteins are present on the parasite surface as hetero-complexes but it is not known how two 6-cys proteins interact together. Here, we present a crystal structure of Pf12 bound to Pf41 at 2.85 Å resolution, two P. falciparum proteins usually found on the parasite surface of late schizonts and merozoites. Our structure revealed two critical interfaces required for complex formation with important implications on how different 6-cysteine proteins may interact with each other. Using structure-function analyses, we identified important residues for Pf12-Pf41 complex formation. In addition, we generated 16 nanobodies against Pf12 and Pf41 and showed that several Pf12-specific nanobodies inhibit Pf12-Pf41 complex formation. Using X-ray crystallography, we were able to describe the structural mechanism of an inhibitory nanobody in blocking Pf12-Pf41 complex formation. Future studies using these inhibitory nanobodies will be useful to determine the functional role of these two 6-cys proteins in malaria parasites.During the different stages of the  Plasmodium falciparum, the most lethal malaria parasite species, is the 6-cysteine protein family. Several members of this family are implicated in critical functions in fertilization, transmission, immune evasion and host cell invasion. Here, using structural biology, we provide an understanding on how two 6-cysteine proteins, Pf12 and Pf41 which are abundant blood stage merozoite proteins, bind to each other using interfaces between their respective 6-cysteine domains and is further anchored by an insertion of a Pf41 loop into the second 6-cysteine domain of Pf12. We found that nanobodies against Pf12 were able to block complex formation and structural analyses showed that a nanobody loop binds to a region, which normally binds the Pf41 insertion loop, thus resulting in steric hindrance in one of the critical interaction sites. Our study provides new structural insights on the 6-cysteine proteins that will be important to progress our understanding on how they function during the malaria parasite life cycle.Malaria is a deadly disease that causes over 400 000 deaths worldwide. During its life cycle, malaria parasites exist in several forms across its different life stages and present diverse surface coat proteins as antigenic targets. One of the more abundant surface coat proteins of  Plasmodium life cycle, the malaria parasite expresses surface coat proteins, which establish key interactions with host proteins and play important roles in parasite survival. The members of the 6-cysteine (6-cys) protein family represent some of the more abundant surface proteins and have crucial roles in host-cell entry, parasite sexual development and immune evasion strategies  and a recombinant fragment of Pf41 encompassing residues 21–368 (Pf41 D1D2) Fig. . AnalytiThe Pf12-Pf41 crystal structure was determined at a resolution of 2.85 Å by molecular replacement and reveals a heterodimeric complex of approximately 80 × 65 × 35 Å in size . The Site 1 interface involves three potential salt bridges, several hydrogen bonds, π-π, as well as Met-π interactions . Site 2 comprises of the Pf12 D2 domain in which its bottom β-sheet and surrounding loops form a concave protein surface that engages with the long α-helix of the Pf41 ID . Here, residues located along the Pf12 β-sheet form a hydrophobic network with residues located at the long α-helix of Pf41 ID .The crystal structure of Pf12-Pf41 shows that both proteins interact through two distinct binding sites (Site 1 and Site 2) with a total surface area of 1051 Å2 Fig. , with Siely Fig. . Site 1 ely Fig.  and Dtopons Fig. top. Site ID Fig. bottom.right), with the main difference being that large regions of the Pf41 ID are resolved in the presence of Pf12.The Pf12 molecule in complex with Pf41 overlays with the existing unbound Pf12 structure (PDB ID 2YMO) with a root mean square deviation (r.m.s.d.) of 2.5 Å (over 1568 atoms), while the individual 6-cys domains align with r.m.s.d. values of 0.99 Å for D1 (over 570 atoms) and 0.58 Å for D2 (over 728 atoms). This finding indicates that the two 6-cys domains adopt different orientations towards each other in the bound and unbound state and provides evidence for potential mobility between the two domains in solution Fig. . Residue1DWe used bio-layer interferometry (BLI) to further determine the role of the individual 6-cysteine domains in Pf12 or Pf41 in complex formation Fig. . Pf41ΔIDWe performed site-directed mutagenesis to identify critical residues at the Pf12–Pf41 interface required for complex formation Fig. . The muttop). To further confirm if the Pf12 residues R109 and R123 were involved in salt bridge formation, we mutated both residues to glutamate to abolish the respective salt bridge and introduce an electrostatic repulsion at the Pf12-Pf41 interface.We examined the mutant Pf12 D1D2 and Pf41 D1D2 proteins for their ability to bind their respective wildtype interaction partner using BLI Fig. . Calcula1Dtop, 1Dtop, For Site 1, Pf12 Y264A and Pf41 F337A did not form a complex with wildtype Pf41 and Pf12 respectively indicating the importance of the π-π interaction as critical for complex formation  and mutant E367A showed a diminished binding signal to Pf12 D1D2 compared to the Pf41 wildtype protein . The distance between them is relatively large, ∼ 5.5 Å. E340A showed a strong binding signal to Pf12 D1D2 suggesting that the potential interaction is not of importance. The guanidino group of Pf12 residue R109 is in closer proximity to other Pf12 residues, such as the side chain of E283 (∼ 4.6 Å) and carbonyl groups of P79 and T80 . The reduced binding signal of R109A and defective binding of R109E to Pf41 might suggest a more important role of R109 in stabilizing the Pf12 region around β-strands β5-β7.We examined the importance of the salt bridge formation at Site 1. While Pf12 mutant R123A reduced binding to wildtype protein, R123E was defective in binding to Pf41 highlighting the additional effect of the electrostatic repulsion   Fig. . We grouR3) Fig. . One memR3) Fig. .left and S6 Fig). The three remaining nanobodies; A6, G11 and G12 bound Pf41 D1D2 . Furthermore, all three Pf41-specific nanobodies bound to Pf41ΔID but not Pf41 D2, indicating that these nanobodies recognise an epitope within the D1 domain of Pf41 but not within the ID .We determined the binding selectivity of the nanobodies for Pf12 and Pf41 using BLI  between 4 and 22 nM. Pf12-specific nanobodies bind their antigen with KD values ranging from pico-to-mid nanomolar (<10 pM and 25 nM). Nanobody G7 is the Pf12-specific nanobody with the weakest affinity with a KD of ∼25 nM. Four anti-Pf12 nanobodies  have KD values < 10 pM, which is below the detection limit.We determined binding kinetics and affinities of the interaction between the nanobodies and Pf12 D1D2 or Pf41 D1D2, respectively using BLI -based assay, we wanted to investigate if the nanobodies were able to disrupt Pf12-Pf41 complex formation Fig. . The res2, with CDR3 providing the major contribution to the paratope  and Pf41 D1D2 (residues K21-S368) were produced and purified as described previously with the following modifications  by IDT or synthesised by Genscript with NheI-HF and NotI-HF restriction enzyme sites, subcloned into the expression vector in-house and sequenced for confirmation. Wildtype and mutant proteins, as well as single domain Pf12 (D1 residues N28-L172 and D2 residues E174-S304) and Pf41 constructs (D2 residues I242-S368) were purified via the same purification protocol. A summary of the different mutants and their sequences can be found in the supplementary.Spodoptera frugiperda 21 cells were used for the generation and amplification of expression viruses as well as for protein production. After three days of infection, growth media was harvested, supplemented with PMSF and a cOmplete protease inhibitor tablet (Roche) and dialysed against 30 mM Tris pH 7.5, 300 mM NaCl. The proteins were purified via Ni-IMAC. The octa-histidine tag was removed by TEV protease cleavage while dialysing into 30 mM Tris pH 7.5, 300 mM NaCl and a following Ni-IMAC. Size exclusion chromatography (SEC) in 20 mM HEPES pH 7.5, 150 mM NaCl was used as a last purification step. In addition, wildtype proteins were also purified as tagged proteins via Ni-IMAC following SEC .Purified, untagged Pf12 D1D2 and Pf41 D1D2 proteins were mixed in a molar ratio of 1.2:1 and incubated for 1 h on ice. SEC in 20 mM HEPES pH 7.5, 150 mM NaCl was performed to separate the formed Pf12-Pf41 complex from excess Pf12. For the purpose of de-glycosylation Glyco buffer 2 and PNGase F (NEB) was added to the mix of Pf12 and Pf41, incubated at RT for 1h prior SEC in 20 mM HEPES pH 7.5, 150 mM NaCl.Purified, untagged Pf12 D1D2 was mixed with Glyco buffer 2 and PNGase F (NEB) and incubated at RT for 1h prior the addition of Nb in a molar ratio of 1:1.5. The proteins were incubated for 1h on ice followed by SEC in 20 mM HEPES pH 7.5, 150 mM NaCl to separate the formed Pf12-Nb complex from excess Nb.Coot and phenix  and interactions, interfaces and buried areas from solvent were analysed using PISA  at 8ºC. Purified Pf12-Pf41 complex with intact glycosylation crystallised as fuzzy needle clusters in 5 mM cadmium chloride, 20% (w/v) PEG 4000, 50 mM HEPES pH 7.5. Complex treated with PNGase F crystallised in the same condition as thin plates. Crystals appeared overnight. Hanging-drop vapour diffusion experiments were set up in-house. Three-dimensional crystals grew in 10 mM cadmium chloride, 14% (w/v) PEG 4000, 25% glycerol, 50 mM HEPES pH 