WO1983004255A1 - Composes informateurs - Google Patents

Composes informateurs Download PDF

Info

Publication number
WO1983004255A1
WO1983004255A1 PCT/US1982/000725 US8200725W WO8304255A1 WO 1983004255 A1 WO1983004255 A1 WO 1983004255A1 US 8200725 W US8200725 W US 8200725W WO 8304255 A1 WO8304255 A1 WO 8304255A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
compound
formula
fluorophore
water
Prior art date
Application number
PCT/US1982/000725
Other languages
English (en)
Inventor
Paul M. Gallop
Mercedes Paz
Original Assignee
The Children's Hospital Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Children's Hospital Medical Center filed Critical The Children's Hospital Medical Center
Priority to PCT/US1982/000725 priority Critical patent/WO1983004255A1/fr
Priority to EP82902137A priority patent/EP0110879A1/fr
Publication of WO1983004255A1 publication Critical patent/WO1983004255A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/06—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K9/00—Tenebrescent materials, i.e. materials for which the range of wavelengths for energy absorption is changed as a result of excitation by some form of energy
    • C09K9/02—Organic tenebrescent materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G01N33/555—Red blood cell

Definitions

  • Such molecules be capable of being transferred from one phase to another; e.g., from an aqueous to a non-aqueous phase, or across biological membranes.
  • the invention features a new class of reagents which we have named the Boronate-Dependent Phase Transfer (BorAdept) compounds.
  • the new compounds allow groups which can 1) report on conditions within living cells and tissues or 2) modify metabolic parameters within living tissues to be presented to and taken up by cells under non-toxic conditions.
  • the new compounds have one of two general formulae:
  • each X and Y independently, is N, O, or S, and r represents a receptor group which, with X and Y, forms adducts or complexes with boronic acids.
  • Cg 1 and Cg 2 are carrier groups which each include a solubilizing group which is substantially ionizable in water
  • Rp 1 and Rp 2 are groups which each include a reporter group which is a fluorophore, a chromophore, an organometallic group, a therapeutic agent, an antigen, or an isotopically-labelled group.
  • the new compounds because of their water-solubility at or near physiological pH, provide reporter groups with greatly enhanced versatility.
  • formula (1) and (2) compounds to exist in pH-dependent equilibria with their dissociated forms renders them useful for a broad range of applications.
  • Many of these compounds have the additional advantage provided by the boronic acid-derived portion of the molecule which, when dissociated from the remainder of the molecule, can exhibit the selective affinity which boronic acids have for organic compounds having reactive hydroxyl, amino, and thiol groups, the aforesaid equilibria also allow some reporter groups to enter environments where their properties are advantageously altered. For example, some of the fluorescent reporter groups, under certain conditions, can be brought into contact with hydrophobic pockets in cells or organic phases where they exhibit enhanced or quenched fluorescence.
  • aspects of the invention feature applications of the new compounds such as the staining of living cells, proteins, and fabrics, and various assay methods.
  • the invention also features new boronic acid containing compounds, which are useful both by themselves, and as starting materials for BorAdept compounds.
  • Figure 1 is a flow chart diagramming an assay method of the invention.
  • Figure 2 is a perspective view of apparatus for carrying out one of said methods.
  • the compounds represented in formula (1) and (2) each is a reaction product between a compound which includes a boronic acid, and a compound which includes a receptor group (r) which, with X and Y, forms adducts or complexes with boronic acids.
  • one of the compounds also includes a group (Rp 1 or Rp 2 ) which includes a reporter group comprising a fluorophore, a chromophore, an organometallic group, a therapeutic agent, an antigen, or an isotopically-labelled group.
  • the other compound includes a carrier group (Cg 1 or Cg 2 ) which includes a strongly basic or acidic solubilizing group which is substantially ionizable in water.
  • Rp 1 is bonded to boronic acid in formula (1)
  • Rp 2 is bonded to the receptor in formula (2).
  • Cg 1 is bonded to the receptor in formula (1), while Cg 2 is bonded to boronic acid in formula (2).
  • the compounds of formula (1) are generally made by the reaction
  • the compounds of formula (2) are generally made by the reaction
  • the compounds of formula (1) and (2) can, if desired, contain more than one reporter group, and in such case the properties of the additional group or groups can influence the dynamic equilibrium of the compound.
  • each R 2 - R 7 is hydrogen, aryl, alkyl, heteroalkyl, hetroaryl, aralkyl, or a solubilizing group which is substantially ionized in water, provided that the compound contains at least one such solubilizing group.
  • n and m independently, is 0, 2, or 3, provided that both cannot be 0.
  • each R 2 - R 5 can be different in successive repeating units. For example, where n is 2, the R 2 attached to the first carbon atom can be hydrogen, while the R 2 attached to the second carbon atom is a solubilizing group.
  • each of groups R 2 - R 7 preferably contains between 0 and about 12 carbon atoms.
  • the solubilizing group is preferably a sulfonic acid, a carboxylic acid, quaternary ammonium, a guanidinium group, or a phosphate group.
  • the first step in making a formula (1) compound is to make the desired ReportaBora. This can be done by reacting a desired organic boronic acid with the desired Rp 1 . Alternatively, the desired Rp 1 precursor can undergo hydroboration with catechol boirane, or can participate in a
  • the reporter group can first be incorporated into a compound having a desired property, e.g., good electron withdrawing power for enhanced activation of the boronate.
  • Table I shows the structures of some ReportaBora compounds which provide the Rp 1 -B portion of the compounds of formula (1) . Each of these can react with any CarrierCeptor, as defined above, to produce a formula (1) compound.
  • Table I also indicates the utility of the reporter group of each listed ReportaBora. This utility, along with other factors, including choice of CarrierCeptor, allows the choice of starting materials which will react to produce a formula (1) compound possessing the properties required for a given application.
  • Table II shows the structures and some properties of some preferred CarrierCeptor
  • CarrierCeptors can be reacted with any ReportaBora, such as those listed in Table I, to make a compound of the general formula (1).
  • Cg 2 typically includes both a solubilizing group and an organic group linking the solubilizing group to the boron atom.
  • Formula (2) compounds thus typically have one of the following general formulae:
