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WO2019013707A1 - Mimétiques de glycosaminoglycane pénétrant des cellules - Google Patents

Mimétiques de glycosaminoglycane pénétrant des cellules Download PDF

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WO2019013707A1
WO2019013707A1 PCT/SG2018/050342 SG2018050342W WO2019013707A1 WO 2019013707 A1 WO2019013707 A1 WO 2019013707A1 SG 2018050342 W SG2018050342 W SG 2018050342W WO 2019013707 A1 WO2019013707 A1 WO 2019013707A1
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group
polyproline
backbone
molecule
glycosaminoglycan
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Song-Gil Lee
Su Seong Lee
Ashwin Kumar
Teck Chuan Lim
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Agency for Science Technology and Research Singapore
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides

Definitions

  • the present invention relates to a glycosaminoglycan mimetic molecule comprising a polyproline backbone, an anionic pendant group and a fiuorophore.
  • the present invention also relates to the use of a glycosaminoglycan mimetic molecule comprising a polyproline backbone and an anionic pendant group for internalizing a target molecule into a cell.
  • the present invention also relates to the method of internalizing a target molecule into a cell, the method comprising the step of contacting a glycosaminoglycan mimetic molecule having a polyproline backbone and an anionic pendant group with a target molecule.
  • the present invention also relates to a method for synthesizing a glycosaminoglycan mimetic molecule.
  • GAGs Intracellular glycosaminoglycans
  • FGF2 Fibroblast Growth Factor 2
  • Intracellular heparan sulfate has been shown to be critical for ⁇ cell survival, and the degradation of intracellular heparan sulfate ultimately resulted in ⁇ cell death, leading to the onset of diabetes.
  • intracellular GAGs are actively involved in regulating the cell proliferation and cell cycle.
  • glycosaminoglycan mimetic molecule comprising a polyproline backbone, an anionic pendant group and a fluorophore.
  • the glycosaminoglycan mimetic molecule is attached to a fluorophore. Accordingly, the presence and absence, as well as the location of the molecule, can be quantified by detecting the resulting fluorescence signal. Further advantageously, the glycosaminoglycan mimetic molecule may facilitate the study of how glycosaminoglycan molecules, in general, behave in a cell. In particular, the glycosaminoglycan mimetic molecule may help to visualize how, to what extent and the colocalization of glycosaminoglycan molecules when glycosaminoglycan molecules, in general, are internalized into cells.
  • the presence of anionic pendant groups of the glycosaminoglycan mimetic molecule may affect the cellular internalization efficiency of the molecule.
  • spatial display of the anionic pendant group may affect the cellular internalization efficiency of the molecule, whereby more internalization may occur if the anionic pendant group is presented on all faces of the polyproline backbone.
  • increasing the chain length of the glycosaminoglycan mimetic molecule may improve the cellular internalization efficiency.
  • the glycosaminoglycan mimetic molecule may advantageously be non-toxic to cells.
  • the glycosaminoglycan mimetic molecule may assist in improving drug design and penetration, be implemented in protein delivery mechanisms, and may potentially modulate intracellular functions. It may therefore have the potential to be a platform for future therapeutic targets.
  • a glycosaminoglycan mimetic molecule comprising a polyproline backbone and an anionic pendant group for internalizing a target molecule into a cell, wherein when the glycosaminoglycan mimetic molecule is covalently attached to the target molecule, the target molecule is internalized into the cell.
  • a method for internalizing a target molecule into a cell comprising the step of contacting a glycosaminoglycan mimetic molecule having a polyproline backbone and an anionic pendant group with a target molecule, wherein when the glycosaminoglycan mimetic molecule is covalently attached to the target molecule, the target molecule is internalized into the cell.
  • the use and method may facilitate efficient internalization of a target molecule into a cell.
  • the anionic pendant group which is highly negatively charged in nature, may allow for ionic interactions with the receptors and other proteins on the plasma membrane of a cell, allowing the glycosaminoglycan mimetic molecule to interface with cell surface proteins, and subsequently internalize.
  • the polyproline backbone with the anionic pendant groups may facilitate the internalization of target molecules such as fluorophores into a cell.