7.5 and were further optimized by micro-seeding. Pf12-Pf41 crystals were harvested from mother liquor and flash frozen in liquid nitrogen. Pf12 in complex to Nb G7 crystallized in 15% (w/v) PEG 4000, 200 mM potassium thiocyanate, 0.05 M sodium cacodylate pH 6, were flash frozen with 30% glycerol in mother liquor. Diffraction data was collected at the MX2 beamline at the Australian synchrotron. The XDS package was used for data processing. The phase problem was solved by molecular replacement using the program Phaser. The atomic coordinates of Pf12 D1D2 (PDB ID 2YMO) and Pf41 D1D2 (PDB ID 4YS4) were used as search models for the Pf12-Pf41 complex structure. PDB ID 2YMO and the coordinates of Nb B5 of PDB ID 7KJH were used to solve the crystal structure of Pf12 bound to Nb G7. Anomalous data collected on Pf12-Pf41 crystal was used to confirm the presence of bound Cd-ions. Alternating rounds of structure building and refinement were carried out using D values and kinetic parameters. This analysis was performed using Octet Data Analysis 10.0 software. We hypothesise that the two separate interaction sites and flexibility of the regions involved in Pf12-Pf41 binding result in a complex association kinetic rate that is better modelled with a heterogeneous 2:1 binding model rather than a 1:1 binding model. Curves that could not be fitted were excluded from the analyses. The maximum response for each mutant relative to wildtype at concentrations of 1000 nM, 500 nM, and 250 nM were calculated by dividing the maximum response of a mutant protein by the maximum response of the wildtype protein at each concentration. The relative maximum response at each concentration was plotted with the mean ± standard deviation (SD).Protein interactions of Pf12 and Pf41 mutants were performed on the Octet RED96e (FortéBio). All assays were performed using NiNTA capture sensor tips (NTA) sensors (FortéBio) with kinetics buffer (PBS pH 7.4 supplemented with 0.1% (w/v) BSA and 0.05% (v/v) TWEEN-20) at 25°C. After a 60 s biosensor baseline step, octa-histidine tagged Pf12 or Pf41 (20 μg/mL) were loaded to a signal of 1.8 nm onto NTA sensors by submerging sensor tips for 200 s and then washed in kinetics buffer for 60 s. Association of Pf12 mutants to Pf41 was observed using a two-fold dilution series of untagged Pf12 mutant from 31–1000 nM for 180 s and dissociation was observed in kinetics buffer for 180 s. Association of Pf41 mutants to Pf12 was observed using the same conditions. Sensor tips were regenerated using a cycle of 5 s in 300 mM imidazole pH 7.5 and 5 s in kinetics buffer repeated five times. Baseline drift was corrected by subtracting the response of a protein loaded sensor not incubated with untagged mutant protein. We observed a fast and slow association rate of the Pf12-Pf41 interaction; therefore, we used a global fit 2:1 heterogeneous model for the curve fitting analysis to determine KD values and kinetic parameters. Curves that could not be fitted were excluded from the analyses. Mean kinetic constants reported are the result of two independent experiments.Affinity measurements were performed on the Octet RED96e using the above method with the following modifications. After a 60 s biosensor baseline step, nanobodies (5 μg/mL) were loaded onto NTA sensors by submerging sensor tips until a response of 0.5 nm and then washed in kinetics buffer for 60 s. Pf12 and Pf41 association measurements were performed using a two-fold dilution series of untagged Pf12 or Pf41 from 6–200 nM for 180 s and dissociation was measured in kinetics buffer for 180 s. Sensor tips were regenerated using a cycle of 5 s in 300 mM imidazole pH 7.5 and 5 s in kinetics buffer repeated five times. Baseline drift was corrected by subtracting the response of a nanobody loaded sensor not incubated with untagged protein. Curve fitting analysis was performed with Octet Data Analysis 10.0 software using a global fit 1:1 model to determine K8 nanobodies with 80% correct sized nanobody insert. The library was cloned into a pMES4 phagemid vector amplified in Escherichia coli TG1 strain and subsequently infected with M13K07 helper phage for recombinant phage expression.One alpaca was subcutaneously immunised six times with approximately 200 μg of recombinant glycosylated Pf12-Pf41 complex. The adjuvant used was GERBU FAMA. Immunization and handling of the alpaca for scientific purposes was approved by Agriculture Victoria, Wildlife & Small Institutions Animal Ethics Committee, project approval No. 26–17. Blood was collected three days after the last immunization for the preparation of lymphocytes. Nanobody library construction was carried out according to established methods . The nanobody was eluted via a linear gradient into 400 mM imidazole, 100 mM NaCl, PBS. The appropriate fractions were concentrated and subjected to SEC (SD200 increase 10/300) pre-equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl.Nanobodies were expressed in Samples were analysed using an XL-I analytical ultracentrifuge  equipped with an AnTi-60 rotor. Protein samples were loaded in the sample compartment of double-sector epon centrepieces, with buffer in the reference compartment. Radial absorbance data was acquired at 20°C using a rotor speed of 50 000 rpm and a wavelength of 280 nm, with radial increments of 0.003 cm in continuous scanning mode. The sedimenting boundaries were fitted to a model that describes the sedimentation of a distribution of sedimentation coefficients with no assumption of heterogeneity (c(s)) using the program SEDFIT Schuck . Data wePf12 was labelled with N-hydroxysuccinimide ester-activated DyLight 488 (DL488) and Pf41 labelled with DyLight 594 (DL594) (Life Technologies). The dyes were dissolved in dimethyl sulfoxide (DMSO) (Sigma) before incubation with the proteins. Pf12 was incubated with DyLight 488 at a 4:1 molar ratio of dye to protein for 30 minutes at room temperature. Pf41 was incubated with DyLight 594 at a 1:1 molar ratio of dye to protein for one hour on ice. Un-conjugated dye was removed using a Zeba Spin Desalting column (7K MWCO) (ThermoFisher). Average dye per protein was ∼ 2.4 dye/protein for Pf12 and ∼ 1.0 dye/protein for Pf41. To test inhibitory nanobodies, Pf12-DL488 and Pf41-DL594 were mixed in a 1:1 molar ratio with five-fold excess of nanobody. The FRET assays were read in Corning 384-well plates and each sample was performed in triplicate. 5 μL of 0.2% SDS, 10% 2-mercaptoethanol in FRET buffer was added to one triplicate sample to denature the proteins and measure the background signal in the absence of complex formation. Fluorescence intensity was measured using EnVision plate reader (PerkinElmer Life Sciences). DL488 (donor) fluorescence was measured with a 485/14-nm excitation filter and 535/25-nm emission filter and DL594 (acceptor) fluorescence was measured with a 590/20-nm excitation filter and 615/9-nm emission filter. Sensitised emission was measured with a 485/14-nm excitation filter and 615/9-nm emission filter. Fluorescence measurements were analysed using Prism 6 software (GraphPad). To account for bleed-through of dyes into the sensitised emission spectra, standard curves for both dyes were measured and the raw data was transformed by multiplying with the slope of the standards. The y-intercept from the standard curves indicated the background fluorescence when no dye was present. The FRET signal was calculated with the following equation: FRET signal = Raw sensitised emission—transformed DL488 emission—transformed DL594 emission—(average of DL488 standard curve y-intercept and DL594 standard curve y-intercept). To compare between independent replicates, percentage FRET signal relative to the no inhibitor control was calculated by dividing the FRET signal with inhibitor by the FRET signal with no inhibitor, multiplied by 100.Plasmodium falciparum parasites were continuously cultured as previously described (Jensen and Trager 3 (Thermo Scientific), 0.5% (w/v) AlbuMAX II (GIBCO)), at 37°C in low oxygen conditions . 3D7 Hyp1-Nluc parasites were used which episomally expresses the exported protein PfHyp1 (PF3D7_0 113 300) tagged with the bioluminescent reporter nanoluciferase (Nluc), under the control of P. berghei ef1α protomer (active from the trophozoite stage)  to trophozoites overnight. Next morning the ML10 was removed and the late stage schizonts were allowed to egress and invade for 4 h. The remaining schizonts were eliminated by sorbitol treatment and the new ring stage parasites were grown for nearly 2 days until late stage schizonts which were enriched from uninfected erythrocytes and younger parasites over a 67% Percoll density gradient. The egress/invasion assay was then performed as previously described , and 473 Å2 , and calculated solvation free energy gains of −10.3 and −11.0 kcal/mol, respectively. The calculated interaction areas and solvation free energy gains indicate that both binding sites contribute to a similar extent to the Pf12-Pf41 engagement. Furthermore, we show that neither Pf12 D2 nor Pf41ΔID were able to form a Pf12-Pf41 complex. These results indicate that the Pf41 ID interaction with Pf12 is necessary but not sufficient for complex formation and that both Site 1 and Site 2 are critical for the assembly of the two 6-cys proteins.Our crystal structure of the Pf12-Pf41 complex indicates that both Site 1 and Site 2 binding sites are important for complex formation. PISA analysis suggests that the heterodimeric complex is a stable biological assembly, and the two binding sites contribute to the interaction surface with 578 ÅThe