WO2004011642A1 - Systeme generique d'ancrage d'une membrane - Google Patents

Systeme generique d'ancrage d'une membrane Download PDF

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Publication number
WO2004011642A1
WO2004011642A1 PCT/US2003/008496 US0308496W WO2004011642A1 WO 2004011642 A1 WO2004011642 A1 WO 2004011642A1 US 0308496 W US0308496 W US 0308496W WO 2004011642 A1 WO2004011642 A1 WO 2004011642A1
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Prior art keywords
group
chemical moiety
membrane
binding
recognition
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PCT/US2003/008496
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English (en)
Inventor
Jugren G. Schmidt
Louis A. Silks, Iii
Basil I. Swanson
Clifford J. Unkefer
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The Regents Of The University Of California
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Priority to AU2003269805A priority Critical patent/AU2003269805A1/en
Publication of WO2004011642A1 publication Critical patent/WO2004011642A1/fr

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    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the present invention relates to chemical moieties including recognition, reporter and anchoring functionalities.
  • Such chemical moieties can be movably anchored to a membrane surface and the resultant membrane assembly can be used, e.g., in highly sensitive sensors.
  • a method for the detection of pre-selected species, e.g., biological and chemical agents, is provided using such sensors.
  • the present invention describes chemical moieties including a recognition functionality, a reporter functionality and an anchoring functionality, such an anchoring functionality allowing for attachment of such chemical moieties to a fluid surface of a membrane.
  • Such chemical moieties can be employed for the ultra-sensitive detection of, e.g., bacterial and viral pathogens and cancer.
  • the present invention provides a chemical moiety including: a central core comprised of an amino acid or an analog thereof having three chemically reactable sites thereon; an anchoring group attached to one of said three chemically reactable sites, said anchoring group adapted for mobile anchoring of said chemical moiety to a membrane surface; a recognition group attached to one of said three chemically reactable sites, said recognition group adapted for binding to a pre-selected species; and, a reporter group attached to one of said three chemically reactable sites, said reporter group adapted for providing a measurable signal upon binding between said recognition group and said preselected species.
  • the chemical moiety further includes one or more spacer groups.
  • spacer groups can be situated, e.g., between said recognition group and one of said three reactable sites of said central core, said spacer group characterized as having a sufficient length so as to provide said recognition group with a spatial orientation away from said membrane surface whereby access to said preselected species within a sample is facilitated, between said reporter group and one of said three reactable sites of said central core, said spacer group characterized as having a sufficient length and chemical functionality so as to provide said reporter group with a spatial orientation whereby said measurable signal in response to binding between said recognition group and said pre-selected species within a sample is enhanced and response from non-specific binding events is minimized, or between said anchoring group and one of said three reactable sites of said central core, said spacer group characterized as having a sufficient length so as to provide said anchoring group with pre-selected orientation and distance to the membrane.
  • the present invention further provides a membrane assembly including a membrane; and, one or more of the chemical moieties of the present invention movably anchored in a surface of said membrane.
  • the present invention further provides a method of detecting a pre-selected species including contacting a sample with a sensor including a substrate having a fluid membrane thereon, a chemical moiety situated at a surface of said fluid membrane, said chemical moiety including a central core comprised of an amino acid or an analog thereof having three chemically reactable sites thereon; an anchoring group attached to one of said three chemically reactable sites, said anchoring group adapted for mobile anchoring of said chemical moiety to a membrane surface; a recognition group attached to one of said three chemically reactable sites, said recognition group adapted for binding to a pre-selected species; and, a reporter group attached to one of said three chemically reactable sites, said reporter group adapted for providing a measurable signal upon binding between said recognition group and said pre-selected species, said chemical moiety characterized as being movable upon said
  • the present invention further provides a sensor for detection of a selected chemical or biological species including a substrate having a fluid membrane thereon; at least two chemical moieties situated at a surface of said fluid membrane, said chemical moieties each including a central core comprised of an amino acid or an analog thereof having three chemically reactable sites thereon; an anchoring group attached to one of said three chemically reactable sites, said anchoring group adapted for mobile anchoring of said chemical moiety to a membrane surface; a recognition group attached to one of said three chemically reactable sites, said recognition group adapted for binding to a pre-selected species; and, a reporter group attached to one of said three chemically reactable sites, said reporter group adapted for providing a measurable signal upon binding between said recognition group and said pre-selected species, said reporter group of a first chemical moiety including a single fluorescence donor molecule and said reporter group of a second chemical moiety including a single fluorescence acceptor molecule, said chemical moieties characterized as being movable upon said surface of said fluid membrane;
  • FIGURE 1 shows a chemical moiety or generic linker system of the present invention.