  • the polyproline backbone with the anionic pendant groups may facilitate the delivery of target molecules such as protein, DNA, lipid, polyethylene glycol, drug, fluorophore into cells.
  • a glycosaminoglycan mimetic molecule comprising a polyproline backbone and an anionic pendant group in the manufacture of a medicament for internalizing a target molecule into a cell.
  • glycosaminoglycan mimetic molecule comprising a polyproline backbone and an anionic pendant group for use in internalizing a target molecule into a cell.
  • a method for synthesizing a glycosaminoglycan mimetic molecule as defined above comprising the step of contacting a polyproline backbone, an anionic pendant group and a fluorophore under reaction conditions.
  • the method of synthesis may be straight-forward while being selective, facilitating facile synthesis of the glycosaminoglycan mimetic molecule, all the while ensuring high yield. Further advantageously, the synthesis may ensure that the product is bio-compatible, by utilizing bio-orthogonal chemistry.
  • glycosaminoglycan refers to any complex polysaccharides having repeating units of either the same saccharide subunit or two different saccharide subunits.
  • Some examples of natural glycosaminoglycans include dermatan sulfates, hyaluronic acid, the chondroitin sulfates, chitin, heparin, keratan sulfates, keratosulfates, heparan sulfates, and derivatives thereof.
  • protein refers to a polymer of at least two amino acids that are covalently linked.
  • the amino acids may be D- or L- amino acids, or mixtures of D- and L-amino acids, as well as naturally occurring or synthetically produced amino acids.
  • glycosaminoglycan refers to a molecule that has a structure, and typically biological properties, that are similar to the molecule it is imitating.
  • glycosaminoglycan refers to a molecule which because of its structural properties, is capable of mimicking the biological function of a glycosaminoglycan.
  • derivative refers to a chemically or biologically modified version of a compound or molecule that is structurally similar to a parent compound or molecule and is derived from that parent compound or molecule.
  • pendant group refers to any functional group that may be attached to, and forms a side -chain of a macromolecule. Typically, the pendant group is attached to the backbone of the macromolecule.
  • the anionic group may form the pendant group that is attached to the polyproline backbone of the glycosaminoglycan mimetic, via linkages such as a covalent bond.
  • Exemplary pendant groups on the polyproline backbone of glycosaminoglycan mimetic include, but are not limited to, hydroxyl, sulfate, phosphate or carboxylate group-containing groups.
  • the glycosaminoglycan mimetic may contain one of the hydroxyl, sulfate, phosphate or carboxylate group-containing groups, or a combination thereof.
  • pre -determined refers to any position along the polyproline backbone that has been selected for attachment of one or more non-saccharide molecules.
  • the position(s) may, for example, have been selected for attachment of one or more non-saccharide molecules to modulate one or more biological functions of the glycosaminoglycan mimetic, for example improved molecular stability, improved selectivity or specificity, improved binding affinity, or the like.
  • attach and “conjugate” and variations of that term including “attaching”, “attachment”, “conjugating” and “conjugation” refers to any form of association of one molecule to another, either directly or indirectly (such as via a linker), via any means including but not limited to a covalent bond, via hybridization, via non-covalent interactions, such as receptor-ligand interactions.
  • face as used herein with reference to the polyproline backbone, refers to a distinct surface on the polyproline backbone to which a pendant group may be attached. Several faces may run roughly parallel along the same polyproline backbone.
  • inhibitor refers to a molecule that interferes (e.g. prevents) with the interaction (e.g. binding) between two or more other molecules.
  • the inhibition can take place in vitro or in vivo, and can be a direct or indirect inhibition.
  • fluorophore and “fluorogen” may be used interchangeably for the purpose of this disclosure, and generally refer to a chemical compound that can re -emit visible light upon excitation by visible light.
  • Alkyl as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a Q-C50 alkyl, preferably a Q-Q2 alkyl, more preferably a Q-Qo alkyl, most preferably C C 6 unless otherwise noted.
  • suitable straight and branched Ci-Ce alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like.
  • the group may be a terminal group or a bridging group.
  • Alkenyl as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain.
  • the group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z.
  • Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl.
  • the group may be a terminal group or a bridging group.
  • Alkynyl as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain.