  • Sol is the aforementioned strongly basic or acidic solubilizing group and A 1 is alkyl, aryl, heteroaryl, heteroalkyl, or aralkyl; most preferably A 1 contains between 3 and about 12 carbon atoms.
  • a 2 - A 7 independently, is hydrogen, aryl, alkyl, hetoralkyl, heteroaryl, aralkyl, or Rp 2 , the group containing a reporter group, provided that the compound contains at least one Rp 2 .
  • Each of A 2 - A 7 , except Rp 2 preferably contains between 0 and about 12 carbon atoms.
  • each of p and q is 0, 2, or 3, provided that both cannot be 0.
  • each A 2 - A 4 can be different in successive repeating units.
  • the A 2 attached to the first carbon atom can be hydrogen, while the A 2 attached to the second carbon atom can be Rp 2 .
  • the first step in making a formula (2) compound is to make the desired ReporterCeptor by
  • Rp 2 can consist entirely of the desired reporter group, or the reporter group can be incorporated into an Rp 2 having a desired property; e.g., good
  • solubilizing groups are suitable compounds.
  • CarrierBoras (putting aside considerations such as cost) in having a wide variety of CarrierBoras to choose from, and one or a few can be reacted with a wide variety of ReporterCeptors.
  • An example of one such versatile CarrierBora is the compound shown below which, although not commercially available, can be made using conventional methods.
  • Compound (11) is particularly useful because its boronate group is highly reactive as a result of the strong electron withdrawing action of the sulfonephenyl group
  • CarrierBoras are m-carboxyphenyl boronic acid, p-sulfophenylboronic acid, m-guanidinophenyl boronic acid, and p-trimethylammoniumphenyl boronic acid. The first two are negatively charged, while the latter two are positively charged.
  • Compound 12 is made by reacting m-aminophenyl boronic acid, in aqueous ethanol at pH 8.0, with
  • a formula (2) compound is typically made by dissolving the desired ReporterCeptor in anorganic, water-miscible solvent, e.g., dimethyl acetamide, acetone, or dimethyl formamide, and then adding an excess of the desired CarrierBora in water or an appropriate aqueous buffer.
  • anorganic, water-miscible solvent e.g., dimethyl acetamide, acetone, or dimethyl formamide.
  • Both formula (1) and formula (2) compounds can, if desired, contain more than one reporter group.
  • a group, Rp 3 including a second reporter group can be attached at any suitable reactive site on formula (1) or (2) compounds, but will most often be attached at a site on Rp 1 or Rp 2 so that the ReportaBora or ReporterCeptor, dissociated from the carrier part of the molecule, bears both reporter groups.
  • Compound 3 of Table III an example of such a compound, bears two staining reporter groups.
  • the compounds of both formulae (1) and (2) in which the reporter group is a chromophore, an antigen, an organometallic group, an isotopically-labelled group, or a fluorophore are useful for staining living cells, proteins, and other materials such as fabrics and paper products.
  • formula (1) compounds in aqueous solution at pH values around physiological pH, exist in dynamic equilibria with their starting materials.
  • the equilibrium permits the presentation of the ReportaBora to cells in media compatible with living cells, and uptake by cell membranes without toxicity.
  • the diagram below illustrates the mechanism of uptake of ReportaBora by cells.
  • the scheme is general, and is not strictly applicable to all formula (1) compounds.
  • a formula (1) compound having a positively-charged solubilizing group such as quaternary ammonium is more likely to enter the cell intact rather than operating as shown below.
  • many ReportaBoras do not exhibit altered reporting properties inside cells.
  • the equation (3) equilibrium permits hydrophobic pockets of the cell membrane to selectively extract ReportaBora, which then enters the cell.
  • the ReportaBora carried by lipophillic components of the cell, is capable of selectively staining cellular components.
  • the boronic acid portion of the molecule binds selectively to certain biological groups within cells which contain appropriately distributed hydroxyl, amino, and thiol groups; binding is particularly favored when such groups are in non-aqueous regions of the cell.
  • Certain complex lipids, mucopolysaccharides, liposaccharides, glycoproteins, and proteins contain such reactive groups.
  • formula (1) compounds make them useful as vital cellular stains in the diagnosis of certain disease states, including certain genetic disorders, particularly those which cause mucopolysaccharidosis, liposaccharidosis, or an increase in either the number or size of cellular lysosomal components.
  • Hurler's Syndrome, Hunter's Syndrome, and ⁇ -glucuronidase deficiency can be diagnosed by culturing a patient's cells, staining them with a fluorescent formula (1) stain, e.g., compound 1 of Table III, and then examining them using a fluorescent microscope.
  • the vital staining patterns and the rate of destaining of certain cellular particulates can provide valuable diagnostic information.
  • the compounds are also useful for gaging the viability and motility of human and animal sperm.
  • the selectivity of formula (1) compounds also makes them useful in comparing staining patterns of various cells in different cell cycle stages.
  • the stains can also be useful for staining catecholamine-rich regions of cells; for example, the compounds stain epinephrine, norepinephrine, dopamine, di-hydroxyphenylalanine, and 3, 4-hydroxphenylacetic acid, compounds which are abundant in nervous and adrenal tissue and are thus useful for the diagnosis of a variety of clinical conditions.
  • Staining of cells can be carried out using any most conventional vital staining techniques, but is preferably carried out according to either of the following two preferred processes.
  • cells to be stained are grown on a coverslip, the culture medium is poured off, the cells are washed with physiological buffer several times, and the coverslip is covered for 5 minutes with a formula (1) compound dissolved in physiological buffer. The coverslip is then washed, turned over onto a slide, and examined using a light or fluorescent microscope, or processed further, e.g., tested for the presence of immune complexes, radioactive label, or heavy metals.
  • ReportaBora is added directly to the cell culture medium in which CarrierCeptor is already present as the buffer. After the desired degree of staining is complete, the medium is poured off and the cells are examined.
  • An additional step may be added to either preferred staining method to obtain additional information, such as the relative dynamic stability of the bonds between the stain and various cellular components.
  • cells are transferred to fresh culture medium and monitored to determine the relative rates at which the stain leaches out of various cellular components.
  • the formula (1) compound contains an organometallic electron opaque reporter group, following vital staining, the cells are fixed and prepared for examination with an electron microscope.
  • any formula (1) compounds can be used for vital staining, some are more useful than others for certain applications.