  • FIGURE 2 shows a synthetic outline setting out individual steps for a customized side chain approach on a chemical moiety in accordance with the present invention.
  • FIGURE 3 shows a synthetic outline for preparation of a generic sulfhydryl-reactive chemical moiety in accordance with the present invention.
  • the present invention is concerned with chemical moieties including three distinct functionalities, i.e., a recognition functionality, a reporter functionality and an anchoring functionality.
  • an anchoring functionality allows for attachment of such chemical moieties to a fluid surface of a membrane.
  • the chemical moieties or molecular assembly of the present invention can be represented by the general formula: (Re)(mA)CgRg where Re is a reporter group, mA is an anchoring group for mobile attachment to a membrane surface, Cg is a trifunctional core, and Rg is a reactive or recognition group.
  • a spacer group (Sp) can be situated between the trifunctional core (Cg) and any of the other functionalities, i.e., Re, mA or Rg.
  • the chemical moieties may also be referred to as a generic membrane anchoring linker system.
  • the chemical moieties or molecular assembly of the present invention are based on a trifunctional central core derived, e.g., from alpha, beta and gamma amino acids such as lysine, homoserine, glutamic acid, serine, cysteine, homocysteine, tyrosine, and analogs thereof.
  • Attached to this trifunctional central core are: (i) reporter groups, e.g., a fluorophore, an isotopic label, a magnetic material or another chemical and biochemical entity or label yielding an externally measurable output signal that can be correlated or assigned with a specific binding event; (ii) an anchoring group, e.g., a long chain alkyl group; and, (iii) attachment arms carrying a reactive group or recognition element to bind to a target species.
  • Such attachment arms can be composed of an amino acid side chain optionally modified or extended by alkyl, ether, thioether, sulfone, phosphate and phosphonate, amide or amine containing spacers.
  • Exemplary spacers include materials such as a polyalkylene glycol, e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG). These molecules can be used in assays towards binding events via the suitable reporter groups.
  • a suitable reporter group e.g., a BODLPY ® fluorophore or fluorescein.
  • a suitable reporter group e.g., a BODLPY ® fluorophore or fluorescein.
  • Exemplary synthetic routes are shown in Figs. 2 and 3.
  • the anchoring groups of the chemical moieties allow anchoring of such moieties to a membrane-containing medium or carrier (including vesicles, immobilized membranes, cell membranes, or membrane mimetic architectures, i.e., materials that mimic the surface of a cell membrane).
  • chemical moieties containing pep tide-binding ligands with an affinity to neuraminidase can be anchored via the anchoring groups to membranes attached to waveguides and may then be used for the highly sensitive and rapid detection of the presence of a virus.
  • the present invention presents the advantages including flexible and rapid access to membrane- anchored chemical moieties including the recognition or binding groups for pre-selected targets, e.g., biological and chemical entities.
  • targets e.g., biological and chemical entities.
  • these chemical moieties or specifically constructed molecules can allow both qualitative and quantitative detection of targets.
  • attachment arm design allows easy and rapid access to modified derivatives thereby allowing specific tailoring of physiochemical properties to accommodate various matrices in the analyte, e.g., a phosphate linked PEG side chain can ensure the necessary hydrophilicity to expose the recognition group into hydrophilic media.
  • This approach can overcome previous limitations of having a target ligand group accessible to the recognition or binding group in any analyte medium.
  • Primary applications can include the rapid, non-invasive and ultra-sensitive detection of multivalent binding molecules of medicinal and diagnostic importance. Examples include point of care detection of viruses, rapid detection of cancer cells and the detection of biological toxins for law enforcement and customs agencies.
  • Biomedical diagnostics and biochemical application of cell membrane bound linkers can facilitate cell modification and binding studies in vitro and in vivo. By immobilization of these molecules in cell membranes valuable tools to investigate binding events on cell surfaces can be provided. With suitable recognition or binding groups, such chemical moieties may be used in conjunction with well known techniques such as liposomes for therapeutic and diagnostic targeting of cells.
