The naturally derived flavonoids are well known to have anticarcinogenic effects. Flavonoids could be an alternative strategy for ovarian cancer treatment, due to existing platinum-based drugs are reported to develop resistance with low survival rates. Inhibition of antiapoptotic proteins, namely B-cell lymphoma (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl), is the key target to stimulate apoptosis process in cancer cells. This study aimed to determine the binding interaction of five naturally derived flavonoids (biochanin A, myricetin, apigenin, galangin, and fisetin) with potential antiapoptotic target proteins (Bcl-2 and Bcl-xl). The molecular docking study was conducted using AutoDock Vina program. The binding affinity and the presence of hydrogen bonds between the flavonoids and target proteins were predicted. Our findings showed that all the flavonoids showed better binding affinity with Bcl-xl than that of Bcl-2 proteins. The highest binding affinity was recorded in fisetin-Bcl-xl protein complex (-8.8 kcal/mol). Meanwhile, the other flavonoids docked with Bcl-xl protein showed binding affinities, ranging from -8.0 to -8.6 kcal/mol. A total of four hydrogen bonds, four hydrophobic contacts, and one electrostatic interaction were detected in the docked fisetin-Bcl-xl complex, explaining its high binding affinity with Bcl-xl. The present results indicate that all flavonoids could potentially serve as Bcl-xl protein inhibitors, which would consequently lead to apoptotic process in ovarian cancers.
Binding of a potent anticancer agent, ponatinib (PTB) to human serum albumin (HSA), main ligand transporter in blood plasma was analyzed with several spectral techniques such as fluorescence, absorption and circular dichroism along with molecular docking studies. Decrease in the KSV value with increasing temperature pointed towards PTB-induced quenching as the static quenching, thus affirming complexation between PTB and HSA. An intermediate binding affinity was found to stabilize the PTB-HSA complex, as suggested by the Ka value. Thermodynamic analysis of the binding phenomenon revealed participation of hydrophobic and van der Waals interactions along with hydrogen bonds, which was also supported by molecular docking analysis. Changes in both secondary and tertiary structures as well as in the microenvironment around Trp and Tyr residues of HSA were anticipated upon PTB binding to the protein, as manifested from circular dichroism and three-dimensional fluorescence spectra, respectively. Binding of PTB to HSA led to protein's thermal stabilization. Competitive ligand displacement experiments using different site markers such as warfarin, indomethacin and ketoprofen disclosed the binding site of PTB as Sudlow's site I in HSA, which was further confirmed by molecular docking analysis.
An efficient microwave-assisted one-step synthetic route toward Mannich bases is developed from 4-hydroxyacetophenone and different secondary amines in quantitative yields, via a regioselective substitution reaction. The reaction takes a short time and is non-catalyzed and reproducible on a gram scale. The environmentally benign methodology provides a novel alternative, to the conventional methodologies, for the synthesis of mono- and disubstituted Mannich bases of 4-hydroxyacetophenone. All compounds were well-characterized by FT-IR, ¹H NMR, 13C NMR, and mass spectrometry. The structures of 1-{4-hydroxy-3-[(morpholin-4-yl)methyl]phenyl}ethan-1-one (2a) and 1-{4-hydroxy-3-[(pyrrolidin-1-yl)methyl]phenyl}ethan-1-one (3a) were determined by single crystal X-ray crystallography. Compound 2a and 3a crystallize in monoclinic, P2₁/n, and orthorhombic, Pbca, respectively. The most characteristic features of the molecular structure of 2a is that the morpholine fragment adopts a chair conformation with strong intramolecular hydrogen bonding. Compound 3a exhibits intermolecular hydrogen bonding, too. Furthermore, the computed Hirshfeld surface analysis confirms H-bonds and π⁻π stack interactions obtained by XRD packing analyses.
