Three new alkaloids were isolated from the bark extract of the Malayan Kopsia arborea, viz., arbophyllidine (1), an unusual pentacyclic, monoterpenoid indole characterized by an absence of oxygen atoms and incorporating a new carbon-nitrogen skeleton, and arbophyllinines A (2) and B (3), two pentacyclic corynanthean alkaloids incorporating a hydroxyethyl-substituted tetrahydrofuranone ring. The structures of the alkaloids were deduced based on analysis of the MS and NMR data and confirmed by X-ray diffraction analyses. The absolute configuration of arbophyllidine (1) was established based on experimental and calculated ECD data, while that of arbophyllinine A was based on X-ray diffraction analysis (Cu Kα). A reasonable biosynthetic route to arbophyllidine (1) from a pericine precursor is presented. Arbophyllidine (1) showed pronounced in vitro growth inhibitory activity against the HT-29 human cancer cell line with IC50 6.2 μM.
A new series of 3-O-substituted xanthone derivatives were synthesised and evaluated for their anti-cholinergic activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The results indicated that the xanthone derivatives possessed good AChE inhibitory activity with eleven of them (5, 8, 11, 17, 19, 21-23, 26-28) exhibited significant effects with the IC50 values ranged 0.88 to 1.28 µM. The AChE enzyme kinetic study of 3-(4-phenylbutoxy)-9H-xanthen-9-one (23) and ethyl 2-((9-oxo-9H-xanthen-3-yl)oxy)acetate (28) showed a mixed inhibition mechanism. Molecular docking study showed that 23 binds to the active site of AChE and interacts via extensive π-π stacking with the indole and phenol side chains of Trp86 and Tyr337, besides the hydrogen bonding with the hydration site and π-π interaction with the phenol side chain of Y72. This study revealed that 3-O-alkoxyl substituted xanthone derivatives are potential lead structures, especially 23 and 28 which can be further developed into potent AChE inhibitors.
3,4-Dimethoxybenzohydrazide derivatives (1-25) have been synthesized and evaluated for their urease inhibitory potential. Among the series, compounds 2, 3, 4 and 5 with IC50 values 12.61 ± 0.07, 18.24 ± 0.14, 19.22 ± 0.21, and 8.40 ± 0.05 µM, respectively, showed excellent urease inhibitory potentials when compared with standard thiourea (IC50 value 21.40 ± 0.21 µM). Compounds 1, 6, 8, 18, 19 and 20 also showed good to moderate inhibition, while the remaining compounds were found to be completely inactive. The structures of compounds 6 and 25 were confirmed through X-ray crystallography while the structures of remaining compounds were confirmed through ESI-MS and 1H NMR. Molecular docking studies were performed understand the binding interactions with enzyme active site. The synthesized compounds were evaluated for cytotoxicity and found to be nontoxic.
Reexamination of the absolute configuration of recently isolated eburnane alkaloids from Malaysian Kopsia and Leuconotis species by X-ray diffraction analysis and ECD/TDDFT has revealed the existence of biosynthetic enantiodivergence. Three different scenarios are discerned with respect to the composition of the enantiomeric eburnane alkaloids in these plants: first, where the new eburnane congeners possess the same C-20, C-21 absolute configurations as the common eburnane alkaloids (eburnamonine, eburnamine, isoeburnamine, eburnamenine) occurring in the same plant; second, where the new eburnane congeners possess opposite or enantiomeric C-20, C-21 absolute configurations compared to the common eburnane alkaloids found in the same plant; and, third, where the four common eburnane alkaloids were isolated as racemic or scalemic mixtures, while the new eburnane congeners were isolated as pure enantiomers with a common C-20, C-21 configuration (20α, 21α). Additionally, the same Kopsia species (K. pauciflora) found in two different geographical locations (Peninsular Malaysia and Malaysian Borneo) showed different patterns in the composition of the enantiomeric eburnane alkaloids. Revision of the absolute configurations of a number of new eburnane congeners (previously assigned based on the assumption of a common biogenetic origin to that of the known eburnane alkaloids co-occurring in the same plant) is required based on the present results.