  • One factor affecting choice of dye is the strength of the bond between the ReportaBora and the CarrierCeptor; very tight complexes, such as those formed with strong CarrierCeptors such as TAPSO, and with certain
  • ReportaBoras containing strong Lewis acid boronic acids tend to make it more difficult for cell membranes to extract appreciable amounts of the ReportaBora. For this reason, it is sometimes preferable, for vital staining, to use a formula (1) stain made with a weaker complexing CarrierCeptor such as TES or MOPSO (Table II). Because Carrier Ceptors which form weak complexes with boronic acids also tend to be less reactive with boronic acids, when it is desired to make a formula (1) compound using a weak CarrierCeptor such as MOPSO, it is often desirable to employ a more reactive ReportaBora; i.e., one in which the boron atom is more electron deficient. Such a reactive ReportaBora is one in which Rp contains a linking group, proximate to the boron atom, which has strong electron withdrawing power. Referring to Table I, FBIII contains the linking group
  • ReportaBoras e.g., FluoraBora I, containing groups such as the sulfamido linkage, which do not exert strong electron withdrawing action, complex to a lesser extent with weakly complexing CarrierCeptors, and formula (1) compounds wherein the ReportaBora is FluoraBora I are thus preferably made with a strong CarrierCeptor such as TAPSO.
  • fluorescent stain for a particular application is whether the fluorophore is enhanced or quenched (inhibited) in hydrophobic environments such as the hydrophobic protein pockets which the ReportaBora is likely to seek within a cell. Still another consideration is whether the fluorophore exhibits a shift in emission wavelength in such an environment; both enhancement and emission shift can advantageously increase detectability of dyes within cells.
  • Formula (2) dyes can also be used for the vital staining of cells.
  • the diagram below illustrates the mechanism of uptake of ReporterCeptor by cells.
  • Formula (2) dyes can also be used for the vital staining of cells.
  • the diagram below illustrates the mechanism of uptake of ReporterCeptor by cells.
  • formula (1) compounds there are exceptions to this general scheme.
  • Cells are contacted with an aqueous solution of the stain, which is in dynamic equilibrium with its respective CarrierBora and ReporterCeptor.
  • the cell selectively extracts ReporterCeptor molecules, which enter and stain the cells, and can exhibit altered reporting properties such as fluorescence enhancement or wavelength shift in hydrophobic regions of the cell.
  • those of formula (2) stains are not boronic acids, and do not form such complexes with particular cellular components; their specificity depends instead on the nature of the receptor portion of the molecule.
  • Formula (2) stains in which the solubilizing group is a positively charged group; e.g., quaternary ammonium, can also be used for vital cell staining.
  • the entire compound may enter the cell and establish local hydrophobicity- dependent equilibria within the cell.
  • a further refinement to vital cell staining using formula (1) and (2) compounds is to employ a stain having more than one staining reporter group.
  • a formula (1) stain can have the structure where Rp 1 and Rp 3 each is a group which includes a fluorophore, chromophore, an antigen, an isotopically-labelled group, or an organometallic compound.
  • Compound 12 of Table I, complexed with TAPSO (Table II) is an example of such a compound, bearing a fluorophore which provides cellular information at the level of the fluorescent microscope, and an electron-opaque organometallic group which provides information on the electron microscope level; the fluorescence pattern of a stained cell indicates to the electron microscopist that that cell took up stain, indicates which portions of the cell were stained at the level of resolution of the light microscope, and, after preparation for electron microscopy, allows a comparison of staining patterns within the same cell at the light and ultrastructural levels.
  • the formula (1) stains can also be used to permanently stain proteins.
  • the preferred method for carrying out such staining is to react the protein to be stained with the dye under mild conditions; e.g., at room temperature or lower, at pH 4 to 10, in the absence of an organic solvent. Excess ReportaBora can then be removed by carrying out dialysis against the CarrierCeptor used to make the stain.
  • compound 9 Table III
  • CBII highly water-insoluble ChromaBora II
  • Table I highly water-insoluble ChromaBora II
  • Serum albumin is then added and the mixture then dialyzed against TAPSO to remove excess CBII.
  • CBII enters the hydrophobic pockets of serum albumin, leaving its CarrierCeptor, TAPSO, in the aqueous phase.
  • Dialysis against TAPSO removes excess CBII as the formula (1) complex, but is unable to remove CBII from the hydrophobic pockets of the serum albumin, which is thus permanently stained.
  • Formula (1) and (2) compounds can also be used to stain or brighten a variety of other materials such as fabrics and paper products.
  • Staining is carried out by simply contacting the material to be stained with an aqueous solution of the desired formula (1) or (2) compound (generally a colored or a fluorescent compound) until the desired degree of staining has been achieved.
  • Brightening of fabrics is carried out using a fluorescent formula (1) or (2) compound, and can often be performed in a conventional clothes washing machine, in soapy or clean water.
  • Many fabrics can be brightened using the new compounds, including cellulose-containing fabrics, such as cotton and linen, and nitrogen-containing fabrics such as wool and silk, and various synthetics.
  • formula (1) compounds because the FluoraBora of the equation (3) equilibrium is attracted to the hydroxyl groups of the fabric.
  • formula (1) dyes are also preferred, and the brightening process may include an additional step. Following the step of contacting the fabric with the fluorescent formula (1) compound, the fabric is contacted with an oxidizing agent such as peroxide, which breaks the carbon-boron bond of the formula (1) compound, releasing Rp 1 - OH, which is then. better locked into the hydrophobic pockets in the fabric. Even greater brightening can be obtained by employing a formula (1) compound in which the fluorescent part of the molecule exhibits enhanced fluorescence in hydrophobic environments.
  • a colored or fluorescent formula (1) compound can be used to measure, in aqueous solution, an unknown amount of a compound which participates in an enzymatic procedure ("procedure", as used herein, includes one or a series of reactions) in which an enzyme acts on a substrate, or a chemical procedure which results in the production of peroxide.
  • peroxide assay excesses of all of the reactants participating in the procedure are introduced into a reaction zone, e.g., a test tube, along with the sample containing an unknown amount of the compound to be assayed. The peroxide produced is brought into contact with a known amount of the formula (1) compound.