The ultimate goal in tissue engineering is to fabricate a scaffold which could mimic the native tissue structure. In this work, the physicochemical and biocompatibility properties of electrospun composites based on polyurethane (PU) with added pepper mint (PM) oil and copper sulphate (CuSO₄) were investigated. Field Emission Electron microscope (FESEM) study depicted the increase in mean fiber diameter for PU/PM and decrease in fiber diameter for PU/PM/CuSO₄ compared to the pristine PU. Fourier transform infrared spectroscopy (FTIR) analysis revealed the formation of a hydrogen bond for the fabricated composites as identified by an alteration in PU peak intensity. Contact angle analysis presented the hydrophobic nature of pristine PU and PU/PM while the PU/PM/CuSO₄ showed hydrophilic behavior. Atomic force microscopy (AFM) analysis revealed the increase in the surface roughness for the PU/PM while PU/PM/CuSO₄ showed a decrease in surface roughness compared to the pristine PU. Blood compatibility studies showed improved blood clotting time and less toxic behavior for the developed composites than the pristine PU. Finally, the cell viability of the fabricated composite was higher than the pristine PU as indicated in the MTS assay. Hence, the fabricated wound dressing composite based on PU with added PM and CuSO₄ rendered a better physicochemical and biocompatible nature, making it suitable for wound healing applications.
The use of antidiabetic agents which control glycemic levels in the blood and simultaneously inhibit oxidative stress is an important strategy in the prevention of Diabetes Mellitus and its complications. In our previous study, malabaricone C (3) and its dimer, giganteone A (5) exhibited significant DPPH free radical scavenging activities which were lower than the activity of the positive control, ascorbic acid. These compounds were evaluated for their α-glucosidase inhibitory activities at different concentrations (0.02-2.5 mM) in the present study. Compounds 3 (IC50 59.61 µM) and 5 (IC50 39.52 µM) were identified as active alpha-glucosidase inhibitors, each respectively being 24 and 37 folds more potent than the standard inhibitor, acarbose. Based on the molecular docking studies, compounds 3 and 5 docked into the active site of the α-glucosidase enzyme, forming mainly hydrogen bonds in the active site.
Activated carbons have been reported to be useful for adsorptive removal of the volatile anaesthetic sevoflurane from a vapour stream. The surface functionalities on activated carbons could be modified through aqueous oxidation using oxidising solutions to enhance the sevoflurane adsorption. In this study, an attempt to oxidise the surface of a commercial activated carbon to improve its adsorption capacity for sevoflurane was conducted using 6 mol/L nitric acid, 2 mol/L ammonium persulfate, and 30 wt per cent (wt%) of hydrogen peroxide (H2O2). The adsorption tests at fixed conditions (bed depth: 10 cm, inlet concentration: 528 mg/L, and flow rate: 3 L/min) revealed that H2O2 oxidation gave desirable sevoflurane adsorption (0.510 ± 0.005 mg/m2). A parametric study was conducted with H2O2 to investigate the effect of oxidation conditions to the changes in surface oxygen functionalities by varying the concentration, oxidation duration, and temperature, and the Conductor-like Screening Model for Real Solvents (COSMO-RS) was applied to predict the interactions between oxygen functionalities and sevoflurane. The H2O2 oxidation incorporated varying degrees of both surface oxygen functionalities with hydrogen bond (HB) acceptor and HB donor characters under the studied conditions. Oxidised samples with enriched oxygen functionalities with HB acceptor character and fewer HB donor character exhibited better adsorption capacity for sevoflurane. The presence of a high amount of oxygen functional groups with HB donor character adversely affected the sevoflurane adsorption despite the enrichment of oxygen functional groups with HB acceptor character that have a higher tendency to adsorb sevoflurane.
DNA polymerase β is a 39 kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31 kDa domain responsible for the polymerase activity and an 8 kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics simulations. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S55/44 and S55 phosphorylation were less dramatic and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is the major contributor to structural fluctuations that lead to loss of enzymatic activity.
