New photoreactive cleavable reagents with (trifluoromethyl)diazirine group

Photoreactive cross-linking reagents that simultaneously contain a trifluoromethyldiazirine and an o-nitrobenzyl groups were synthesized for the first time. Photochemical properties of the reagents were studied, and the possibility of separate activation of the diazirine group and o-nitrobenzyl linker was shown.     

A novel biotinylated heterobifunctional cross-linking reagent bearing an aromatic diazirine

The synthesis of a p-[(3-trifluoromethyl)diazirine-3-yl]benzoic acid derivative is described as a new carbene generating heterobifunctional cross-linking reagent. The cross-linker carries a biotin moiety in order to make use of avidin—biotin technology for specific manipulation of cross-linked components. To evaluate the ability of this reagent, the inter-subunit cross-linking of egg-white avidin tetramer was investigated. As a typical application of avidin—biotin technology for cross-linking experiments, a chemiluminescent detection method was examined to identify photobiotinylated components. A cross-linked dimeric product with an apparent molecular mass of 38 kDa was clearly visualized by the combined use of a horseradish peroxidase—streptavidin conjugate and a luminol-based chemiluminescent system.     

Chromogenic diazirine: A new spectrophotometric approach for photoaffinity labeling

[[2-Nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]]phenoxy]acetic acid and its derivatives have been synthesized as a special carbene precursor with a chromogenic group. Photolysis of the diazirine in methanol and cyclohexane gave intermolecular O---H and C---H insertion products, respectively. Spectroscopic properties of the diazirine derivatives and the photo-products revealed that irradiation and detection can be performed in a spectral region where the absorption due to most biological macromolecules is negligible. The application of this reagent will provide a useful approach for simple spectrophotometric detection of labeled products without recourse to conventional radioactive techniques in the photoaffinity labeling methodology.     

Efficient approach for profiling photoaffinity labeled peptides with a cleavable biotinyl photoprobe

Based on the application of our recent biotinyl photoprobe with a cleavable N-acylsulfonamide, an efficient process has been developed for profiling photoaffinity labeled peptides among a large excess of unlabeled concomitants. N-acylsulfonamide group was found to be stable under the usual S-pyridylethylation condition of cysteine residues whereas the group was easily cleaved by N-alkylation with iodoacetic acid in acidic condition. The selective nature between two common protein alkylation reactions was evaluated with l-glutamate dehydrogenase (GDH) using an acidic amino acid photoprobe with biotinylated acylsulfonamide function. The labeled GDH was successfully subjected to S-pyridylethylation keeping the biotin tag intact, and then was easily released from streptavidin matrix with high purity via iodoacetic acid-mediated alkylation under mild condition at pH 5.0.     

A novel biotinylated diazirinyl ceramide analogue for photoaffinity labeling

A novel photoreactive ceramide analogue, which contains (3-trifluoromethyl)phenyldiazirinyl lipid and biotinylated sphingosine, was synthesized. The probe was recognized as an antigen by anti-ceramide antibody and as a substrate for sphingolipid ceramide N-deacylase. These results indicate that the probe may be useful as a photoaffinity-biotinylating agent in sphingolipid studies.     

Synthesis of biotinylated photoaffinity probes based on arylsulfonamide gamma-secretase inhibitors

Synthesis and biological evaluation of an arylsulfonamide class of gamma-secretase inhibitors are described. Design, synthesis, and biological evaluation of multifunctional molecular probes harboring a benzophenone photophore as a cross-linking group and a biotin tag are also reported.     

Synthesis of biotinylated bis(D-glucose) derivatives for glucose transporter photoaffinity labelling

New diazirine based bis-glucose derivatives for tagging glucose transporters have been synthesised. These included two biotinylated compounds linked either by an aminocaproate or by a cleavable dithiol link. These compounds have been derivatised via a key skeleton compound that can be easily used for introduction of additional tags. Studies on the erythrocyte glucose transporter (GLUT1) and the insulin-stimulated adipose cell transporter (GLUT4) have revealed the biotinylated photoreactive bis-glucose compounds are effective labelling reagents.     

Development of a ligand blot assay using biotinylated live cells

Adhesive interactions between cells are critical to a variety of processes, including host-pathogen relationships. The authors have developed a new technique for the observation of binding interactions in which molecules obtained from excised tissues are resolved by gel electrophoresis and transferred to a membrane. Biotinylated live cells are then kept in contact with that membrane, and their interactions with proteins of interest are detected by peroxidase-labeled streptavidin, followed by a biotin-streptavidin detection system. The adhesion proteins can eventually be identified by cutting the relevant band(s) and performing mass spectrometry or other amino acid-sequencing methods. The technique described here allows for the identification of both known and novel adhesion molecules capable of binding to live cells, among a complex mixture and without previous isolation or purification. This is especially important for the analysis of host-parasite interactions and may be extended to other types of cell-cell interactions.     