crystal structure of the Pf12-Pf41 complex suggests that binding of Pf12 to Pf41 causes conformational changes within the proteins. We found that the D1 and D2 domains of bound and unbound Pf12 adopt different orientations towards each other. Furthermore, a region of 33 residues spanning 84–116, which includes the β-sheets β5 to β7 of the Pf12 D1 domain, which was unstructured in the unbound Pf12 molecule, was well resolved in the presence of Pf41, suggesting that binding of Pf41 possibly reduces the flexibility within Pf12 and stabilises the junction of the Pf12 D1-D2 domains. The Pf41 ID consists of only ∼ 40% secondary structure elements and its inherent flexibility is supported by higher B-factors of the non-coordinated regions and unresolved residues that connects it to the core structure of the 6-cys domain. The long α-helix which interacts with Pf12 has lower B-factors than the remaining parts of the Pf41 ID suggesting a stabilizing effect caused by Pf12-binding.Plasmodium spp (Aurrecoechea et al. Using site directed mutagenesis, we observed that on both binding sites either aromatic or solely hydrophobic residues played crucial roles in the complex assembly. Alanine substitutions of Pf12 residues Y264 or F283 abolished binding to Pf41. On Pf41, alanine mutants of F337, L159, and L163 were defective for binding to Pf12. These residues, which appear critical for Pf12 and Pf41 binding, are also conserved in orthologous proteins and may also play critical roles in P12-P41 complex formation in other 2 compared to the ID binding site on Pf12, which comprises 473 Å2. We propose that G7 inhibits Pf12-Pf41 complex assembly by binding Pf12 with higher affinity and blocking the binding site for Pf41 ID.All Pf12-specific nanobodies, except for H2, block Pf12-Pf41 complex formation. Furthermore, the inhibitory nanobodies bind to the Pf12 D2 domain, while the non-inhibitory H2 nanobody binds to the Pf12 D1 domain. The crystal structure of Pf12 in complex with inhibitory nanobody G7 showed that the CDR loops on G7 interact with the bottom β-sheet of the Pf12 D2 domain. The CDR3 loop folds into a small α-helix which binds to the Pf12 β-sheet that normally accommodates the long α-helix of the Pf41 ID. G7 binds to Pf12 with higher affinity compared to Pf12-Pf41 binding (25 nM vs 120 nM, (Dietrich et al. The CDR3 loops of nanobodies are often longer compared to conventional antibodies, which allows them to bind to groove-like structures (Bannas et al. in vitro. This is consistent with previous findings showing that Pf12 and Pf41 could be knocked-out without impacts in parasite growth (Taechalertpaisarn et al. P. vivax, another Pv12-interaction partner, PVX_110 945, was identified in an AVEXIS screen (Hostetler et al. Our inhibitory nanobodies were able to block Pf12-Pf41 complex formation, but they did not inhibit egress or invasion of blood stage parasites xtac005_Supplemental_FilesClick here for additional data file.


O-glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3-O-glucoside tested. Results of this study provide insight into the structure–function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases.Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3- Anthocyanins are a diverse group of pigments that are ubiquitous in the plant kingdom. They impart most of the orange to red and purple to blue colors present in plant species. Due to the prevalence of these compounds in many plant species and due to the attractiveness of their hues, anthocyanins are becoming popular natural substitutes for synthetic dyes. Purple corn is becoming an increasingly popular choice for anthocyanin extraction. The economy of scale for purple corn and the utilization of anthocyanins as co-products in the maize supply chain make purple corn an attractive alternative to other natural colorant sources currently on the market ,2. AnthoA major obstacle for adopting anthocyanins as natural colorants, however, is the inferior stability compared to synthetic dyes. Stability of anthocyanins is controlled in part by the inherent structure of the molecule. Adding acyl groups to the anthocyanin glycone is one method plants use to enhance the stability of these pigments. Acylation is defined as the esterification of organic acids to the sugar of the anthocyanin molecule. Two types of acylation are possible: aliphatic and aromatic. Aromatic acylation generally confers the greatest stability probably due to intramolecular stacking of pigment molecules . AliphatAnthocyanin acyltransferase1 (Aat1) [Dendranthema × morifolium) Dm3Mat2 [In maize, acylated anthocyanins account for a majority of the total anthocyanins produced, with aliphatic malonic acid derivatives being the most represented form . In part1 (Aat1) . Nearly  Dm3Mat2 . In thisOryza sativa, OsMat2, is most similar to Aat1 in maize and shares a clade in the phylogenetic tree. However, they only share 51% sequence identity. Maize Aat1 shares 28% to 31% identity with other malonyltransferases in eudicots, which is typical of acyltransferase members. Previous studies report that the minimal amount of sequence similarity among acyltransferases is 25% to 34% [O-glucoside and 7-O-glucoside forms equally, which is unique for a flavonoid malonyltransferase [Phylogenetic analysis of known flavonoid malonyltransferases is similar to previously reported analyses with other acyltransferase enzymes . Another% to 34% . Despite% to 34% ,13. The % to 34% . Motifs % to 34% . Both Aa% to 34% . Manjase% to 34% . Howevernsferase . Future Aat1 has been revised since it was first discovered [O-glucoside. In addition, maize Aat1 can catalyze the subsequent addition of a malonyl group to form dimalonylglucosides  [Specificity and reaction kinetics of Aat1 varied widely from the other anthocyanin dimalonyltransferase, Dm3Mat2 . The mos10−3·s−1 . This ist shown) ,22. The O-glucoside, delphinidin 3-O-glucoside, pelargonidin 3-O-glucoside, and peonidin 3-O-glucoside were purchased from Extrasynthese . Malonyl-CoA, succinyl-CoA, and acetyl-CoA were purchased from CoALA Biosciences . Quercetin 3-O-glucoside standard was purchased from Sigma-Aldrich. Anthocyanins were reconstituted in 0.1% HCl in water, while acyl donor substrates were reconstituted in water and used within a day. To prepare malonylglucoside standards, an enzyme assay (see Enzyme Assays) was performed under standard conditions and run on a semi-preparative HPLC (see Liquid Chromatography and Mass Spectrometry) to isolate the malonylglucoside peak. The purified aliquots were combined, concentrated in a SpeedVac® AES2010 concentrator  at 43 °C and then lyophilized. Powder was reconstituted with 0.1% HCl in water and the concentration was adjusted to 600 µM based on the A520 of cyanidin 3-O-glucoside given that the molar extinction coefficients are similar [Liquid Chromatography and Mass Spectrometry).Analytical standards of cyanidin 3- similar . Purity Aat1 transcript (NCBI Reference Sequence NP_001148286.2) was amplified using PCR with primers 5′-CATGATTCGAATTCATGGCGGCAGCAACGGCAACT-3′ and 5′-GCTACGATAAGCTTTCACAGCAACCGGAGCCACTCCA-3′ that introduce EcoRI and HindIII sites (underlined). Restriction enzyme cloning introduced the transcripts into the pET-30a(+) vector  as described by the supplier [4 to assist growth [v/v) glycerol. After cultivating overnight at 37 °C, two mL of overnight culture was added to 100 mL fresh media and grown at 37 °C to an OD600 of 0.6 to 0.8. Cultures were put on ice for five minutes before being induced with 0.4 mM IPTG overnight at 18 °C. Overnight cultures were centrifuged at 1600× g for 10 min. Media were decanted and pellets were resuspended in 100 mM potassium phosphate (pH 8.0), 10% glycerol, 1 mM EDTA, and 5 mM 2-mercaptoethanol (Buffer A) with an addition of 1 mM PMSF. Bacterial lysis by sonication was done on ice with a 20 kHz probe set to 50% power with five 10 s bursts with 20 s rests between pulses. Full length supplier . Vectorst growth  and 50 µ® TGX™ gel with 50 µL wells .To capture 6x-His-tagged recombinant Aat1, one mL HisPur™ Ni-NTA agarose resin  was equilibrated with 10 column volumes ultrapure water and 20 column volumes of Buffer A + 10 mM imidazole before the addition of the protein extract. Whole crude protein extract was filtered in a 0.45 µm PES syringe filter  and equilibrated with constant shaking for 1 hr. The resin with captured protein was washed with 30 column volumes of Buffer A + 10 mM imidazole. The 6x-His-tagged protein was eluted in 3 column volumes of Buffer A + 500 mM imidazole. Purified protein was desalted in a G-25 MidiTrap  desalting column according to manufacturer’s instruction using 100 mM potassium phosphate (pH 8.0) + 10% glycerol as the equilibration buffer. SDS-PAGE analysis was done on a 10% Mini-ProteanO-glucoside as the acyl acceptor in 100 mM potassium phosphate (pH 8.0). To calculate reaction kinetics, acyl acceptors were varied between 5 and 120 µM, acyl donors were varied between 5 and 30 µM, and cyanidin 3-O-6”-malonylglucoside was varied between 2.5 and 30 µM. Anthocyanins, which were diluted with 0.1% HCl in water, were no more than 5% of the reaction volume so as to not change the pH of the reaction. The final reaction volume was 100 µL and each assay was replicated three times. Reactions were run for 30 min at 30 °C before being halted with 100 µL ice cold 4% (v/v) formic acid. Enzyme assays determining pH preference used 100 mM sodium acetate (pH 3.0 to 5.5), 100 mM potassium phosphate (pH 6.0 to 8.0), 100 mM Tris (pH 8.5 and 9.0), and 100 mM CAPS (pH 10 and 11). Kinetics constants were determined using the rearranged Michaelis–Menten equation proposed by K. A. Johnson (2019) [cat and Ksp (Kcat/Km) were solved using the “nls” non-linear modeling function in R [A standard reaction consisted of 60 µM malonyl-CoA as the acyl donor and 120 µM cyanidin 3-n (2019) . Constanion in R . Velocit18  column  kept at 50 °C. The wavelengths used for detection were 520 nm for all anthocyanins or 280 nm for quercetin 3-O-glucoside. The mobile phase consisted of 2% formic acid (A) and acetonitrile (B) at a rate of 1.7 mL/min in a gradient from 4% B from 0 min to 1 min, 4% to 20% B from 1 to 10 min, then 20% to 100% in 30 s and back to 4% B in 30 s. The column was equilibrated with 4% B for 4 min between samples. Anthocyanin standards were varied in a linear range to calibrate peak areas. To confirm compounds formed in enzyme assays, representative samples were analyzed on a Waters Synapt G2-Si ESI/LC-MS/MS at the Mass Spectrometry Lab at the University of Illinois  using the same column and temperature as the enzyme assays. The gradient was adjusted to run on the machine. