WO2010071772A2 - Synthèse de 3b-amino-5-cholestène et de 3b-halogénures apparentés mettant en jeu des réarrangements de 1-stéroïde et rétro-1-stéroïde - Google Patents

Synthèse de 3b-amino-5-cholestène et de 3b-halogénures apparentés mettant en jeu des réarrangements de 1-stéroïde et rétro-1-stéroïde Download PDF

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WO2010071772A2
WO2010071772A2 PCT/US2009/067897 US2009067897W WO2010071772A2 WO 2010071772 A2 WO2010071772 A2 WO 2010071772A2 US 2009067897 W US2009067897 W US 2009067897W WO 2010071772 A2 WO2010071772 A2 WO 2010071772A2
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receptor
synthetic
cells
receptors
cholestene
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WO2010071772A3 (fr
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Blake R. Peterson
Qi Sun
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The Penn State Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
    • C07J41/0011—Unsubstituted amino radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring the nitrogen atom being directly linked to the cyclopenta(a)hydro phenanthrene skeleton
    • C07J41/0027—Azides

Definitions

  • the present invention relates to new synthetic receptors and the use of the synthetic receptors for delivering a protein, peptide, drug, prodrug, nucleic acid, or a small molecule into a target cell via receptor-mediated endocytosis.
  • the synthetic receptors comprise a membrane-binding element that is a cholesterol derivative and novel methods for stereoselective preparation of 3 ⁇ -amino-cholestene and related 3 ⁇ - halides are also disclosed for use according to the invention.
  • Receptor-mediated endocytosis involves the movement of ligands bound to membrane receptors into the interior of an area bounded by the membrane through invagination of the membrane. The process is initiated or activated by the binding of a receptor- specific ligand to the receptor.
  • the internal pH of these endosomes decrease by the action of proton pumps, changing the conformation of the receptor and/or ligand.
  • the result is the release of the ligand and the formation of separate receptor-containing vesicles and ligand-containing ones.
  • the receptor-bearing vesicles are delivered to the cell membrane where they are released and "recycled" for additional use.
  • the resulting vesicles, along with the internalized ligand are delivered to and fused with lysosomes where the eventual breakdown likely takes place.
  • Another destination of some ligands internalized by RME including certain drugs, viral proteins, and protein toxins is escape from endosomes and entry into the cellular cytoplasm or nucleus.
  • Receptor-mediated endocytotic activity has been utilized for delivering exogenous molecules such as proteins and nucleic acids to cells.
  • exogenous molecules such as proteins and nucleic acids
  • a specified ligand is chemically conjugated by covalent, ionic or hydrogen bonding to an exogenous molecule of interest (i.e. the exogenous compound), where the modified ligand is still able to bind to its cognate receptor.
  • this method limits delivery of the exogenous molecule to cells that display a particular receptor.
  • agents including pharmaceuticals, small molecules, peptides and oligonucleotides
  • Cholesterol mimics have been utilized to facilitate delivery of siRNA, enhance DNA transfection, probe cellular membrane subdomains, and have been proposed for tumor targeting applications. See e.g., Wolfram, C. et al, Nat. Biotechnol. 2007, 25, 1149- 1157; Pitard, B. et al, Proc. Natl. Acad. ScL U.S.A. 1999, 96, 2621-2626; Sato, S. et al, J. Biol. Chem. 2004, 479, 23790-23796; Firestone, R.A., Bioconjug. Chem. 1994, 5, 105-113.
  • synthetic receptors are provided in which the receptor includes the following regions: at least one binding, chelating, or mimicking motif linked to a membrane-binding element that anchors the receptor to a plasma membrane of a cell.
  • the binding, chelating, or mimicking motif and membrane-binding element are linked to the other via a linker region.
  • the membrane-binding element includes at least of one the following: cholesterol, dihydrocholesterol, ergosterol, brassicasterol, derivatives of cholesterol, dihydrocholesterol, ergosterol, brassicasterol, and related compounds thereof.
  • the membrane-binding element includes at least one of the following: cholesterylamine, dihydrocholesterylamine, ergosterylamine, brassicasterylamine, derivatives of cholesterylamine, dihydrocholesterylamine, ergosterylamine, brassicasterylamine, and related compounds thereof.
  • synthetic receptors include synthetic protein- binding receptors, synthetic metal-chelating receptors, synthetic immunoglobulin Fc- binding receptors, synthetic cytokine or growth factor-binding receptors, synthetic drug- binding receptors, synthetic lipid mimicking receptors, and synthetic transmembrane receptors.
  • Another embodiment includes the loading of low-density lipoprotein (LDL) with synthetic receptors for the delivery of proteins, carbohydrates, nucleic acids, lipids, or drugs, to cells, tissues, or tumors.
  • LDL low-density lipoprotein
  • a protein into a cell that includes contacting the cell with a synthetic cell receptor so that the synthetic receptor inserts into the cell plasma membrane on the cell's surface.
  • the synthetic receptor has a protein- binding motif and a membrane-binding element that anchors the synthetic receptor into the cell plasma membrane.
  • the inserted synthetic receptor binds a protein via its protein- binding motif, thereby triggering receptor-mediated endocytosis of the protein-bound synthetic receptor and delivery of the protein into the cell.
  • the present invention provides pharmaceutical compositions comprising a synthetic receptor and a pharmaceutically acceptable carrier.
  • the present invention further includes methods of using the synthetic receptors for various applications.
  • the synthetic receptors may be used as cellular probes.
  • the present invention provides a method of treating a disease, disorder, or condition comprising delivering a therapeutically effective amount of a protein, peptide, drug, prodrug, or small molecule into a target cell using a synthetic cell receptor.
  • the synthetic receptors may be used in enzyme replacement therapy or to treat viral, yeast, or bacterial infections, cancer, inflammation, or autoimmune diseases.
  • the invention includes methods of modulating the immune response of a subject by removing a protein of interest from circulation in a subject, in particular extracellular ligands.
  • a method for inducing apoptosis by contacting a synthetic lipid-mimicking receptor with a cell is provided.
  • methods of using synthetic metal chelating receptors as a contrast agent in a patient in need of an MRI by delivering a synthetic metal chelating receptor into a cell population either in vivo, ex vivo or in vitro. Methods of this invention are useful for in vitro and in vivo applications.
  • the present invention provides methods for synthesizing preferred membrane-binding elements, preferably cholesterylamine derivatives, including 3 ⁇ -amino-5-cholestene (3 ⁇ -cholesterylamine) and related 3 ⁇ -halides.
  • preferred membrane-binding elements preferably cholesterylamine derivatives, including 3 ⁇ -amino-5-cholestene (3 ⁇ -cholesterylamine) and related 3 ⁇ -halides.
  • the synthesis methods convert inexpensive cholesterol to 3 ⁇ -amino-5- cholestene and related 3 ⁇ -halides through a mechanism involving i-steroid and retro-j- steroid rearrangements.
  • Fig. 1 Structures of the small natural receptor ganglioside GMl, novel synthetic receptors, and related control compounds.
  • Fig. 2 The synthetic receptor targeting approach for enhancing cellular uptake of impermeable ligands. Synthetic mimics of cell surface receptors are added to living mammalian cells. These cells internalize cognate ligands such as macromolecular antibodies (IgG) bound to bacterial protein A (PrA) by synthetic receptor-mediated endocytosis.
  • IgG macromolecular antibodies
  • PrA bacterial protein A
  • Fig. 3 The construction of synthetic receptors by modifying a previously reported synthesis of 3beta-cholesterylamine.
  • the reagents and the conditions that correspond with the figure are as follows: (a) Oxalyl chloride, DMSO, CH 2 Cl 2 , TEA, -78 0 C. (b) L- Selectride, THF, -78 0 C. (c) PPh 3 , HN 3 , DEAD, benzene, (d) LiAlH 4 , Et 2 O, 0 0 C. (e) 2- Nitrobenzensulfonyl chloride, DIEA, THF. (f) Boc-3-chloropropylamine, K 2 CO 3 , DMA, 120 0 C.
  • Fig. 4 Determination of the apparent affinities of DNP (27, Panel A) and NBD (28, Panel B) derivatives for rabbit polyclonal anti-DNP IgG (Sigma) by fluorescence polarization.
  • the fluorescein moiety of 27 is shown in the dianionic form that predominates at pH 7.4. l Fixed concentrations of 27 (20 nM) and 28 (100 nM) in PBS (pH 7.4, 100 ⁇ L) were employed.
  • the data shown in Panel B was corrected to compensate for partial binding-induced quenching of the NBD fluorophore. 2 ' 3 These experiments were run in triplicate, and errors reflect 95% confidence intervals.
  • Fig. 5 Analysis of uptake of red fluorescent anti-DNP / PrA-AF633 by cells treated with the green fluorescent receptors 6-9.
  • Jurkat lymphocytes were treated with receptors 6-9 (10 ⁇ M) for 1 h followed by addition of anti-DNP / PrA-AF633 for 4 h.
  • Cells were washed with media containing 6-(2,4-dinitrophenyl) aminohexanoic acid (100 ⁇ M) to remove any non-internalized protein prior to analysis by flow cytometry.
  • Panel A Median green (NBD) and red (AF633) cellular fluorescence resulting from treatment with 6-9 and anti-DNP / PrA-AF633.
  • Panels B-E the distribution of green and red cellular fluorescence after treatment with anti-DNP / PrA-AF633 alone or receptor 6 (10 ⁇ M) and anti-DNP / PrA-AF633. UV absorbance measurements were employed to equalize receptor concentrations prior to addition to cells. Total cellular fluorescence from the NBD groups of receptors 6-9 was within two-fold.
  • Fig. 6 Confocal laser scanning and DIC microscopy of living Jurkat lymphocytes treated with green fluorescent receptor 9 (10 ⁇ M) and red fluorescent BODIPY TR ceramide (5 ⁇ M) as a probe of the golgi apparatus and nuclear membrane. Yellow pixels indicate colocalization of green and red fluorophores. These experiments revealed that the ineffective receptor 9 (the iV-acyl analogue) exhibits a unique intracellular localization compared with receptors 6-8.
  • Fig. 7. Confocal laser scanning and DIC microscopy of living Jurkat lymphocytes transfected with the green fluorescent late endosome / lysosome marker EGFP-lgpl20 4 and subjected to ligand uptake mediated by receptor 2. Transfected cells were treated with receptor 2 (10 ⁇ M) for 1 h followed by addition of red fluorescent anti-DNP / PrA-AF594 for 4 h. Yellow pixels indicate colocalization of green and red fhiorophores.
  • Fig. 8 Synthetic receptor-mediated uptake of protein ligands in Jurkat lymphocytes. Cells were pretreated with receptors for one hour at 37 0 C prior to addition of an anti-DNP IgG/PrA complex.
  • Panel A Confocal laser scanning and differential interference contract (DIC) microscopy of cells treated with green fluorescent receptor 6 (10 ⁇ M) followed by the addition of red anti-DNP/PrA-AF594 (for 4 hours). Yellow pixels indicate the localization of green and red fluorophores.
  • Panel B Dose-dependent magnitude of uptake of anti-DNP/PrA-AF488 quantified by flow cytometry after 4 hours.
  • Fig. 9 Subcellular localization of synthetic receptors in living Jurkat lymphocytes.
  • Panel A DIC (left images) and epifluorescence micrographs (right images) of cells treated for 1 hour with green fluorescent receptors 6-9 (10 ⁇ M).
  • Panel B Quantification of cell surface fluorescence by sodium dithionite quenching of exposed fluorophores. Cells treated as shown in Panel A were washed with sodium dithionite (5 mM) for 10 seconds at 22 0 C and residual fluorescence quantified by flow cytometry.
  • Panel C Quantification of the cellular half- life of receptor 2.
  • Fig. 11 Quantification of efflux kinetics of receptor 10 from endosomes to the plasma membrane of Jurkat lymphocytes by back exchange.
  • Cells treated with 10 (1 ⁇ M, 1 hour) were cooled to 4 0 C and cell surface fluorophores removed (>90%) by repeated washing with ice-cold back exchange media containing BSA (1%) and methyl- ⁇ - cyclodextrin (2 mM).
  • Cells were resuspended in back exchange media at 37 0 C to resume recycling, cellular fluorescence was quantified by flow cytometry, and loss of fluorescence was evaluated with a one-site exponential decay model.
  • Fig. 12 Association of receptor-ligand complexes with putative lipid raft functions of plasma membranes of Jurkat lymphocytes.
  • Panel A Cells were treated with AF488- labeled protein ligand for 30 minutes at 4 0 C.
  • Panel B Cells were treated with receptors 2- 5 (1 ⁇ M) for 1 hour at 37 0 C followed by anti-DNP-AF488 for 30 minutes at 4 0 C. Cells fractionated by ultracentrifugation for 20 hours at 4 0 C in a 2% to 40% sucrose step gradient containing 0.2% Triton-X.
  • CT-B Cholera toxin B subunit.
  • Fig. 13 A simple model of synthetic receptor-mediated endocytosis. Synthetic receptors embedded in cellular plasma membranes rapidly cycle between the cell surface and intracellular endosomes. Binding of the ligand (IgG) results in association with lipid rafts and uptake of the complex by endocytosis. Dissociation in endosomes frees the receptor to return to the cell surface. The protein ligand is sorted to late endosomes and lysosomes.
  • Fig. 14 Structures of the antibiotic vancomycin (1) and a green fluorescent derivative oregon green vancomycin (2).
  • Fig. 15 Structures of a vancomycin-binding synthetic receptor bearing a D-Phe-D- Ala motif (3), a related L-Phe-L-Ala analogue that does not bind vancomycin (4), and an amide analogue that binds vancomycin but exhibits a lower affinity for the cellular plasma membrane (5).
  • Fig. 16 The synthetic receptor targeting strategy for endocytic delivery of vancomycin (1).
  • Fig. 17 Confocal laser scanning and differential interference contrast (DIC) microscopy of living J-774 macrophages alone (Panel A) and infected with L. monocytogenes (Panel B). Prior to microscopy, receptor 2 (10 ⁇ M) was added to cells for 1 h, cells were washed to remove excess receptor, and fluorescent vancomycin (3, 3 ⁇ M) was added for 4 h. Scale bar: 10 ⁇ m.
  • Fig. 18 Flow cytometric analysis of cellular uptake of fluorescent vancomycin 2 promoted by synthetic receptor 3 and control compounds.
  • Preload conditions Receptor 3 or analogues (4, 5) were added to cells for 1 h to load the plasma membrane, cells were washed, and 2 (3 ⁇ M) was added for 4 h.
  • Premix conditions Receptor 3 or analogues (4, 5) were preequilibrated with 2 (3 ⁇ M) at 23 0 C for 1 h followed by addition of this mixture to cells for 4 h.
  • Fig. 19 Effects of premixed receptor 3 and vancomycin (1) on HeLa cells infected with L. monocytogenes. Infected cells were treated with 1 and 3 for 6 h at the concentrations indicated, then washed with media lacking antibiotics.
  • Panel A Viability of L. monocytogenes cultured from infected HeLa cells. BHI media was added, and bacterial growth was quantified by absorbance measurements after an additional 18 h.
  • Panel B DMEM media was added and viability of infected HeLa cells after an additional 30 h in culture quantified by flow cytometry.