Design and synthesis of cleavable biotinylated dideoxynucleotides for DNA sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Knowledge of the anti-drug antibody (ADA) status is necessary in early research studies. Because specific assay materials are sparse and time is pressing, a generic assay format with drug tolerance for detection of ADAs in serum samples from mice exposed to immunoglobulin G (IgG) or antigen-binding fragments (Fabs) is highly desirable. This article describes a generic immune complex assay in the sandwich enzyme-linked immunosorbent assay (ELISA) format based on (i) transformation of free ADAs to immune complexes by preincubation with excess drug, (ii) the use of a murine anti-human Fab constant domain Fab as capture reagent, (iii) detection of the immune complexes by a peroxidase-labeled rabbit anti-murine Fc antibody, and (iv) ADA-positive control conjugates consisting of human Fab and murine IgG. Results of the experiments suggest that the generic immune complex assay for mouse serum samples was at least equivalent to specific ADA immune assays and even superior regarding drug tolerance. The generic immune complex assay confers versatility as it detects ADAs in complex with full-length IgG as well as with Fabs independent of the target specificity in mouse serum samples. These features help to save the sparse amounts of specific antibodies available in early research and development and speed up drug candidate selection.     

A novel photoaffinity ligand for the dopamine transporter based on pyrovalerone

Non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored in contrast to tropane-based compounds such as cocaine. In order to fill this knowledge gap, a ligand was synthesized in which the aromatic ring of pyrovalerone was substituted with a photoreactive azido group. The analog 1-(4-azido-3-iodophenyl)-2-pyrrolidin-1-yl-pentan-1-one demonstrated appreciable binding affinity for the DAT (K_i = 78 ± 18 nM), suggesting the potential utility of a radioiodinated version in structure-function studies of this protein.     

Para-azidoclonidine: a novel photoaffinity ligand for the alpha 2-receptor

Reversible and irreversible interactions of the photoreactive clonidine analogue p-azidoclonidine (PAZC) with brain alpha 2-adrenergic receptors were examined. In the absence of light, PAZC displayed selective, high affinity, competitive interactions with sites labeled by the alpha 2-agonist 3H-p-aminoclonidine (3H-PAC). Reversible binding characteristics resembled those of other alpha 2-agonists. Preincubation of bovine frontal cortex membranes with 100 nM PAZC followed by ultraviolet irradiation and thorough washing decreased alpha 2-receptor density 42% relative to controls receiving irradiation alone. The loss of receptors could be prevented by inclusion of 500 nM phentolamine in the preincubation medium. Alpha 1- and beta-adrenergic receptors were relatively unaffected. PAZC is a potential photoaffinity ligand for the alpha 2-adrenergic receptor.     

Mitochondrial single nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using cleavable biotinylated dideoxynucleotides

Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC-SBE approach based on chemically cleavable biotinylated dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.     

Preparation and properties of an improved photoaffinity ligand for the N-formyl peptide receptor

A new superior photoaffinity ligand for the N-formyl peptide receptor was prepared by derivatization of N-formyl-Met-Leu-Phe-Lys with a commercially available heterobifunctional crosslinking agent. The product, N-formyl-Met-Leu-Phe-N epsilon-(2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionyl)-Lys was readily synthesized and radiolabelled, and had increased specificity and stability as compared to previously used photoaffinity ligands. The ligand rapidly associated with the receptor with high affinity (Kd = 0.28 nM). Once bound, it was virtually non-dissociable (in the absence of photolysis) in an experimental time-frame (t1/2 (off) = 154 min). The covalent incorporation of the photoaffinity ligand into the receptor upon irradiation was complete within 5 min, minimizing cell damage, and the efficiency of covalent incorporation was approx. 40%. The derivative had enhanced biological activity, having an ED50 for superoxide anion production of 0.23 nM, 27-fold lower than its parent peptide. This derivative of the N-formyl peptide was useful for up to 3 months after radiolabelling, showing a progressive decline in specific activity during storage in the dark at 4 degrees C. The enhanced stability, reproducibility and solubility of the photoaffinity ligand is expected to aid in the purification of the N-formyl peptide receptor and will prove a useful tool for analysing receptor-mediated processes.     

Identification of ligand binding site of phytosulfokine receptor by on-column photoaffinity labeling

Phytosulfokine (PSK), an endogenous 5-amino-acid-secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner with a nanomolar dissociation constant. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand binding pocket. Here, we have identified the PSK binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK analog [(125)I]-[N(epsilon)-(4-azidosalicyl)Lys(5)]PSK with DcPSKR1 using UV irradiation and mapped the cross-linked region using chemical and enzymatic fragmentation. We also established a novel "on-column photoaffinity labeling" methodology that allows repeated incorporation of the photoaffinity label to increase the efficiency of the photoaffinity cross-linking reactions. We purified a labeled DcPSKR1 tryptic fragment using anti-PSK antibodies and identified a peptide fragment that corresponds to the 15-amino-acid Glu(503)-Lys(517) region of DcPSKR1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Deletion of Glu(503)-Lys(517) completely abolishes the ligand binding activity of DcPSKR1. This region is in the island domain flanked by extracellular LRRs, indicating that this domain forms a ligand binding pocket that directly interacts with PSK.     