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) at a rate of 1 mL/min in a gradient of 4% B from 0 to 1 min, 4% B to 25% B from 1 to 25 min, then a 95% B hold for 5 min and back to equilibration with 4% B for 5 min. Aliquots of 10 µL were injected for each sample. To prepare semi-pure malonylglucoside standards, standard enzyme assays were run on an Agilent 1100 Series HPLC with a semi-preparative fraction collector. Aliquots totaling 75 µL were separated in a Grace Prevail RP-C18  column  heated to 30 °C. The diode array detector was monitored at 280 nm. Mobile phases used were 2% formic acid (A) and acetonitrile (B) with a flowrate of 1 mL/min in a gradient of 10% B at 0 min, 30% B at 35 min, 100% B from 36 to 38 min, and equilibration for 5 min at 10% B.Anthocyanins in enzyme assays were quantified using an Agilent 1290 series UHPLC. An aliquot of 20 µL of enzyme assay was separated in an Agilent InfinityLab Poroshell 120 SB-CO-glucoside anthocyanin substrates. Future studies should investigate the effect of the residues that may be important for dimalonyltransferase activity, especially the Lys substitution in Motif 2 (Maize Aat1 is an anthocyanin dimalonyltransferase responsible for synthesizing the majority of anthocyanins in maize grain. It can utilize malonyl-CoA, succinyl-CoA and a variety of 3- Motif 2 . In addi


Comoclathris. The effect of different fermentation conditions on the production of tyrosinase inhibitory activity was examined, in order to identify the optimum cultivation conditions. LCMS based metabolomics was applied to determine significant differences between the strains and fermentation conditions, and to identify potential bioactive secondary metabolites. Bioassay-guided purification of the main active components led to the isolation of three new compounds (1–3), along with the known compounds graphostrin B (4) and brevianamide M (5). Compound 1 (Comoclathrin) demonstrated the strongest anti-tyrosinase activity (IC50 0.16 μΜ), which was 90-times higher than kojic acid (IC50 14.07 μΜ) used as positive control. Additionally, comoclathrin showed no significant cytotoxicity against a panel of cancer cell lines  and normal BJ fibroblasts. These properties render comoclathrin an excellent development candidate as whitening agent.As part of our screening program for the discovery of molecules of microbial origin with skin-whitening activity, 142 diverse fungal endophytes from a wide variety of Andalusia arid plants were screened, applying the OSMAC approach. The fungal strains CF-090361 and CF-090766, isolated from xerophytic plants, were selected as the most promising, while phylogenetic analysis revealed that both strains could represent a new species within the genus  During this complex relationship with host plants endophytic fungi produce various biologically active secondary metabolites to increase their adaptability to both biotic and abiotic stresses5. Over the past two decades, endophytic fungi have attracted attention owing to their ability to produce many valuable compounds with high chemical diversity, including alkaloids, terpenoids, steroids, quinones, lignans, phenols and lactones7. Among them, several antimicrobial, immunosuppressant, insecticidal, cytotoxic, anticancer and antioxidant secondary metabolites have been reported7. However, and besides the wide range of bioactive substances produced by the endophytic microorganisms their potential to inhibit the adverse effects of tyrosinase enzyme has not been studied. Tyrosinase is the key enzyme of melanin biosynthesis in microorganisms, plants, and animals, playing an important role in the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and in the oxidation of L-DOPA to dopaquinone8. The only reported whitening agent isolated from endophytic fungi is kojic acid, which has been produced from strains of Colletotrichum gloeosporioides and Aspergillus niger isolated from Sonneratia apetala and from the roots of Entandrophragma congoënse, respectively10. Moreover, high levels of kojic acid are also produced by other endophytic fungal isolates belonging to the genera Aspergillus, Petromyces, Penicillium, Chaetomium, Emericella and Pleospora, and associated with common medicinal plants in Egypt11.The term endophyte was first introduced by De Bary in 1866 and refers to an endosymbiotic group of microorganisms—including both bacteria and fungi—colonizing inter- and/or intracellular locations of plants, without causing any immediate pathogenic effect on its host(s)12, 142 diverse fungal endophytes from a wide variety of Andalusian arid plants were selected to be screened, applying the OSMAC (One Strain Many Compounds) approach by using four different fermentation media16. The two fungal strains CF-090361 and CF-090766, isolated from the xerophytic plants Sedum sediforme and Nerium oleander, were found to exhibit the most potent anti-tyrosinase effect when grown in LSFM and MMK2 media and were selected to be further studied. Their anti-tyrosinase activity was further confirmed when they were further tested in mouse melanocytes (B16F10 cell line) . The bioassay-guided isolation and structural elucidation of the main active components 1–5, led to the identification of three new compounds  and the assessment of their in vitro whitening and cytotoxic activity.This work describes the novelty of the producer strains as a new potential species of the genus Comoclathris according to their ITS and 28S rDNA sequences and their phylogenetic position based on Bayesian inference : 37 (1909)), and until now 44 species have been described [http://www.indexfungorum.org/names/Names.asp]. Comoclathris was originally wrongly associated with the genus Alternaria18 and it has been recently accommodated within the Pleosporaceae upon morphology and phylogenetic analysis. The consensus phylogenetic tree used in the study includes 11 Comoclathris endophytic strains isolated from arid plants of Andalusia and 17 GenBank™ sequences of representative Comoclathris species  was chosen in order to compare the secondary metabolite production of the two strains.The strongest anti-tyrosinase effect was observed when the strain CF-090361 was cultivated in the fermentation media LSFM and MKK2 and when the strain CF-090766 was cultivated in the medium LSFM for 14 days14. In order to investigate in a time course the production of tyrosinase inhibitory activity, the strains were grown in LSFM medium during 7, 14 and 21 days using as fermentation formats 40 mL EPA vials and 250 mL Erlenmeyer flasks, containing 10 and 50 mL of medium, respectively.Different culture conditions, with variations of cultivation parameters , were investigated, in order to define the optimum conditions for inducing the anti-tyrosinase effect of the two strainsEvaluation of the anti-tyrosinase effect of the extracts showed that the best results were obtained for both strains when they were incubated for 7 days in flasks with high percentage of inhibition in the range of 80% Table . This ac22. Volcano plots are scatter-plots used for the visualization of statistical results of omics data. In our case, they are used to visually describe how two different experimental conditions statistically affect a large set of components23. Thus, extracts generated from fermentations performed in ten replicates in both formats were analyzed by LC–MS and the metabolite differences between the two fermentation formats for each strain were visualized using Volcano Plots  and in more than twofold increase in flasks than in EPA vials by the strains Overexpressed ions in the active fermentation condition, as indicated by Volcano Plots, were used as a discrimination tool, in combination with the bioassay-guided isolation, for the fast identification of secondary metabolites exhibiting the most significant anti-tyrosinase activity.For the isolation of the active components, the fermentation of the strains CF-090361 and CF-090766 was scaled-up to 1 L in LSFM medium and each strain was grown in flasks for 7 days, which are the selected optimum conditions. The purification of the active components was performed by parallel evaluation of the anti-tyrosinase activity and the LCMS profile of the fractions of the extracts of the two strains obtained by flash chromatography. The fractions that exhibited the most significant anti-tyrosinase activity were subjected to HRMS and LRMS analysis, in order to detect the pseudomolecular ions and fragment ions of interest, that were overexpressed in the active