nitrophenols and 4-nitrophenyl-phosphonate

This paper describes the synthesis of giant cyclic molecules having diameters of 10-20 nm. The molecules are prepared through the reactions of a fusion protein building block with small molecule linkers that are terminated in irreversible inhibitors of enzyme domains present in the fusion. This building block has N-terminal cutinase and C-terminal SnapTag domains that react irreversibly with p-nitrophenyl phosphonate (pNPP) and benzylguanine (BG) groups, respectively. We use a bis-BG and a BG-pNPP linker to join these fusion proteins into linear structures that can then react with a bis-pNPP linker that joins the ends into a cyclic product. The last step can occur intramolecularly, to give the macrocycle, or intermolecularly with another equivalent of linker, to give a linear product. Because these are coupled first- and second-order processes, an analysis of product yields from reactions performed at a range of linker concentrations gives rate constants for cyclization. We determined these to be 9.7 × 10

Topics: Carboxylic Ester Hydrolases; Cross-Linking Reagents; Cyclization; Guanine; Macrocyclic Compounds; Models, Molecular; Nitrophenols; O(6)-Methylguanine-DNA Methyltransferase; Organophosphonates; Protein Domains; Protein Multimerization

This work illustrates a strategy for the design of molecularly defined immunotherapies, using a blend of supramolecular peptide self-assembly and active site-directed protein capture.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Antibodies; Antigens; Green Fluorescent Proteins; Mice; Mice, Inbred C57BL; Nanofibers; Nitrophenols; Organophosphonates; Peptides; Vaccines, Synthetic

Reactive phosphonates are important probes to target the active site of serine hydrolases, one of the largest and most diverse family of enzymes. Developing such inhibitory probes is of special importance in activity based protein profiling, a strategy that is increasingly used to gain information about a certain class of enzymes in complex proteosomes. Therefore, gaining detailed information about these inhibition events on the individual protein level is important since it affords information that can be used to fine-tune the probe for a specific task. Here, we report a novel and versatile synthesis protocol to access a variety of functionalised p-nitrophenyl phosphonate (PNPP) inhibitors from a common azide functionalised precursor using click chemistry. The obtained PNPPs were successfully used to covalently label serine hydrolases in their active sites with molecular tags. Furthermore, a model study is described in which we developed straightforward protocols that can be used to study protein inhibition events. Kinetic studies using UV-Vis and fluorescence spectroscopy techniques revealed that these PNPPs possess different inhibition rates for various proteins and were shown to be suitable probes to discriminate between various lipases. Additionally, we demonstrate that PNPPs are highly selective for serine hydrolases, making these probes very interesting as diagnostic or affinity probes for studying proteins in complex proteosomes.

Topics: Affinity Labels; Binding Sites; Dansyl Compounds; Hydrolases; Kinetics; Nitrophenols; Organophosphonates; Proteome; Serine; Spectrometry, Fluorescence; Substrate Specificity

The real-time observation of protein dynamics in living cells and organisms is of fundamental importance for understanding biological processes. Most approaches to labeling proteins exploit noncovalent interactions, unsuitable to long-term studies, or genetic fusion to naturally occurring fluorescent proteins that often have unsatisfactory optical properties. Here we used the fungal enzyme cutinase and its suicide substrate p-nitrophenyl phosphonate to covalently attach a variety of labels to the integrin lymphocyte function-associated antigen-1 (LFA-1) on the surface of living cells. Cutinase was embedded in the extracellular domain of LFA-1 with no appreciable influence on integrin function and conformational regulation. p-nitrophenyl phosphonate-conjugated fluorochromes, including the very bright and stable quantum dots, bound efficiently and specifically to LFA-1/cutinase. The availability of a genetically encoded tag that binds covalently to quantum dots could foster the development of new experimental strategies for the study of protein dynamics in vivo.

Topics: Allosteric Regulation; Animals; Binding Sites; Carboxylic Ester Hydrolases; Cell Line; Fluorescent Dyes; Fungal Proteins; Humans; Integrins; Intercellular Adhesion Molecule-1; Kidney; Lymphocyte Function-Associated Antigen-1; Membrane Proteins; Mice; Models, Molecular; Nitrophenols; Organophosphonates; Protein Binding; Protein Conformation; Protein Engineering; Protein Structure, Tertiary; Protein Subunits; Quantum Dots; Recombinant Fusion Proteins; Staining and Labeling; Substrate Specificity

The activity of human placental alkaline phosphatase (PLAP) is downregulated by a number of effectors such as l-phenylalanine, an uncompetitive inhibitor, 5'-AMP, an antagonist of the effects of PLAP on fibroblast proliferation and by p-nitrophenyl-phosphonate (PNPPate), a non-hydrolysable substrate analogue. For the first two, such regulation may be linked to its biological function that requires a reduced and better-regulated hydrolytic rate. To understand how such disparate ligands are able to inhibit the enzyme, we solved the structure of the complexes at 1.6A, 1.9A and 1.9A resolution, respectively. These crystal structures are the first of an alkaline phosphatase in complex with organic inhibitors. Of the three inhibitors, only l-Phe and PNPPate bind at the active site hydrophobic pocket, providing structural data on the uncompetitive inhibition process. In contrast, all three ligands interact at a remote peripheral site located 28A from the active site. In order to extend these observations to the other members of the human alkaline phosphatase family, we have modelled the structures of the other human isozymes and compared them to PLAP. This comparison highlights the crucial role played by position 429 at the active site in the modulation of the catalytic process, and suggests that the peripheral binding site may be involved in the functional specialization of the PLAP isozyme.