  • Fig. 20 Synthetic receptor targeting of fluorescent vancomycin 2 to specific tissues in vivo. Mice were injected i.p. with compounds, tissues were isolated after 8 h, and cellular fluorescence was analyzed by flow cytometry. In the spleen, liver, heart, kidney, and pancreas, two different populations of cells were analyzed.
  • Fig. 21 SDS PAGE analysis of oligohistidine-tagged AcGFP proteins.
  • Lane 1 Protein molecular weight marker.
  • Lane 2 Crude lysate from cells expressing AcGFP(His) 10 .
  • Lane 3 Purified AcGFP(His) 10 .
  • Lane 4 Crude lysate from cells expressing AcGFP(His) 6 .
  • Lane 5 Purified AcGFP(His) 6 .
  • Fig. 22 Delivery of oligohistidine-tagged AcGFP to endosomes and lysosomes.
  • Jurkat lymphocytes were treated with synthetic receptor 1 (10 ⁇ M) at 37 0 C for 1 h, the cells were washed with media, and Ni(OAc) 2 (100 ⁇ M), green fluorescent AcGFP(HiS) 1 O (3.2 ⁇ M), and red fluorescent Dil-loaded LDL (50 ⁇ g / mL ( ⁇ 0.1 ⁇ M)) were added at 37 0 C for an additional 4 h.
  • Fig. 23 Inhibition of protein uptake at 4 0 C.
  • Jurkat lymphocytes were treated with synthetic receptor 1 (10 ⁇ M) at 37 0 C for 1 h, washed to remove unincorporated receptors, the cells were cooled to 4 0 C, and AcGFP(His) 10 (3.2 ⁇ M) and Ni(OAc) 2 (100 ⁇ M) were added at 4 0 C for 4 h.
  • Fig. 24 Confocal laser scanning (left) and differential interference contrast (right) micrographs of living Jurkat lymphocytes.
  • Cellular plasma membranes were preloaded with receptor 1 (10 ⁇ M) for 1 h at 37 0 C, cells were washed with fresh media, and media containing AcGFP-HiS 10 (3.2 ⁇ M) and Ni(OAc) 2 (100 ⁇ M) was added for an additional 4 h at 37 0 C.
  • Cells shown in Panel A were imaged immediately after this treatment.
  • Cells shown in Panel B were washed with NTA (400 ⁇ M) prior to microscopy to remove non- internalized protein from the cell surface.
  • Scale bar 10 ⁇ m.
  • Fig. 25 Analysis of cellular uptake of His-tagged AcGFP proteins by flow cytometry. Each bar represents the average fluorescence of 10,000 cells. Unless otherwise noted, cellular plasma membranes of Jurkat lymphocytes were preloaded with receptor 1 (10 ⁇ M) for 1 h at 37 0 C, cells were washed with fresh media, and His-tagged AcGFP (3.2 ⁇ M) / metal diacetate solution (100 ⁇ M) was added for 4 h at 37 0 C. Prior to analysis, cells were washed with NTA (400 ⁇ M, 30 min) in PBS (pH 7.4) to remove bound surface protein. Panel A: Dose dependence of receptor-mediated uptake.
  • Premix conditions a solution containing receptor 1, Ni(OAc) 2 , and AcGFP(His) 10 was added to cells for 4 h.
  • Panel B Dependence on [Ni(OAc) 2 ].
  • Panel C Omission control experiments.
  • Panel D Uptake of (His) 6 and (HiS) 1O fusion proteins promoted by different metal diacetates.
  • Fig. 26 Uptake of fluorescent human IgG by FcR + and FcR " human cells. Micrographs shown on the top. Confocal laser scanning and DIC images of human THP-I monocytes (left image) and human Jurkat lymphocytes (middle and right images) treated with green fluorescent human IgG.
  • sFcR synthetic Fc receptor. Histogram shown on the bottom: Flow cytometric quantitation of cellular uptake of fluorescent human IgG by THP-I monocytes and Jurkat lymphocytes. sFcR: synthetic Fc receptor. PrA: Protein A, a competitor that binds to the Fc region of the IgG. Fig. 27. Uptake of synthetic receptors by certain mammalian cells requires low- density lipoprotein (LDL).
  • LDL low- density lipoprotein
  • Fig. 28 Examination of the conversion of mesylate (2) to 3 ⁇ -azido-5-cholestene (3) using 1 H NMR (400 MHz).
  • Fig. 28A shows 1 H NMR spectra of mesylate (2) before (bottom spectrum) and at selected time points after the addition of TMSN 3 (1 equiv) and BF 3 -OEt 2 (2 equiv) to mesylate (2) (0.5 mmol) in CDCl 3 (0.5 mL).
  • the arrows point to signals identified as the C6 proton of 6 ⁇ -azido-3 ⁇ , 5-cyclo-5 ⁇ -cholestane (12).
  • Fig. 28B shows the observed chemical shifts of mesulate (2), 6 ⁇ -azido-3 ⁇ ,5-cyclo-5 ⁇ -cholestane (12), and 3 ⁇ -azido-5-cholestene (3).
  • the present invention relates to new synthetic receptors. More particularly, the invention relates to methods for synthesizing preferred cholesterylamine-derived membrane-binding elements, preferably 3 ⁇ -amino-5-cholestene (3 ⁇ -cholesterylamine) and related 3 ⁇ -halides through a mechanism involving i-steroid and retro-j-steroid rearrangements.
  • preferred cholesterylamine-derived membrane-binding elements preferably 3 ⁇ -amino-5-cholestene (3 ⁇ -cholesterylamine) and related 3 ⁇ -halides through a mechanism involving i-steroid and retro-j-steroid rearrangements.
  • Sun, Q. et ah Practical Synthesis of 3beta-Amino-5-Cholestene and Related 3beta-Halides Involving i-Steroid and Retro-i-Steroid Rearrangements, Org. Lett. 2009, 11, 567-570.
  • the present invention relates to the use of the synthetic receptors for delivering a polymer, protein, peptide, drug, prodrug, nucleic acid, or small molecule into a target cell via receptor-mediated endocytosis.
  • one aspect of the present invention is to deliver a desired protein, peptide, prodrug, or small molecule from the outside of a cell to the inside of a cell.
  • the present inventors contemplate the use of synthetic receptors to treat a variety of diseases, conditions, and disorders, including those for which the conventional therapeutic alternatives are not very effective or are non- existent.
  • the present inventors are the first to recognize that synthetic receptors comprising membrane-binding elements of cholesterol, dihydrocholesterol, ergosterol, brassicasterol, derivatives of cholesterol, dihydrocholesterol, ergosterol, brassicasterol, and related compounds thereof linked to a protein binding group provide a novel mechanism for receptor mediated endocytosis of the synthetic receptor bound to at least one specific protein and delivery of the protein to the endosome/lysosome.
  • the synthetic receptors' membrane-binding elements insert into the cell's plasma membranes, project a protein-binding motif from the cell surface so that the protein-binding motif binds a cognate protein.
  • the synthetic receptor undergoes receptor-mediated endocytosis allowing the cell to internalize the conjugate of protein bound to the synthetic receptor.
  • the synthetic receptors allow for cycling between plasma membranes and intracellular endosomes.
  • the synthetic receptor according to the invention may, in addition, be linked or conjugated to other molecules, including but not limited to for example, a labeling molecule which makes it possible, for example, to visualize the distribution or localization of the synthetic receptor after administration in vitro or in vivo.
  • a labeling molecule which makes it possible, for example, to visualize the distribution or localization of the synthetic receptor after administration in vitro or in vivo.
  • the protein that binds to the protein-binding motif may be labeled to visualize the distribution or localization of the protein-bound synthetic receptor after administration in vitro or in vivo.
  • LDL low-density lipoprotein
  • / or other lipoproteins prior to their uptake by cells.
  • LDL low-density lipoprotein
  • Certain mammalian cells take up LDL loaded with synthetic receptors via natural LDL receptors expressed on the cell surface. This delivery mechanism enables targeting of these compounds to specific cells or tissues in vivo. Because tumors often over express natural LDL receptors, the synthetic receptors described here enable tumor or tissue targeting in vivo. Cell lines that lack LDL receptors or that possess other cholesterol uptake mechanisms can take up synthetic receptors through other mechanisms of action.
  • a synthetic receptor comprises a membrane- binding element and a target-binding motif.
  • a synthetic receptor comprises a membrane-binding element, a linker region, and a target-binding motif.
  • the term "membrane-binding element” includes molecules that can be inserted into a lipid membrane through a hydrophobic anchor and allows the molecule to reside on the cell surface or reside on the cell surface and cycle between endosomes and cell surface.
  • the membrane-binding element comprises proteins, peptides, peptidomimetics, phospholipids, sphingolipids, steroids, cholesterol, dihydrocholesterol, ergosterol, brassicasterol, derivatives of proteins, peptides, peptidomimetics, phospholipids, sphingolipids, steroids, cholesterol, dihydrocholesterol, ergosterol, brassicasterol, and related compounds thereof.
  • the membrane-binding element comprises cholesterylamine, dihydrocholesterylamine, ergosterylamine, brassicasterylamine, derivatives of cholesterylamine, dihydrocholesterylamine, or ergosterylamine, brassicasterylamine, and related compounds thereof.
  • the membrane-binding element comprises 3- cholesterylamine, 3-dihydrocholesterylamine, 3-ergosterylamine, derivatives of 3- cholesterylamine, 3-dihydrocholesterylamine, 3-ergosterylamine, or 3-brassicasterylamine, and related compounds thereof.
  • the term “derivative” refers to a chemical substance related structurally to another, i.e., an "original" substance, which can be referred to as a "parent” compound.
  • the term “derivative” as used herein with reference to proteins, peptides, peptidomimetics, phospholipids, sphingolipids, steroids, cholesterol, dihydrocholesterol, ergosterol, brassicasterol, derivatives of cholesterol, dihydrocholesterol, ergosterol, brassicasterol, of the membrane-binding element of the present invention refers to a molecule that contains at least the portion of the proteins, peptides, peptidomimetics, phospholipids, sphingolipids, cholesterol, dihydrocholesterol, ergosterol, brassicasterol, derivatives of cholesterol, dihydrocholesterol, ergosterol, brassicasterol, and related compounds thereof that allows the molecule to insert into a cell plasma membrane.
  • derivatives of cholesterol include cholesteryl esters and cholesteryl carbamates.
  • derivative as used herein with reference to 3- cholesterylamine, 3-dihydrocholesterylamine, or 3-ergosterylamine of the membrane- binding element of the present invention refers to a molecule that contains at least the portion of the 3-cholesterylamine, 3-dihydrocholesterylamine, 3-ergosterylamine, or 3- brassicasterylamine, that allows the molecule to insert into a cell plasma membrane.
  • derivatives of the membrane- binding element of the present invention refers to a molecule that contains at least the portion of the 3 ⁇ -cholesterylamine, 3 ⁇ -dihydrocholesterylamine, 3 ⁇ -ergosterylamine or 3 ⁇ - brassicasterylamine that allows the molecule to insert into a cell plasma membrane.
  • derivatives of 3 ⁇ -cholesterylamine include, without limitation, N-alkyl, N- aryl, and N-acyl 3 ⁇ -cholesterylamines.
  • the term "related compound” refers to a chemical molecule that associates with the cellular plasma membrane, is hydrophobic and has a positive charge, for example, a positive charge from an amine group, or has a polar functional group that allows the molecule to reside on the cell surface and yet cycle between the endosome and cell surface without becoming trapped intracellularly.
  • Examples include without limitation a protonated cholesterylamine or a cholesteryl ester, cholesteryl amide, cholesteryl ether, or cholesteryl carbamate linked to another polar or non-polar headgroup.
  • the membrane-binding element as described above can be used with any of the synthetic receptors of the present invention regardless of the various binding, chelating or mimicking motifs and/or use of a linker region.
  • the present inventors also contemplate that a membrane-binding element with an affinity for membranes can be created by enhancing the hydrophobicity of the membrane- binding element domain (e.g., increasing the amount of hydrophobic residues or functional groups, replacing non-hydrophobic residues with hydrophobic residues, and swapping in a hydrophobic domain that consists of entirely hydrophobic residues).
  • the synthetic receptor comprises a linker region that links the target-binding motif of the synthetic receptor to the membrane-binding element of the synthetic receptor.
  • the linker region is structurally variable and can have one or multiple functions depending on the intended use of the receptor.
  • the linker region (shown as position X 1 in the embodiments above) may include but is not limited to zero, one, or more atoms selected from the group of atoms, C, N, S, and O and/or other chain-extending atoms, zero, one, or more alkane, alkene, alkyne, aryl, ketone, amine, amide, ester, ether, urea, carbamate, heterocyclic, and related functional groups, either linear or cyclic, zero, one, or more alpha, beta, gamma, and delta amino acids as well as aminohexanoic acid and related structures.
  • amino acid subunits may also include side-chains such as those found in natural L- and nonnatural D-configuration alpha amino acids or other amino acids.
  • the linker may serve as a spacing group between the membrane-binding element and the protein-binding motif so as to minimize the possibility that the membrane-binding element will interfere with the interaction or binding of the binding motif with the target molecule, e.g., a peptide, protein, carbohydrate small molecule, drug, prodrug or nucleic acid that is to be internalized.
  • the target molecule may be noncovalently bound or covalently linked to the target-binding motif.
  • the linker may also facilitate association with LDL, with the cell surface, and rapid cycling between the cell surface and endosomes, as demonstrated by the present inventors in Example 11 and as discussed below.
  • the linker region may contribute to receptor localization and ligand uptake efficiency. See Examples 8 and 9.
  • the linker as described above can be used with any of the synthetic receptors of the present invention regardless of the differing membrane-binding elements or the various target-binding, chelating or mimicking motifs.
  • the linker comprises zero, one or more atoms that may be selected from the group of atoms, C, N, S, and O and/or other chain- extending atoms repeated 0 to 100 or more times as indicated by the symbol x ⁇ . ⁇ .
  • the linker region comprises one or more groups selected from the following: alkane, alkene, alkyne, aryl, ketone, amine, amide, ester, ether, urea, carbamate, heterocyclic, and related functional groups in position X 1 . These functional groups can be linear or cyclic.
  • the linker may comprise 0 to 100 or more subunits such as alpha, beta, gamma, and delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and carboxylic acid.
  • These amino acid subunits may also include side-chains such as those found in natural L- and nonnatural D- configuration alpha amino acids or other amino acids.
  • the linker comprises one or more alpha amino acids, beta amino acids, gamma amino acids, delta amino acids, 6 aminohexanoic acid or related compounds to facilitate association with the cell surface and rapid cycling between the cell surface and endosomes.
  • the linker region is located between the last amine of the cholesterylamine and the first amino acid of the protein-binding or other binding element.
  • the linker region has at least three carbon atoms between the cholesterylamine and the first polar functional group (such as an amide).
  • the present inventors have found that modifying the linker region of the synthetic receptor effects the receptor localization and ligand uptake efficiency.
  • the present inventors have found that insertion of amide derivatives of 3 ⁇ -cholesterylamine in the linker region of the receptor elicits certain effects in trafficking and expression of the receptor on the cell surface.
  • the present inventors have found that N-acyl derivatives of 3 ⁇ -cholesterylamine in the linker region results in decreased expression of the receptor on the cell surface, especially when compared with the N-alkyl receptors containing ⁇ -alanine subunits in the linker.