  • the peroxide an oxidizing agent, breaks the carbon-boron bond of the ChromoBora or FluoroBora, releasing Rp 1 - OH, which is then contacted with a substance having a greater affinity for Rp 1 - OH than for the formula (1) compound.
  • the fluorescence or color intensity of the substance is then measured as a measure of the unknown amount of the compound being assayed.
  • the selective affinity substance can be a water-immiscible organic solvent which is capable of extracting Rp 1 - OH away from the formula (1) compound.
  • the solvent forms a fluorescent organic layer separate from the aqueous layer; if desired the two layers can then be separated to facilitate quantification of the fluorescence of the organic layer.
  • Table V shows the extent to which three FluoraBoras, complexed with six CarrierCeptors in aqueous solution, are extracted into ethyl acetate.
  • those made with comparatively tightly-complexing TAPSO are most preferred for the peroxide assay because, absent peroxide (i.e., in a blank reaction), a comparatively large proportion of the fluorescence remains in aqueous solution, unextracted by ethyl acetate.
  • the reporter group of the formula (1) compound is a fluorophore
  • the manner in which the organic solvent being used affects the properties of the fluorophore is the manner in which the organic solvent being used affects the properties of the fluorophore.
  • Some fluorophores are enhanced, i.e., their fluorescence is made more intense, in a given organic solvent, while others exhibit diminished fluorescence (quenching).
  • Some fluorophores may also exhibit altered emission spectra in certain organic solvents.
  • any fluorescent formula (1) compound can be used, provided its fluorophore is not totally quenched in the organic solvent, both enhancement and spectrum alteration desirably heighten detectability.
  • Table VI shows the relative fluorescence of three FluoraBoras combined with TAPSO, compared to the fluorescence of equal concentrations of FluoraBora in the water-immiscible organic solvent ethyl acetate. Because FBPA and FBI are enhanced (two and seven-fold, respectively), formula (1) compounds made with them are suitable for use in the peroxide assay described above. FBIII, on the other hand, is quenched to a large extent in ethyl acetate, and formula (1) compounds made with it are therefore not desirable for use in the assay in which ethyl acetate is employed.
  • the selective affinity substance in the peroxide assay can be a water-miscible protein which has a greater affinity for Rp 1 than for the corresponding formula (1) compound being used.
  • Preferred proteins are those, e.g., serum albumin, which posses hydrophobic pockets which can attract Rp 1 .
  • the formula (1) compound be fluorescent, and that the protein be capable of producing an observable effect, preferably enhancement or, less preferably, quenching, of fluorescence, of Rp 1 , there being no easily observed water/protein phase separation.
  • Table VII shows the relative enhancement or inhibition with serum albumin, of the three fluorophores of eighteen formula (1) compounds. Table VII shows that, in general, FBI and, to a lesser extent, FBPA, are enhanced in serum albumin, while FBIII exhibits diminished fluorescence.
  • each of the three fluorophores of Table VII is enhanced or inhibited depends, to a significant extent, on the CarrierCeptor with which it is combined.
  • enhancement, in serum albumin, of FBPA and FBI, and inhibition of FBIII is much greater if the formula (1) compound has been made with loosely complexing MOPSO than with tightly complexing TAPSO. This is because the loose Rp 1 -MOPSO complex favors an equilibrium in which more free Rp 1 is available to seek the hydrophobic pockets of serum albumin, where enhancement or inhibition of fluorescence occurs.
  • a blank should always be run in which the peroxide-generating reagents are not added, so that enhancement or inhibition in the absence and presence of those reagents can be compared.
  • the other observable effect which a water-miscible protein such as serum albumin can have on a fluorophore is a shift in the peak emission spectrum; such a shift can, as in the case of a water-immiscible organic solvent, aid detectability.
  • FBPA for example, normally emits at 460 nm, while its dissociated fluorophore, in the hydrophobic pockets of serum albumin, emits at 427 nm.
  • Glucose is an example of a compound which can be measured using the above-described method.
  • a sample containing an unknown amount of glucose is placed in a test tube with a known amount of Compound 1 (Table III) and a known amount of serum albumin in aqueous solution at pH between 8 and 9.
  • An excess of glucose oxidase is then added, and the increse in fluorescence compared to that of a no-enzyme, no-substrate blank is measured as a measure of the amount of peroxide produced, which in turn is a measure of the amount of glucose in the sample.
  • glucose oxidase catalyzes the oxidation of glucose to gluconic acid by oxygen, forming H 2 O 2 .
  • the H 2 O 2 oxidizes the C-B bond of FBI in the formula (1) complex, forming dansylamido phenol, which enters the serum albumin pockets, where it exhibits enhanced fluorescence.
  • certain formula (1) compounds can also be used to measure an unknown amount of a compound which participates in an enzymatic procedure in which an enzyme acts on a substrate.
  • the method employs fluorescent formula (1) compounds in which Rp, includes a group, positioned between the boron atom and the fluorophore, capable of acting as a substrate for the enzyme.
  • a desired enzyme substrate can be incorporated into a desired formula (1) compound using coventional techniques.
  • the formula (1) compound being used is introduced into a reaction zone, along with excesses of all of the other participating reactants except the compound to be measured, and a sample containing the compound to be measured.
  • the enzymatic reaction releases modified fluorophore which, after completion of the reaction, is contacted with a water-immiscible organic solvent or a water-miscible protein, as described above for the peroxide assay.
  • the extent of fluorophore extraction or fluorescence enhancement or inhibition is then measured versus a blank, as described above, as a measure of the unknown compound. If a water-miscible protein is used, the rate of the enzymatic reaction can also be followed by measuring the rate at which fluorescence enhancement increases.
  • Esterases such as Upases are examples of compounds which can be assayed using this method.
  • a test tube in aqueous solution, is placed a known amount of compound 6 (Table III), of the formula
  • a sample containing an unknown amount of lipase is then added. Lipase present in the sample breaks the ester bond (as indicated by the dotted line), releasing modified Rp 1 , containing the fluorescent dansyl group, which is then extracted or contacted with a known amount of serum albumin and fluorescence measured, as described above for the peroxide assay.
  • Antibody Assay Another new assay employing formula (1) compounds is an antibody assay employing red blood cells. (Red Blood cells are commonly used in other antibody assays.) A formula (1) compound is used in which the reporter group of Rp 1 is a hapten capable of complexing with the antibody being assayed.