Dehalogenase E (DehE) is a non-stereospecific enzyme produced by the soil bacterium, Rhizobium sp. RC1. Till now, the catalytic mechanism of DehE remains unclear although several literature concerning its structure and function are available. Since DehE is non-stereospecific, the enzyme was hypothesized to follow a 'direct attack mechanism' for the catalytic breakdown of a haloacid. For a molecular insight, the DehE modelled structure was docked in silico with the substrate 2-chloropropionic acid (2CP) in the active site. The ideal position of DehE residues that allowed a direct attack mechanism was then assessed via molecular dynamics (MD) simulation. It was revealed that the essential catalytic water was hydrogen bonded to the 'water-bearer', Asn114, at a relatively constant distance of ∼2.0 Å after 50 ns. The same water molecule was also closely sited to the catalytic Asp189 at an average distance of ∼2.0 Å, signifying the imperative role of the latter to initiate proton abstraction for water activation. This reaction was crucial to promote a direct attack on the α-carbon of 2CP to eject the halide ion. The water molecule was oriented favourably towards the α-carbon of 2CP at an angle of ∼75°, mirrored by the formation of stable enzyme-substrate orientations throughout the simulation. The data therefore substantiated that the degradation of a haloacid by DehE followed a 'direct attack mechanism'. Hence, this study offers valuable information into future advancements in the engineering of haloacid dehalogenases with improved activity and selectivity, as well as functionality in solvents other than water.
Effective novel peptide inhibitors which targeted the domain III of the dengue envelope (E) protein by blocking dengue virus (DENV) entry into target cells, were identified. The binding affinities of these peptides towards E-protein were evaluated by using a combination of docking and explicit solvent molecular dynamics (MD) simulation methods. The interactions of these complexes were further investigated by using the Molecular Mechanics-Poisson Boltzmann Surface Area (MMPBSA) and Molecular Mechanics Generalized Born Surface Area (MMGBSA) methods. Free energy calculations of the peptides interacting with the E-protein demonstrated that van der Waals (vdW) and electrostatic interactions were the main driving forces stabilizing the complexes. Interestingly, calculated binding free energies showed good agreement with the experimental dissociation constant (Kd) values. Our results also demonstrated that specific residues might play a crucial role in the effective binding interactions. Thus, this study has demonstrated that a combination of docking and molecular dynamics simulations can accelerate the identification process of peptides as potential inhibitors of dengue virus entry into host cells.
This work introduces a new additive named 4,4'-trimethylenedipiperidine for the practical and ecofriendly preparation of ethyl 5-amino-7-(4-phenyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate derivatives. This chemical is commercially available and easy to handle. It also possesses a low melting point and a broad liquid range temperature, high thermal stability, and good solubility in water. Based on green chemistry principles, the reaction was performed in a) a mixture of green solvents i.e. water and ethanol (1:1 v/v) at reflux temperature, and b) the additive was liquefied at 65 °C and the reaction was conducted in the liquid state of the additive. High yields of the desired triazolo-pyrimidines were obtained under both aforementioned conditions. Our results demonstrated that this additive, containing 2 Lewis base sites and able to act as an acceptor-donor hydrogen bonding group, is a novel and efficient alternative to piperidine, owing to its unique properties such as its reduced toxicity, nonflammable nature, nonvolatile state, broad liquid range temperature, high thermal stability, and ability to be safely handled. Furthermore, this additive could be completely recovered and exhibited high recyclability without any change in its chemical structure and no significant reduction in its activity. The current methodology has several advantages: (a) it avoids the use of hazardous materials, as well as toxic, volatile, and flammable solvents, (b) it does not entail tedious processes, harsh conditions, and the multistep preparation of catalysts, (c) it uses a metal-free and noncorrosive catalyst, and (d) reduces the generation of hazardous waste and simple work-up processes. The most important result of this study is that 4,4'-trimethylenedipiperidine can be a promising alternative for toxic, volatile, and flammable base reagents in organic synthesis owing to its unique properties.