  • Exemplary structures include, but are not limited to, ethynyl and propynyl.
  • the group may be a terminal group or a bridging group.
  • Amino refers to groups of the form -NR a R b wherein R a and R b are individually selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl groups.
  • Aryl as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring.
  • aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C 5 _ 7 cycloalkyl or C 5 _ 7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.
  • the group may be a terminal group or a bridging group.
  • an aryl group is a C 6 -C 18 aryl group.
  • Halogen represents chlorine, fluorine, bromine or iodine.
  • optionally substituted means the group to which this term refers may be unsubstituted, or unless otherwise specified, may be substituted with one or more groups independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl, optionally substituted amine, optionally substituted acylamino, optionally substituted alkyloxy, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or any mixture thereof.
  • the terms “comprising” and “comprise”, and grammatical variants thereof, are intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, unrecited elements.
  • the term “about”, in the context of concentrations of components of the formulations typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • range format may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Certain embodiments may also be described broadly and generically herein.
  • glycosaminoglycan mimetic molecule comprising a polyproline backbone, an anionic pendant group and a fiuorophore.
  • the anionic pendant group and the fiuorophore may be independently covalently bonded to the polyproline backbone.
  • the anionic pendant groups may be bound to one or more prolines on the proline backbone.
  • the anionic pendant group may be attached at a pre -determined position along the polyproline backbone.
  • the anionic pendant group may be attached at equal distances along the polyproline backbone.
  • the anionic pendant group may be attached along more than one different face of the polyproline backbone.
  • the anionic pendant group may be attached along the same face of the polyproline backbone or attached along three different faces of the polyproline backbone.
  • the polyproline backbone may repeat precisely every three units to form a polyproline backbone helix having three faces.
  • the anionic pendant group may be distributed on all three sides of the polyproline backbone helix.
  • the anionic pendant group may only appear on one face of the polyproline backbone helix.
  • glycosaminoglycan mimetic molecule may have the following general formula (I) or (la):
  • y and z may independently be 0, 1 or 2.
  • an anionic pendant group within the bracket bound by n or m, respectively, may be attached to every proline group in the polyproline background, and the anionic pendant group may be distributed on all three sides of the polyproline backbone helix.
  • n and m may independently be an integer of at least 1.
  • n and m may independently be an integer of from 1 to 30.
  • n and m may independently be an integer from 1 to 23.
  • n and m may independently be an integer of from 6 to 24.
  • n and m may independently be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
  • n and m may independently preferably be an integer selected from 6, 12, 18 or 24.
  • the value of n + m may be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
  • the value of n + m may be an integer selected from 6, 12, 18 or 24.
  • Q 1 and Q 2 may independently be an anionic group.
  • Q 1 and Q 2 may independently comprise primary, secondary or tertiary negatively charged groups, or a combination thereof.
  • the negatively charged group may be selected from the group consisting of hydroxyl, sulfate, carboxylate and phosphate.
  • the negatively charged group may be sulfate.
  • Q 1 and Q 2 may be an anionic group.
  • Q 1 and Q 2 may independently comprise primary, secondary or tertiary negatively charged groups, or a combination thereof.
  • the negatively charged group may be selected from the group consisting of hydroxyl, sulfate, carboxylate and phosphate.
  • the negatively charged group may be sulfate.
  • Q 1 and Q 2 may
  • a 3 may independently be a linker comprising a group selected from the group consisting of alkyl, alkenyl, alkynyl, amide, disulfide, ester, ether, triazole, and any mixture thereof.
  • the linker may be an alkyl-amide, amide-alky, alkyl-triazole or triazole-alkyl.
  • a 1 and A 2 a triazole.
  • a 1 and A 2 may independently have the
  • s p may be 0 or an integer of 1 to 10. p may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • a 3 may comprise an amide bond.
  • R may be H, an alkyl or aryl.
  • the alkyl may be methyl.
  • the fluorophore may be Alexa Fluor® 488, having the following structure:
  • glycosaminoglycan mimetic molecule may have the following general formula (II) or (Ila):
  • glycosaminoglycan mimetic molecule may have the following formula (III) or (Ilia):
  • glycosaminoglycan mimetic molecule comprising a polyproline backbone and an anionic pendant group for internalizing a target molecule into a cell, wherein when the glycosaminoglycan mimetic molecule is covalently attached to the target molecule, the target molecule is internalized into the cell.