Enzyme occupancy measurement of intracellular protein tyrosine phosphatase 1B using photoaffinity probes

Protein tyrosine phosphatase 1B (PTP1B) is believed to be one of the enzymes involved in down-regulating the insulin receptor and is a drug target for the treatment of type II diabetes. To better understand the in vitro and in vivo behavior of PTP1B inhibitors, a cell-based assay to directly measure enzyme occupancy of PTP1B by inhibitors using photoaffinity labeling was developed. Two photoaffinity probes were synthesized containing the photolabile diazirine moiety. These photoprobes were specific for PTP1B and T-cell protein tyrosine phosphatase over CD45, with the most potent photoprobe having an IC(50) value of 0.2nM for PTP1B. Activation of the photoprobes with a 40-W UV lamp in the presence of purified AspTyrLysAspAspAspAspLys (Flag)-PTP1B formed a 1:1 irreversible adduct with the enzyme. The photolabeling was competed by known PTP1B inhibitors, vanadate, and the peptide inhibitor N-benzoyl-l-glutamyl-[4-phosphono(difluoromethyl)]-l-phenylalanyl-[4-phosphono(difluoromethyl)]l-phenylalanineamide (BzN-EJJ-amide). In HepG2 (human hepatoma cell line) cells, endogenous PTP1B was labeled by the UV-activated photoprobes in both lysed and intact cells. Enzyme occupancy measurements were conducted with a series of PTP1B inhibitors using the photoprobe affinity assay. Several compounds were shown to bind to endogenous PTP1B in the HepG2 intact cells.     

Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes

The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme.     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Atrial natriuretic factor receptor on cultured Leydig tumor cells: ligand binding and photoaffinity labeling

Atrial natriuretic factor (ANF) is a peptide hormone discovered recently from the heart atrium that possesses potent natriuretic and vasorelaxant activities. Recently we found that ANF markedly stimulates intracellular cGMP and almost completely inhibits cAMP accumulation in testicular interstitial tumor cells [Pandey, K. N., Kovacs, W. J., & Inagami, T. (1985) Biochem. Biophys. Res. Commun. 133, 800-806]. These actions of ANF suggest the presence of ANF receptors in testicular interstitial cells. In this study, cultured murine Leydig tumor cells have been shown to contain specific binding sites for ANF. Saturation binding studies indicated a single class of binding sites with a Kd of 5 X 10(-9) M at a density of 2 X 10(6) sites/cell. The binding of mono[125I]iodo-ANF (125I-ANF) was competed by unlabeled ANF in a dose-dependent manner. Hormones unrelated to ANF such as angiotensin I, bovine luteinizing hormone, and human chorionic gonadotropin were not able to compete against 125I-ANF. The binding of 125I-ANF was rapid, reaching maximum levels in 15 min at 4 degrees C. At 37 degrees C, the cell-bound 125I label was quickly decreased. Pretreatment of cells with NH4Cl, chloroquine, or NaN3 resulted in significant increases in maximum levels of the cell-bound 125I radioactivity. A photoaffinity reagent for ANF receptor was prepared by reacting ANF with succinimido 4-azidobenzoate, and resultant 4-azidobenzoyl- (AZB-) ANF was purified by high-performance liquid chromatography (HPLC). AZB-ANF was radioiodinated by use of chloramine T and purified again by HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and evaluation of oligodeoxyribonucleotides containing an aryl(trifluoromethyl)diazirine moiety as the cross-linking probe: photoaffinity labeling of mammalian DNA polymerase beta.

Photolabile 2'-deoxy- E -5-[4-(3-trifluoromethyl-3 H-diazirin-3-yl)styryl]uridine and its protected phosphoramidite derivatives have been synthesized and introduced into DNA oligomers through solid-phase DNA synthesis. The (trifluoromethyldiazirinyl)stylyl moiety of this nucleoside was found to be sufficiently stable for automated DNA synthesis. In addition, this moiety was found to be stable at 60 degrees C in aqueous solution under the annealing conditions for duplex formation with complementary strands, since >95% of the photolabile nucleoside remained after heating for 1 h. The oligo(dT) 15mer analog bearing the photolabile residue was activated/decomposed by near-UV irradiation. In photoaffinity cross-linking experiments with recombinant rat DNA polymerasebeta, constituted from a 40 kDa polypeptide, using oligo(dT) 15mer analogs bearing the photolabile residue near the 3'-terminus, a covalently bound complex of 45 kDa was obtained in the presence of complementary templates. Thus it was demonstrated that our method for synthesis of photolabile oligodeoxyribonucleotides may be useful for studies of DNA-related enzymes and DNA binding proteins.