fermentation condition as indicated by Volcano Plots.m/z 219 and 220 (in red colour) and at m/z 223, 241 and 263 (in yellow colour), were allocated in the upper-right part (flask) of the generated Volcano plots  that it is produced in higher amounts in the flask format. Thus, our focus has been on the isolation, structure elucidation and biological evaluation of compounds containing this specific ion.To this end, we found that the active fractions contained the ions at 1), one new sorbicillin analogue, namely 4,6-demethylsorbicillin (2) and one new violapyrone analogue, namely violapyrone L (VLP L) (3), together with two known compounds (4 and 5)  and the indole alkaloid dimer brevianamide M (5), that has been isolated before from fungi belonging to the genus Graphostroma and Aspergillus, respectively29. To the best of our knowledge the genus Comoclathris has not been thoroughly studied regarding its chemical content, as only one compound, namely altersetin, has been isolated from strains belonging to this genus30.Targeted chromatographic separations led to the isolation in both strains of one novel furan derivative, namely comoclathrin (1 gave a [M + H]+ ion at m/z 219.0651 in the (+)ESI-TOF MS appropriate for a molecular formula of C12H10O4  requiring 8 sites of unsaturation. Based on 1 and 2D NMR experiments , the 12 carbon atoms were assigned as five methines, one methyl and six quaternary carbons. The 1H NMR spectra of 1 (in DMSO-d6) showed characteristic signals of a 1,2,4-trisubstituted benzene ring at δH 7.57 , 6.37  and 6.44 , two olefinic protons at δH 7.46  and 6.89 , attributed to a 2,5-disubstituted furan ring and one singlet at δH 2.42  corresponding to the hydrogens of the acetyl group. Two additional singlets at δH 10.32 and 9.78 were identified, attributed to the exchangeable phenolic hydrogens at C-2’ and C-4’. The position of the hydroxy group at δH 10.32 was deduced by the HMBC correlations with the quaternary carbon at δC 108.34 (C-1’) and the methine C-3’ (δC 103.3). Likewise, the hydroxy group at δH 9.78 was assigned at the position C-4’ showing correlation peaks with C-5’ (δC 108.05) and C-3’. The cross peak between H-5’ and H-6’ in the 1H-1H COSY, their characteristic H–H coupling relationship (J = 8.7 Hz) of vicinal protons, and the heterocorrelation peaks with the quaternary carbons C-1’ and C-5 (δC 155.8) of the furan ring confirmed their relative position in the molecule. The presence of an additional methyl group at δH 2.42  of the 1H NMR spectrum and its HMBC correlations with the carbonyl carbon 2-COCH3 (δC 185.1) and the aromatic oxygenated carbon of the furan ring at δC 149.7 (C-2) suggested the position of the acetyl group. Finally, the key HMBC correlations of C-2 and C-5 with methine protons H-3 and H-4 as well as their cross peak in the 1H-1H COSY spectra and coupling constant (3JH/H) of 8.7 Hz confirmed the presence of the 2,5-disubstituted furan ring. According to the above evidence, structure of compound 1 was elucidated as 1- furan-2-yl) ethan-1-one and given the trivial name comoclathrin.Compound 2 gave a [M + H]+ ion at m/z 205.0869 in the (+)ESI-TOF MS, appropriate for a molecular formula of C12H12O3  requiring 7 sites of unsaturation. The 1H NMR spectrum (in CD3OD) displayed characteristic signals of a 1,2,4-trisubstituted benzene ring at δH 7.82 , 6.38  and 6.27 , of a conjugated trans double bond system at δH 7.11 , δH 7.42 , δH 6.44  and δH 6.33 , and of a methyl group at δH 1.90 . Analysis of the 13C NMR spectrum revealed twelve signals. Based on an HSQC-DEPT experiment, the aforementioned signals were assigned as seven methines and one methyl carbon. The remaining four signals in the 13C NMR spectrum were deduced as quaternary, corresponding to the carbonyl carbon C-1’ at δC 192.4, to the oxygenated aromatic carbons C-1 and C-5 at δC 165.1 and 167.0, and to the quaternary carbon C-2 (δC 113.2). Comparison of HRMS, 1H- and 13C-NMR spectra -ESI-TOF MS, accounting for a molecular formula of C11H16O3  requiring 4 sites of unsaturation. Analysis of chemical shifts, peak multiplicities and peak areas of the 1H NMR spectrum suggested characteristic signals of a linear C-5 alkyl chain at δH 2.40 , 1.53 , 1.29 , 1.27 , 0.88 , an olefinic methine proton at δH 5.98 , and a methyl group at δH 1.74 . One additional singlet at δH 11.14 was identified, attributed to the exchangeable hydroxy group at C-4. The hypothesis of a linear penta-alkyl chain was further confirmed by 2D NMR. Analysis of the 1H-1H COSY experiment suggested a proton spin system from H-8 to H-12 while the HSQC-DEPT experiments unambiguously assigned relative carbons (C-8 to C-12) as four methylenes at δC 32.9 (C-8), δC 26.3 (C-9), δC 22.1 (C-10), δC 30.8 (C-11) and one methyl carbon at δC 14.1 (C-12). The relative position of the olefinic proton H-5 and of the methyl group at δH 1.74 (H3-7) was established by the common HMBC correlation with the quaternary carbon C-3 at δC 96.3 and by the key correlation of the hydroxy proton at C-4 (δC 165.17) with C-3 and C-5 (δC 99.6). The 13C-NMR revealed one more signal of oxygenated carbon (δC 165.06) which was attributed to C-2 of the compound. Further HMBC correlations of the methyl group at δH 1.74 with the oxygenated carbons C-2 and C-4 together with the λmax at 289.0 and the fact that compound 3 needs to form a ring to satisfy the unsaturation number indicated the presence of a typical α-pyrone chromophore. The α-pyrone ring was identified as 3-methyl-4-hydroxypyran-2-one with a linear pentyl moiety at C-6 established by the HMBC correlation of H-8 with the methine carbon C-5. Thus, the structure of 3 was determined as 4-hydroxy-3-methyl-6-pentyl-2H-pyran-2-one and represents a new violapyrone analogue, named violapyrone L (VLP L)34.Compound 4 and 5 were identified as graphostrin B and brevianamide M respectively, based on NMR and HRMS data, and by comparison with those reported in the literature29.Compounds 1–5 were evaluated for their tyrosinase inhibitory activity using mushroom tyrosinase. The highest anti-tyrosinase activity was presented by compounds 1, 2 and 5, with IC50 values ranging from 0.16 to 6.81 μΜ , exhibited the most potent activity, showing an 87-times higher inhibitory effect (IC50 = 0.16 μΜ) as compared to the positive control kojic acid (IC50 = 14.07 μΜ). On the other hand, compounds 3 and 4 did not demonstrate a significant inhibitory effect (IC50 > 100 μΜ) (Table Purified compounds Μ) Table .1–5, five different cancer cell lines were chosen . Compounds 2 and 5 showed slight cytotoxic activity against A2058 (90.00 uΜ) and HepG2 (25.00 uΜ), while compound 1, having the most potent anti-tyrosinase effect, did not present activity against any of the cancer cell lines tested  was also found to exert no cytotoxicity on normal human dermal fibroblast cell line, which suggests that this compound could be safely used for its whitening activity.To evaluate the cytotoxicity and possible specificity of compounds m/z 219, which was overexpressed in the active culture conditions in both strains and found abundant in several active fractions of the extracts of both strains  production is 64-fold higher in the strain CF-090766 than in the strain CF-09036, while compound 4 (ions highlighted in yellow) is produced 4 times more by the strain CF-090361.Volcano plots presented in Figs. ns Figs. , corresptes Fig. . The actComoclathris strains CF-090766 and CF-090361, highlighting the optimum conditions for the production of metabolites with anti-tyrosinase activity. Bioassay-guided isolation led to the identification of the three new compounds (compounds 1–3), which demonstrated a high anti-tyrosinase activity without showing toxicity against a panel of cancer and normal cell lines. Furthermore, our findings highlighted that conspecific strains of the genus Comoclathris, isolated from different plants and geographic areas, demonstrated a relevant whitening effect by producing the same active metabolites. More importantly the furan derivative comoclathrin (1) has been shown to be a potent tyrosinase inhibitor devoid of cytotoxic effect with excellent properties and development potential as whitening agent.In conclusion, our approach was successfully applied to the fungal endophytic 1H and 13C NMR spectra were obtained at 500 MHz, using a Bruker Avance III 500 MHz spectrometer (500 MHz and 125 MHz for 1H and 13C NMR respectively) equipped with a low volume 1.7 mm inverse detection microcryoprobe. HRESIMS and LC-UV–MS data were measured using a Bruker maXis QTOF mass spectrometer coupled to an Agilent 1200 HPLC system and on an Agilent 1100 single quadrupole LC–MS system, as previously described35. Preparative HPLC was performed on a Gilson 322 System using a Xbridge™ C18  column at a flow rate of 14 mL/min. Semipreparative HPLC was performed on the same system using a Xbridge™ C18  column or a Xbridge Prep Phenyl,  column at a flowrate of 3.6 mL/min. Evaporation of solvents was performed on a vacuum rotary evaporator . The acetone employed for extraction, as well as the solvents used for isolation were of analytical and HPLC grade, respectively.Sedum sediforme  and Nerium oleander , respectively16. Frozen stock cultures in 10% glycerol (− 80 °C) are maintained in the culture collection of Fundación MEDINA. DNA extraction, PCR amplification, DNA sequencing and Bayesian phylogenetic analysis were performed following an already described process36. Sequences of the complete ITS1-5.8S-ITS2-28S region or independent ITS and partial 28S rDNA sequences were compared with sequences at GenBank®, the NITE Biological