The disease Tuberculosis (TB) is caused by a bacterium called Mycobacterium tuberculosis (Mtb). The bacterial cell-wall consists of peptidoglycan layer maintains the cellular integrity and cell viability. The main problem resides in the cell cycle of Mycobacterium tuberculosis in its quiescent form which is not targeted by any drugs hence there is an immediate need for new antibiotics to target the cell wall. The current study deals with the dTDP-4-dehydrorahmnose reductase (RmlD) which is the final enzyme in the series of cell-wall proteins of Mtb. The RmlD is a part of Carbohydrate biosynthesis has been considered as a good drug target for the novel class of antibiotics. Our study begins with the protein structure prediction, Homology studies were conducted using the Phyre2 web server. The structure is then refined and subjected to molecular dynamics simulations for 50 ns using GROMACS. The clustering analysis has been carried out and generated 41 clusters with 2 Å as the cut-off. Blind docking virtual screening was performed against RmlD protein using the Super Natural-II database with AutoDock4.0. its results helped to screen top ligands based on best binding energies. In both dockings, there are some common residues in which the ligands are interacting and forming the Hydrogen bonds such as Asp-105, Val-158, Thr-160, Gly-161, Arg-224, Arg-256. The ligand-567 giving the best results by being in the top-3 of all the clusters in both blind docking as well as the active-site docking. Hence ligand-567 can be a potential inhibitor of RmlD which can further inhibit the cell-wall synthesis of Mycobacterium tuberculosis.Communicated by Ramaswamy H. Sarma.
In this study, we explored the possibility of determining the synergistic interactions between nucleotide-binding domain (NBD) of Homo sapiens heat-shock 70 kDa protein (Hsp70) and E1A 32 kDa of adenovirus serotype 5 motif (PNLVP) in the efficiency of killing of tumor cells in cancer treatment. At present, the protein interaction between NBD and PNLVP motif is still unknown, but believed to enhance the rate of virus replication in tumor cells. Three mutant models (E229V, H225P and D230C) were built and simulated, and their interactions with PNLVP motif were studied. The PNLVP motif showed the binding energy and intermolecular energy values with the novel E229V mutant at -7.32 and -11.2 kcal/mol. The E229V mutant had the highest number of hydrogen bonds (7). Based on the root mean square deviation, root mean square fluctuation, hydrogen bonds, salt bridge, secondary structure, surface-accessible solvent area, potential energy and distance matrices analyses, it was proved that the E229V had the strongest and most stable interaction with the PNLVP motif among all the four protein-ligand complex structures. The knowledge of this protein-ligand complex model would help in designing Hsp70 structure-based drug for cancer therapy.
We developed a step-by-step experimental protocol using differential scanning calorimetry (DSC), dynamic vapour sorption (DVS), polarized light microscopy (PLM) and a small-scale dissolution apparatus (μDISS Profiler) to investigate the mechanism (solid-to-solid or solution-mediated) by which crystallization of amorphous drugs occurs upon dissolution. This protocol then guided how to stabilize the amorphous formulation. Indapamide, metolazone, glibenclamide and glipizide were selected as model drugs and HPMC (Pharmacoat 606) and PVP (K30) as stabilizing polymers. Spray-dried amorphous indapamide, metolazone and glibenclamide crystallized via solution-mediated nucleation while glipizide suffered from solid-to-solid crystallization. The addition of 0.001%-0.01% (w/v) HPMC into the dissolution medium successfully prevented the crystallization of supersaturated solutions of indapamide and metolazone whereas it only reduced the crystallization rate for glibenclamide. Amorphous solid dispersion (ASD) formulation of glipizide and PVP K30, at a ratio of 50:50% (w/w) reduced but did not completely eliminate the solid-to-solid crystallization of glipizide even though the overall dissolution rate was enhanced both in the absence and presence of HPMC. Raman spectroscopy indicated the formation of a glipizide polymorph in the dissolution medium with higher solubility than the stable polymorph. As a complementary technique, molecular dynamics (MD) simulations of indapamide and glibenclamide with HPMC was performed. It was revealed that hydrogen bonding patterns of the two drugs with HPMC differed significantly, suggesting that hydrogen bonding may play a role in the greater stabilizing effect on supersaturation of indapamide, compared to glibenclamide.