  • the linker plays a role in maintaining or increasing a synthetic receptor population on the cell surface by keeping the synthetic receptors cycling from the cell surface to the endosome back to the cell surface.
  • the linker region of the synthetic receptor comprises ⁇ -alanine subunits.
  • the linker comprises an N-acyl derivative of 3 ⁇ -cholesterylamine.
  • the linker comprises an N-aryl derivative of 3 ⁇ -cholesterylamine.
  • the linker comprises an N-alkyl derivative of 3 ⁇ -cholesterylamine.
  • the synthetic receptor comprises a target- binding motif.
  • target-binding motif refers to a region in the receptor that recognizes and binds non-covalently or is covalently linked to a protein, a polypeptide, a peptide, an antibody, an immunoglobulin, a ligand, a cytokine, a growth factor, a nucleic acid, a lipid, membrane, a carbohydrate, a drug, a prodrug, a small molecule or a fragment thereof.
  • the synthetic receptor comprises a protein-binding motif.
  • protein-binding motif refers to a region in the receptor that recognizes and binds non-covalently or is covalently linked to specific amino acid residues in the context of variable surrounding peptide or protein sequences.
  • protein includes polypeptides, peptides, antibodies, and other protein ligands, including, for example, cytokines, growth factors, immunoglobulins, cell surface receptors, antigens, or drugs or medicine, for example, peptide drugs, glycopeptide antibiotics such as vancomycin and teicoplanin.
  • the protein-binding motif comprises a protein, peptide, nucleic acid, or synthetic polymer that disrupts membranes of intracellular endosomes.
  • the protein includes but is not limited to a viral or bacterial fusogenic protein such as hemagglutinin or listeriolysin O or a fragment thereof.
  • the peptide includes but is not limited to the peptide termed GALA or endoporter. See Summerton JE. Endo-porter: a novel reagent for safe, effective delivery of substances into cells. Ann N Y Acad Sci.
  • the synthetic polymer includes but is not limited to poly (alkylacrylic acids) such as poly (2-propylacrylic acid) and salts thereof.
  • the present invention provides a synthetic receptor that is a synthetic immunoglobulin Fc receptor.
  • the synthetic immunoglobulin Fc receptor has a Fc binding region and a membrane-binding element.
  • the synthetic immunoglobulin Fc receptor has a Fc binding region, a linker region, and a membrane-binding element.
  • membrane-binding element has been described above, and examples of the membrane-binding element include but are not limited to N-alkyl, N-aryl, or N-acyl derivatives of 3-cholesterylamine, 3- dihydrocholesterylamine, 3-ergosterylamine, 3-brassicasterylamine and related compounds.
  • the Fc binding region includes a protein including but not limited to the IgG- binding proteins: Protein A (from S. aureus), Protein G, Protein L, or Protein Z, a peptide, a cyclic peptide including but not limited to the minimized protein A variant termed Z34C, or a small molecule that binds the Fc fragment of immunoglobulins.
  • Protein A from S. aureus
  • Protein G from S. aureus
  • Protein L Protein L
  • Protein Z Protein Z
  • a peptide a cyclic peptide including but not limited to the minimized protein A variant termed Z34C
  • Z34C minimized protein A variant
  • small molecule that binds the Fc fragment of immunoglobulins is shown below:
  • the linker comprises atoms that may be selected from the group of atoms, C, N, S, and O and/or other chain-extending atoms repeated 0 to 100 or more times as indicated by the symbol n ⁇ .
  • the linker region comprises one or more groups selected from the following: alkane, alkene, alkyne, aryl, ketone, amine, amide, ester, ether, urea, carbamate, heterocyclic, and related functional groups in position X 1 . These functional groups can be linear or cyclic.
  • the linker may comprise 0 to 100 or more subunits such as alpha, beta, gamma, and delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and carboxylic acid.
  • These amino acid subunits may also include side-chains such as those found in natural L- and nonnatural D-configuration alpha amino acids or other amino acids.
  • These and related synthetic immunoglobulin Fc receptors promote the cellular endocytosis of immunoglobulins by recruiting these proteins to cell surfaces.
  • the synthetic immunoglobulin Fc receptor may also be used to control natural receptors on cell surfaces by providing additional stabilization with the cell surface of antibodies that target natural cell surface receptors.
  • the synthetic immunoglobulin Fc receptor may also be used to deliver therapeutic antibodies into cells. These antibodies include neutralizing antibodies against intracellular bacteria, viruses, and other pathogens. Other therapeutic antibodies that target antigens involved in cellular proliferation may be delivered to interfere with the proliferation of cancer cells. Synthetic immunoglobulin Fc receptors can also be used to deliver IgG into cells in combination with an endosome disruptive agent such as membrane fusogenic proteins, peptides, or synthetic polymers to enable live cell immunolabeling of antigens such as immunofluorescence experiments or to control intracellular processes by binding of targeted biomolecules. Other Fc-binding motifs such as substituted triazines (See: J. Comb. Chem. 2004, 6, 862-868) are known to those skilled in the art, and these and related Fc-binding compounds linked to membrane- binding elements are claimed in this application.
  • the present invention provides a synthetic receptor that is a synthetic receptor for cytokines and growth factors.
  • a synthetic receptor for cytokines and growth factors comprises a cytokine or growth factor -binding motif and a membrane-binding element as described above.
  • a synthetic receptor for cytokines and growth factors comprises a cytokine or growth factor- binding motif, a linker region as described above, and a membrane-binding element as described above.
  • the cytokine or growth factor-binding motif may comprise a small molecule, an oligonucleotide, a carbohydrate, a peptide, or a protein-based binding motif that binds an extracellular cytokine or a growth factor.
  • a synthetic receptor for binding cytokines and growth factors is shown below:
  • n ⁇ comprises 0 to 100 or more atoms
  • X 1 comprises one or more linker atoms or functional groups such as amines, amides, carbamates, ureas, esters, or ethers.
  • X 1 comprises 0 to 100 linker subunits such as alpha, beta, gamma, or delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and the carboxylic acid.
  • the present invention provides a synthetic receptor that is a synthetic drug delivery receptor.
  • the synthetic drug delivery receptor has a drug-binding motif and a membrane-binding element.
  • the synthetic drug delivery receptor has a drug-binding motif, a linker region, and a membrane-binding element.
  • a membrane -binding element's structure and function has been described above.
  • the drug-binding motif may bind non-covalently to drugs or prodrugs or be covalently linked to drugs or prodrugs.
  • the drug-binding motif comprises a protein, a linear or cyclic peptide, a small molecule, a carbohydrate or oligosaccaride, or a nucleic acid or oligonucleotide.
  • the drug-binding motif is capable of binding non-covalently to or is covalently linked to a drug, a prodrug, a small molecule, a linear or cyclic peptide, a protein, a carbohydrate or oligosaccaride, or a nucleic acid such as a DNA or RNA oligonucleotide, plasmid DNA, siRNA, or ribozyme.
  • Binding of nucleic acids by the synthetic receptor may allow delivery of linear or plasmid DNA into cells for transfection as used in gene therapy, diagnostic, or other applications. Binding of RNA may allow delivery of RNA into cells for antisense or siRNA gene silencing applications.
  • the drug- binding motif can include DNA or RNA intercalators such as ethidium bromide and related compounds or major-groove or minor-groove binding agents that bind nucleic acids.
  • RNA intercalators such as ethidium bromide and related compounds or major-groove or minor-groove binding agents that bind nucleic acids.
  • ni comprises 0 to 100 or more atoms
  • X 1 comprises one or more linker atoms such as O, N, C, or S, or functional groups such as amines, amides, carbamates, ureas, esters, or ethers.
  • X 1 comprises 0 to 100 linker subunits such as alpha, beta, gamma, or delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and the carboxylic acid.
  • a synthetic drug delivery receptor has one or more drug- binding motif linked to N-alkyl, N-aryl, or N-acyl derivatives of cholesterylamine, dihydrocholesterylamine or ergosterylamine.
  • Synthetic drug delivery receptors having more than one drug-binding motif can bind multiple drugs or increase the affinity for a specific drug through multivalent interactions to enhance efficacy.
  • one aspect of the synthetic drug delivery receptor includes a vancomycin-binding motif as a drug-binding motif.
  • the vancomycin-binding motif is ⁇ -Ahx-D-Phe-D-Ala.
  • the vancomycin- binding motif is D-AIa-D-AIa.
  • the vancomycin-binding motif includes D-amino acids.
  • the protein-binding motif of the receptor comprises 2,4-dinitrophenyl (DNP).
  • the protein-binding motif of the receptor comprises 7-nitrobenz-2-oxa-l,3-diazole (NBD) derivatives.
  • NBD 7-nitrobenz-2-oxa-l,3-diazole
  • the protein-binding motif of the synthetic receptor may selectively bind almost any desired protein or peptide.
  • the protein-binding embodiments of the invention include partial or complete amino acid sequences and functional equivalents to such molecules including, but not limited to, polypeptides having conservative and nonconservative amino acid substitutions, mutants and peptidomimetics that resemble these molecules.
  • the term "functional equivalents” refers to any modified version of a nucleotide or polypeptide which retains the basic function of its unmodified form. As an example, it is well-known that certain alterations in amino acid or nucleic acid sequences may not affect the polypeptide encoded by that molecule or the function of the polypeptide. It is also possible for deleted versions of a molecule to perform a particular function as well as the original molecule. Even where an alteration does affect whether and to what degree a particular function is performed, such altered molecules are included within the term "functional equivalent” provided that the function of the molecule is not so deleteriously affected as to render the molecule useless for its intended purpose.
  • substitutions refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution conserves the character of the amino acid residue.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity and hydrophilicity.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, 1984, Proteins).
  • the protein-binding motif of the synthetic receptor may be modified to enhance its binding affinity for the cognate protein using standard molecular techniques. See Maniatis and Sambrook et al, In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989. Additionally, methods of preparing the protein-binding embodiments of the invention through chemical synthesis and recombinant techniques are disclosed.
  • the synthetic receptors may be used to construct vaccines. Lipids are often linked to peptides or proteins to create vaccines (see J. Immunol. 2002, 169, 4905-4912).
  • novel membrane anchors described in this invention By linking the novel membrane anchors described in this invention to peptides, proteins, lipids, carbohydrates, or nucleic acids, new synthetic vaccines can be constructed.
  • the novel membrane anchor comprises a derivative of cholesterol, dihydrocholesterol, cholesterylamine, or dihydrocholesterylamine.
  • the synthetic receptor of the present invention comprises a synthetic metal chelating receptor.
  • the synthetic metal chelating receptor comprises a metal-binding motif and a membrane-binding element as described above.
  • the synthetic metal chelating receptor comprises a metal-binding motif, a linker region as described above, and a membrane -binding element as described above.
  • the metal binding motif includes metal chelating groups, for example, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), 10-(2-hydroxypropyl)-l,4,7,10- tetraazacyclododecane-l,4,7-triacetic acid (HP-DO3A), and (carboxymethyl) imino] bis (ethyleneitrilo) tetra-acetic acid (DTPA) that bind metal including but not limited to gadolinium, aluminum, lead, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium,
  • x ⁇ . ⁇ comprises 0 to 100 or more atoms
  • X 1 comprises one or more linker functional groups such as amines, amides, carbamates, ureas, esters, or ethers.
  • X 1 comprises 0 to 100 linker subunits such as alpha, beta, gamma, or delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and the carboxylic acid.
  • one aspect of the synthetic metal-chelating receptor includes a NTA-binding motif as a metal-chelating motif.
  • synthetic metal chelating receptors may be useful for MRI and other imaging applications.
  • synthetic metal chelating receptors may be delivered into a cell population ex vivo/in vitro and injected into a patient or delivered to a cell population in vivo in a patient in need of an MRI.
  • the cell population displaying the synthetic metal chelating receptors or the synthetic metal chelating receptors themselves may be administered a site of interest in the patient, for example, by injection.
  • the synthetic metal chelating receptors may be bound to metal ions prior to administration to the patient or they may bind metal ions in the patient' s blood stream or any combination thereof, thereby acting as a MRI contrast agent.
  • the synthetic metal chelating receptor can be used to reduce the level of iron or other metals in cells in need of such a reduction. In one aspect, the synthetic metal chelating receptor can be used to inhibit tumor cell growth. It is contemplated that the synthetic metal chelating receptor can be used in the treatment and prevention of the following medical conditions including, but are not limited to, cancer, inflammatory and infectious conditions, vasoreactive and vasoocclusive conditions, coronary and peripheral athlerosclerosis, parasitic diseases, neurologic and neuromuscular conditions, and viral conditions including AIDS.
  • Additional medical conditions further include vasospasm, Parkinson's disease, Alzeihmer's disease, malaria, tuberculosis, arthritis, allergic and asthmatic conditions, hepatitis, coronary and peripheral vascular ischemia-reperfusion injury of blood vessels.
  • the synthetic receptor of the present invention comprises synthetic a lipid-mimicking receptor.
  • the synthetic lipid-mimicking receptor comprises a lipid-mimicking motif and a membrane-binding element as described previously.
  • the synthetic lipid-mimicking receptor comprises a lipid- mimicking motif, a linker region as described previously, and a membrane -binding element as described previously.
  • C-reactive protein is a protein that circulates in the human bloodstream and is a non-specific but sensitive marker of the acute inflammatory response. CRP binds to lipids on the surface of dead or dying cells.
  • CRP Normally lipids are packed very tightly on the surface of a cell so that CRP does not have access to bind these extracellular surface lipids. But in dead and dying cells, phosphatidylcholine lipids protrude allowing the CRP to bind to the cell, triggering an immune response. CRP can bind to complement factor CIq and factor H and activate the classic pathway of complement activation. In addition, CRP plays a part in the innate immunity (opsonization) and in the removal of membrane and nuclear material from necrotic cells.
  • the present inventors have found that when a synthetic lipid-mimicking receptor comprising phosphatidylcholine linked to a membrane-binding element is contacted with cells, the CRP recognizes the cells as dead or dying cells, binds the lipid-mimicking receptor, triggering an immune response, thereby promoting apoptosis of the cell.
  • the lipid-mimicking motif includes lipids that are found on dead and/or dying cells.
  • the lipid-mimicking motif includes without limitation phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine or components thereof such as the headgroups phosphocholine, phosphoserine, and phosphoethanolamine.
  • a synthetic lipid-mimicking receptor of the present invention is shown below:
  • xs. ⁇ comprises 0 to 100 or more atoms
  • X 1 comprises one or more linker atoms or functional groups such as amines, amides, carbamates, ureas, esters, or ethers.
  • X 1 comprises 0 to 100 linker subunits such as alpha, beta, gamma, or delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and the carboxylic acid.
  • the synthetic lipid-mimicking receptor can be used to induce apoptosis.
  • the synthetic lipid-mimicking receptor that mimics phosphatidylcholine shown above inserted into a cell membrane binds C-reactive protein to induce cellular apoptosis.
  • the present invention provides a method for stimulating an immune response against cells.
  • stimulating an immune response means increasing the amount of a component of the immune system or the activity by which a component of the immune system is characterized, increasing the amount of a receptor present on the surface of an immune cell, or increasing the number of immune cells present in the mammal.
  • the present invention provides a method for inducing apoptosis by contacting the synthetic lipid-mimicking receptor with a cell.
  • the cell is a tumor cell.
  • the following standard techniques can be used to measure the induction of apoptosis caused in a target cell after it is contacted with a synthetic lipid- mimicking receptor of the present invention.
