Receptors on the surface of mammalian cells promote the uptake of cell-impermeable ligands by receptor-mediated endocytosis. To mimic this process, we synthesized small molecules designed to project anti-dinitrophenyl antibody-binding motifs from the surface of living Jurkat lymphocytes. These synthetic receptors comprise N-alkyl derivatives of 3beta-cholesterylamine as the plasma membrane anchor linked to 2,4-dinitrophenyl (DNP) and structurally similar fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD) headgroups. Insertion of two beta-alanine subunits between a DNP derivative and 3beta-cholesterylamine yielded a receptor that avidly associates with cell surfaces (cellular t(1/2) approximately 20 h). When added to Jurkat cells at 10 microM, this receptor enhanced uptake of an anti-DNP IgG ligand by approximately 200-fold in magnitude and approximately 400-fold in rate within 4 h (ligand internalization t(1/2) approximately 95 min at 37 degrees C). This non-natural receptor mimics many natural receptors by dynamically cycling between plasma membranes and intracellular endosomes (recycling t(1/2) approximately 3 min), targeting of protein ligands to proposed cholesterol and sphingolipid-enriched lipid raft membrane microdomains, and delivery of protein ligands to late endosomes/lysosomes. Quantitative dithionite quenching of fluorescent extracellular NBD headgroups demonstrated that other 3beta-cholesterylamine derivatives bearing fewer beta-alanines in the linker region or N-acyl derivatives of 3beta-cholesterylamine were less effective receptors due to more extensive trafficking to internal membranes. Synthetic cell surface receptors have potential applications as cellular probes, tools for drug delivery, and methods to deplete therapeutically important extracellular ligands.

Fluorescent small molecules are powerful tools for exploring cellular biology. As a more hydrophobic, photostable, and less pH sensitive alternative to fluorescein, we synthesized Pennsylvania Green, a bright, monoanionic fluorophore related to Oregon Green and Tokyo Green.Comparison of membrane probes comprising N-alkyl-3 -cholesterylamine linked to 4-carboxyTokyo Green (pKa ~ 6.2) and 4-carboxy-Pennsylvania Green (pKa ~ 4.8)revealed that only Pennsylvania Green was highly fluorescent in acidic early and recycling endosomes within living mammalian cells.Molecular probes derived from fluorescein (1, Figure 1) are widely used as tools for studies of cellular biology. This green fluorophore is particularly suited for cellular analysis by confocal laser scanning microscopy and flow cytometry due to its excitation maximum at 490 nm, closely matching the 488 nm spectral line of the argon-ion laser. In addition, fluorescein has a high molar absorptivity and excellent quantum yield (0.92 at pH 9). Under physiological conditions (pH 7.4), fluorescein is predominantly a highly hydrophilic dianion. However, the monoanionic form of fluorescein exhibits the relatively high pKa of 6.5, rendering this dye much less fluorescent in acidic solutions. 1 Fluorescein is also relatively susceptible to photobleaching, which is thought to involve reactions with molecular oxygen and proximityinduced reactions of the dye. 2 Oregon Green, a more acidic 2', 7'-difluoro derivative of fluorescein (2, Figure 1), was developed as a less pH-sensitive fluorophore. 3 The appended fluorine atoms reduce the pKa of this dye to 4.8, substantially improving fluorescence at low pH. This compound is also significantly more photostable than fluorescein. However, the high cost and high polarity of Oregon Green limits its utility as a building block for hydrophobic molecular probes.As a more hydrophobic alternative to fluorescein, recent pioneering work by Urano, Nagano, and coworkers replaced the carboxylate of fluorescein with a methyl group. 4 This structural modification yielded a highly fluorescent monoanionic fluorophore termed Tokyo Green (3, Figure 1). This analogue of fluorescein provides a new platform for the design of fluorescent probes.We report here the synthesis of a novel fluorophore termed Pennsylvania Green (4). This fluorophore melds the pH-insensitivity and photostability of Oregon Green with the hydrophobicity of Tokyo Green. To demonstrate the utility of the Pennsylvania Green fluorophore, we compared cellular membrane probes derived from 4-carboxy-Tokyo Green brpeters@chem.psu.edu. (5) and 4-carboxy-Pennsylvania green (6). The lower pKa of the Pennysylvania Green-derived probe enables visualization of early / recycling endosomes within living mammalian cells, and this fluorophore provides a usful tool for analysis of these and related acidic intracellular compartments. NIH Public AccessThe synthesis of 4-carboxy-Pennsylvania Green (6) was accomplished in 10-steps from commercially available 1,2,4-trifluoro-5-nitrobenzene (7)...

The emergence of multidrug-resistant tuberculosis (TB) and problems with the BCG tuberculosis vaccine to protect humans against TB have prompted investigations into alternative approaches to combat this disease by exploring novel bacterial drug targets and vaccines. Phosphatidylinositol mannosides (PIMs) are biologically important glycoconjugates and represent common essential precursors of more complex mycobacterial cell wall glycolipids including lipomannan (LM), lipoarabinomannan (LAM), and mannan capped lipoarabinomannan (ManLAM). Synthetic PIMs constitute important biochemical tools to elucidate the biosynthesis of this class of molecules, to reveal PIM interactions with host cells, and to investigate the function of PIMs as potential antigens and/or adjuvants for vaccine development. Here, we report the efficient synthesis of all PIMs including phosphatidylinositol (PI) and phosphatidylinositol mono- to hexa-mannoside (PIM1 to PIM6). Robust synthetic protocols were developed for utilizing bicyclic and tricyclic orthoesters as well as mannosyl phosphates as glycosylating agents. Each synthetic PIM was equipped with a thiol-linker for immobilization on surfaces and carrier proteins for biological and immunological studies. The synthetic PIMs were immobilized on microarray slides to elucidate differences in binding to the dendritic cell specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) receptor. Synthetic PIMs served as immune stimulators during immunization experiments in C57BL/6 mice when coupled to the model antigen keyhole-limpet hemocyanin (KLH).