Resource Center (http://www.nbrc.nite.go.jp) and CBS strain database (http://www.westerdijkinstitute.nl) by using the BLAST® application. Species affinities of Comoclathris was inferred from Bayesian analysis using the Markov Chain Monte Carlo (MCMC) approach with MrBayes 3.0137. Akaike Information Criterion (AIC) of the nucleotide substitution models was calculated using MrModeltest® 2.2 software38, being GTR + I + G was the selected model for the alignment.The endophytic fungi CF-090361 and CF-090766 were isolated by using standard indirect isolation techniques from stems of 20. In order to evaluate the impact of different nutritional conditions (OSMAC approach) on the rate of secondary metabolites production by this fungal strain, four different fermentation media 20 were used for the liquid-state cultures. These four media formulations were selected based on their reported ability to induce the production of high chemical diversity in taxonomically diverse fungal strains20. The cultures and the small scale extractions were performed by following an already described procedure20.142 diverse fungal strains were revived from cryotubes containing fungal mycelia discs in 10% (v/v) glycerol following a procedure as described by González-Menéndez20. After 7 days of incubation at 22 °C, two sets of fermentations were performed and the strains were inoculated in the culture medium LSFM20, where they exhibited the most significant tyrosinase inhibitory activity, as indicated during the screening process.The fungal strains were revived from cryotubes containing fungal mycelia discs in 10% (v/v) glycerol following a procedure as described by González-Menéndez39. All fermentation broths were incubated for 7, 14 and 21 days, in order to investigate the best time course of the production of the bioactive metabolites. Biological assay revealed that 7 days is the optimum time of fungal incubation.The first set of fermentations was performed in duplicate in vials  and 250 mL Erlenmeyer flasks containing 10 and 50 mL of medium respectively. Fungal inocula cultured in SMYA medium during 7 days at 22 °C, 220 rpm and 70% relative humidity were used to inoculate EPA vials and Flasks at 3% of final volume (v/v) following previously described protocols20.The second set of ten repetitions of each fermentation condition was performed also in 40 mL EPA vials and in 250 mL flasks containing 10 mL and 50 mL of LSFM medium respectively, for 7 day, as this was the optimum time of the fungal incubation. All fermentation broths were extracted with acetone, and samples were finally prepared in a final 20% dimethyl sulfoxide (DMSO)/ water solution at two whole broth equivalent (WBE) concentration according to the procedure previously described40.The culture extracts (2 µL) were analyzed by HPLC–UV-LRMS. LC analysis was performed on an Agilent 1200 , using a Zorbax SB-C8 column  with guard column, maintained at 40 °C with a flow rate of 300 µL/min and 210 nm UV detection. Mass spectrometry acquisition was performed on an Agilent MSD 1100 mass low resolution spectrometer to generate the metabolomic raw data. The solvents and gradient system used, as well as the statistical analyses by t-test and metabolomic charts (volcano-plots) were performed by following an already described process20. Inoculated flasks were incubated during seven days at 22 °C, 220 rpm and 70% relative humidity in a shaking incubator .1 L scale up fermentation of both strains CF-090361 and CF-090766 was performed by inoculating aliquots of 1.5 mL of the each inoculum into twenty 250 mL flasks containing 50 ml of LSFM medium obtained following previously described protocol−1 on an automatic flash chromatography system , using a linear gradient from 5 to 20% acetone in water (in 15.8 min) with a final 100% acetone step (for 19.2 min), collecting 35 fractions of 20 mL. For the extract of the strain CF-090766 the elution was performed at 18 mL min−1 on an automatic flash chromatography system , using an isocratic at 5% acetone in water (for 6 min), followed by an isocratic at 20% acetone in water (for 6 min), an isocratic at 40% acetone in water (for 6 min), an isocratic at 60% acetone in water (for 6 min), an isocratic at 80% acetone in water (for 6 min), and an isocratic at 100% acetone (for 20 min), collecting 48 fractions of 20 mL. Fractions were concentrated to dryness on a centrifugal evaporator, tested for their tyrosinase inhibitory activity and forwarded for further chemical investigation.The scale up fermentation broths (1 L) were extracted with acetone (1 L) under continuous shaking at 220 rpm for 1 h and centrifugation was followed. The remaining mixture (ca. 2 L) was concentrated to ca. 1 L under a nitrogen flow. The solution was loaded, with continuous 1:1 water dilution, keeping the flow-through on a column packed with SP-207ss reversed-phase resin  previously equilibrated with water. The loaded column was further washed with water (2 L). For the extract of the strain CF-090361, the elution was performed at 10 mL min1 (1 mg) was obtained from fraction F24 of CF-090766 using a Xbridge Prep Phenyl,  column and a gradient method as follows: 0–20 min, 40% MeOH (solvent B) (isocratic); 20–40 min 100% B (linear gradient); 40–45 min, 100% B (isocratic); 45–47 min, 40% B (linear gradient). Flow rate was set at 3.6 ml/min, detection wavelength at 210 and 350 nm and the retention time was 11.8 min.Compound 2 (0.5 mg) was obtained from fraction F30 of CF-090766 using a ReproSil 100 C18,  column and a gradient method as follows: 0–18 min, 40 to 47% CH3CN (solvent C) (isocratic); 23–28 min 100% C (linear gradient). Detection wavelengths were set at 316 and 346 nm and the retention time was 19.23 min.Compound 3 (0.9 mg) was obtained from fraction F26 of CF-090766 using a Xbridge™ C18  column and a gradient method varying as follows: 0–2 min, 20% C (isocratic); 32–35 min 60% C (linear gradient); 40–50 min, 100% B (linear gradient). Flow rate was set at 3.6 ml/min, detection wavelength was set at 254 nm and the retention time of the fraction containing compound 3 was 18 min. Then compound 3 was further purified by using the same column and the following gradient method: 0–5 min, 20% B (isocratic), 10–30 min, 30% (linear gradient), 35–40 min, 40% B (linear gradient), 50–55 min, 100% B (linear gradient). Detection wavelength was set at 210 nm and the retention time of the pure compound 3 was 23 min.Compound 4 (2.7 mg) was obtained from fraction F19 of CF-090361 using a Xbridge™ C18  column and a gradient method as follows: 0–5 min, 5% C (isocratic); 20–25 min 100% C (linear gradient). Flow rate was set at 14 ml/min, detection wavelength was set at 254 nm and the retention time was 15.85 min.Compound 5 (0.2 mg) was obtained from fraction F21 of CF-090361 using a Xbridge™ C18  column and a gradient method varying as follows: 0–2 min, 2% C (isocratic); 40–45 min 100% C (linear gradient). Flow rate was set at 3.6 ml/min, detection wavelength was set at 210 nm and the retention time of the fraction containing compound 5 was 21.5 min. Then compound 5 was further purified by using the same column and the following gradient method: 0–5 min, 50% B (isocratic), 10–20 min, 70% (linear gradient), 30–35 min, 100% B (linear gradient). Detection wavelength was set at 210 nm and the retention time of the pure compound 5 was 15 min.Compound 1): brownish solid; UV (MeOH) λmax 210 nm and 350 nm; 1H NMR and 13C NMR data see Table m/z: [M + H] + calcd for C12H11O4 219.0657; found 219.0651.1-furan-2-yl)ethan-1-one (2): yellow powder; UV (MeOH) λmax 316 nm and 346 nm; 1H NMR and 13C NMR data see Table m/z: [M + H] + calcd for C12H13O3 205.0865; found 205.0869.-1-hexa-2,4-dien-1-one (3): brown solid; UV (MeOH) λmax 316 nm and 346 nm; 1H NMR and 13C NMR data see Table m/z: [M + H] + calcd for C11H17O3 197.1177; found 197.1191.4-hydroxy-3-methyl-6-pentyl-2H-pyran-2-one (4): white powder; 1H NMR and 13C NMR data were consistent with those previously reported29; HRMS (ESI-Q-TOF) m/z: [M + H] + calcd for C12H17O5 241.1076; found 241.1068.Graphostrin B (5): colorless crystals; 1H NMR and 13C NMR data were consistent with those previously reported28; HRMS (ESI-Q-TOF) m/z: [M + H]+ calcd for C18H16N3O3 322.1191; found 322.1183.Brevianamide M , was employed41. Kojic acid (5 to 30 μM) and potassium buffer were used as a positive and negative controls, respectively. All experiments were performed in triplicate.The effects of the isolated compounds 12. Analysis were performed in triplicates and the initial crude extracts were tested at 0.01xWBE dilution. Cytotoxic evaluation. The cytotoxic activity of compounds 1–5 was measured through the MTT -2,5-diphenyl-2H-tetrazolium bromide) assay against HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, following an already described process44. Doxorubicin (8 point dose–response curve with 1/3 dilutions starting at 50 μM) and DMSO (0.5%) were used as a positive and negative controls, respectively. All experiments were performed in triplicate.During the screening process, the anti-tyrosinase effect of the extracts was also evaluated on mouse skin melanoma cells (B16-F10) as previously described1 on the viability of the human foreskin fibroblasts (BJ), frequently used for testing of skin active natural compounds, obtained from the American Tissue Culture Collection (ATTC), was examined, using the MTT method, at concentrations 0.5 μΜ, 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ and 5 μΜ, using an already described process45.Moreover, the effect of the active compound Supplementary Information 1.