Four new compounds, (+)- and (-)-ecarlottone (1), (±)-fislatifolione (5), (±)-isofislatifolione (6), and (±)-fislatifolic acid (7), and the known desmethoxyyangonin (2), didymocarpin-A (3), and dehydrodidymocarpin-A (4) were isolated from the stem bark of Fissistigma latifolium, by means of bioassay-guided purification using an in vitro affinity displacement assay based on the modulation of Bcl-xL/Bak and Mcl-1/Bid interactions. The structures of the new compounds were elucidated by NMR spectroscopic data analysis, and the absolute configurations of compounds (+)-1 and (-)-1 were assigned by comparison of experimental and computed ECD spectra. (-)-Ecarlottone 1 exhibited a potent antagonistic activity on both protein-protein associations with Ki values of 4.8 μM for Bcl-xL/Bak and 2.4 μM for Mcl-1/Bid.
β-Glucosidases and β-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 β-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the -1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant β-glycosidases.
HU proteins belong to the nucleoid-associated proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcription etc. No data are available on the structures of HU proteins from mycoplasmas. To this end, the HU protein from the parasitic mycoplasma Spiroplasma melliferum KC3 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Prismatic crystals of the protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 Å resolution (the best resolution ever obtained for a HU protein). The diffraction data were indexed in space group C2 and the structure of the protein was solved by the molecular-replacement method with one monomer per asymmetric unit.
Organotin complexes are recognized as the biologically active compounds in inducing cancerous cells death at very low doses. To date, organotin compounds currently appear among the most potent candidates in research related to the new anticancer drugs. In this study, new organotin(IV) N-butyl-N-phenyldithiocarbamate compounds have been successfully synthesized between the reaction of N-butylaniline amine with organotin(IV) chloride in 1:2/1:1 molar ratio. All compounds were characterized using the elemental analysis, FT-IR and NMR spectroscopy. The single crystal structure was determined by X-ray single crystal analysis. The elemental analysis showed good agreement with the suggested formula (C4H9)2Sn[S2CN(C4H9)(C6H5)]2 (Compound 1 and 2), (C6H5)2Sn[S2CN(C4H9)(C6H5)]2 (Compound 3) and (C6H5)3Sn[S2CN(C4H9)(C6H5)] (Compound 4). The important infrared absorbance peaks, v (C = N) and v(C = S) were detected in range between 1457-1489 cm(-1) and 951-996 cm(-1), respectively. The chemical shift of carbon in NCS2 group obtained from 13C NMR was found in range 198.86-203.53 ppm. The crystal structure of compound 4 showed that the dithiocarbamate ligand coordinates in a monodentate fashion. It crystallized in monoclinic P2(1)/n space group with the crystal cell parameter: a = 10.0488(1) angstroms, b = 18.0008(2) angstroms, c = 15.2054(2) angstroms, beta = 102.442(1) degrees and R = 0.044. The cytotoxicity (IC50) of these compounds against Jurkat E6.1 and K-562 leukemia cells were in the range between 0.4-0.8 and 1.8-5.3 microM, respectively as assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazholium bromide (MTT) assay. In conclusion, our study demonstrate that all compounds showed potent cytotoxicity towards both cell lines tested with the triphenyltin(IV) compound displayed the greatest effect.