  • the cell may be in vitro or in vivo.
  • the cell may be in vitro.
  • glycosaminoglycan mimetic molecule may have the following general formula (IV) or (IVa):
  • the broken bond ( ) of general formula (III) may indicate where the glycosaminoglycan mimetic molecule may be attached to the target molecule.
  • the glycosaminoglycan mimetic molecule may have the following general formula (V) or (Va):
  • the target molecule may be selected from the group consisting of protein, DNA, lipid, polyethylene glycol, drug, fluorophore, and any mixture thereof.
  • the target molecule may be a fluorophore.
  • the target molecule may be an Alexa Fluor® fluorophore.
  • glycosaminoglycan mimetic molecule attached to the target molecule may have the following formula (III) or (Ilia):
  • a method for internalizing a target molecule into a cell comprising the step of contacting a glycosaminoglycan mimetic molecule having a polyproline backbone and an anionic pendant group with a target molecule, wherein when the glycosaminoglycan mimetic molecule is covalently attached to the target molecule, the target molecule is internalized into the cell.
  • the contacting step may comprise the step of covalently bonding the glycosaminoglycan mimetic molecule to the target molecule.
  • glycosaminoglycan mimetic molecule comprising a polyproline backbone and an anionic pendant group in the manufacture of a medicament for internalizing a target molecule into a cell.
  • glycosaminoglycan mimetic molecule comprising a polyproline backbone and an anionic pendant group for use in internalizing a target molecule into a cell.
  • a method for synthesizing a glycosaminoglycan mimetic molecule comprising the step of contacting a polyproline backbone, an anionic pendant group and a fluorophore under reaction conditions.
  • the polyproline backbone may be attached to the fluorophore before the anionic pendant group is attached to the polyproline backbone.
  • the polyproline backbond may comprise an amino group and the fluorophore may comprise a N-hydroxysuccinimide.
  • Each proline in the polyproline backbone may be independently a proline or a proline derivative.
  • the proline derivative may comprise a functional group for conjugation to an anionic pendant group, wherein the proline derivative is optionally selected from the group consisting of azidoproline, aminoproline, mercaptoproline, prolinecarboxylic acid, hydroxyproline and any mixture thereof.
  • the proline derivative may be azidoproline.
  • the anionic pendant group may be optionally substituted with a functional group for conjugation to the proline backbone, wherein the functional group is optionally selected from the group consisting of alkyne, carboxylic acid, sulfhydryl, amine, hydroxyl and any mixture thereof.
  • the anionic pendant group may be functionalized with an alkyne.
  • the anionic pendant group may be conjugated or covalently bonded to the proline backbone by a click reaction.
  • the polyproline backbone may comprise an azido group and the anionic pendant group may comprise an alkyne group.
  • the polyproline backbone may comprise an alkyne group and the anionic pendant group may comprise an azido group.
  • FIG. 1 is a schematic representation of the structures of the GAG mimetics synthesized.
  • Fig. 2 is a schematic representation of the structures of the GAG mimetics synthesized.
  • FIG. 2 is a schematic representation of the process for synthesizing the azidoprolines.
  • Fig. 3 is a schematic representation of the process in which the GAG mimetics are synthesized.
  • A synthesis of azidopolyprolines,
  • B synthesis of the conjugation to fluorophore and
  • C click reaction to attach the sulfated groups.
  • FIG. 4 is a schematic representation of the spatial display parameters (A) distributed design and (B) single-facial design of the pendant groups.
  • Fig. 5 is a schematic representation of the spatial display parameters (A) distributed design and (B) single-facial design of the pendant groups.
  • FIG. 5 refers to graphs showing flow cytometry data for mimetic internalization. HCF cells were incubated with 10 ⁇ GAG mimetic for 24 hours, and shows the (A) effect of pendant spatial display on internalization, (B) effect of sulfated pendants compared to unsulfated pendants on internalization rate and (C) effect of backbone chain length on the internalization rate.