Isonicotinamide-4-methoxybenzoic acid co-crystal (1), C6H6N2O·C8H8O3, is formed through slow evaporation from methanol solution and it undergoes a first-order isosymmetry (monoclinic I2/a ↔ monoclinic I2/a) structural phase transition at Tc= 142.5 (5) K, which has been confirmed by an abrupt jump of crystallographic interaxial angle β from variable-temperature single-crystal XRD and small heat hysteresis (6.25 K) in differential scanning calorimetry measurement. The three-dimensional X-ray crystal structures of (1) at the low-temperature phase (LTP) (100, 140 and 142 K) and the high-temperature phase (HTP) (143, 150, 200, 250 and 300 K) were solved and refined as a simple non-disordered model with final R[F2> 2σ(F2)] ≃ 0.05. The asymmetric unit of (1) consists of crystallographically independent 4-methoxybenzoic acid (A) and isonicotinamide (B) molecules in both enantiotropic phases. Molecule A adopts a `near-hydroxyl' conformation in which the hydroxyl and methoxy groups are positioned on the same side. Both `near-hydroxyl' and `near-carbonyl' molecular conformations possess minimum conformational energies with an energy difference of hydrogen bonds which were preserved in both phases. However, these ABBA arrays are displaced from planarity upon LTP-to-HTP transition and the changes in inter-array interactions are observed in two-dimensional fingerprint plots of their Hirshfeld surfaces. The PIXEL energies of each molecular pair in both phases were calculated to investigate the difference in intermolecular interaction energies before and after the displacement of ABBA arrays from planarity, which directly leads to the single-crystal-to-single-crystal phase transition of (1).
Binding studies between a multi-targeted anticancer drug, sunitinib (SU) and human serum albumin (HSA) were made using fluorescence, UV-vis absorption, circular dichroism (CD) and molecular docking analysis. Both fluorescence quenching data and UV-vis absorption results suggested formation of SU-HSA complex. Moderate binding affinity between SU and HSA was evident from the value of the binding constant (3.04×104M-1), obtained at 298K. Involvement of hydrophobic interactions and hydrogen bonds as the leading intermolecular forces in the formation of SU-HSA complex was predicted from the thermodynamic data of the binding reaction. These results were in good agreement with the molecular docking analysis. Microenvironmental perturbations around Tyr and Trp residues as well as secondary and tertiary structural changes in HSA upon SU binding were evident from the three-dimensional fluorescence and circular dichroism results. SU binding to HSA also improved the thermal stability of the protein. Competitive displacement results and molecular docking analysis revealed the binding locus of SU to HSA in subdomain IIA (Sudlow's site I). The influence of a few common ions on the binding constant of SU-HSA complex was also noticed.
The crystallins are a family of monomeric proteins present in the mammalian lens and mutations in these proteins cause various forms of cataracts. The aim of our current study is to emphasize the structural characterization of aggregation propensity of mutation R58H on γD crystallin using molecular dynamics (MD) approach. MD result revealed that difference in the sequence level display a wide variation in the backbone atomic position, and thus exhibits rigid conformational dynamics. Changes in the flexibility of residues favoured to increase the number of intra-molecular hydrogen bonds in mutant R58H. Moreover, notable changes in the hydrogen bonding interaction resulted to cause the misfolding of mutant R58H by introducing α-helix. Principal component analysis (PCA) result suggested that mutant R58H showed unusual conformational dynamics along the two principal components when compared to the wild-type (WT)-γD crystallin. In a nutshell, the increased surface hydrophobicity could be the cause of self-aggregation of mutant R58H leading to aculeiform cataract.
Congenital myopathy is a broad category of muscular diseases with symptoms appearing at the time of birth. One type of congenital myopathy is Congenital Fiber Type Disproportion (CFTD), a severely debilitating disease. The G48D and G48C mutations in the D-loop and the actin-myosin interface are the two causes of CFTD. These mutations have been shown to significantly affect the structure and function of muscle fibers. To the author's knowledge, the effects of these mutations have not yet been studied. In this work, the power stroke structure of the head domain of myosin and the wild and mutated types of actin were modeled. Then, a MD simulation was run for the modeled structures to study the effects of these mutations on the structure, function, and molecular dynamics of actin. The wild and mutated actins docked with myosin showed differences in hydrogen bonding patterns, free binding energies, and hydrogen bond occupation frequencies. The G48D and G48C mutations significantly impacted the conformation of D-loops because of their larger size compared to Glycine and their ability to interfere with the polarity or hydrophobicity of this neutralized and hydrophobic loop. Therefore, the mutated loops were unable to fit properly into the hydrophobic groove of the adjacent G-actin. The abnormal structure of D-loops seems to result in the abnormal assembly of F-actins, giving rise to the symptoms of CFTD. It was also noted that G48C and G48D did not form hydrogen bonds with myosin in the residue 48 location. Nevertheless, in this case, muscles are unable to contract properly due to muscle atrophy.