  • the multifunctional membrane anchor and recognition carrier of the invention as shown in Fig. 1 is derived from a trifunctional central core amino acid, hi one embodiment, the reporter group can be generally attached to the ⁇ -amine, the anchor group can be generally attached to the carboxyl-group and the recognition group attached the sidechain of the amino acid.
  • the central core amino acid is chosen preferentially from serine, homoserine, glutamic acid, cysteine, homocysteine, tyrosine and lysine.
  • the anchor group can be attached preferentially by amide or ester formation with the carboxyl of the amino acid and generally consists of long aliphatic chains, polyaromatics or other natural and commercially available long "fatty" lipids (e.g., dioleoyl-phosphatylethanolamine available from, e.g., Avanti Polaris Lipids, Inc.) suited to retain the lipid character necessary for anchoring in vesicles, natural and manmade membranes by hydrophobic interaction and insertion.
  • the anchoring group shown in Fig. 1 represents a dialkylamino- anchor (e.g., dioctadecylamine from, e.g., Sigma- Aldrich as a Fluka brand).
  • the reporter group can be preferably chosen from the following classes of commercially available detectable entities: fluorophores (e.g., BODIPY ® dyes from Molecular Probes), radioisotopes and chelated derivatives thereof (e.g., Tc), stable isotope labeled entities, magnetic particles or spin labeled carriers (e.g., for magnetic resonance imaging via nuclear magnetic resonance).
  • fluorophores e.g., BODIPY ® dyes from Molecular Probes
  • radioisotopes and chelated derivatives thereof e.g., Tc
  • stable isotope labeled entities e.g., magnetic particles or spin labeled carriers
  • magnetic particles or spin labeled carriers e.g., for magnetic resonance imaging via nuclear magnetic resonance
  • other entities attached to the ⁇ -amine may be chosen for therapeutic purposes rather than imaging purposes and can include cytotoxic entities, carriers of radioisotopes and ligands for metals commonly used in treatment and diagnosis of tumor tissue.
  • the reactive attachment site for the recognition group can be chosen from standard linking moieties, e.g., iodoacetamide, pyridinium disulfide, hydrazine, N-hydrosuccinamide ester and the like to allow attachment of biomolecules and manmade receptors using techniques practiced in the field as state of the art (see, e.g.,
  • the ligand or reactive site in this approach is either directly attached to the amino acid core sidechain or to a suitable spacer, e.g., a spacer consisting of alkylene glycol repeating units such as ethylene glycol repeating units, i.e., PEG n where n ranges from about 1 to about 30 or propylene glycol repeating units, i.e., PPG n where n ranges from about 1 to about 30.
  • a suitable spacer e.g., a spacer consisting of alkylene glycol repeating units such as ethylene glycol repeating units, i.e., PEG n where n ranges from about 1 to about 30 or propylene glycol repeating units, i.e., PPG n where n ranges from about 1 to about 30.
  • the attachment of the spacer and the repeating units can be derivatized to contain amino, sulfone, sulfonamide, phosphate, phosphonate, amide, carbonate and carbamate units starting, ending or inserted in repeats of spacer units.
  • These units can be further directly modified to terminate in amine, sulfhydryl, carboxylate, carbonyl, hydrazine, or halogenide functionalities.
  • the anchoring group for mobile attachment into a membrane surface can be attached to an amino acid core using standard peptide chemistry methods using activated esters or in situ activation.
  • One exemplary anchoring group consists of dialkylamino- (e.g. dioctadecylamine).
  • This lipid residue is exemplary only and is but one of a variety of suitable anchoring groups that contain alkyl, alkenyl-, alkynyl and polyaromatic chains of a carbon length from about 4 to about 30 carbons.
  • Such moieties are well suited for intruding into hydrophobic assemblies such as phospholipid membranes via insertion and hydrophobic interaction.
  • Attachment of a spacer e.g., a PEG spacer group
  • Polyethylene glycol units have been previously reported to be beneficial in reducing nonspecific interaction with proteins and are further intended to increase the hydrophihcity of this part of the anchoring group.
  • defined length and selectively protected PEG units can be obtained by solid phase immobilization and derivatization using a published procedure (Nash et al., Tetrahedron Letters, v. 37, 3625 (1996)).