Syzygium campanulatum Korth is an equatorial, evergreen, aboriginal shrub of Malaysia. Conventionally it has been used as a stomachic. However, in the currently conducted study dimethyl cardamonin or 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) was isolated from S. campanulatum Korth, leaf extract. The structural characterization of DMC was carried out by making use of various techniques including UV, IR, NMR spectral followed by LC-MS, and X-ray crystallographic techniques. For determining the purity of compound, highly effective techniques including TLC, HPLC, and melting point were used. The cytotoxicity of DMC and three different extracts of S. campanulatum was evaluated against human colon cancer cell line (HT-29) by three different assays. DMC and ethanolic extract revealed potent and dose-dependent cytotoxic activity on the cancer cell line with IC50 12.6 and 90.1 µg/mL, respectively. Quite astonishingly to our knowledge, this is the very first report on S. campanulatum as being a rich source (3.5%) of DMC, X-ray crystallography, and anticancer activity on human colon cancer cells.
Schiff bases of 3,4-dimethoxybenzenamine 1-25 were synthesized and evaluated for their antioxidant activity. All the synthesized compounds were characterized by various spectroscopic techniques. In addition, the characterizations of compounds 13, 15 and 16 were supported by crystal X-ray determinations and their geometrical parameters were compared with theoretical DFT calculations at the B3LYP level of theory. Furthermore, the X-ray crystal data of two non-crystalline compounds 8 and 18 were theoretically calculated and compared with the practical values of compounds 13, 15, 16 and found a good agreement. The compounds showed good DPPH scavenging activity ranging from 10.12 to 84.34 μM where compounds 1-4 and 6 showed stronger activity than the standard n-propyl gallate. For the superoxide anion radical assay, compounds 1-3 showed better activity than the standard.
A new isomers of thiourea derivatives, namely N-(4-chlorobutanoyl)-N'-(2-methylphenyl)-thiourea (1a), N-(4-chlorobutanoyl)-N'-(3-methylphenyl)thiourea (1b) and N-(4-chlorobutanoyl)-N'-(4-methylphenyl)thiourea (1c) have been synthesized by refluxing mixture of equimolar amounts of 4-chlorobutanoylisothiocyanate with 2, 3 or 4-toluidine, respectively. The three isomers were characterized by spectroscopic (UV/vis, FT-IR and NMR) and X-ray crystallography techniques. To investigate the isomerization effect on spectroscopic data, DFT and TD-DFT calculations have been carried out using five hybrid functionals (B3LYP, B3P86, CAM-B3LYP, M06-2X and PBE0) to predict UV/vis absorption bands (n→π∗ and π→π∗), (1)H and (13)C NMR chemical shifts, FT-IR vibration modes and X-ray parameters (bonds, bond angles and torsion angles) for 1a, 1b and 1c isomers. The results showed that the isomerization effect is significant on λ(MAX) absorption bands, while for IR and NMR the effect is negligible. In accordance with previous studies, B3LYP, B3P86 and PBE0 gave the most reliable to predict the excitation energies of thiourea derivatives.
A truncated haemoglobin (tHb) has been identified in an acidophilic and thermophilic methanotroph Methylacidiphilium infernorum. Hell's Gate Globin IV (HGbIV) and its related tHbs differ from all other bacterial tHbs due to their distinctively large sequence and polar distal haem pocket residues. Here we report the crystal structure of HGbIV determined at 1.96 Å resolution. The HGbIV structure has the distinctive 2/2 α-helical structure with extensions at both termini. It has a large distal site cavity in the haem pocket surrounded by four polar residues: His70(B9), His71(B10), Ser97(E11) and Trp137(G8). This cavity can bind bulky ligands such as a phosphate ion. Conformational shifts of His71(B10), Leu90(E4) and Leu93(E7) can also provide more space to accommodate larger ligands than the phosphate ion. The entrance/exit of such bulky ligands might be facilitated by positional flexibility in the CD1 loop, E helix and haem-propionate A. Therefore, the large cavity in HGbIV with polar His70(B9) and His71(B10), in contrast to the distal sites of other bacterial tHbs surrounded by non-polar residues, suggests its distinct physiological functions.