  • FIG. 6 refer to confocal images of live HCF cells incubated with 10 ⁇ P E NC24 for 72 hours on a Zeiss LSM 510 microscope. Images were taken at 40x magnification.
  • A DAPI image showing cell nuclei
  • B FITC channel showing AF-488 tagged GAG mimetic
  • C Rhodamine channel showing endoplasmic reticulum staining
  • D Bright-field image showing cell outline
  • E Overlap image of (A), (B) and (C) and showing co-localisation of GAG mimetic with the endoplasmic reticulum and cell nuclei
  • F Overlap image of (B), (C) and (D), showing co- localisation of GAG-mimetic with the endoplasmic reticulum and cell outline.
  • FIG. 7 refers to a graph showing the cytotoxicity assay conducted with HCF cells incubated with various concentrations of P E NC24 for 72 hours/
  • Cells were suspended in fresh growth culture medium and 100 ⁇ of cell suspension were seeded on 96-well plates (ThermoFisher Scientific 167008) at a density of about 625 cells per mm 2 , and were allowed to attach overnight at 37 °C with 5% C0 2 in a humidified chamber. Fluorescent-tagged GAG mimetics (final concentration 10 ⁇ ) were dissolved in 100 ⁇ fresh growth culture medium. Cells were first rinsed with PBS, before adding the GAG mimetics dissolved in growth culture medium to the cells. After 24 hours incubation at 37 °C with 5% C0 2 in a humidified chamber, cells were processed for flow cytometry analysis.
  • the cells were rinsed with 100 ⁇ PBS, and incubated with 100 ⁇ Trypsin-EDTA (0.05%/0.02%) in PBS for 5 minutes at 37 °C with 5% C0 2 in a humidified chamber. Trypsin was neutralised upon mixing with 100 ⁇ Foetal Bovine Serum (10%) in PBS. Prior to FACS analysis on a BD FACSCelesta system, an additional 5 ⁇ trypan blue (Sigma -Aldrich T8154) was added and mixed in to the cell suspension to quench any cell-surface bound fluorophores. Flow cytometry data was processed using FlowJo (Treestar). Single, live cells were first identified and gated, and the mean fluorescence values were calculated.
  • Cells were suspended in fresh growth culture medium and 100 ⁇ of cell suspension were seeded on 96-well plates (ThermoFisher Scientific 167008) at a density of approximately 625 cells per mm 2 , and were allowed to attach overnight at 37 °C with 5% C0 2 in a humidified chamber. Fluorescent GAG mimetics (final concentration 10 ⁇ ) were dissolved in 100 ⁇ growth culture medium and were added to the cells. After 72 hours incubation at 37 °C with 5% C0 2 in a humidified chamber, cells were rinsed with 100 ⁇ PBS.
  • PrestoBlue Cell Viability Reagent Invitrogen
  • 100 ⁇ growth culture medium was also added to an additional well without any cells to measure background fluorescence. Fluorescence was measured at 590 nm using a fluorescent plate reader with gain set to 75 (Tecan Infinite M200 Pro) and the values were normalized to the control well using the following formula, where the control cells are incubated without GAG mimetics. / n 11 v K ' i t - ⁇ 590 - - ⁇ 590 ⁇ No Cells ⁇
  • reaction mixture was stirred at 50 ⁇ 55 °C for 6 hr.
  • the reaction mixture was diluted with 100 mL of EtOAc and extracted with brine.
  • the reaction was then concentrated and purified by flash chromatography (10 ⁇ 30% hexanes:EtOAc) to afford the desired product (1, 0.182 g, 84.2%) as a colorless oil.
  • Boc- azidopolyproline(l)-azidopolyproline(2)-OCH 3 was dissolved in CH 2 C1 2 /TFA (1 :1) at 0 °C, and stirred for 2 hours.
  • Pent-4-yne-l,2-diyl bis(sulfate) (40.0 equiv.), AF488-azidopolyprolines (1.0 equiv.), and TBTA (0.3 equiv. per azide) were added into a vial. Under argon atmosphere, the mixture was dissolved in anhydrous DMSO (final concentration: 0.1 M) and copper (I) iodide stock solution in DMSO (0.6 mol% per azide) and DIPEA (120.0 equiv.) were sequentially added. The reaction mixture was then stirred for 7 to 10 days at room temperature under argon atmosphere.