  • Coupling to the anchoring group can be achieved by a variety of standard amino acid and peptide chemical methods either in solution or to the immobilized PEG unit on a solid support. Both intermediate active ester shown hereinafter as Example 1 and in situ activation shown hereinafter as Example 2 have been employed.
  • a second variant uses pre-derivatized core amino acid membrane anchors to obtain halogenides, phosphate and phosphonates as amino acid side chains. These functionalities can be further derivatized into a wide diversity of sidechain functions of sulfur (S), selenium (Se), metals with or without ethylene glycol or other polymeric chain and modifiers.
  • S sulfur
  • Se selenium
  • metals with or without ethylene glycol or other polymeric chain and modifiers.
  • a synthetic outline is given below for phosphates (Example 4) and halogenides (Example 5) and exemplified in the syntheses of the homoserine precursors.
  • Halogenides can be later converted to, e.g., sulfones, sulfoxides or sulfides. Installation of reactive attachment site for the recognition moiety
  • crosslinking reagents are suited to be incorporated in the outlined reaction schemes.
  • attaching a sulfhydryl reactive endgroup is shown, which is of particular interest to conjugate cysteine-terminated peptides, antibodies and antibody fragments.
  • Other reactive groups easily incorporated in the linker scheme include, but are not limited to, hydrazines, activated esters, photoactivated (azide or Affymax photolinkers) and halogenides.
  • these moieties are described as heterobifunctional crosslinkers with an amine reactive group that can be used for attachment to an amine terminated PEG, and a differentiated second functionality to attach, e.g., saccharides, oligonucleotides, peptides, proteins or chemically derived binding ligands (e.g., neuraminidase inhibitors).
  • amine reactive group that can be used for attachment to an amine terminated PEG
  • a differentiated second functionality to attach, e.g., saccharides, oligonucleotides, peptides, proteins or chemically derived binding ligands (e.g., neuraminidase inhibitors).
  • Examples of these types of crosslinking reagents are shown by Bioconjugate Techniques, G.T. Hermanson, Academic Press 1996. Commercially available crosslinking reagents are available from, e.g., Pierce/PerBio.
  • the ⁇ amine; and/or the ⁇ - amine (in the case of lysine), of the amino acid core of the membrane anchor can be protected by standard peptide synthetic protecting groups such as Boc or Fmoc. These protecting groups can be easily removed using the standard deprotection conditions of piperidine for Fmoc or TFA for Boc respectively.
  • the released amine is generally not long term stable to the reactive site installed previously on the PEG spacer arm and should therefore be converted immediately to the reporter containing moiety shown hereinafter as Example 11.
  • the synthetic approach of the present invention allows installation of a large variety of reporter groups.
  • the examples given are representative but non-exclusive to a general moiety suited to report on a binding event by physical, chemical and electrochemical means.
  • Such a report can be direct as an immediate readout or indirect as a cascade event within a suitable transfer or amplification mechanism (e.g., FRET, ELISA, or induction of coupled cell cascade response).
  • the chemical moieties of the present invention can be attached to a membrane. Subsequently, after attachment of the chemical moieties to a membrane, such a system can be used to test a sample for a pre-selected species within the sample as described by Song et al., U.S. Patent No. 6,297,059, such description incorporated herein by reference. While the examples shown in the chemical syntheses examples are for fluorophores, the chemistry shown is universal adaptable to other reporter and diagnostic entities practiced in the field of bioconjugate techniques.
  • DIPEA diisoproylethylamine
  • DCC N-N'-dicylohexylcarbodiimide
  • HV high vacuum (to 10 mT)
  • M molar MALDI: matrix assisted laser desorption ionization
  • NMR nuclear magnetic resonance spectrometry
  • PEG n polyethylene glycol, n indicates the length in ethylene glycol units
  • RV rotary evaporator RP: reversed phase chromatography SPDP: (N-succinimidyl 3-[2-pyridyldithio]propionate)
  • the reaction was stirred at room temperature (rt) for 8h, when TLC control (CH Cl /Et 2 O 10%) showed disappearance of the starting material and a product at Rf 0.4-0.5 (PMA).
  • the reaction was diluted with 200 mL diethyl ether and stored at 4°C overnight to precipitate the NHS. After filtration the solvents were removed on a RV followed by HV (2h).
  • the product was purified by FLC (EA/hexanes of 1 :9 to 1 :5) on silica gel followed by RV and HV (12h) to obtain the dioctadecylamide of the glutamic acid (8.842 g, 10.5 mmol, 91.4 %) as a waxlike, white solid.