C-5-bromo-2-hydroxyphenylcalix[4]-2-methylresorcinarene (I) was synthesized by cyclocondensation of 5-bromo-2-hydroxybenzaldehyde and 2-methylresorcinol in the presence of concentrated HCl. Compound I was characterized by infrared and nuclear magnetic resonance spectroscopic data. X-ray analysis showed that this compound crystallized in a triclinic system with space group of Pī, a = 15.9592(16)Å, b = 16.9417(17)Å, c = 17.0974(17)Å, α = 68.656(3)°, β = 85.689(3)°, γ = 81.631(3)°, Z = 2 and V = 4258.6(7)Å3. The molecule adopts a chair (C2h) conformation. The thermal properties and antioxidant activity were also investigated. It was strongly antiviral against HSV-1 and weakly antibacterial against Gram-positive bacteria. Cytotoxicity testing on Vero cells showed that it is non-toxic, with a CC50 of more than 0.4 mg/mL.
Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.
Three new limonoids, chisomicines A-C (1-3), have been isolated from the bark of Chisocheton ceramicus. Their structures were determined by 2D NMR, CD spectroscopic methods, and X-ray analysis. Chisomicine A (1) exhibited NO production inhibitory activity in J774.1 cells stimulated by LPS dose-dependently at high cell viability.
Leucofoline and leuconoline, representing the first members of the aspidospermatan-aspidospermatan and eburnane-sarpagine subclasses of the bisindole alkaloids, respectively, were isolated from the Malayan Leuconotis griffithii. The structures of these bisindole alkaloids were established using NMR and MS analysis, and in the case of leuconoline, confirmed by X-ray diffraction analysis. Both alkaloids showed weak cytotoxicity towards human KB cells.
A total of 25 alkaloids were isolated from the leaf and stem-bark extracts of Alstonia spatulata, of which five are new alkaloids of the strychnan type (alstolucines A-E, 1-5) and the other, a new alkaloid of the secoangustilobine A type (alstolobine A, 6). The structures of these alkaloids were established using NMR and MS analysis and, in the case of alstolucine B (2), also confirmed by X-ray diffraction analysis. A reinvestigation of the stereochemical assignment of scholaricine (13) by NMR and X-ray analyses indicated that the configuration at C-20 required revision. Alkaloids 1, 2, 6, 7, 9, 10, and 13 reversed multidrug resistance in vincristine-resistant KB cells.
A series of ternary metal(ii) complexes {M(phen)(edda); 1a (Cu), 1b (Co), 1c (Zn), 1d (Ni); H(2)edda = N,N(')-ethylenediaminediacetic acid} of N,N'-ethylene-bridged diglycine and 1,10-phenanthroline were synthesized and characterized by elemental analysis, FTIR, UV-visible spectroscopy and magnetic susceptibility measurement. The interaction of these complexes with DNA was investigated using CD and EPR spectroscopy. MTT assay results of 1a-1c , screened on MCF-7 cancer cell lines, show that synergy between the metal and ligands results in significant enhancement of their antiproliferative properties. Preliminary results from apoptosis and cell cycle analyses with flow cytometry are reported. seems to be able to induce cell cycle arrest at G(0)/G(1). The crystal structure of 1a is also included.
Multiple sequence alignment was performed against eight proteases from the Flaviviridae family using ClustalW to illustrate conserved domains. Two sets of prediction approaches were applied and the results compared. Firstly, secondary structure prediction was performed using available structure prediction servers. The second approach made use of the information on the secondary structures extracted from structure prediction servers, threading techniques and DSSP database of some of the templates used in the threading techniques. Consensus on the one-dimensional secondary structure of Den2 protease was obtained from each approach and evaluated against data from the recently crystallised Den2 NS2B/NS3 obtained from the Protein Data Bank (PDB). Results indicated the second approach to show higher accuracy compared to the use of prediction servers only. Thus, it is plausible that this approach is applicable to the initial stage of structural studies of proteins with low amino acid sequence homology against other available proteins in the PDB.
Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.