  • Red blood cells are contacted, in aqueous solution, with the formula (1) compound, which is in an equation (3)-type equilibrium with its starting materials.
  • the red blood cell membrane selectively extracts hapten-containing ReportaBora, thus becoming sensitized.
  • the sensitized red blood cells are contacted with a sample containing an unknown amount of antibody, together with complement which is capable of participating in the complexation of the hapten with the antibody.
  • the antibody in the sample complexes with the hapten in the sensitized cell membrane, fixing complement and causing cell lysis and the release of hemoglobin from the cell.
  • the amount of hemoglobin released is a measure of the amount of antibody in the sample.
  • the formula (1) compound used contains two reporter groups; a hapten and a fluorophore, both of which become incorporated into the membranes of sensitized red cell ghosts by the BorAdept process.
  • the ghosts are contacted with antibody and complement, lysis of the ghost stroma occurs, causing the release of soluble fragments containing fluorophore.
  • the solution can contain a CarrierCeptor to facilitate solubilization of the fluorescent fragments. The fluorescence in solution is then measured as a measure of the antibody titer. Solubilization of Therapeutic Agents
  • the BorAdept system of the invention can greatly facilitate the administration of therapeutic agents such as drugs which have heretofor been difficult to administer because of poor water solubility or difficult to test in cell culture systems.
  • a desired therapeutic agent can be incorporated into either a formula (1) or (2) compound. In either case, by virtue of the equation (3) or (4) equilibrium, the agent is not only solubilized, but is in a form which enables the agent to be presented to and taken up by cells to which it is desired to deliver the agent, by the same mechanism by which fluorophore reporter groups are taken up by cells to be stained.
  • Therapeutic formula (1) compounds are generally made in the same way fluorescent formula (1) compounds are made; i.e., the de-sired therapeutic agent or a suitable precursor or derivative thereof is first reacted with the desired boronic acid to form a ReportaBora (in this case, a TheraBora), which is then reacted with the desired CarrierCeptor.
  • the boronic acid is preferably chosen to include a group which renders the boronic acid reactive with the therapeutic agent being used.
  • Compound 4 of Table III is an example of a formula (1) compound containing a derivative of therapeutic agent, the water-insoluble anti convulsant diphenyl hydantoin.
  • Another example is compound 2 of Table III, containing a derivative of the water-soluble anti-epileptic drug carbamazepine. This new compound is made according to the reaction scheme illustrated on the following page.
  • Therapeutic formula (2) compounds are generally made in the same way that fluorescent formula (2) compounds are made, i.e., the desired therapeutic agent or a suitable precursor or derivative thereof is, if necessary, incorporated into a compound having a desired property to make Rp 2 , which is then reacted with a compound of the
  • Compound 5 of Table IV is made according to the reaction scheme illustrated on the following pages:
  • Table I lists some preferred ReportaBoras of the invention. These new compounds, as is discussed above, are useful in making formula (1) BorAdept compounds. In addition, we have discovered that they have many applications by themselves, even when not complexed with a CarrierCeptor in a formula (1) compound. Many of these applications, e.g., the peroxide assay, are similar in principle to applications of formula (1) compounds.
  • the ReportaBoras of the invention are generally made by reacting a desired reporter group-containing compound (Rp 4 ) with a desired organic boronic acid capable of reacting with that
  • organic boronic acid has the general formula
  • R o is aryl, alkyl, heteroalkyl, heteroaryl, or aralkyl
  • Z is a group capable of chemically combining with the Rp 4 being used.
  • R o contains between 3 and about 25 carbon atoms.
  • a preferred (because of ready availability) boronic acid is m-aminophenyl boronic acid, in which Z is
  • Rp 4 usually has the formula where Re is the reporter group and Y is a group capable of chemically combining with the Z portion of the boronic acid being used.
  • the ReportaBoras made by reacting the aforesaid boronic acids and Rp 4 's thus either have the formula
  • Z is NH 2 , OH, SH, or COOH, and in some preferred
  • X being any halogen.
  • the carbon or sulfur atom of Y to which X was bonded bonds to Z replacing an H.
  • Z' is thus Z minus an H
  • Y' is Y minus X. (there is no Y' when Y consists entirely of X).
  • the resulting ReportaBoras have the formula.
  • FBPA ReportaBoras
  • the first step in our synthesis of FBPA is to make the sulfonyl chloride of Ilford's compound by reacting it with PCl 5 .
  • the new sulfonyl chloride has the formula:
  • the new compound has a chemically stable crystalline structure with a melting point of 119°C. Besides being useful for making ReportaBoras, it is, also useful as a fluorescent reporter for labelling proteins such as antibodies and other compounds such as amino acids and polyamines. Because of its azuxe fluorescence, it can be used in conjunction with red and yellow fluorescent dyes in immunofluorescent labelling experiments.
  • Compound 21 is a diaryl sulfonyl chloride, and is one of a family of fluorescent compounds which we refer to as DARPSYL chlorides. These compounds are the sulfonyl chloride derivatives of compounds of the general formula
  • Ar 1 or Ar 2 is an aromatic group containing a sulfonyl chloride group and Q is hydrogen or an alkyl group.
  • the compounds in which Ar 1 contains the sulfonyl chloride group are the "A” compounds, and are derived from the DARPSYL compounds described in British Pat. No. 670,857.
  • the sulfonyl chloride derivatives in which Ar 2 contains a sulfonyl chloride group are the "B” compounds, and are also useful fluorescent stains.
  • the new A-DARPSYL chloride, (compound 21), as well as other DARPSYL-chlorides, can be combined with organic boronic acids to make useful FluoraBoras.
  • FBPA (Table I)
  • A-DARPSYL chloride described above is combined with m-aminophenyl boronic acid in DMF-H 2 O.
  • the mixture is then acidified, water is added causing FBPA to precipitate out and recrystallization is carried out, from cyclohexane.
  • Another FluoraBora listed in Table I, FBI is made by adding an acetone solution of dansyl chloride to an aqueous acetone solution of m-aminophenyl boronic acid hemisulfate containing 2.5 equivalents of sodium bicarbonate, and then acidifying the solution, adding more water causing FBI to precipitate out and recrystallizing from benzene.
  • FBII another FluoraBora
  • m-aminophenyl boronic acid is made by adding solid fluorescein isothiocyanate to an aqueous alkaline solution of m-aminophenyl boronic acid.