  • Boc-glutamic acid dioctadecylamide 500 mg, 665 ⁇ mol was dissolved in 20 mL dry dichloromethane.
  • HOBT 108 mg, 0.8 mmol
  • DIPEA 285 ⁇ L, 1.66 mmol
  • DCC 343 mg, 1.66 mmol
  • TFA salt of FmocPeg-NH 2 381 mg, 732 ⁇ mol
  • DIPEA 150 ⁇ L, 0.87 mmol
  • Customized attachment sites such as ⁇ -bromo-homoalanine from homoserine is shown in Fig. 2 (step IN — > step Va) and can be accomplished as follows.
  • the Fmoc homoserine ⁇ -alcohol membrane anchor (169 mg, 0.2 mmol) was dissolved in 2 mL of dry dichloromethane.
  • Triphenylphosphine 80 mg, 0.3 mmol
  • carbon tetrabromide 100 mg, 0.3 mmol
  • the reaction was stirred at room temperature and monitored by TLC (dichlormethane: diethyl ether 2%), which showed conversion of the starting material at Rf 0.15 to a new product at Rf 0.6-0.7.
  • the volume was reduced to 2 mL, 5 mL diethyl ether was added and the precipitate of triphenylphosphine oxides removed by filtration.
  • the filtrate on solvent evaporation was purified by FLC on silica gel (dichloromethane: diethyl ether 2%).
  • the solid white product (144 mg, 0.144 mmol, 72 %) was light-sensitive and should be stored at -20°C shielded from light.
  • Customized attachment sites such as di-tertiary-butyl phosphono-Hse is shown in Fig. 2 (step IN --> step N) and can be accomplished as follows.
  • ⁇ -Fmoc-homoserine alcohol membrane anchor (169 mg, 0.2 mmol) was suspended in 5 mL tetrazole/AC ⁇ activator mix (ABI; D ⁇ A synthesizer reagent and di-tertiary-butylphosporamidate (0.25 mmol, 80 ⁇ L) were added.
  • the Fmoc protected PEG-glutamic acid dioctadecylamide was deprotected using standard peptide synthetic methods in dichloromethane with 20 % piperidine to give the free amine in 78% yield as white solid after FLC purification.
  • the Boc-glutamic acid dioctadecylamide-PEG-amine (93 mg, 105 ⁇ mol) was dissolved in 3 mL dry dichloromethane and the NHS ester of iodoacetic acid (50 mg, 176 ⁇ mol) dissolved in 3 mL dry dichloromethane and DIPEA (85 ⁇ L, 0.5 mmol) were added.
  • the reaction was shielded from light and stirred at room temperature for 30 minutes. The volume was reduced on RV to 2 mL.
  • the residue was purified by FLC using a gradient elution dichloromethane/methanol 5% to 10%.
  • the product was obtained as a waxlike, white solid (96 mg, 92 ⁇ mol, 87%), that should be stored at -20°C shielded from light.
  • EXAMPLE 8 An approach incorporating a lactose receptor was as follows. A solution of the pentafluorophenol- ⁇ -ester of N-Fmoc glutamic acid di-octadecylamide (200 mg, 0.193 mmol), the per-acetylated lactose derivative (made following the published procedure in the Journal of Organic Chemistry (Vol. 66, No. 9, 2001, pp. 2948-2956); 225 mg, 0.289 mmol), and diisopropylethylamine (25 mg, 0.193 mmol) was prepared in 2.5 mL of dichloromethane.
  • the Boc protecting group of the membrane anchor Glutamic acid iodoacetamide was removed in a 1 :1:1 mixture of dichloromethane:TFA:anisole.
  • the TFA salt of the anchor glutamic acid iodoacetamide was obtained in 98% yield after HV as a white salt and was sufficient in purity as determined by NMR for further reaction.
  • BODIPY ® -TRX NHS ester (10 mg, 15.7 ⁇ mol) was added to the TFA salt of the Glutamic acid membrane anchor -PEG- iodoacetamide (16.7 mg, 15.7 ⁇ mol) in 1 mL 0.1 M phosphate buffer at pH 8.5.
  • the residue in the BODIPY ® vials was dissolved in 2 mL dioxane and added to the reaction.