In this work, a novel nitrogen-doped porous carbon material was fabricated by carbonizing the precursor of melamine-resorcinol-formaldehyde resin/graphene oxide (MR/GO) composites with KOH as the activation agent. Detailed characterization results revealed that the fabricated MR(0.25)/GO-500 porous carbon (0.25 represented the amount of GO added in wt.% and 500 denoted activation temperature in °C) had well-defined pore size distribution, high specific surface area (1264 m2·g−1) and high nitrogen content (6.92 wt.%), which was mainly composed of the pyridinic-N and pyrrolic-N species. Batch adsorption experiments demonstrated that the fabricated MR(0.25)/GO-500 porous carbon delivered excellent CO2 adsorption ability of 5.21 mmol·g−1 at 298.15 K and 500 kPa, and such porous carbon also exhibited fast adsorption kinetics, high selectivity of CO2/N2 and good recyclability. With the inherent microstructure features of high surface area and abundant N adsorption sites species, the MR/GO-derived porous carbon materials offer a potentially promising adsorbent for practical CO2 capture.CO Recently, the United Nations Environment Program’s  annual Emissions Gap Report 2020 reported that global greenhouse gas (GHS) emissions continued to increase and reached a record high of 52.4 ± 5.2 Gt CO2e without land-use change (LUC) emissions and 59.1 ± 5.9 Gt CO2e with LUC in 2019. Fossil CO2 emissions dominate total GHS emissions with 65% and reached a record 38.0 ± 1.9 GtCO2 on the basis of preliminary data [2 emission [Carbon dioxide CO emission2 adsorbents [2 capture because of their low cost, abundant source, excellent CO2 adsorption performance and physicochemical stability [2. The introduction of nitrogen atoms into the framework of PCM will further enhance CO2 capture and selectivity by the interaction between basic nitrogen sites and acidic CO2 molecules [2 molecules, the polar CO2 molecules were preferred to be adsorbed by electronegative N species, thus enhancing the selectivity for CO2 capture and separation of CO2 from N2/CO2 gas mixture. Therefore, different methods have been developed to prepare the nitrogen-doped porous carbon material (NPCM). Over the past decades, different porous solid materials such as carbon materials, zeolite, metal-organic frameworks, porous polymers and their composite materials have been investigated as COsorbents ,7,8,9. Ptability ,11,12. Iolecules ,14,15. M2 capture performance. Both inorganic such as ammonium [2 adsorption. Generally, the micropores enhance the interaction between CO2 molecules and adsorbent, whilst the mesopores can improve the utilization efficiency of the nitrogenous adsorption sites and surface area and accelerate the diffusion of CO2 in the pore channels. Therefore, it is of great importance to optimize pyrolysis and activation temperature for balancing the surface area and nitrogen content.Up to now, two commonly used ways have been reported to synthesize the NPCM, one is direct pyrolysis and activation of nitrogenous organic precursors, the other is to introduce nitrogen element by using nitrogenous activating agents during the activation at elevated temperatures ,17,18,19ammonium  and sodiammonium  and orgaammonium , dopaminammonium , pyrroleammonium , melaminammonium , ethylenammonium , benzeneammonium , dicyandammonium , hexametammonium , benzimiammonium , carbazoammonium , cyanuriammonium , polyacrammonium , polyimiammonium , polyindammonium  and chitammonium  have beeammonium . Tseng eammonium . In our ammonium . However2CO3 and ZnCl2 and its adding ratio to carbon precursor also have significant effect on the pore structure, surface area and nitrogen content of the NPCM, and then affect the CO2 capture performance [2/g with nitrogen content of 0.75 wt.%, the NPCM prepared at 700 °C with ZnCl2 had the highest nitrogen content of 7.27 wt.% and highest CO2/N2 adsorption selectivity of 42.6, whilst the NPCM prepared at 700 °C with KOH had the nitrogen content of the highest CO2 uptake of 5.45 mmol·g−1 [2 as both activation agent and nitridation reagent, Wang et al. found that the nitrogen content for the NPCM increased from 1.56 to 5.94 wt.% with the increase in the NaNH2/PR ratio from 1 to 4 and the NPCM with the nitrogen content of 4.25 wt.% has the highest surface area of 1432 m2/g and CO2 uptake of 4.64 mmol·g−1 at 25 °C at 1 atm [The selection of activating agents such as KOH, Naformance ,40. For mmol·g−1 . By usinat 1 atm .2 capture [2 capture capacity of 1.77 mmol·g−1 and very high CO2/N2 adsorption selectivity of 100 at 25 °C at 1 atm [2 adsorption capacity of 3.64 mmol·g−1 at 25 °C and 100 kPa using the activated dopamine-melamine copolymer [2 adsorption capacity of 4.95 mmol·g−1 at 25 °C and 100 kPa [2 adsorption behavior on melamine-based NPCM at low temperature (0–25 °C) or low pressure (0–100 kPa). However, few studies have been documented to study the CO2 adsorption on melamine-based NPCM at a relatively high temperature and high pressure, which is closer to the industrial conditions applied in CO2 capture.Melamine, as a nitrogen-rich organic molecule and low-cost raw material in the chemical industry, has been extensively employed in the preparation of NPCM for the CO capture . Lee et at 1 atm . Wu et aopolymer . Rehman  100 kPa . These s2 adsorption in a temperature range of 25–50 °C and pressure range of 0–500 kPa. The effects of pyrolysis temperature (400–700 °C) and the amount of GO added on the pore structure and surface features of NPCM were systematically investigated. The CO2 adsorption behaviors including adsorption isotherms, thermodynamics, kinetics and cyclic adsorption property were also investigated. The unique pore structure and doped nitrogen species for MR/GO-based NPCM significantly facilitated CO2 adsorption and selectivity, exhibited highly potential values in CO2 capture.Herein, the aim of the study is to prepare a novel NPCM derived from the composite material composed of melamine-resorcinol-formaldehyde resin and graphene oxide(MR/GO) for CO® Co. . Formaldehyde (37% aqueous solution), ethanol (AR), and potassium hydroxide  were obtained from Chengdu Kelong Reagent Co. . Deionized water (18 MΩ·cm) was produced by an ultrapure water system from Ultrapure Technol. Co. .Melamine (AR), resorcinol (AR) and graphene oxide  were purchased from AladdinThe synthesis process of GO/MR composite and derived NPCM is illustrated in −1 and a N2 flow rate of 60 mL·min−1. After pyrolysis treatment, the carbon materials were washed with ethanol and ultra-pure water alternately until the pH of filtrate reached around 7, then dried in vacuum under 80 °C. The finally obtained carbon materials with activation temperature of 500 °C and 0.250 wt.% GO added was named GO(0.25)/MR-500.To obtain GO/MR-derived NPCM with a high surface area and abundant pores, the activation was carried out under a nitrogen stream with KOH as activator. Briefly, 1 g of GO/MR was mixed with 2 g of KOH in ultra-pure water and then freeze-dried. Subsequently, the obtained solid was activated at 400–700 °C for 2 h with a ramp rate of 5 °C·min−1 on a Perkin-Elmer FTIR spectrometer. Raman spectra were collected using a DXR Raman Microscope  with excitation wavelength of 455 nm. The X-ray photoelectron spectroscopy  with a monochromatic Al Ka X-ray source  was used to analyze the surface structure of all samples. The specific surface area was calculated by using the Brunauer–Emmett–Teller (BET) method on a Micromeritics analyzer ASAP 2460.The morphology of all samples was analyzed by scanning electron microscope  and transmission electron microscopy , respectively. The crystalline structure of the samples was measured by using X-ray diffractometer  with Cu Ka radiation (k = 1.5418 Å). The Fourier transform infrared spectroscopy (FTIR) was collected in a range of 400–4000 cm2 adsorption isotherms of all samples were measured at 25–50 °C and 0–500 kPa using a self-made static adsorption apparatus, as shown in 2/N2 selectivity. For each measurement, the sample was degassed at 100 °C under a high vacuum for more than 12 h.The CO2 adsorption-desorption isotherms and pore size distributions of GO and GO(0.25)/MR-derived