  • apoptosis can be examined or measured in a variety of ways including the detection of morphological indicia of apoptosis (e.g., membrane blebbing, chromatin condensation and fragmentation, and formation of apoptotic bodies), TUNEL (Terminal end-labeling of broken DNA fragments with labeled nucleotides ) staining, measuring of DNA laddering, measuring known caspase substrate degradation (e.g., PARP; Taylor et al, J. Neurochem. 68:1598-605, 1997) and counting dying cells, which have become susceptible to dye uptake.
  • morphological indicia of apoptosis e.g., membrane blebbing, chromatin condensation and fragmentation, and formation of apoptotic bodies
  • TUNEL Terminal end-labeling of broken DNA fragments with labeled nucleotides staining
  • measuring of DNA laddering measuring known caspase substrate degradation (e.g., PARP; Taylor et al, J.
  • kits useful for the measurement of apoptosis by various methods.
  • the method of contacting the cell with a synthetic lipid-mimicking receptor, the amount of the synthetic lipid-mimicking receptor administered, the appropriate incubation time with the synthetic lipid-mimicking receptor are well known to those of ordinary skill in the art.
  • known inhibitors of apoptotic pathways for instance caspase inhibitors, can be used to compare the effectiveness of synthetic lipid-mimicking receptors of this invention.
  • Appropriate inhibitors include viral caspase inhibitors like crmA and baculovirus p35, and peptide-type caspase inhibitors including zVAD-fink, YVAD- and DEVD-type inhibitors. See Rubin, British Med. Bulle., 53:617-631, 1997.
  • related synthetic receptors incorporating phosphoserine and phosphoethanolamine derivatives can be transported to the inner leaflet of cellular plasma membranes (or displayed on the cell surface, depending on the receptor structure) and by projecting target-binding, chelating, or mimicking groups into the extracellular environment or cellular cytoplasm can be useful for controlling cellular signal transduction, cellular proliferation, and other biological processes.
  • the synthetic receptors of the present invention comprise a synthetic transmembrane receptor.
  • the synthetic transmembrane receptor comprises at least two binding motifs and at least two membrane-binding elements.
  • the synthetic transmembrane receptor comprises a binding motif linked to a first linker region that is in turn linked to a first membrane-binding element.
  • the first membrane-binding element is linked to a second linker region linked to a second membrane-binding element which is linked to a third linker region.
  • the third linker region is linked to a second binding motif.
  • the binding motif comprises a small molecule, an oligonucleotide, a carbohydrate, a peptide, or a protein capable of binding noncovalently or covalently linked to a biomolecule, including a small molecule, an oligonucleotide, a carbohydrate, a peptide, a protein, or a drug.
  • the binding motif includes but is not limited to a protein-binding motif, a drug-binding motif, a lipid-binding motif, or a metal-binding motif as described above. Suitable linker regions and membrane-binding elements for use with the synthetic receptors and synthetic transmembrane receptors of the present invention have been described above in detail.
  • a synthetic transmembrane receptor of the present invention is shown below:
  • n ⁇ and n 2 comprise 0 to 100 or more atoms
  • the Linker comprises zero, one, or more linker functional groups such as amines, amides, carbamates, ureas, esters, or ethers. These functional groups can be linear or cyclic.
  • the Lipid region is a natural or nonnatural phospholipid, sphingolipid, or sterol or derivative thereof.
  • X 1 , X 2 , or X 3 Linker regions comprise 0 to 100 linker subunits such as natural or nonnatural alpha, beta, gamma, or delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and the carboxylic acid.
  • X 1 , X 2 , or X 3 Linker regions may comprise a fully saturated alkane of zero to 100 carbon atoms or an alkane of zero to 100 carbon atoms that includes one or more alkene or alkyne functional groups.
  • the linker regions comprise zero, one or more groups selected from the following: alkane, alkene, alkyne, aryl, ketone, amine, amide, ester, ether, urea, carbamate, heterocyclic, and related functional groups in positions X 1 , X 2 , or X 3 .
  • X 1 , X 2 , or X 3 linker regions may comprise 0 to 100 or more subunits such as alpha, beta, gamma, and delta amino acids as well as aminohexanoic acid and related structures with 0 to 100 or more atoms between the amine and carboxylic acid. These amino acid subunits may also include side-chains such as those found in natural L- and nonnatural D-configuration alpha amino acids or other amino acids.
  • At least one binding motif is located intracellularly.
  • the intracellular binding motif may bind or mimic proteins or or other biomolecules or bind or associate with cytoplasmic domains of other proteins, lipids, carbohydrates, or receptors known to activate various messenger systems.
  • at least one binding motif is located extracellularly.
  • the extracellular binding motif as described previously may bind noncovalently or be covalently linked to proteins, drugs, prodrugs, carbohydrates, nucleic acids, lipids or other biomolecules, including those associated with ligand binding and/or signal transduction.
  • the synthetic transmembrane receptor may be part of a protein which is monomeric, homodimeric, heterodimeric, or associated with a larger number of proteins in a non-covalent or disulfide-bonded complex.
  • the receptors of the present invention can be useful for modifying or mediating cellular signal transduction, proliferation, apoptosis, endocytosis, cellular transfection, and drug delivery.
  • CTLs cytotoxic CD8+ T cells
  • CTLs which have synthetic transmembrane receptors inserted to their cell membrane where the synthetic transmembrane receptors contain an extracellular binding domain which recognizes specific antigens can be used to augment proliferation and/or killing of infected cells in a variety of viral, and parasitic diseases, where the infected cells express the antigens from the pathogen.
  • the membrane-binding element may be prepared by chemical synthesis. (See E. J. Corey. THE LOGIC OF CHEMICAL SYNTHESIS, Wiley-Interscience, New Ed edition (1995) and K. C. Nicolaou. CLASSICS IN TOTAL SYNTHESIS, Wiley- VCH (1996)) See also Examples section.
  • the linker region may be prepared using standard techniques known to those skilled in the art or purchased from commercially available sources. See, for example, Thermo Electron Corporation, world wide web at thermo.com. A wide variety of linkers are known in the art for linking two molecules together, particularly, for linking a moiety such as cholesterylamine or its derivative to a peptide, all of which are included within the scope of the present invention.
  • a cholesterylamine membrane-binding element may be synthesized as described above and then a linker added as an intact unit using standard techniques, for example, amide bond formation reactions or the linker may be sequentially added to the membrane-binding element using standard chemical synthesis methods, for example, using protecting groups such as trifluoro acetyl or Fmoc or Boc, and selective deprotection of a protecting group to couple the linker to the membrane-binding element.
  • linkers examples include but are not limited to amino acids, such as those described in Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B. R. /. Am. Chem. Soc. 2004, 126, 16379-16386.
  • the determination of an appropriate linker that allows for cycling between the cell surface and the endosomes can be determined using the assays detailed in the present disclosure.
  • linkers may influence receptor localization and ligand uptake efficiency which may be determined by performing standard assays including but not limited to fluorescence based assays, microscopy, flow cytometry to measure the amount of the synthetic receptor on the cell surface using an antibody directed against the protein-binding motif or an antibody directed against a protein bound by the synthetic receptor as described in Examples 6 and 22.
  • the protein-binding domain may be prepared using standard techniques. Appropriate molecular biological techniques may be found in Maniatis and Sambrook et al, In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989.
  • the protein binding domain can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using techniques known in the art such as those set forth by Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA, 82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis, Pierce Chem Co., Rockford, 111. (1984), and Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N. Y. herein incorporated by reference.
  • the protein-binding motif can be coupled to the linker using standard techniques, including, for example, standard techniques such as amide bond formation reactions or the linker may be sequentially added to the membrane-binding element using standard chemical synthesis methods, for example, using protecting groups such as trifluoroacetyl or Fmoc or Boc, and selective deprotection of a protecting group, followed by addition of a coupling reagent, to couple the linker to the membrane-binding element.
  • standard techniques including, for example, standard techniques such as amide bond formation reactions or the linker may be sequentially added to the membrane-binding element using standard chemical synthesis methods, for example, using protecting groups such as trifluoroacetyl or Fmoc or Boc, and selective deprotection of a protecting group, followed by addition of a coupling reagent, to couple the linker to the membrane-binding element.
  • the protein-binding motif can bind known binding proteins, such as vancomycin, or used to identify new binding proteins or partners not yet identified.
  • the synthetic receptor is allowed to contact the cell surface of a cell and fluorescence based assays or functional assays are used to detect proteins that bind to the protein-binding motif of the synthetic receptor inserted into the cell's membrane. See Examples 6 and 22. Protein-protein interactions between a protein and the protein binding motif of the synthetic receptor can be determined using two-hybrid systems (Field & Song, Nature 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci.
  • the receptors of the present invention When the receptors of the present invention are contacted with living mammalian cells, these receptors insert in the cellular plasma membrane, project the protein-binding motifs from the cell surface, bind the proteins and internalize them through endocytosis.
  • the various forms of the term "contact”, “contacting”, “contacted”, etc. i.e., contacting a synthetic receptor with a cell
  • incubating the synthetic receptor and the cell together in vitro e.g., adding the synthetic receptor to cells in culture
  • administering the agent to a subject such that the synthetic receptor and cells of the subject are contacted in vivo. Any suitable method of contacting the synthetic receptor with cells may be used.
  • the present invention also provides a method for delivering a protein, peptide, nucleic acid, or small molecule into a cell.
  • the term "delivering” includes but is not limited to causing the protein to enter the cell.
  • the method comprises contacting the synthetic receptors with the target cells.
  • Target cells according to the invention is understood to include, without limitation, bacterial, fungal, plant, insect, avian, reptilian, amphibian, and mammalian cells, including human or animal cells. Any suitable duration of growth of the target cells of contacting of the target cells with a synthetic receptor may be used in the present invention.
  • the synthetic cell surface receptors exhibit stability, dynamic cycling between the plasma membrane and intracellular endosomes, targeting of ligands to proposed cholesterol and sphingolipid-enriched lipid raft membrane microdomains, and delivery of protein or drug ligands to late endosomes/lysosomes. Therefore, the synthetic receptors with modified linker regions have potential as cellular probes, as they confer dramatic differences in subcellular localization, and would provide useful as probes for cellular mechanisms that regulate the segregation, localization, and sorting of membrane-associated biomolecules.
  • the synthetic receptor conjugated to a label or tag with or without the linker region described previously or constructed as a tagged small molecule, fusion protein, peptide, nucleic acid, or carbohydrate can also be utilized as a tool for visualizing cellular or subcellular localization, such as labeling of the cellular plasma membrane, endosomes, or other subcellular compartments, and protein trafficking or cycling from the cell surface to the endosome.
  • the synthetic receptor conjugated to a fluorophore or otherwise tagged or labeled or a synthetic receptor analog can also be used to track or analyze cells in vitro or in vivo.
  • synthetic receptors conjugated to a fluorophore such as fluorescein or fluorescein analogues may be added to cells ex vivo and administered to a recipient.
  • synthetic receptor conjugated to a fluorophore can be inserted into a cell or cell population and detected using standard techniques known to one skilled in the art, including for example, flow cytometry, fluorescence activated cell sorting, fluorescence microscopy, and other histological methods.
  • the synthetic receptors can be labeled with a detectable moiety. Detection of a synthetic receptor of the present invention can be facilitated by coupling (i.e., physically linking) the synthetic receptor to a detectable moiety.
  • detectable moieties include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I 125 , 1 131 , S 35 or H 3 .
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. Any method known in the art for conjugating the synthetic receptor to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).
  • the synthetic receptor comprises a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag may be placed at the amino- or carboxyl-terminus of the synthetic receptor.
  • epitope-tagged forms of the synthetic receptor can be detected using an antibody against the tag polypeptide.
  • provision of the epitope tag enables the synthetic receptor to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., MoI. Cell.
  • tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an ⁇ -tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. ScL USA, 87:6393-6397 (1990)].
  • synthetic cellular receptors may be used as mechanistic probes.
  • the probe is a fluorescent analog of a synthetic receptor.
  • These fluorescent analogs may contain diverse fluorophores, including for example, without limitation, NBD, fluorescein and its derivatives, for example, the red- shifted hexachlorofluorescein, texas red, rhodamine, coumarin derivatives, Cy3, AlexaFluor dyes, and fluorescent quantum dots.
  • the invention also relates to a method for delivering in vitro, ex vivo or in vivo at least one protein into target cells according to which the cell is brought into contact with a synthetic receptor bound to a protein according to the invention.
  • the present invention provides a method of treating a disease, disorder, or condition comprising administering a therapeutically effective amount of a protein into a target cell using a synthetic cell receptor to a subject in need thereof.
  • the synthetic receptor for delivering a protein into a cell comprises a protein-binding motif and a membrane-binding element.
  • the membrane-binding element may include, for example, cholesterylamine, dihydrocholesterylamine, ergosterylamine, derivatives of cholesterylamine, dihydrocholesterylamine, ergosterylamine and related compounds thereof wherein the element anchors the receptor into a cell plasma membrane.
  • terapéuticaally effective amount refers to an amount that is effective in reducing, eliminating, treating, preventing or controlling the symptoms of diseases and conditions.
  • controlling is intended to refer to all processes wherein there may be a slowing, interrupting, arresting, or stopping of the progression of the diseases and conditions described herein, but does not necessarily indicate a total elimination of all disease and condition symptoms, and is intended to include prophylactic treatment.
  • the method additionally comprises a synthetic receptor as described above and pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use. Combinations of carriers may also be used.
  • Pharmaceutically acceptable carriers include but are not limited to cyclodextrins, Low-Density Lipoprotein (LDL), and High-Density Lipoprotein (HDL).
  • LDL Low-Density Lipoprotein
  • HDL High-Density Lipoprotein
  • a pharmaceutical composition for treating a disease, disorder, or condition comprises a pharmaceutically acceptable carrier and a synthetic cell receptor for delivering a protein into a cell where the receptor comprises a protein-or target-binding motif and a membrane-binding element.
  • the membrane-binding element may include cholesterylamine, dihydrocholesterylamine, ergosterylamine, derivatives of cholesterylamine, dihydrocholesterylamine, ergosterylamine and related compounds thereof to anchor into a cell plasma membrane.
  • the synthetic receptor bound to a protein according to the invention can be used to treat a disease, disorder, or condition, or as a prophylactic.
  • the subject of the invention may be used in enzyme replacement therapy or to treat a variety of diseases, disorders, or conditions, including without limitation, viral, yeast, and bacterial infections, cancer, inflammation, and autoimmune diseases.
  • the synthetic receptors may be used to deliver an enzyme to a cell in need of the enzyme replacement therapy.
  • treating refers to: (i) preventing a disease, disorder or condition from occurring in an animal or human that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and/or (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and/or condition.
  • synthetic receptors may be employed to deliver proteins, peptides, or small molecules that inhibit viruses that replicate in the cytoplasm.
  • proteins include, for example, nucleic acid aptamers or antibodies that bind proteins important in viral life cycles.
  • viruses that replicate in the cytoplasm belong to the families: Picornaviridae, Caliciviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Reoviridae, Bunyaviridae, Arenaviridae, Paramyxoviridae, Rhabdoviridae, Poxviridae, and Hepadnaviridae. This is important since many viruses do not have vaccines to provide immunity. Thus, the present invention offers new prospects for applications as tools for drug delivery.