  • ReportaBoras listed in Table I are not fluorescent, but rather contain reporter groups such as chromophores or derivatives of therapeutic agents.
  • the reporter group can also be an antigen or an isotopically-labelled group, or it can be an organometallic group, as in compounds 8 and 15 of Table III.
  • Compound 12 of Table III also contains an organometallic group, and in addition contains the fluorescent dansyl reporter group.
  • Compound 8 is made according to the reaction diagrammed below.
  • any desired organometallic complex can be used to make a ReporterBora, which in turn can be combined with a CarrierCeptor to make a BorAdept reagent.
  • other useful organometallic complexes contain heavy, electron-opaque metals such as osmium, which are useful in staining for electron microscopy.
  • ReportaBoras of Table I possess such poor water solubility that they cannot be presented to and taken up by living cells under non-toxic, physiological conditions. Such compounds, as has been discussed, are rendered useful for vital staining only when coupled to a CarrierCeptor.
  • ReportaBoras e.g., FBI
  • Such ReportaBoras are generally used for selective vital staining in the same manner as are BorAdept vital stains, described above.
  • ReportaBoras and ReporterCeptors can also be used to selectively stain fixed cells, using conventional techniques and solvents. As with vital staining, the selectivity provided by the boronic acid portion of the ReportaBoras can provide valuable diagnostic information.
  • ReportaBoras are useful for vitally staining the surfaces of cells and extracellular areas, and are also useful for staining the eye surfaces of living animals to determine the location of surface irregularities such as scratches and lesions; the negatively charged ReportaBoras are particularly useful for the latter purpose.
  • the desired Reporta3ora dissolved in a physiological buffer or CarrierCeptor as a formula (1) complex, is applied in any conventional manner, e.g., with an eyedropper.
  • FluroaBoras can be bound to certain water-insoluble supports and used in peroxide and other assay methods.
  • the supports which can be used include all organic, water-insoluble polymeric materials having hydroxyl groups which can complex with boronic acids.
  • suitable supports are polymers containing polyglycerol methacrylate, and hydrophilic fibrous polyethylene containing attached polyvinyl alcohol; the latter support is available from Crown Zellerbach under the name "Fybrel".
  • Support-bound FluoraBoras preferably are made by reacting a solubilized FluoraBora (in acetone, benzene, DMF, or the like) with the powdered form of an appropriate support material and then washing the powder with a solvent to remove excess FluoraBora.
  • a solubilized FluoraBora in acetone, benzene, DMF, or the like
  • the FluroaBora being used is water-insoluble, it is first complexed with a suitable CarrierCeptor to form a formula (1) BorAdept compound, as described above.
  • a CarrierCeptor it and the support should be chosen so that the support tends to form stronger complexes with the boronate of the FluoraBora being used, at the pH under which the reaction is carried out, then does the CarrierCeptor at that pH.
  • the equation (3) equilibrium then allows the support to act as a separatory funnel, complexing with dissociated FluoraBora.
  • a weakly complexing buffer such as MOPSO will be used in this situation.
  • FluoraBoras bonded to Fybrel are particularly preferred embodiments of this aspect of the invention.
  • the structure of FBI so bound is shown below. It can be seen that the boron atom forms a stable 6-membered ring with three carbons and two oxygens of the polyvinyl alcohol.
  • FBI bound to Fybrel is made as follows. FBI is dissolved in acetone. Fybrel is ground to coarse powder and shaken with the dissolved FBI for 5 minutes. After exhaustive washing with acetone, support-bound FBI is stored in the dark at 5°C until used, either dry or in an organic solvent. Peroxide Assay
  • Flow Chart I (Fig. 1) diagrams a method for assaying, in aqueous solution, a compound which participates in a chemical procedure which results in the production of peroxide.
  • the basic principle is the same as that of the BorAdept peroxide assay, previously described.
  • Excesses. of all of the reactants participating in the procedure are introduced into a reaction zone, e.g., a test tube, along with the sample containing an unknown amount of the compound to be assayed.
  • the peroxide produced is brought into contact with a support-bound FluoraBora, described above.
  • the peroxide breaks the carbon-boron bond of the boronic acid, releasing fluorescent compound into solution, where it can be measured directly using, e.g., a fluorometer, enhanced using a protein such as serum albumin, or extracted using a water-immiscible organic solvent such as ethyl acetate.
  • the rate of release of fluorescent compound can be increased by maintaining the pH of the reaction zone in the range of about 8 to 9.
  • the assay can be further modified by separating fluorescent compound in solution from the insoluble support folllowing the oxidation of the carbon-boron bonds by the peroxide. This separating can be carried out by filtering, by removing the insoluble support using centrifugation, or by extracting the dissolved fluorescent compound in an organic, water-immiscible solvent.
  • Glucose is an example of a compound which can be assayed using the above-described method.
  • a sample in aqueous solution, containing an unknown amount of glucose are added aqueous buffer of pH 8 to 9, glucose oxidase, and an excess of FBI bound to Fybrel.
  • the Fybrel is filtered out and fluorophore is measured, using a fluorometer, as a measure of glucose in the sample.
  • tube 2 contains an excess of a support-bound FluoraBora, described above.
  • tube 1 Into tube 1 are introduced, in aqueous solution, excesses of all of the reactants, except the compound to be assayed, necessary to produce peroxide in the procedure being measured.
  • tube 1 is pushed down to allow the fluorophore in solution to pass upward through filter 4.
  • the fluorescence of the solution, or of an extract of the solution, a measure of the compound being assayed in the sample is measured with a fluorometer.
  • the stable bond formed between FluoraBoras and supports containing hydroxyl groups and between FluoraBoras and certain other compounds, e.g., mucopolysaccharides, provides the basis for an assay for compounds having an affinity for boronic acids.
  • the desired FluoraBora is contacted with an unknown amount of a compound to be assayed having an affinity for boronic acids.
  • Another material having an affinity for boronic acids e.g., one of the supports discussed above, is then added to the mixture. This second material binds with FluoraBora which did not complex with the compound being assayed.
  • the amount of FluoraBora bound to this second material is inversely proportional to the amount of unknown present.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Materials Engineering (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Emergency Medicine (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)