Recent outbreaks of highly pathogenic influenza strains have highlighted the need to develop new anti-influenza drugs. Here, we report an in silico study of carvone derivatives to analyze their binding modes with neuraminidase (NA) active sites. Two proposed carvone analogues, CV(A) and CV(B), with 36 designed ligands were predicted to inhibit NA (PDB ID: 3TI6) using molecular docking. The design is based on structural resemblance with the commercial inhibitor, oseltamivir (OTV), ligand polarity, and amino acid residues in the NA active sites. Docking simulations revealed that ligand A18 has the lowest energy binding (∆Gbind) value of -8.30 kcal mol-1, comparable to OTV with ∆Gbind of -8.72 kcal mol-1. A18 formed seven hydrogen bonds (H-bonds) at residues Arg292, Arg371, Asp151, Trp178, Glu227, and Tyr406, while eight H-bonds were formed by OTV with amino acids Arg118, Arg292, Arg371, Glu119, Asp151, and Arg152. Molecular dynamics (MD) simulation was conducted to compare the stability between ligand A18 and OTV with NA. Our simulation study showed that the A18-NA complex is as stable as the OTV-NA complex during the MD simulation of 50 ns through the analysis of RMSD, RMSF, total energy, hydrogen bonding, and MM/PBSA free energy calculations.
Polyethylene glycol functionalized with oxygenated multi-walled carbon nanotubes (O-PEG-MWCNTs) as an efficient nanomaterial for the in vitro adsorption/release of curcumin (CUR) anticancer agent. The synthesized material was morphologically characterized using scanning electron microscopy, Fourier transform infrared spectroscopy and transmission electron microscopy. In addition, the CUR adsorption process was assessed with kinetic and isotherm models fitting well with pseudo-second order and Langmuir isotherms. The results showed that the proposed O-PEG-MWCNTs has a high adsorption capacity for CUR (2.0 × 103 mg/g) based on the Langmuir model. The in vitro release of CUR from O-PEG-MWCNTs was studied in simulating human body fluids with different pHs (ABS pH 5, intestinal fluid pH 6.6 and body fluid pH 7.4). Lastly, to confirm the success compliance of the O-PEG-MWCNT nanocomposite as a drug delivery system, the parameters affecting the CUR release such as temperature and PEG content were investigated. As a result, the proposed nanocomposite could be used as an efficient carrier for CUR delivery with an enhanced prolonged release property. Graphical Abstract ᅟ.
5M mutant lipase was derived through cumulative mutagenesis of amino acid residues (D43E/T118N/E226D/E250L/N304E) of T1 lipase from Geobacillus zalihae. A previous study revealed that cumulative mutations in 5M mutant lipase resulted in decreased thermostability compared to wild-type T1 lipase. Multiple amino acids substitution might cause structural destabilization due to negative cooperation. Hence, the three-dimensional structure of 5M mutant lipase was elucidated to determine the evolution in structural elements caused by amino acids substitution. A suitable crystal for X-ray diffraction was obtained from an optimized formulation containing 0.5 M sodium cacodylate trihydrate, 0.4 M sodium citrate tribasic pH 6.4 and 0.2 M sodium chloride with 2.5 mg/mL protein concentration. The three-dimensional structure of 5M mutant lipase was solved at 2.64 Å with two molecules per asymmetric unit. The detailed analysis of the structure revealed that there was a decrease in the number of molecular interactions, including hydrogen bonds and ion interactions, which are important in maintaining the stability of lipase. This study facilitates understanding of and highlights the importance of hydrogen bonds and ion interactions towards protein stability. Substrate specificity and docking analysis on the open structure of 5M mutant lipase revealed changes in substrate preference. The molecular dynamics simulation of 5M-substrates complexes validated the substrate preference of 5M lipase towards long-chain p-nitrophenyl-esters.