  • the present inventors have found a method for treating microbes that elude antibiotics and related glycopeptide antibiotics administered in a traditional manner.
  • Listeria moncytogenes L. monocytogenes
  • the present inventors have found that the present invention may be used to deliver vancomycin, a glycopeptide antibiotic, and/or other related glycopeptide antibiotics into the cytoplasm of cells infected with L. moncytogenes.
  • the present inventors treated non-infected mammalian cells with a synthetic receptor containing an enhanced vancomycin-binding motif linked to 3 ⁇ -cholesterylamine.
  • the synthetic receptor bound the vancomycin derivative, underwent endocytosis, released the vancomycin derivative in the endosome/lysosome where it was degraded, allowing the synthetic receptor to recycle to the cell surface to recommence the delivery process.
  • the endosome/lysosome in particular, the protein is released from the synthetic receptor.
  • the pathogen secretes membrane- disruptive proteins that disrupt the integrity of the endosome so that the pathogen can escape from the endosome and replicate in the cytoplasm.
  • the present invention capitalizes on this event so that when the synthetic receptor bound with vancomycin undergoes endocytosis, the vancomycin is delivered to the endosome, but escapes into the cytoplasm using the same route that the pathogen created. In this way, the antibiotic is able to encounter and bind L. monocytogenes, inhibiting the construction of the bacterial cell walls. Therefore, in another aspect, once the protein-bound synthetic receptor has entered the cell, the protein is released from the synthetic receptor and may enter the cytoplasm. The receptor is then free to recycle to the cell surface for additional rounds of delivery. Therefore, in one aspect of the present invention, the synthetic receptors have bacteriocidal or bacteriostatic effects as shown in viability assays. Examples 15-19.
  • these synthetic receptors with modified linker regions have unlimited potential to treat bacteria and viruses.
  • the present inventors have identified a composition and method that effectively enhances the delivery of poorly permeable antibiotics, such as vancomycin or teicoplanin, to cells.
  • This invention will provide an effective tool for the war on drug-resistant bacteria. Once internalized, the drug can accumulate within the cells where it has a therapeutic effect. Not only does the current invention deliver an antibiotic to the pathogenic replicative source but also does so in an efficient manner using any "custom-designed" binding motif.
  • the synthetic receptor contains an enhanced binding motif for vancomycin, resulting in improved affinity for the antibiotic.
  • a synthetic receptor having an ergosterol-derived membrane-binding element can be used to treat bacteria or yeast infections in a subject.
  • the method comprises contacting a synthetic receptor with yeast or bacterial cells in vitro or in vivo, causing the synthetic receptor to insert into the cells' membranes, so that the yeast or bacterial cells displaying the synthetic receptors in vivo, for example, in a mammal, would be recognized as foreign by the mammal's immune system and destroyed.
  • a synthetic receptor having an ergosterol-derived or brassicasterol-derived membrane -binding element can be used as probe in avian, reptilian, amphibian, insect, yeast, bacterial, or plant cells.
  • synthetic receptors may be used to modulate therapeutically important extracellular ligands.
  • These ligands include but are not limited to cytokines, growth factors, hormones, antibodies, and angiogenic factors. These ligands are secreted by a number of cells and act through cell surface receptors to elicit biochemical responses in their target cells, such as cell differentiation and proliferation.
  • the present invention contemplates the removal of extra-cellular ligands from circulation in physiological fluids, including, for example, blood, using synthetic receptors. Receptors displaying the desired ligand-binding motif would bind the cognate ligand, deliver the ligand to the endosome, where it would be released and degraded.
  • the receptor would then be recycled back to the surface to bind and remove more ligands from circulation. This has been demonstrated by removal of antibodies from cell culture media using synthetic receptors bearing antibody-binding motifs. Therefore, use of the present invention to remove targeted ligands from circulation would allow for the modulation of the immune response, cellular proliferation such as cancer proliferation, or cellular differentiation.
  • modulating of the immune response means increasing or decreasing either the amount of a component of the immune system or the activity by which a component of the immune system is characterized, increasing or decreasing the amount of receptor present on the surface of an immune cell, or increasing or decreasing the number of immune cells present in the mammal. In the context of the use of a method of treatment in vivo according to the present invention, it is, in addition, a treatment designed deplete a patient of a particular protein.
  • the synthetic receptors may be used to treat cancer.
  • Many tumor cells do not express the necessary cell surface receptor to "take in" anti-cancer drugs or express the cell surface receptor at insufficient levels.
  • the drug can still be delivered.
  • cancer cells may become resistant to methotrexate by stopping production of the folate receptors.
  • synthetic folate receptors can be expressed on the surface of tumor cells.
  • the protein-binding motif of the synthetic folate receptor can then bind the anti-cancer drug methotrexate and undergo receptor-mediated endocytosis, thereby inhibiting the enzyme dihydrofolate reductase.
  • Calixarenes can be prepared by those skilled in the art to bind the vascular epidermal growth factor (VEGF), platelet-derived growth factor (PDGF), and other proteins. These compounds could be used to remove these and related cancer-promoting growth factors from circulation to stop tumor growth or conversely could be used to activate receptors for these proteins by promoting the association of growth factors with cell surfaces.
  • VEGF vascular epidermal growth factor
  • PDGF platelet-derived growth factor
  • Cyclic peptides can be prepared by those skilled in the art to bind immunoglobulins and other proteins. This would allow for modulation the immune system by promoting the endocytic destruction of antibodies such as antibodies involved in autoimmune diseases such as lupus and pemphigus.
  • cysteine-disulfide constrained cyclic peptides containing the sequence DCAWHLGELVWCT exhibit high affinity for the hinge region of human immunoglobulins.
  • Other cyclic peptides can be identified by techniques such as phage display that bind to specific proteins.
  • the effective dose and method of administration of a particular synthetic receptor thereof can vary based on the individual needs of the patient and the treatment or preventative measure sought.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population).
  • the synthetic receptors discussed above can be administered to the mice and the effect on certain tissues or cells can be determined using histology, flow cytometry, ELISA and other assays. The data obtained from these assays is then used in formulating a range of dosage for use with other organisms, including humans. The dosage varies within this range depending upon type of synthetic receptor or binding protein, the dosage form employed, sensitivity of the organism, and the route of administration.
  • Normal dosage amounts of various synthetic receptors can vary from approximately 1 to 100,000 micrograms, up to a total dose of about 10 grams, depending upon the route of administration.
  • a constant infusion of the synthetic receptors can also be provided so as to maintain a stable concentration in the tissues as measured by blood levels.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that can be taken into account include the severity of the disease, age of the organism, and weight or size of the organism; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Synthetic receptors may be administered hourly, daily, weekly, monthly or as needed.
  • Routes of administration of the synthetic receptors of the invention include, but are not limited to, topical, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar.
  • Transdermal administration is accomplished by application of a cream, rinse, gel, etc. capable of allowing the pharmacologically active compounds to penetrate the skin.
  • Parenteral routes of administration include, but are not limited to, electrical or direct injection such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.
  • Gastrointestinal routes of administration include, but are not limited to, ingestion and rectal.
  • Transbronchial and transalveolar routes of administration include, but are not limited to, inhalation, either via the mouth or intranasally.
  • Synthetic receptors of this invention that are suitable for transdermal or topical administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams, and ointments applied directly to the skin or incorporated into a protective carrier such as a transdermal device ("transdermal patch").
  • a transdermal device such as a transdermal device ("transdermal patch").
  • suitable creams, ointments, etc. can be found, for instance, in the Physician's Desk Reference.
  • suitable transdermal devices are described, for instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen, et al., herein incorporated by reference.
  • Synthetic receptors of this invention that are suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions.
  • Such solutions include, but are not limited to, saline and phosphate buffered saline for injection into a central venous line, intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection.
  • Synthetic receptors of this invention that are suitable for transbronchial and transalveolar administration include, but not limited to, various types of aerosols for inhalation.
  • Devices suitable for transbronchial and transalveolar administration of these are also embodiments.
  • Such devices include, but are not limited to, atomizers and vaporizers. Many forms of currently available atomizers and vaporizers can be readily adapted to deliver compositions having the pharmacologically active compounds of the invention.
  • Synthetic receptors of this invention that are suitable for gastrointestinal administration include, but not limited to, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration. Once the synthetic receptors has been obtained, it can be administered to a organism in need to treat or prevent a disease, disorder, or condition.
  • the synthetic receptors according to the present invention can be administered orally, by subcutaneous or other injection, intravenously, intracerebrally, intramuscularly, parenternally, transdermally, nasally or rectally.
  • the appropriate route of administration and dosage vary according to various parameters, for example the individual or disease to be treated or alternatively the polynucleotide to be transferred.
  • the form in which the synthetic receptor is administered depends at least in part on the route by which the synthetic receptor is administered.
  • the synthetic receptors described according to the present invention comprise a membrane-binding element linked to a protein-binding or other binding group in order to provide a novel mechanism for receptor-mediated endocytosis of the synthetic receptor and bound ligands.
  • the membrane-binding element is preferably cholesterol, dihydrocholesterol, ergosterol, brassicasterol, or derivatives of cholesterol, dihydrocholesterol, ergosterol, brassicasterol, and related compounds thereof.
  • the membrane-binding element according to the present invention may preferably be derived from the inexpensive and readily available source of cholesterol.
  • cholesterol is derived to form the cholesterol derivatives 3 ⁇ -amino-5-cholestene, and related 3 ⁇ -halides, including 3 ⁇ -chloro-5-cholestene, 3 ⁇ -bromo-5-cholestene and 3 ⁇ -iodo-5-cholestene.
  • the present invention relates to the use of regiospecific, stereoselective and stereo specific sequential i-steroid and retro-j-steroid rearrangements for the synthesis of 3 ⁇ -amino-5-cholestene, 3 ⁇ -chloro-5-cholestene, 3 ⁇ -bromo-5-cholestene, and 3 ⁇ -iodo-5- cholestene from cholesterol.
  • the w-alkyl derivatives of 3 ⁇ -amino-5-cholestene are preferably selected for the ability to insert into plasma membranes of living mammalian cells and cycle between the cell surface and early/recycling endosomes, mimicking the membrane trafficking of many cell surface receptors, and/or delivering molecules directly to the cytosol and nucleus of cells when linked to endosome disruptive peptides. See Peterson, B., Org. Biomol. Chem. 2005, 3, 3607-3612; Sun et al, J. Am. Chem. Soc. 2008, 130, 10064-10065.
  • the methods for synthesizing membrane-binding elements that are 3 ⁇ -amino-5- cholestene, and related 3 ⁇ -halides may comprise synthesizing 3 ⁇ -azido-5-cholestene by Vorbruggen-type coupling of electrophilic cholesterol derivatives.
  • the synthesis comprises reacting cholesterol or a cholesterol bearing a leaving group at the 3-position (including for example, cholesterol triflate, cholesterol mesylate, cholesterol tosylate or cholesteryl esters) with a Lewis acid in the presence of an azide.
  • a Lewis acid catalyst may further be utilized at a temperature between minus 150 0 C to plus 200 0 C to obtain a reaction mixture containing 3 ⁇ -azido-5-cholestene.
  • a preferred Lewis acid catalyst is boron trifluoride.
  • Lewis acid may include for example, boron trifluoride, tin tetrachloride, aluminum trichloride, titanium tetrachloride, trimethylsilyl triflate, BBr 3 , SnCl 4 , ZnCl 2 , MgCl 2 , or MgBr 2 Et 2 O or any salt thereof.
  • azide refers to any compound or derivative having the N 3 " ; moiety therein, including azide derivatives or salts thereof.
  • the azide can be a silyl azide such as trimethylsilyl azide or a metal azide wherein the metal is an alkali metal such as potassium, sodium, lithium, rubidium or cesium.
  • the metal can be a transition metal such as, but not limited to, iron, cobalt, nickel, copper or zinc.
  • the azide of the present invention can also be an organic azide or ammonium azide.
  • the 3 ⁇ -azido-5-cholestene (3) produced from the reaction with the Lewis acid and azide is next reacted with a reducing agent in a solvent to produce the desired 3 ⁇ -amino-5-cholestene or related 3 ⁇ -halide.
  • a reducing agent in a solvent to produce the desired 3 ⁇ -amino-5-cholestene or related 3 ⁇ -halide.
  • various reducing agents and solvents may be utilized.
  • Preferred reducing agents according to the synthesis methods described herein are lithium aluminium hydride or catalytic hydrogenation.
  • the solvent used for the synthesis methods is dichloromethane.
  • benzene or chloroform may be utilized.
  • the solvent utilized does not result in diminishing the yield of the 3 ⁇ -azido-5- cholestene or related 3 ⁇ -halides.
  • Solvents having heteroatoms that function as Lewis bases including for example, tetrahydrofuran, acetone, diethyl ether, or DMF, are not preferred solvents according to the invention.
  • One skilled in the art may ascertain additional solvents that may be utilized according to the present invention.
  • the reaction time for synthesis according to the invention may vary from a few minutes to 18 hours, preferably from 2 hours to 12 hours. The synthesis reactions preferably occur at ambient temperature.
  • the methods of synthesis described herein provide high yields of the 3 ⁇ -amino-5- cholestene and related 3 ⁇ -halides for membrane -binding elements for use with the synthetic receptor according to the invention.
  • the Lewis acid BF3*OEt 2 provides optimal yields of 3 ⁇ -azido-5-cholestene.
  • yields of 3 ⁇ -azido-5-cholestene in excess of 90%, 91%, 92%, 93%, 94%, 95% and 96% may be observed.
  • use of varying Lewis acids result in the synthesis of alterative related 3 ⁇ - halides.
  • SnCl 4 , TiCl 4 , and AlCl 3 preferrably generate 3 ⁇ -chloro-5- cholestene.
  • yields of 3 ⁇ -amino-5-cholestene and the related 3 ⁇ -halides for use as membrane-binding elements according to the invention exceed at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95% and 96%.
  • i- steroid and retro-j- steroid rearrangements are promoted by a Lewis acid to synthesize the halides.
  • the Lewis acid converts the cholesterol derivative to a homoallylic carbocation that rapidly undergoes i-steroid rearrangement and/or retro-j- steroid rearrangement (Fig. 28).
  • i-steroid rearrangement and/or retro-j- steroid rearrangement Fig. 28.
  • These novel synthesis methods are highly efficient, demonstrating high retention of 3 ⁇ -stereochemistry configurations in high yields as a result of the non-classical carbocation forming a partial bond between C5 and C3 only on the alpha face of the steroid. Accordingly, the synthesis methods may be utilized for large-scale preparation of such halides with 3 ⁇ - stereochemistry, demonstrating significant improvement over the substitution reactions of cholesterol and derivatives which result in poor stereoselectivity, elimination, and rearrangement.
  • Embodiments of the present invention are further defined in the following non- limiting Examples. While the present invention is disclosed with reference to embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and the scope of the appended claims. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions.
  • EXAMPLE 1 Design and Synthesis of Receptors for Anti-DNP IgG Ligands.
  • the numbered receptors in examples 1-14 refer to those shown in Figures 1-13 and in examples 1-14.
  • Synthetic receptors were designed that incorporate 2,4-dinitrophenyl (DNP) and structurally similar green fluorescent 7-nitrobenz-2-oxa-l,3-diazole (NBD) headgroups. These headgroups were linked to N-alkyl and N-acyl derivatives of 3 ⁇ - cholesterylamine via tethers containing 6-aminohexanoic acid and ⁇ -alanine subunits as seen in FIG. 1.