Abstract

Composés ayant l'une des deux formules I ou II. Dans la formule I, Rp1 est un groupe qui comprend un groupe informateur comprenant un fluorophore, un chromophore, un groupe organométallique, un agent thérapeutique, un antigène ou un groupe marqué par isotope, X et Y représentent, indépendamment, N, O ou S; Cg1 est un groupe porteur qui comprend un groupe de solubilisation sensiblement ionisé dans l'eau, et r est un groupe récepteur qui, avec X et Y, forme des complexes avec des acides boriques. Dans la formule II, Rp2 est un groupe qui comprend un groupe informateur contenant un fluorophore, un chromophore, un groupe organométallique, un agent thérapeutique, un antigène ou un groupe marqué par isotope; X et Y représentent indépendamment M, O ou S; Cg2 est un groupe porteur qui comprend un groupe de solubilisation sensiblement ionisé dans l'eau, et r est un groupe récepteur qui, avec X et Y, forme des complexes avec des acides boriques.
PCT/US1982/000725 1982-05-26 1982-05-26 Composes informateurs WO1983004255A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1982/000725 WO1983004255A1 (fr) 1982-05-26 1982-05-26 Composes informateurs
EP82902137A EP0110879A1 (fr) 1982-05-26 1982-05-26 Composes informateurs

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1982/000725 WO1983004255A1 (fr) 1982-05-26 1982-05-26 Composes informateurs

Publications (1)

Publication Number Publication Date
WO1983004255A1 true WO1983004255A1 (fr) 1983-12-08

Family

ID=22168016

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1982/000725 WO1983004255A1 (fr) 1982-05-26 1982-05-26 Composes informateurs

Country Status (2)

Country Link
EP (1) EP0110879A1 (fr)
WO (1) WO1983004255A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0282448A2 (fr) * 1987-03-05 1988-09-14 Ciba-Geigy Ag Composés de pyrazoline
EP0414730A1 (fr) * 1988-03-31 1991-03-06 Randell L. Mills Categories de produits pharmaceutiques luminide et macroluminide
WO1991004488A1 (fr) * 1989-09-21 1991-04-04 Russell Anthony P Procede et moyen de detection de composes de polyhydroxyle
EP0424168A1 (fr) * 1989-10-19 1991-04-24 Nippon Oil And Fats Company, Limited Complexe de polymère du type à réponse en sucre
WO2002099077A2 (fr) * 2001-06-06 2002-12-12 Proteologics, Inc. Methodes et compositions liees au marquage des proteines de la surface membranaire
US7317000B2 (en) 2001-12-02 2008-01-08 Novo Nordisk A/S Glucose-dependent insulins
FR2966153A1 (fr) * 2010-10-19 2012-04-20 Commissariat Energie Atomique Utilisation d'azaboronates cycliques comme materiaux sensibles dans des capteurs destines a detecter la presence de peroxydes dans un milieu gazeux