Binding of ligands to macromolecular receptors on the surface of mammalian cells often results in ligand uptake through receptor-mediated endocytosis. Certain human leukocytes and epithelial cells express Fc receptors (FcRs) that bind and internalize antibodies through this mechanism. To mimic this process, we synthesized an artificial FcR comprising the membrane anchor N-alkyl-3beta-amino-5alpha-cholestane linked to a disulfide-constrained cyclic peptide, termed FcIII, known to exhibit high affinity and specificity for the Fc region of human IgG. Treatment of human Jurkat lymphocytes that lack natural FcRs with the synthetic FcR (1 microM, 1 h) installed an average of approximately 6.2 x 10(5) synthetic receptor molecules per cell surface. These treated cells gained the capacity to internalize human IgG at levels greater than human THP-1 cells that express the natural receptors FcgammaRI and FcgammaRII. By linking binding motifs for circulating ligands to membrane anchors that cycle between the cell surface and intracellular endosomes, minimalistic cell surface receptors can be used to destroy targeted ligands by endocytosis. These small mimics of macromolecular receptors may be useful for controlling the extracellular abundance of ligands involved in disease.

Extracellular domains of internalizing cell surface receptors are often targeted to enable drug delivery through the mechanism of receptor-mediated endocytosis. To circumvent natural receptors required for endocytic drug delivery, we constructed a small artificial cell surface receptor comprising the membrane anchor N-alkyl-3β-cholesterylamine linked to a D-Phe-D-Ala motif that binds the glycopeptide antibiotic vancomycin. By mimicking membrane association and trafficking properties of cholesterol, this cholesterol-derived synthetic receptor functions as a prosthetic molecule, inserting into plasma membranes of mammalian cells, and rapidly cycling between the cell surface and intracellular endosomes. Human HeLa cells treated with this receptor gained the ability to internalize vancomycin by endocytosis, enabling this cell-impermeable antibiotic to eradicate the lethal intracellular pathogen Listeria monocytogenes, and preserving the viability of the host cells in vitro. Moreover, when combined with the synthetic receptor, vancomycin penetrated the blood brain barrier, accumulating in the brain and other tissues of mice in vivo. Because of their ability to define new pathways across biological membrane barriers, small synthetic cell surface receptors represent promising tools for drug delivery.Receptor-mediated drug delivery is a Trojan horse approach for the release of therapeutics into cells, tissues, or organs. By coupling drugs to ligands of cell surface receptors, ligand-drug conjugates can accumulate in specific tissues and can be internalized by cells through the mechanism of receptor-mediated endocytosis. 1 Receptors targeted in this way include the low density lipoprotein (LDL) receptor, 2 transferrin receptors, 3 and folate receptors. 4 Receptors involved in the passage of nutrients across the blood-brain barrier through the related mechanism of receptor-mediated transcytosis have been similarly targeted to deliver drugs and imaging agents into the brain. 5 This latter strategy is of significant interest because less than 2% of all drugs are capable of penetrating the blood-brain barrier. 5We report here synthetic receptor-mediated drug delivery. This approach was applied to the glycopeptide antibiotic vancomycin (1, Figure 1, Panel A), a drug of last resort against bacterial pathogens such as methicillin-resistant Staphylococcus aureus. 6 By binding to peptidoglycan precursors that terminate in the peptide sequence D-Ala-D-Ala, vancomycin (1) blocks crosslinking of the bacterial cell wall, providing an effective mode of action against gram-positive bacteria. However, the high polarity and consequent low cell permeability of glycopeptide E-mail: brpeters@chem.psu.edu. antibiotics render these drugs ineffective against bacteria capable of replicating either in the cytoplasm of mammalian host cells or in organs such as the brain that are protected by membrane barriers. NIH Public AccessWe hypothesized that a synthetic receptor capable of promoting the endocytosis of vancomycin (1) might allow this...

Cell penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3β-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 micromolar) for one hour displayed ~ 8,400,000 NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 micromolar) and Ni(OAc) 2 (100 micromolar) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within four hours at 37 °C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology.Cell penetrating peptides and proteins (CPPs) have emerged as important new tools for the delivery of impermeable molecules into living mammalian cells. These delivery systems have been constructed from basic segments of HIV Tat, 1 oligoguanidine-containing peptoids, 2 β-peptides, 3 and other oligocationic motifs. 4 The mechanism of cellular penetration by CPPs is primarily thought to involve endocytosis, 5 particularly with larger cargo, 6 but varies depending on the delivery system and the cell type. 7Oligohistidine peptides are often fused to proteins to provide affinity tags ("His tags") that facilitate protein purification. To enable these peptides to function as CPPs, we synthesized an artificial cell surface receptor (1) comprising the plasma membrane anchor N-alkyl-3β-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). N-Alkyl-3β-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-occurring cell surface receptors. 8,9 The 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. 10 Other lipids 11,12 and polymers 13 linked to NTA headgroups have also been reported. Receptor 1 was synthesized from 3β-cholesterylamine (2) 9 as shown in Scheme 1. Alkylation of 2 with ethyl 5-bromovalerate, followed by Boc protection to afford 3, provided an improved route to protected N-alkyl derivativ...

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Glycosyl tricyclic orthoesters provide a versatile basis for the efficient generation of glycosyl phosphates, which are used in the automated synthesis of lipomannan backbone alpha(1-6) hexa-mannoside.