Topics: Adenosine Monophosphate; Alkaline Phosphatase; Binding Sites; Catalytic Domain; Cell Proliferation; Crystallography, X-Ray; Down-Regulation; Fibroblasts; Humans; Hydrolysis; Ligands; Models, Molecular; Molecular Conformation; Nitrophenols; Organophosphonates; Phenylalanine; Placenta; Protein Conformation; Protein Isoforms; Protein Structure, Tertiary

Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase-inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis. Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active site of the enzyme in different solvents. The respective compounds are hexyl and methyl dimethylamino-naphthalenecarbonylethylmercaptoethoxy-phosphonates. The hexyl phosphonate derivative was reacted with lipases from Rhizopus oryzae (ROL), Chromobacterium viscosum (CVL), and Pseudomonas cepacia (PCL). The resulting lipid-protein complexes were characterized in solution with respect to water penetration into the lipid binding site and the associated conformational changes of the proteins as a consequence of solvent polarity changes. We found that the accessibility of the lipid-binding site in all lipases studied was lowest in water. It was much higher when the protein was dissolved in aqueous ethanol. These biophysical effects may contribute to the previously observed dramatic changes of enzyme functions such as activity and stereoselectivity depending on the respective solvents.

Topics: 2-Naphthylamine; Binding Sites; Burkholderia cepacia; Enzyme Inhibitors; Fluorescent Dyes; Lipase; Lipids; Molecular Structure; Naphthalenes; Nitrophenols; Organophosphonates; Protein Conformation; Rhizopus; Serine; Solvents; Spectrometry, Fluorescence

A detailed characterization of p-nitrophenyl phosphate as energy-donor substrate for the sarcoplasmic reticulum Ca(2+)-ATPase was undertaken in this study. The fact that p-nitrophenyl phosphate can be hydrolyzed in the presence or absence of Ca(2+) by the purified enzyme is consistent with the observed phenomenon of intramolecular uncoupling. Under the most favorable conditions, which include neutral pH, intact microsomal vesicles, and low free Ca(2+) in the lumen, the Ca(2+)/P(i) coupling ratio was 0.6. A rise or decrease in pH, high free Ca(2+) in the lumenal space, or the addition of dimethyl sulfoxide increase the intramolecular uncoupling. Alkaline pH and/or high free Ca(2+) in the lumen potentiate the accumulation of enzyme conformations with high Ca(2+) affinity. Acidic pH and/or dimethyl sulfoxide favor the accumulation of enzyme conformations with low Ca(2+) affinity. Under standard assay conditions, two uncoupled routes, together with a coupled route, are operative during the hydrolysis of p-nitrophenyl phosphate in the presence of Ca(2+). The prevalence of any one of the uncoupled catalytic cycles is dependent on the working conditions. The proposed reaction scheme constitutes a general model for understanding the mechanism of intramolecular energy uncoupling.

Topics: Calcium; Calcium-Transporting ATPases; Dimethyl Sulfoxide; Hydrogen-Ion Concentration; Hydrolysis; Nitrophenols; Organophosphonates; Sarcoplasmic Reticulum; Thapsigargin; Vanadates

Polyclonal antibodies catalyzing the hydrolysis of carbonate ester were generated by immunizing a rabbit with hapten(4-nitrophenyl phosphate II) conjugated to keyhole limpet hemocyanin. The hydrolytic activity of IgG purified from antisera exhibited plateu one month later than the simple hapten-binding. The affinity of IgG with substrate increased even after the hapten-binding reached plateu. These suggest a strategy to generate good polyclonal catalytic antibodies and the day to fuse spleen cell with myeloma cell to get good monoclonal catalytic antibodies.

Topics: Adjuvants, Immunologic; Animals; Binding, Competitive; Carbonates; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Haptens; Hemocyanins; Hydrolysis; Immune Sera; Immunoglobulin Fab Fragments; Immunoglobulin G; Immunoglobulin M; Kinetics; Nitrophenols; Organophosphonates; Rabbits; Spectrophotometry, Ultraviolet; Spleen; Vaccination

A prerequisite to the design and engineering of catalytic antibodies is the knowledge of their structure and in particular which residues are involved in binding and catalysis. We compared the structure and catalytic properties of a series of six monoclonal antibodies which were all raised against a p-nitrophenyl (PNP) phosphonate and which catalyze the hydrolysis of p-nitrophenyl esters. Three of the antibodies (Group I) have similar light and heavy chain variable regions. The other three antibodies have similar VL regions of which two (Group II) have VH regions from the MOPC21 gene family and the remaining one (Group III) a VH from the MC101 gene family making a total of three different groups based on their V region sequences. The structural division into groups is paralleled by the differences in binding constants to hapten analogs, substrate specificity and the susceptibility of the catalytic activity of the antibodies to chemical modification of tryptophan and arginine residues. The relative binding of a transition state analog to the binding of substrate is much higher for the Group I antibodies than for the other groups. Only the Group I antibodies can catalyze the hydrolysis of a carbonate substrate. However all of the antibodies lose catalytic activity upon specific tyrosine modification which highlights the importance of tyrosine in the active site of the antibodies. Thus, antibodies raised against a single hapten can give antibodies with different structures, and correspondingly different specificities and catalytic properties.

Topics: Amino Acid Sequence; Animals; Antibodies, Catalytic; Antibodies, Monoclonal; Base Sequence; Cloning, Molecular; DNA; Esters; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Kinetics; Mice; Molecular Sequence Data; Nitrophenols; Organophosphonates; Sequence Homology, Amino Acid