  • DNP 2,4-dinitrophenyl
  • NBD green fluorescent 7-nitrobenz-2-oxa-l,3-diazole
  • EXAMPLE 3 Scheme Showing the Synthesis of 3 ⁇ -Cholesterylamine and Derivatives from Cholesterol.
  • Reagents and conditions (a) MsCl, DIEA, CH 2 Cl 2 , 4°C; (b) BF3 etherate, TMSN 3 , CH 2 Cl 2 ; (c) LiAlH 4 , ether, 4°C; (d) 3-bromopropyl phthalimide, K 2 CO 3 , DMF, 55°C; (e) (Boc)2O, DIEA, CH 2 Cl 2 ; (f) NH 2 NH 2 , EtOH, 50 0 C; (g) ethyl 5-bromovalerate, K 2 CO 3, 6O 0 C; (h) (BoC) 2 O, CH 2 Cl 2 ; (i) LiOH, MeOH, THF.
  • the present inventors have discovered a novel method for synthesis of 3 ⁇ -azido-5- cholestene by Vorbruggen-type coupling of electrophilic cholesterol derivatives (shown as step b in the above scheme).
  • the method for synthesis of 3 ⁇ -azido-5- cholestene by Vorbruggen-type coupling of electrophilic cholesterol derivatives comprises reacting cholesterol or a cholesterol bearing a leaving group at the 3-position, for example, cholesterol triflate, cholesterol mesylate, cholesterol tosylate or cholesteryl esters, with a Lewis acid, for example, boron trifluoride etherate, in the presence of an azide, for example, a silyl azide such as trimethylsilyl azide, at a temperature between minus 150 0 C to plus 200 0 C and a Lewis acid catalyst to obtain a reaction mixture containing 3 ⁇ -azido-5- cholestene.
  • a Lewis acid for example, boron trifluoride etherate
  • the present inventors have discovered a novel method for synthesis of N-[3- (phthalimido)propyl]-N-cholesterylamine by SN2 reaction of cholesterylamine with an electrophile (shown as step d in the above scheme).
  • the method for synthesis of N-[3-(phthalimido)propyl]-N-cholesterylamine by SN2 reaction of cholesterylamine with an electrophile comprises reacting cholesterylamine with 3-bromopropyl phthalimide, K 2 CO 3 , and DMF at a temperature between -78°C and 250 0 C.
  • the present inventors have discovered a novel method for synthesis of N-[3- (phthalimido)propyl]-N-(t-butyloxycarbonyl)-3 ⁇ -cholesterylamine by nucleophilic acyl substitution of N-[3-(phthalimido)propyl]-3 ⁇ -cholesterylamine (shown as step e in the above scheme).
  • the method for synthesis of N-[3-(phthalimido)propyl]-N-(t- butyloxycarbonyl)-cholesterylamine by nucleophilic acyl substitution of N-[3- (phthalimido)propyl]-N-cholesterylamine comprises reacting N-[3-(phthalimido)propyl]- N-cholesterylamine with (Boc)2O, DIEA, CH 2 Cl 2 .
  • the present inventors have discovered a novel method for synthesis of ethyl 5-
  • the method for synthesis of ethyl 5- ⁇ (tert-butoxycarbonyl)[(3beta)-cholest-5-en-3-yl] amino ⁇ pentanoate (3) comprises adding to DMF (10 mL) 3 ⁇ -amino-5-cholestene (2, 386 mg, 1.0 mmol), ethyl 5 -bromo valerate (174 microL, 1.1 mmol) and K2CO3 (276 mg, 2.0 mmol), heating the solution to 60 0 C, stirring the solution for 24 hours, cooling the reaction to 23°C, removing the DMF in vacuo to generate a solid residue, adding CH 2 Cl 2 (10 mL) to the resulting solid residue, removing insoluble salts from residue by filtration, and washing the solids with additional CH 2 Cl 2 (5 niL) to generate a
  • Lewis acids may be used in these synthesis methods as exemplified in Table 1.
  • Additional Lewis acids that may be used according to the invention include, without limitation, boron trifluoride, tin tetrachloride, aluminum trichloride, titanium tetrachloride or trimethylsilyl triflate, BBr 3 , SnCl 4 , ZnCl 2 , MgCl 2 , or MgBr 2 Et 2 O.
  • Table 1 Varying Lewis Acids entry substrate Lewis acid reaction yield (%) conditions 1 M 3b% SnCl 4 -20 0 C, 1 h to 54 (3); 36
  • Table 2 summarizes the effect of the leaving group on the reactivity of 3 ⁇ - cholesterol derivatives. Reactions were run on a 1 mmol scale in anhydrous CH 2 Cl 2 containing TMSN 3 (1.1 equiv). Recovery of starting material (4) is additionally shown in entry 2. NR indicates no reaction was observed.
  • EXAMPLE 4 Construction of Synthetic Receptors.
  • Various synthetic receptors were constructed by modifying a 3 ⁇ -cholesterylamine as shown in Example 3 above. Cholesterol may alternatively be modified to become a nosyl-protected 3 ⁇ - cholesterylamine via a Swern oxidation, reduction to epicholesterol, and synthesis of 3beta-azido-5-cholestene via Mitsunobu reaction with hydrazoic acid.
  • the compound underwent further modification to become a receptor by reduction, protection as a nosyl sulfonamide, and use of Fukuyama's amine synthesis methodology and/or deprotection and sequential amide bond formation reactions.
  • EXAMPLE 5 General. Chemical reagents were obtained from Acros, Aldrich, Alfa Aesar, or TCI America. Solvents were from EM Science. Media and antibiotics were purchased from Gibco BRL. Cholera toxin B subunit-Alexa Fluor 488 (CT-B-AF488), Transferrin-Alexa Fluor-488, Protein A Alexa Fluor 488 (PrA- AF488), Protein A Alexa Fluor 594 (PrA- AF594), Protein A Alexa Fluor 633 (PrA- AF633), and BODIPY TR ceramide were from Molecular Probes. Rabbit polyclonal antidinitrophenyl (anti-DNP) IgG was from Sigma.
  • Flash column chromatography employed ICN SiliTech Silica Gel (32-63 ⁇ m).
  • Infrared spectra were obtained with a Perkin Elmer 1600 Series FTIR.
  • NMR spectra were obtained with Bruker AMX-360, DRX-400, or AMX-2-500 instruments with chemical shifts reported in parts per million (ppm, ⁇ ) referenced to either CDCl 3 ( 1 H 7.27 ppm; 13 C 77.23 ppm), DMSOd 6 ( 1 H 2.50 ppm; 13 C 39.51 ppm), or (CH 3 ) 4 Si.
  • High-resolution mass spectra were obtained from the University of Texas at Austin Mass Spectrometry Facility (ESI and CI). Peaks are reported as m/z.
  • the organic layer was separated and the aqueous layer was extracted with diethyl ether (60 mL x 2). The organic layers were combined, washed with deionized water (100 mL), dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to give the crude product as light yellow solid.
  • 3 ⁇ -Amino-5-cholestene A solution of 3 ⁇ -azido-5-cholestene (6.18 g, 15.0 mmol) in anhydrous diethyl ether (100 mL) was cooled to 4 0 C by ice-water bath. To this solution, LiAlH 4 (855 mg, 22.5mmol) was added in four equal portions. The reaction was maintained at 4 0 C for 30 min, warmed to 22 0 C and stirred for 2 h. When the starting material disappeared by TLC analysis (hexanes), the reaction was cooled to 4 0 C and carefully quenched by slow drop wise addition of cold deionized water (10 mL).
  • N-cholesteryl-2-nitrobenzenesulfonamide (15). To 3 ⁇ -azido-5-cholestene (14, 3.30 g, 8.02 mmol) in dry diethyl ether (150 mL) at 4 0 C was added lithium aluminum hydride
  • This aminosteroid was dissolved in dry THF (150 rnL), and diisopropylethylamine (2.61 rnL, 15.03 mmol) was added.
  • 2-Nitrobenzenesulfonyl chloride (2.20 g, 9.92 mmol) in dry THF (40 mL) was added by addition funnel over 30 min. After 2 h at 23 0 C, solvents were removed in vacuo, CH 2 Cl 2 was added (75 mL), and this organic solution was washed with aqueous NaHCO 3 (10%, 1 x 100 mL) and saturated aqueous NaCl (2 x 100 mL).
  • N-[3-(tert-butoxycarbonylamino)propyl]-N-cholesteryl-2-nitrobenzenesulfonamide (16).
  • iV-cholesteryl-2-nitrobenzenesulfonamide (15, 3.50 g, 6.14 mmol)
  • potassium carbonate (12.50 g, 90.44 mmol)
  • Boc-3-chloropropylamine (2.50 g, 12.91 mmol, prepared as previously reported 5 ) were added to dimethylacetamide (200 mL). This mixture was heated to 12O 0 C for 20 h. The reaction was filtered to remove excess potassium carbonate and the solvent was removed in vacuo.
  • N- [3-(te/t-butoxycarbonylamino) propyl]- ⁇ f-cholesteryl-2-nitrobenzenesulfonamide (16, 38 mg, 0.053 mmol) was treated with trifluoroacetic acid in CH 2 Cl 2 (2:25, 10 mL). After 2 h at 23 0 C, TLC analysis (MeOH / CH 2 Cl 2 , 1:50) revealed conversion to the more polar primary amine.
  • CI m/z 1093.4837 (MH + , C 60 H 70 F 2 N 4 O 11 S requires 1093.4809).
  • N- ⁇ 3-[5-fluoresceinisothiourea]propyl-6-[(2,4- dinitrophenyl)amino]hexanamide (27).
  • EXAMPLE 6 Biological Assays.
  • Jurkat lymphocytes acute T-cell leukemia, ATCC #TIB-152
  • RPMI 1640 media supplemented with Fetal Bovine Serum (FBS, 10%), penicillin (100 units / mL), and streptomycin (100 ⁇ g / mL).
  • FBS Fetal Bovine Serum
  • penicillin 100 units / mL
  • streptomycin 100 ⁇ g / mL
  • RPMI media used for cell culture and wash steps contained antibiotics and FBS unless otherwise noted.
  • OPTI-MEM media employed for transfection was not supplemented with FBS or antibiotics.
  • Epifluorescence micrographs were captured through a Zeiss Fluar (100 X) objective by a Zeiss Axiocam digital camera interfaced to a Zeiss Axiovert SlOOTV microscope. Fluorophores were excited with FITC/Bodipy (Ex. 480/40, Em. 535/50) or Texas Red (Ex. 560/55, Em. 645/75) filter sets (Chroma). Images were processed with Adobe Photoshop 7.0.
  • Flow cytometry Analyses were performed with Beckman-Coulter XL-MCL bench-top and EPICS Elite flow cytometers. Forward- scatter (FS) and side-scatter (SSC) dot plots afforded cellular physical properties of size and granularity that allowed gating of live cells. After gating, 10,000 cells were counted.
  • FS Forward- scatter
  • SSC side-scatter
  • AF-488 was excited at 488 nm with a 15 mW air- cooled argon-ion laser, the emission was split with a 550 nm dichroic and filtered through a 510 nm long pass filter and 530/30-nm band pass filter using the XL-MCL cytometer.
  • the PMT voltage for this instrument was set to 724 for detection of AF488.
  • Calibration with Sphere Rainbow Calibration particles (Spherotech) bearing 330,000 molecules of fluorescein/particle provided a fluorescence of 15.6 at this voltage.
  • treated cells Prior to analysis, treated cells were washed with media (0.5 mL), washed again with media containing 6-(2,4- dinitrophenyl)aminohexanoic acid (100 ⁇ M, 3 x 0.5 mL x 20 min, 1% DMSO) to compete away any non-internalized protein, washed again with media (0.5 mL), and resuspended in media (0.6 mL) for analysis by epifluorescence microscopy, confocal microscopy, or flow cytometry.
  • Jurkat lymphocytes were subjected to the antibody uptake assay (cells were treated with receptors 2 - 5 at 10 ⁇ M for 1 h followed by addition of a pre-equilibrated complex (preincubated at 23 0 C for 1 h) of anti-DNP-IgG / PrA- AF488 for 15 min to 4 h) and internalized cellular fluorescence was quantified by flow cytometry. Doubling the concentration of pre-equilibrated anti-DNP-IgG / PrA-AF488 did not affect the rate or magnitude of uptake (data not shown), indicating that synthetic receptors were saturated under these conditions.
  • the rate constant and the ligand internalization half-life shown on the right of Figure 8C were determined by treatment of Jurkat lymphocytes (in RPMI media, 500 ⁇ L) with receptor 2 (1 ⁇ M) for 1 h at 37 0 C. These cells were washed with ice-cold media (500 ⁇ L) and maintained at 4 0 C. A pre-equilibrated (1 h at 23 0 C) ice-cold solution of RPMI media (100 ⁇ L) containing anti-DNP-IgG (7.2 ⁇ g) and PrA-AF488 (2 ⁇ g) was added. After 1 h at 4 0 C, the cells were washed with ice-cold RPMI media (2 x 500 ⁇ L).
  • Intracellular fluorescence was quantified by flow cytometry.
  • Jurkat lymphocytes (4 x 10 6 ) were suspended in media (2 mL, 1% DMSO) containing receptor 6 (10 ⁇ M) and incubated for 1 h at 37 0 C. Cells were washed with media (2 x 2 mL), and cooled on ice (2 min) to block plasma membrane recycling. Ice-cold media containing sodium dithionite (30 mM, 1 mL) was added and the cells were maintained at 4 0 C for 5 min to irreversibly quench cell surface fluorophores.
  • PrA633 (2 ⁇ g) in phosphate buffered saline (PBS, 8 ⁇ L, pH 7.4) was added to tubes A and C.
  • PBS 8 ⁇ L, pH 7.4 was added to tubes B and D as control experiments.
  • the four tubes were maintained at 4 0 C for an additional 15 min, washed with ice-cold serum free recycling media (1 mL), resuspended in ice-cold serum free recycling media (1 mL), and the fluorescence of bound anti-DNP/PrA-AF633 was analyzed by flow cytometry.
  • the background fluorescence of cells in tubes B and D was subtracted from values obtained from cells in tubes A and C respectively to correct for minor spectral overlap ( ⁇ 10%) of the NBD and AF633 fluorophores.
  • Receptor-treated cells were washed with ice-cold serum free recycling media (2 x 0.5 mL), resuspended in ice- cold serum free recycling media (1.5 mL), and analyzed by flow cytometry.
  • Cells were isolated by centrifugation, resuspended in ice-cold back-exchange media (serum free recycling media containing 2 mM Me- ⁇ -cyclodextrin and 1% BSA, 1.5 mL), maintained on ice (2 min), and analyzed by flow cytometry (this single wash with back-exchange media resulted in a 44% loss of total cellular fluorescence).
  • the cells were maintained on ice, washed again with ice-cold back exchange media (3 x 10 min x 1.5 mL), and analyzed by flow cytometry (this combination of four total wash steps with back-exchange media resulted in a 84% loss of total cellular fluorescence; additional wash steps resulted in >90% removal of receptor from the cell surface).