  • the reaction was stirred at room temperature under argon and shielded from light and monitored by TLC (dichloromethane-.methanol 5%) until complete conversion of the BODIPY ® starting material (Rf 0.8) to one major product at Rf 0.2-0.3.
  • the reaction mixture was frozen in liquid nitrogen and lyophilized to dryness.
  • the product was first purified by FLC on silica gel using a gradient from 5 to 10% methanol in dichloromethane and after concentration on RV the product was finally purified by HPLC on C18 RP using a gradient from methanol to 10% dichloromethane.
  • the product fractions were pooled then concentrated on RV and dried on HV to yield a blue solid (15.8 mg, 10.8 ⁇ mol, 68%), which should be stored at -70° C and handled under argon and shielded from light.
  • BODIPY -TMRX-NHS ester (5 mg, 8.2 ⁇ mol) was added to a solution of the lactose - Glutamic acid membrane linker (19.2 mg, 15.8 ⁇ mol) in 3 mL DMF.
  • DIPEA/NMP (2M, 2 mmol) were added and the reaction was stirred at room temperature shielded from light and monitored by HPLC on C18 Rp using a three-solvent gradient from water to acetonitrile to acetonitrile/THF. The reaction lead to formation of one predominant product within 3 hours at which point side-product formation became evident.
  • reaction mixture was frozen in liquid nitrogen and concentrated, then purified by HPLC on C18 Rp using the same three-solvent gradient from water to acetonitrile to acetonitrile/THF.
  • product-containing fractions were pooled, concentrated and dried on RV then HV to obtain a red crystalline solid (11.4 mg, 7.0 ⁇ mol, 85%).
  • the reaction volume was reduced to 1 mL and the product was purified by chromatography on basic alumina oxide with diethyl ether.
  • the greasy residual oil after evaporation of the solvent at RV was highly viscous and was kept for 3 days on HV until the weight stays constant.
  • the product was obtained as a greasy, wax-like dark blue solid (10 mg; 8.4 ⁇ mol; 84 %).

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Abstract

L'invention concerne un fragment chimique, et des détecteurs et des procédés basés chacun sur un tel fragment chimique. Le fragment chimique comprend un noyau central d'un acide aminé ou d'un analogue dudit acide aminé présentant trois sites capables de réaction chimique; un groupe d'ancrage fixé à l'un des trois sites capables de réaction chimique, le groupe d'ancrage étant adapté pour assurer un ancrage mobile du fragment chimique à la surface d'une membrane; un groupe de reconnaissance fixé à l'un des trois sites capables de réaction chimique, le groupe de reconnaissance étant adapté pour se lier à une espèce présélectionnée; et un groupe rapporteur fixé à l'un des trois sites capables de réaction chimique, le groupe rapporteur étant adapté pour produire un signal mesurable après liaison entre le groupe de reconnaissance et l'espèce présélectionnée.
PCT/US2003/008496 2002-03-21 2003-03-20 Systeme generique d'ancrage d'une membrane WO2004011642A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010099200A1 (fr) * 2009-02-24 2010-09-02 Nektar Therapeutics Conjugués oligomère-acides aminés

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6235535B1 (en) * 1996-06-28 2001-05-22 Valtion Teknillinen Tutkimuskeskus Fluorescent energy transfer ligand interaction assay on a lipid film
US6297059B1 (en) * 1998-06-22 2001-10-02 The Regents Of The University Of California Triggered optical biosensor
US6627396B1 (en) * 1999-10-28 2003-09-30 The Regents Of The University Of California Influenza sensor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6235535B1 (en) * 1996-06-28 2001-05-22 Valtion Teknillinen Tutkimuskeskus Fluorescent energy transfer ligand interaction assay on a lipid film
US6297059B1 (en) * 1998-06-22 2001-10-02 The Regents Of The University Of California Triggered optical biosensor
US6627396B1 (en) * 1999-10-28 2003-09-30 The Regents Of The University Of California Influenza sensor

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US20120053237A1 (en) * 2009-02-24 2012-03-01 Nektar Therapeutics Oligomer-Amino Acid Conjugates
US9192681B2 (en) 2009-02-24 2015-11-24 Nektar Therapeutics Oligomer-amino acid conjugates
US9487473B2 (en) 2009-02-24 2016-11-08 Nektar Therapeutics Oligomer-amino acid conjugates

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