porous carbon materials at different activation temperatures. The isotherms of GO and GO(0.25)/MR-derived carbon materials display type IV isotherms according to IUPAC classification, which indicates the existence of micropores and mesopores. A slow climb for N2 adsorption at low relative pressure (P/P0 < 0.01) and a steep rise in N2 adsorption at high relative pressure (P/P0 > 0.4) are observed. In addition, there is a large hysteresis loop between 0.4 and 0.9 of the relative pressure P/P0, suggesting the abundant mesopores for GO. When the activation temperature is 500–700 °C, all the isotherms of carbon materials show mix of type I and type VI isotherms, which indicates that these carbon materials process abundant micropores and mesopores. As shown in 2·g−1, pore volume of 2.20 cm3·g−1 and mean pore size of 20.8 nm. After in situ polymerization of melamine-resorcinol resin (MR) on GO, the BET surface area, pore volume and mean pore size of GO(0.25)/MR decreased to 6.7 m2·g−1, 0.003 cm3·g−1 and 8.5 nm, respectively. The results indicated the pore filling and surface coverage of MR on the GO. With the activation temperature at 400 °C and 500 °C, the BET surface area increased to 32 m2·g−1 for GO(0.25)/MR-400 and the highest BET surface area of 1264 m2·g−1 was obtained for GO(0.25)/MR-500, respectively. As shown in 2·g−1, respectively. Meanwhile, the number of micropores also decreases. Furthermore, the effect of adding amount for GO on the pore structure was also studied and all the samples also display type IV isotherms /MR and GO(0.25)/MR-500 was characterized by using SEM and TEM. As shown in −1 and 1618/1638 cm−1 can be assigned to the stretching vibration of –OH of GO and adsorbed H2O, respectively [−1 can be attributed to C=C stretching vibration, tertiary alcoholic C–OH bending and C–O stretching vibration, respectively [−1 appear on GO(0.25)/MR composite, which are associated to N–H in-plane deformation vibration, aromatic C–C stretching vibration, C–N stretching vibration and s-triazine rings in the carbon matrix [−1 (D band) and 1590 cm−1 (G band) for all samples. The D band and G band are related to the defects or disorder in carbon and the vibration of sp2 hybridized carbon atoms in graphitic layer. Generally, the ID/IG value increases with the increase in activation temperature, indicating that more defects were generated. Generally, the adsorption capacity is correlated with ID/IG ratio and increases with increasing ID/IG ratio. The thermal stability of GO(0.25)/MR-derived carbon materials was also studied by using TGA and shown in ectively ,46. The ectively ,48,49. Cn matrix ,51. Howe2 atmosphere at high temperature. 2 (a Lewis acid) capture [2 due to the stronger interactions that occur between pyrrolic-N and CO2 molecules [XPS was used to characterize the chemical information of GO(0.25)/MR composite and its derived carbon material. The wide XPS spectra  of all s capture . Moreoveolecules .2 adsorption isotherms of GO(0.25)/MR composite and its derived carbon materials with different activation temperatures. 2 at 298.15 K and 100/500 kPa. The adsorption capacity of all the samples increases with the increasing of pressure. Among them, GO(0.25)/MR-500 exhibits the best performance for CO2 adsorption with a capacity of more than 2.27/5.21 mmol·g−1 at 100/500 kPa. However, the GO(0.25)/MR without activation only has CO2 adsorption capacity of 0.40/0.76 mmol·g−1 at 100/500 kPa. With the increase in the activation temperature from 500 °C to 600 °C and 700 °C, the CO2 adsorption capacity of GO(0.25)/MR-600 and GO(0.25)/MR-700 decrease to 1.33/4.29 mmol·g−1 and 0.87/1.89 mmol·g−1 at 100/500 kPa, respectively. The MR-500 without GO and GO(0.375)/MR-500 has CO2 adsorption capacity of 0.19/0.49 mmol·g−1 and 0.74/2.81 mmol·g−1 at 100/500 kPa, respectively. This trend agrees well with the surface area and micropore volume of samples, which indicates that the CO2 adsorption capacity would increase with increasing surface area and micropore volume.2 adsorption mechanism of GO(0.25)/MR-500, the Langmuir, Freundlich, and Redlich–Peterson models were applied to simulate the CO2 adsorption at temperatures of 298.15–323.15 K /MR-500 at 323.15 K. This is also the main reason why the adsorption decreases with the increase in temperature. Hence, both the micropore volume and properties of the surface have strong effects on the CO2 adsorption capacity at low temperatures, while the role of the micropore structure gradually weakened and the properties of surface play dominant role influencing the CO2 adsorption capacity at high temperature. This is closely related to the chemical state and exposed content of N-doping sites at the surface.To understanding the CO323.15 K  and the 2 uptake for GO(0.25)/MR-500 is shown in −1 . The results are similar with adsorption of CO2 on other carbon adsorbents. Moreover, the value of Qst decreases from 29.88 to 24.43 kJ·mol−1 with the increase in CO2 uptake from 0.5 to 3.5 mmol·g−1, revealing the gradual decrease in the interaction between CO2 and GO(0.25)/MR-500 and the surface heterogeneity of GO(0.25)/MR-500.The isosteric heat of adsorption (Qst) was calculated based on the isotherms at 298.15–323.15 K by employing the Clausis–Claperyon equation, and the plots of ln(P) as a function of 1/T are shown in wn in −1 , indicat2 adsorption were observed on GO(0.25)/MR-500, which reached 90 within 2 min and 97% within 10 min, respectively. In addition, the Fickian diffusion model was found more accurate to describe the kinetics of CO2 adsorption on GO(0.25)/MR-500 than the LDF model in 2 is calculated to be 2.11 min−n and 4.87 mmol∙g−1, respectively. These fast kinetics revealed that GO(0.25)/MR-500 is beneficial in shortening the adsorption cycle time for potential practical applications.As shown in 2/N2 selectivity is also a very important property of adsorbent for CO2 capture. 2 adsorption isotherm and CO2/N2 selectivity of GO(0.25)/MR-500. The adsorption capacity of N2 is very low, only 0.492 mmol·g−1 at 298.15 K and 500 kPa. Highest CO2/N2 selectivity of 58 was reached at 20 kPa, while the CO2/N2 selectivity gradually decrease to 39 and 19 at 100 kPa and 500 kPa, respectively, which is still at a high level for high-pressure CO2 adsorption compared with the porous carbons reported previously. Surface-doped N, especially for pyrrolic-N, offer abundant sites for high selective adsorption for CO2 at low pressure.Apart from fast adsorption kinetics, the CO2 adsorption in practical industrial application. To study the stability and recyclability of GO(0.25)/MR-500 in CO2 adsorption, the adsorption was operated at 298.15 K and high pressure (500 kPa), while the in situ regeneration was carried out at 373 K and in a vacuum. It should be note that the adsorbent in our study need to undergo a wide pressure change, which is more likely to destroy the structure of adsorbent compared with the test method of stability reported in literatures. This procedure is also much more similar with the vacuum pressure swing adsorption (VPSA) applied in industry. As shown in 2 could be maintained at 4.66 mmol·g−1 with a recovery rate of 98.5% even after five adsorption-desorption cycles.The cyclic performance of adsorbent are very important features for CO2·g−1. Appropriate addition of GO is beneficial to the formation of pores and increases the BET surface area. In addition, high contents of pyridinic-N and pyrrolic-N were formed on the surface of samples after activation, providing abundant adsorption sites for CO2. Therefore, GO(0.25)/MR-500 exhibited the highest CO2 capture ability of 5.21 mmol·g−1 at 298 K and 500 kPa. Detailed studies also revealed the moderate heat of adsorption, fast adsorption kinetics, high selectivity of CO2/N2 and good recyclability. These results demonstrate the strong potential of MR/GO-derived carbon in this practical application.Herein, we report the synthesis of high surface area and high N content porous carbons from MR/GO composite with KOH chemical activation. The optimum activation temperature is found to be 500 °C to obtain a porous carbon with a suitable pore structure and the highest BET surface area of 1264 m