  • Jurkat lymphocytes (3 x 10 6 ) were resuspended in serum free recycling media containing 10 (1 ⁇ M, 1% DMSO, 1.5 niL) and incubated for 10 min at 37 0 C. These cells were washed with serum free recycling media (2 x 1 mL) to remove unincorporated receptor (10). These receptor- treated cells were cooled on ice (5 min) to halt recycling and washed with ice-cold back-exchange media (6 x 1.5 mL x 10 min) to selectively remove 10 from the cell surface without affecting the population of 10 in intracellular endosomes.
  • Serum free recycling media This media was prepared as previously described 7 and included NaCl (150 mM), KCl (5 mM), CaCl 2 (1 mM), MgCl 2 (1 mM), glucose (2g / L), and Hepes (20 mM, pH 7.4).
  • Cells were maintained at 4 0 C for 30 min and washed with ice-cold media (2 x 0.5 mL). Cells were lysed at 4 0 C in an ultracentrifugation tube (ultra clear 14x89 mm, Beckmann Instruments) by addition of TNEV lysis buffer (2 mL, 0.2% Triton X-100 (v/v), 150 mM NaCl, 5 mM EDTA, 5 mM Na 3 VO 4 , 25 mM Tris-HCl, pH 7.4) followed by vortex mixing for 20 seconds every 20 min for 1 h.
  • TNEV lysis buffer 2 mL, 0.2% Triton X-100 (v/v), 150 mM NaCl, 5 mM EDTA, 5 mM Na 3 VO 4 , 25 mM Tris-HCl, pH 7.4
  • This lysate was combined with a solution of 80% sucrose in TNEV lysis buffer (2 mL, w/v) to yield 4 mL of a 40% sucrose solution at the bottom of the ultracentrifugation tube.
  • This 40% sucrose solution was overlayed with 30% sucrose in TNEV lysis buffer (4 rnL), followed by 5% sucrose in TNEV lysis buffer (2 rnL), and a final solution of 2% sucrose in TNEV lysis buffer (2 rnL) by syringe.
  • Masses of ultracentrifugation tubes were balanced to within 1 mg by the addition of 2% sucrose solution in TNEV lysis buffer.
  • Tubes were centrifuged at 33,000 rpm (134,409 g) for 20 h at 4 0 C in a Beckman OptimaTM LE-80K preparative ultra centrifuge using a SW41 swinging bucket rotor. Fractions (12 x 1 rnL) were harvested by aspiration with a capillary tube and small pump beginning with the highest density fraction at the bottom of the ultracentrifugation tube. The fluorescence of these fractions were quantified with a Packard Fusion microtiterplate reader fitted with a 480 nm excitation filter and 535 nm emission filter.
  • Fluorescence polarization assays ( Figure 9). Fluorescence polarization binding experiments employed a Packard Fusion microtiterplate reader equipped with a 485 nm excitation filter and a 535 nm polarization emission filter. Apparent dissociation constants for binding of NBD and DNP derivatives to rabbit polyclonal anti-DNP IgG were determined by analyzing a fixed concentration of FITC-DNP (27, 20 nM) or 6-(7- nitrobenzofuran-4-ylamino)hexanoic acid (28, 100 nM) in PBS (pH 7.4, 100 ⁇ L).
  • Fluorescent probes were equilibrated for 30 min with varying concentrations of rabbit anti- DNP IgG in black 96 well plates (Costar) prior to fluorescence polarization measurements in triplicate. Dissociation constants were calculated by nonlinear regression using a sigmoidal dose-response model with variable slope (GraphPad Prism 3.0 software).
  • the 24-well plate was incubated at 37 0 C in a humidified CO 2 incubator for 4 h.
  • RPMI media 400 ⁇ L
  • FBS FBS
  • PHA-L 1 ⁇ g / mL
  • PMA 5 ng / mL
  • Maximal transfection efficiency 40%) was obtained after incubation for 24 to 36 h (37 0 C, 5% CO 2 ).
  • Analysis of receptor cytotoxicity Jurkat lymphocytes were treated with receptor 3 (10 ⁇ M) for 1 h followed by Anti-DNP-IgG / PrA488 for 4 h under the antibody uptake assay conditions. These cells were washed twice with media and incubated for an additional 24 h at 37 0 C.
  • the dead-cell stain propidium iodide (10 ⁇ g / mL) was added to cells prior to analysis, and viability was quantified from flow cytometry forward and side- scatter dot plots. Greater than 95% of cells remained viable under these conditions. Additionally, cells were examined after treatment with receptor 2 (10 ⁇ M) for 1 h, removal of unincorporated 2 by resuspension in receptor-free cell culture media, and longer-term growth in culture. After an additional 96 h in culture, >90% of these cells remained viable and were morphologically indistinguishable from untreated cells.
  • EXAMPLE 7 Kinetics of Ligand Uptake for Synthetic Receptors. Synthetic receptors were evaluated as mediators of cellular uptake of protein ligands in living Jurkat lymphocytes, a human T cell line. Confocal laser scanning microscopy and flow cytometry were employed to analyze cells treated with compounds 2-9 for 1 hour followed by addition of anti-DNP IgG complexed with fluorescent conjugates of the IgG-binding protein A (PrA) from S. aureus.
  • PrA IgG-binding protein A
  • the IgG-loaded intracellular compartments were identified as late endosomes and lysosomes by co-localization studies in cells transfected with EGFP- lgp 120, a fluorescent protein marker of these compartments.
  • Excess anti-DNP IgG/PrA-AF594 added to cells treated with 6 did not appreciably deplete this receptor from the cell surface despite extensive uptake of this protein complex.
  • EXAMPLE 8 Efficiency of Uptake Mediated by the DNF '-based receptors 2-5.
  • the magnitude and kinetics of delivery of a green fluorescent anti-DNP/PrA-AF488 complex was quantified by flow cytometry as shown in FIG. 8., panels B and C.
  • EXAMPLE 9 Effect of Linker Structure on Subcellular Localization of Synthetic Receptors.
  • Cells were treated with structurally similar green fluorescent receptors (6-9).
  • Epifluorescence microscopy and fluorescence quenching assays were employed to investigate the differences in cellular fluorescence and subcellular localization.
  • Panel A the overall fluorescence resulting from treatment with 10 ⁇ M of these compounds was within 2-fold.
  • the subcellular localization of these compounds differed dramatically.
  • Receptor 6 bearing two ⁇ -alanine linker subunits was observed predominantly on the cell surface.
  • the single ⁇ -alanine containing receptor 7 exhibited lower cell surface localization, and the absence of B-alanine in the linker of 8 rendered this compound primarily intracellular.
  • the amide analog 9 was essentially completely associated with internal membranes which explain the inability of this analog to mediate significant cellular uptake of the anti-IgG ligand.
  • This amide analog nearly completely localized with a red fluorescent probe of the cellular golgi apparatus and nuclear membrane, indicating a unique intracellular destination of this receptor.
  • the fraction of receptors 6-9 at the cell surface was quantified by irreversible quenching of exposed NBD fluorophores on the cell surface. This was accomplished by brief treatment of cells with relatively cell-impermeable reducing agent (sodium hypodisulfite, NaO 2 S-SO 2 Na), which rapidly reduces the nitro functionality of extracellular NBD headgroups to the amine, enabling quantification of differences in cell surface and intracellular fluorescence.
  • cell-impermeable reducing agent sodium hypodisulfite, NaO 2 S-SO 2 Na
  • Quantitative dithionite quenching assays demonstrated dramatic differences in receptor subcellular localization that paralleled the magnitude and kinetics of IgG uptake observed with receptors 2-5, as shown in FIG. 10, Panel B. Thus, differences in trafficking to intracellular membranes control the efficiency of these synthetic receptors.
  • EXAMPLE 10 Temporal Stability of Receptor 2. To evaluate the temporal stability of receptor 2 on the cell surface, the half- life of this compound on Jurkat lymphocytes was quantified by addition of a soluble antibody. Cells were treated with this receptor for 1 hour (10 ⁇ M), unincorporated receptor was removed by washing cells with fresh media, and the abundance of 2 as a function of time was detected with a green fluorescent anti-DNP/PrA-AF488 complex. As shown in FIG. 10., Panel C, curve fitting of the flow cytometry data revealed a cell surface half-life of 20 hours. This value is similar to the 24 hour half- life of natural folate receptor that is anchored to the cell surface by covalently attached GPI lipids.
  • EXAMPLE 11 Cycling of Receptor 6 Between the Cell Surface and Intracellular Endosomes in Jurkat Lymphocytes.
  • a fluorescent-quenching assay with sodium dithionite was employed. As shown in FIG 11, Jurkat cells were treated with receptor 6 (10 ⁇ M) and cooled to 4 0 C to stop plasma membrane recycling. Fluorescence at the cell surface was quenched by treatment with ice-cold sodium dithionite, and cells were washed with cold media to remove this reducing agent (FIG. 11, Panel A).
  • This cell culture was split into two equal portions; one portion was maintained at 4 0 C, shown in FIG 11, Panel B, for 30 minutes, and the other portion was allowed to return to 37 0 C for 30 minutes to reactivate plasma membrane recycling as shown in FIG.l 1, Panel C.
  • Significant return of green fluorescence back to the plasma membrane was observed only in the panel warmed to 37 0 C. Compare Panels B and C in FIG 11. NBD headgroups that have returned to the cell's surface were detected with an NBD-binding red fluorescent anti-DNP/PrA-AF633 complex.
  • EXAMPLE 12 Quantifying the rate of plasma membrane recycling of receptor 10.
  • oregon green-derived receptor 10 was employed. Unlike the more strongly cell-associated DNP and NBD-based receptors, the compound 10 binds proteins in cell culture media and can be rapidly and efficiently depleted from cell surfaces by washing cells with "back exchange" media containing bovine serum albumin (BSA, 1%) and methyl-beta-cyclodextrin (Me-beta-CD, 2 mM). This concentration of Me-beta-CD is known to affect neither endocytosis nor plasma membrane recycling.
  • BSA bovine serum albumin
  • Me-beta-CD methyl-beta-CD
  • the oregon green-fluorophore of 10 was also chosen for these studies because unlike structurally similar carboxydfluorescein the fluorescence of oregon green is not appreciably quenched in acidic environment of endosomal compartments.
  • Cells treated with 10 (1 ⁇ M) exhibited bright green fluorescence both at the cell surface and in intracellular (endosomal) compartments; patterns of cellular fluorescence were similar to treatment with the structurally related receptor 7 (data not shown).
  • Cells were treated with 10 and cooled to 4 0 C to stop dynamic plasma recycling. These cells were washed four times with ice-cold back exchange media to extensively deplete 10 from the cell surface without affecting the population of this compound in intracellular endosomes.
  • EXAMPLE 13 Synthetic receptor/ligand complexes co-fractionate with lipid raft components of cellular plasma membranes. Another hallmark of many natural cell surface receptors is the proposed association of these biomolecules with cholesterol and sphingolipid-enriched lipid raft subdomains of cellular plasma membranes. These subdomains are generally biochemically characterized as low density fractions of the plasma membrane insoluble in buffers containing non-ionic detergents such as Triton-X at 4 0 C.
  • Ganglioside GMl (1) is prototypical example of a small lipid raft- associated cell surface receptor, and complexes of this glycolipid with a fluorescent cholera toxin protein are often used as a marker for lipid rafts.
  • macromolecules receptors including B-cell receptors, T cell receptors, and growth factor receptors are thought to associate with these membrane subdomains.
  • binding of ligands to receptors initiates formation of lipid rafts. This ligand-mediated concentration of specific membrane lipids has been proposed to recruit intracellular raft-associated kinases to activate cellular signaling pathways.
  • EXAMPLE 14 References for examples 1-13. (1) Sjoback, R.; Nygren, J.; Kubista, M. Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy 1995, 57, L7-L21.
  • EXAMPLE 15 Design and synthesis of receptors for vancomycin and related glycopeptide antibiotic ligands.
  • the numbered receptors in examples 15-21 refer to those shown in Figures 14-19 and in Examples 15-21.
  • vancomycin (1) into endosomes of cells infected with pathogenic bacteria might enable access of this cell-impermeable antibiotic to the cytoplasm.
  • vancomycin (2) and an artificial cell surface receptor (3) comprising the vancomycin-binding motif D-Phe-D-Ala linked to 3 ⁇ - cholesterylamine.
  • the D-Phe-D-Ala dipeptide was chosen because of its improved affinity for vancomycin (K d ⁇ 7 ⁇ M) compared with the natural D-AIa-D-AIa substrate. 6 This binding group was coupled to 3 ⁇ -cholesterylamine to add a membrane anchor previously shown by our laboratory to access a membrane trafficking pathway involving rapid cycling between the plasma membrane and endosomes of mammalian cells. 7 Control compounds that do not bind vancomycin (L- Ala- L- Ala analogue 4) or that exhibit lower affinity for the cellular plasma membrane (amide analogue 5) were also synthesized for comparison.
  • EXAMPLE 16 Confocal laser scanning and differential interference contrast (DIC) microscopy of living J -774 murine macrophages. Confocal laser scanning microscopy was used to examine the delivery of fluorescent vancomycin derivative 2 into J774 mouse macrophage cells. As shown in Figure 17, cells treated with both receptor 3 and ligand 2 exhibited substantial uptake of this fluorescent probe. In healthy (non- infected) cells, this intracellular fluorescence was localized to endosomes and lysosomes as evidenced by colocalization with a red fluorescent marker of these compartments (supporting information). However, when these cells were also infected by L. monocytogenes, the fluorescent vancomycin (2) was distributed throughout the cytoplasm and nucleus.
  • DIC differential interference contrast
  • EXAMPLE 17 Dose-dependent effectiveness of delivery of receptor 2 into mouse J-774 cells and human HeLa cells.
  • the dose-dependent effectiveness of delivery of 2 into mouse J-774 cells and human HeLa cells mediated by receptor 2 was examined quantitatively by flow cytometry.
  • preequilibration of the receptor (3) with the ligand (2) prior to addition to cells was ca. 2-fold more effective for delivery compared with preloading of cellular plasma membranes with the receptor (3) followed by addition of the ligand (2).
  • both of these conditions engendered substantial receptor- mediated enhancements of ligand uptake.
  • EXAMPLE 18 Delivery of vancomycin via synthetic receptor 3 into HeLa cells infected by L. monocytogenes to determine effects on antibiotic activity against this pathogen.
  • the synthetic receptor 3 was employed to deliver vancomycin (1) into HeLa cells infected by L. monocytogenes to investigate effects on antibiotic activity against this pathogen.
  • treatment of HeLa cells with this receptor (10 ⁇ M) enabled vancomycin (50 ⁇ M) to eliminate this intracellular parasite and rescue HeLa cells from the lethal effects of this pathogen.
  • EXAMPLE 19 Delivery of fluorescent Vancomycin derivative 2 by synthetic receptor 3 in vivo to mice.
  • the delivery of fluorescent Vancomycin derivative 2 by synthetic receptor 3 was investigated in vivo ( Figure 20).
  • Figure 20 synthetic receptor-mediated targeting of the fluorescent vancomycin (2) to specific tissues was observed, indicating that N-alkyl-3b-cholesterylamine derivatives such as 3 provide novel tools for drug delivery in vivo.
  • a synthetic cell surface receptor can enhance the in vitro effectiveness of a clinically used drug against an intracellular pathogen.
  • This receptor also enables targeting of the drug to specific tissues in vivo. This approach provides a novel tool to combat microorganisms that invade mammalian cells.