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB790090A (en) * 1952-12-02 1958-02-05 Bataafsche Petroleum Cyclic boron compounds
US3038926A (en) * 1958-09-17 1962-06-12 Roussel Uclaf Organic diol aryl boronates
US3131208A (en) * 1962-05-07 1964-04-28 United States Borax Chem Glycol monoborates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB790090A (en) * 1952-12-02 1958-02-05 Bataafsche Petroleum Cyclic boron compounds
US3038926A (en) * 1958-09-17 1962-06-12 Roussel Uclaf Organic diol aryl boronates
US3131208A (en) * 1962-05-07 1964-04-28 United States Borax Chem Glycol monoborates

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS Volume 88, No. 17 issued 24 April 1978, (Columbus, Ohio, U.S.A.) A.G. SMITH et al, 'Gas Chromotography Chemical Ionization Mass Spectrometry of Prostaglandin Fa Cyclic Bornate Derivatives' see page 459, column 1, the Abstract No. 120142m Biomed Spectom. 1977, 4(4) 258-64 (Eng). (section 22) *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0282448A3 (fr) * 1987-03-05 1988-09-21 Ciba-Geigy Ag Composés de pyrazoline
EP0282448A2 (fr) * 1987-03-05 1988-09-14 Ciba-Geigy Ag Composés de pyrazoline
EP0414730A4 (en) * 1988-03-31 1993-06-16 Randell L. Mills Luminide and macroluminide class of pharmaceuticals
EP0414730A1 (fr) * 1988-03-31 1991-03-06 Randell L. Mills Categories de produits pharmaceutiques luminide et macroluminide
AU650425B2 (en) * 1989-09-21 1994-06-23 Anthony P. Russell Method and means for detecting polyhydroxyl compounds
WO1991004488A1 (fr) * 1989-09-21 1991-04-04 Russell Anthony P Procede et moyen de detection de composes de polyhydroxyle
EP0424168A1 (fr) * 1989-10-19 1991-04-24 Nippon Oil And Fats Company, Limited Complexe de polymère du type à réponse en sucre
US5478575A (en) * 1989-10-19 1995-12-26 Nippon Oil & Fats Co., Ltd. Polymers having benzeneboronic acid groups and insulin complexes of same of a sugar response type
WO2002099077A2 (fr) * 2001-06-06 2002-12-12 Proteologics, Inc. Methodes et compositions liees au marquage des proteines de la surface membranaire
WO2002099077A3 (fr) * 2001-06-06 2003-11-13 Proteologics Inc Methodes et compositions liees au marquage des proteines de la surface membranaire
US7317000B2 (en) 2001-12-02 2008-01-08 Novo Nordisk A/S Glucose-dependent insulins
FR2966153A1 (fr) * 2010-10-19 2012-04-20 Commissariat Energie Atomique Utilisation d'azaboronates cycliques comme materiaux sensibles dans des capteurs destines a detecter la presence de peroxydes dans un milieu gazeux
WO2012052399A1 (fr) * 2010-10-19 2012-04-26 Commissariat à l'énergie atomique et aux énergies alternatives Utilisation d'azaboronates cycliques comme materiaux sensibles dans des capteurs destines a detecter la presence de peroxydes dans un milieu gazeux
US9194853B2 (en) 2010-10-19 2015-11-24 Commissariat A L'energie Atomique Et Aux Energies Alternatives Use of cyclic azaboronates as sensitive materials in sensors for detecting the presence of peroxides in a gaseous environment

Also Published As

Publication number Publication date
EP0110879A1 (fr) 1984-06-20

Similar Documents

Publication Publication Date Title
US4496722A (en) Reporter compounds
US4659817A (en) Reporter compounds containing boron
US11740233B2 (en) Antibody-nanoparticle conjugates and methods for making and using such conjugates
DE3788356T2 (de) Monomere phthalocyanin-reagenzien.
Thompson et al. Fluorescent zinc indicators for neurobiology
CA1337754C (fr) Composes fluorescents a base de phtalocyanine et de tetrabenztriazaporphyrine, avec decalage vers le rouge
US5135717A (en) Tetrabenztriazaporphyrin reagents and kits containing the same
US8389223B2 (en) Probes for anionic cell surface detection
CN114478473B (zh) 一种亮氨酸氨基肽酶化学发光检测试剂的合成及应用
CN112500386B (zh) 基于吡罗红肟的近红外HClO荧光探针、制备及其应用
CN113956265B (zh) 一种基于丙二醛响应的近红外分子探针、制备方法及其应用
EP0297303B1 (fr) Fluorophores pour encapsulation dans des liposomes
US8968997B2 (en) Benzoxazole-based fluorescent metal ion indicators
US9920020B2 (en) Using squaraine dyes as near infrared fluorescent sensors for protein detection
WO1983004255A1 (fr) Composes informateurs
US11045559B2 (en) Reactive fluorogenic compound and nanocomposite for sensing hydrogen sulfide comprising the same
EP0277139A1 (fr) Derives de 7-amino-4-methyle-coumarine-3-carboxyalkyle et leurs conjugues fluorescents.
CN108699091B (zh) pH响应性荧光化合物和应用其的线粒体自噬检测用组合物及细胞内线粒体自噬的检测方法
CN112694469B (zh) 基于吡罗红肼的HOCl荧光探针、制备方法及应用
CN103435625A (zh) 红光发射罗丹明类离子荧光探针及其应用
CA2266405A1 (fr) Composition de coloration immunohistochimique
CN108610270B (zh) 双氰基二苯代乙烯型双光子荧光溶剂生色水溶性探针及其合成方法和应用
US4970074A (en) Fluorophores for encapsulation into liposomes
IE870866L (en) Rainbow text device (composition for visual determination of¹analyte in fluids).
Dubicki Insights Towards Optimizing Palladium-Catalyzed Bioorthogonal Reactions

Legal Events

Date Code Title Description
AK Designated states

Designated state(s): DE GB JP

AL Designated countries for regional patents

Designated state(s): AT BE CH DE FR GB LU NL SE

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642