  • EXAMPLE 20 General information for histidine/metal chelators. Chemical reagents were obtained from Acros, Aldrich, Alfa Aesar, or TCI America. Solvents were from EM Science. Media and antibiotics were purchased from Mediatech. Dil-loaded human low-density lipoprotein was from Invitrogen. Commercial grade reagents were used without further purification unless otherwise noted. Anhydrous solvents were obtained after passage through a drying column of a solvent purification system from GlassContour (Laguna Beach, CA). All reactions were performed under an atmosphere of dry argon or nitrogen. Reactions were monitored by analytical thin-layer chromatography on plates coated with 0.25 mm silica gel 60 F 254 (EM Science).
  • Agilent 1100 preparative pump / gradient extension instrument equipped with a Hamilton PRP-I (polystyrene-divinylbenzene) reverse phase column (7 ⁇ m particle size, 21.5 mm x 25 cm).
  • Melting points were measured with a Thomas Hoover capillary melting point apparatus and are uncorrected. Infrared spectra were obtained with a Perkin Elmer 1600 Series FTIR.
  • NMR spectra were obtained with Bruker CDPX-300, DPX-300, AMX-360, or DRX-400 instruments with chemical shifts reported in parts per million (ppm, ⁇ ) referenced to either CDCl 3 ( 1 H 7.27 ppm; 13 C 77.23 ppm), DMSOd 6 ( 1 H 2.50 ppm; 13 C 39.51 ppm), or (CH 3 ) 4 Si.
  • High-resolution mass spectra were obtained from the University of Texas at Austin and Penn State University Mass Spectrometry Facilities (ESI and CI). Peaks are reported as m/z.
  • N-Fmoc- ⁇ -alanine- ⁇ -alanine ethyl ester (5). ./V-Fmoc- ⁇ -alanine (311 mg, 1.0 mmol) in anhydrous CH 2 Cl 2 (20 niL) under N 2 was cooled to O 0 C. HOBt (168 mg, 1.1 mmol) and EDC (230 mg, 1.2 mmol) were added and the solution was stirred for 30 min at O 0 C. ⁇ - Alanine ethyl ester hydrochloride (169 mg, 1.1 mmol) in anhydrous CH 2 Cl 2 (10 mL) and DIEA (180 ⁇ L, 1.1 mmol) were added.
  • the reaction was warmed to 23 0 C and stirred for 16 h.
  • the reaction solution was diluted with CH 2 Cl 2 (30 mL) and washed with aqueous HCl (5%, 30 mL), followed by aqueous NaOH (0.1 M, 30 mL), and deionized H 2 O (30 mL).
  • the organic layer was dried over anhydrous Na 2 SO 4 and concentrated in vacuo.
  • Aqueous LiOH (10 mL, 0.5 M) was added dropwise to a solution of 3 (315 mg, 0.51 mmol) in a mixture of MeOH (15 mL) and THF (10 mL). The solution was stirred for 4 h at 23 0 C and the organic solvents were removed in vacuo. The remaining aqueous solution was acidified with aqueous HCl (10%) and the resulting carboxylic acid precipitated as a white solid. This solid was collected by vacuum filtration, washed with cold water, and dried in vacuo. The dried solid was dissolved in anhydrous CH 2 Cl 2 (20 ml) under dry N 2 and cooled to O 0 C.
  • This amine was prepared from N a ,N a -Bis[(tert- butoxycarbonyl)methyl]-./V- ⁇ -benzyloxycarbonyl-L-lysine tert-butyl ester (the Cbz precursor) as previously reported, 2 but the Cbz precursor (3.4 g) in EtOH (50 mL) was deprotected by catalytic hydrogenation (Pd(C), 10%) using 300 psi H 2 for 12 h to afford 2.51 g of amine (97%). The reaction was warmed to 23 0 C and stirred for 16 h.
  • numbered receptors in examples 22-24 refer to those shown in Figures 20-25 and in Examples 22-24.
  • Jurkat lymphocytes human acute T-cell leukemia, ATCC #TIB- 152 were maintained in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with Fetal Bovine Serum (FBS, 10%), penicillin (100 units / mL), and streptomycin (100 ⁇ g / mL).
  • RPMI media used for cell culture and wash steps contained antibiotics and FBS unless otherwise noted.
  • Microscopy A Zeiss LSM 5 Pascal confocal laser- scanning microscope fitted with a Plan Apochromat objective (63 X) was employed. Alexa Fluor 488 was excited with a 488 nm Argon ion laser and emitted photons were collected through 505 nm LP filter.
  • the dead-cell stain propidium iodide (10 ⁇ g / mL) was added to cells prior to analysis, and viability was quantified by flow cytometry forward and side- scatter dot plots. No significant effects on viability or cellular morphology were observed under these conditions.
  • the gene encoding AcGFP was amplified by PCR using primers 5'-EcoRI-XhoI-AcGFP (5 ' - AGTCGAATTCGGTCTCGAGATGGTGAGCAAGGGC-S ' ), 3 ' -SalINoStop- AcGFP (5'-GACTGTCGACCTTGTACAGCTCATC-S'), and pAcGFPl(BD biosciences) as the template to append flanking EcoRI and Sail restriction sites.
  • This PCR product was digested with EcoRI and Sail and inserted into EcoRI / X/zoI-digested vectors pSA4 and pSLH2, derivatives of vector pLM 3 that add decahistidine (pSA4) or hexahistidine (pSLH2) peptides, followed by a stop codon, to the C-terminus of the gene.
  • the resulting AcGFP(His) 10 and AcGFP(HiS) 6 genes were digested with EcoRI I Sail and ligated to the EcoRI I S ⁇ /I-digested vector pSMl, a derivative of pLM 3 modified by S.
  • HA hemagglutinin
  • the cultures were shaken at 3O 0 C for 5 h and cells harvested by centrifugation (4400 rpm, 10 min). The cell pellets were resuspended in Bacterial Protein Extraction Reagent (BPER, Pierce, 3 mL). The cells were shaken at 3O 0 C for 30 min, centrifuged (10,000 rpm, 5 min) and the supernatant was applied to packed Talon resin (7.5 mL, Clontech / BD biosciences) prewashed with phosphate buffered saline (DPBS, pH 8).
  • BPER Bacterial Protein Extraction Reagent
  • the resin was washed with cold DPBS (5 x 5 mL), and the protein was eluted from the resin by washing with DPBS containing imidazole (300 mM, 2 x 2 mL).
  • the eluate was concentrated by centrifugation against a protein-impermeable membrane (Millipore centricon concentrator, 10K) and washed at 4 0 C with cold DPBS (5 x 1.5 mL) to remove the imidazole.
  • proteins were analyzed by SDS PAGE on a 15% polyacrylamide gel (Cambrex) and detected by staining with coomassie dye.
  • the concentration of the protein was quantified using the Coomassie Plus protein assay reagent (Pierce).
  • the purified proteins were stored at final concentrations of: 1.19 mg / mL (AcGFP(His)io, 1000 ⁇ L) and 1.09 mg / mL, (AcGFP(His) 6 , 750 ⁇ L). Amino acid sequences of AcGFP proteins expressed in E. coli.
  • these proteins also include an N- terminal hemagglutinin (HA) epitope tag (sequence: YPYDVPDYA).
  • HA hemagglutinin
  • AcGFP(HJs) n MRGSGTELQLMASYPYDVPDYASPEFGLEMVSKGAELFTGIVPILIELNGDVNGHK FSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQH DFFKSAMPEGYIQERTIFFEDDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILG NKMEYNYNAHNVYIMTDKAKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGP VLLPDNHYLSTQSALSKDPNEKRDHMIYFGFVTAAAITHGMDELYKVEGTHHHH
  • EXAMPLE 23 Oligohistidine peptides are often fused to proteins to provide affinity tags that facilitate protein purification.
  • an artificial cell surface receptor (1) comprising the plasma membrane anchor iV-alkyl-3 ⁇ -cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA).
  • NTA metal chelator nitrilotriacetic acid
  • iV-Alkyl-3 ⁇ -cholesterylamine derivatives can function as prosthetic molecules active on the surface of living mammalian cells because this steroid can insert into cellular plasma membranes, project linked headgroups from the cell surface, and rapidly cycle between the plasma membrane and intracellular endosomes, similar to many naturally- occuring cell surface receptors.
  • NTA motif binds tightly to nickel, cobalt, copper, and zinc dications, and immobilized metal chelate chromatography (IMAC) with NTA- linked supports is widely used to purify proteins fused to His-tags. Other lipids linked to NTA headgroups have also been reported. 9 ' 10
  • Receptor 1 was synthesized from 3 ⁇ -cholesterylamine (2) 8 as shown in Scheme 1. Alkylation of 2 with ethyl 5-bromovalerate, followed by Boc protection of the secondary amine to afford 3, was found to provide an improved route to protected iV-alkyl derivatives of the membrane anchor. Addition of a linker shown to increase the population of these compounds on the cell surface, 8 acylation with a protected NTA derivative, and final deprotection afforded 1 in 47% overall yield. As representative His-tagged proteins, we overexpressed the monomeric green fluorescent protein AcGFP fused to C-terminal (His) 6 and (HiS) 1O peptides and purified these proteins by IMAC.
  • the (His) 10 peptide binds Ni-NTA complexes ⁇ 6-fold more tightly than (His) 6 .
  • human Jurkat T-lymphocytes were treated with 1 (10 ⁇ M) for one hour to load this compound into the outer leaflet of the cellular plasma membrane. These cells were washed and subsequently treated with a solution of AcGFP(His) 10 (3.2 ⁇ M) and Ni(OAc) 2 (100 ⁇ M) for an additional four hours. As shown in Figure 24 (panel A), examination of these cells by confocal laser scanning microscopy revealed fluorescent protein both on cell surface and in defined intracellular compartments.
  • cellular viability was examined by flow cytometry.
  • Jurkat lymphocytes were treated with receptor 1 (10 ⁇ M, 1 h), followed by AcGFP(His)i 0 (3.2 ⁇ M), and Ni(OAc) 2 (100 ⁇ M, 4 h) to enhance protein uptake by 600-fold.
  • Cells were washed with disodium NTA to remove cell surface protein and were cultured for an additional 48 h.
  • EXAMPLE 24 References for examples 16-23. (1) Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B. R. /. Am. Chem.
  • EXAMPLE 25 A Synthetic Scheme Describing the Synthesis of the Lipid- mimicking synthetic receptors, e.g. CRP receptors (lipid mimics that promote cellular apoptosis in the presence of C-reactive protein)
  • CRP receptors lipid mimics that promote cellular apoptosis in the presence of C-reactive protein
  • EXAMPLE 28 Synthesis of 3 ⁇ -amino-5-cholestene and Derivatives from cholesterol (yields represented on a 1 mmol scale).
  • the method for synthesis of 3 ⁇ -amino-5-cholestene (7) by i-steroid and retro-j-steroid rearrangements comprises reacting cholesterol (1) or a cholesterol bearing a leaving group at the 3- position, for example, cholesterol mesylate (2), cholesterol triflate, cholesterol tosylate or cholesterol esters, with a Lewis acid, for example, boron trifluoride etherate, in the presence of an azide, for example, a silyl azide such as trimethlysilyl azide, at a temperature between minus 150 0 C to plus 200 0 C and a Lewis acid, such as boron trifluoride etherate, in a solvent such as dichloromethane, to obtain a reaction mixture comprising 3 ⁇ -azi
  • 3 ⁇ -azido-5-cholestene (3) is then reacted with a reducing agent such as lithium aluminium hydride in a solvent such as diethyl ether to produce 3 ⁇ -amino-5-cholestene (7).
  • a reducing agent such as lithium aluminium hydride
  • a solvent such as diethyl ether
  • the reduction of 3 ⁇ -azido-5-cholestene (3) by catalytic hydrogenation may be performed in tetrahydrofuran over 10% palladium on carbon or another suitable catalyst.
  • the novel method for synthesis of 3 ⁇ -chloro-5-cholestene (4) by i-steroid and retro- i- steroid rearrangements was further examined.
  • the method for synthesis of 3 ⁇ -chloro-5-cholestene (4) comprises reacting cholesterol (1) or a cholesterol-bearing a leaving group at the 3-position, for example, cholesterol mesylate (2), cholesterol triflate, cholesterol tosylate or cholesterol esters, with titanium (IV) chloride, trimethylsilyl chloride, and dichloromethane, achieving a 95% yield.
  • the synthesis methods for 3 ⁇ - bromo-5-cholestene (5) by i-steroid and retro-j-steroid rearrangements may comprise reacting cholesterol (1) or a cholesterol bearing a leaving group at the 3-position, for example, cholesterol mesylate (2), cholesterol triflate, cholesterol tosylate or cholesterol esters, with boron trifluoride-diethyl ether complex, trimethylsilyl bromide, and dichloromethane, achieving a 92% yield.
  • novel method for synthesis of 3 ⁇ - iodo-5-cholestene (6) by i-steroid and retro-j-steroid rearrangements may comprise reacting cholesterol (1) or a cholesterol bearing a leaving group at the 3-position, for example, cholesterol mesylate (2), cholesterol triflate, cholesterol tosylate or cholesterol esters, with boron trifluoride-diethyl ether complex, trimethylsilyl iodide, and dichloromethane, achieving an 82% yield.
  • EXAMPLE 29 Additional Synthetic procedures and compound characterization data. Chemical reagents were obtained from Acros, Aldrich or Alfa Aesar. Solvents were from EM Science. Commercial reagents were used without further purification unless otherwise noted.
  • EI spectra were obtained on a ZAB HS mass spectrometer (VG Anlytical Ltd, Manchester, UK) or an Agilent 6890N GC interfaced with quadrupole mass analyzer (Quattro Micro GC, Waters corp., Milford, MA); ESI spectra were obtained on a LCT Premier time of flight mass spectrometer (Waters Corp., Milford, MA). Peaks are reported as m/z.
  • Compound 7 could alternatively be prepared by hydrogenation of 3 as follows: To a solution of 3 ⁇ -azido-5a-cholestene (3, 10.5 g, 25.5 mmol) in anhydrous THF (120 mL) in a round bottom flask (250 mL) was added Pd (10%) on activated carbon (2.65 g, 2.5 mmol). The suspension was stirred under one atmosphere of hydrogen for 24 h at ambient temperature (22 0 C).
  • EXAMPLE 30 References for Example 29. (1) Poza, J. et al, Bioorg. Med. Chem. 2007, 15, 4722-4720.

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Abstract

La présente invention porte sur des procédés de synthèse d’éléments de liaison membranaire préférés, de préférence des dérivés de cholestérylamine, comprenant le 3-amino-5-cholestène (3-cholestérylamine) et les 3-halogénures apparentés par des réarrangements de 1-stéroïde et rétro-1-stéroïde.
PCT/US2009/067897 2008-12-15 2009-12-14 Synthèse de 3b-amino-5-cholestène et de 3b-halogénures apparentés mettant en jeu des réarrangements de 1-stéroïde et rétro-1-stéroïde WO2010071772A2 (fr)

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JP7080826B2 (ja) 2016-05-16 2022-06-06 ザ ボード オブ リージェンツ オブ ザ ユニバーシティー オブ テキサス システム カチオン性スルホンアミドアミノ脂質および両親媒性両性イオンアミノ脂質
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