  • Table 1 Summary of the mean fluorescent intensities (MFI) of the synthesized GAG mimetics.
  • the efficacy of the pendant unit display along the polyproline backbone of the GAG mimetics was evaluated as it has been previously suggested that the sulfation display can affect the internalization efficiency.
  • the polyproline helix repeats precisely every three units, this was used to create specific designs with pendants distributed on all three sides of the polyproline helix (hereafter described as the distributed design, Fig. 4A).
  • the prolines can be interspaced with pendants with two unadorned proline molecules, resulting in chemical motifs on only one side of the molecule (hereafter described as the single -facial design, Fig. 4B).
  • the effect of spatial display of these chemical motifs were investigated on cellular internalization, to elucidate how critical this is.
  • Full length heparin has a greater binding affinity to cell surface proteins compared to low- molecular weight heparin. Additionally, GAGs with a greater number of sulfated groups are able to interact with cell surface proteins and internalize better than less sulfated GAGs. This prompted the investigation into the effect of extending the chain length of the GAG mimetic and increasing the number of sulfation units, in an effort to increase the rate of internalization.
  • GAG mimetics of various lengths with 6, 12 18 and 24 repeating units were synthesized.
  • Table 1 and Fig. 5C when the GAG mimetic design was extended from P E NC 12 to P E _ NC 18 and P E _ NC 24, significant increase in the internalization potential was observed. This resulted in a 50% increase in fluorescence between P E NC 12 and P E NC 24. Conversely, while reducing the length to P E N c6, there was an overall reduction in internalization as a 56% decrease in fluorescence was observed.
  • endocytosis In the internalization of fluorescently tagged GAG mimetics was investigated. From the four major modes of endocytosis, which are phagocytosis, macropinocytosis, caveolin mediated endocytosis, and clathrin mediated endocytosis, only clathrin mediated endocytosis and caveolin mediated endocytosis are implicated in small molecule internalization.
  • HCF cells live HCF cells were imaged using confocal microscopy (Zeiss LSM 510). Cells were grown for 12 hours on glass coverslips before a 72 hour incubation period with the GAG mimetc (P E NC 12 at a concentration of 10 ⁇ ).
  • FIG. 6 shows DAPI staining for cell nuclei
  • B shows FITC staining for AF 488 conjugated GAG mimetic
  • C shows rhodamine staining for endoplasmic reticulum
  • D shows bright-field image for cell outline
  • E shows overlap image of A, B, and C for co-localization of GAG mimetic with the endoplasmic reticulum and cell nuclei
  • F shows the overlap image of B, C, and D for co-localization of GAG mimetic with the endoplasmic reticulum and cell outline.
  • the glycosaminoglycan mimetic molecule comprising a polyproline background, an anionic pendant group and a fluorophore may be useful in studying how glycosamine molecules, in general, behave when they become internatlized in cells.
  • the glycosamine molecule may be further useful in studying what properties of the glycosaminoglycan molecule affects their internalization efficiency.
  • glycosaminoglycan mimetic molecule comprising a polyproline background and an anionic pendant group may be further useful in internalizing a target molecule into a cell.
  • the glycosaminoglycan mimetic molecule may be attached to any target molecule whereby internalization into the cell is desired, to ensure that the target molecule is internalized into the cell.
  • the glycosaminoglycan mimetic molecule may be useful in delivering drugs into cells that may otherwise be difficult to internalize, to enhance treatment of diseases, and for delivering fluorophores into cells to enhance imaging capabilities.

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Abstract

La présente invention concerne une molécule mimétique de glycosaminoglycane comprenant un squelette polyproline, un groupe anionique pendant et un fluorophore. L'invention concerne également l'utilisation de la molécule mimétique de glycosaminoglycane pour l'internalisation d'une molécule cible dans une cellule, un procédé d'internalisation d'une molécule cible dans la cellule, et un procédé de synthèse d'une molécule mimétique de glycosaminoglycane. [Pas de figure donnée à titre d'exemple]
PCT/SG2018/050342 2017-07-11 2018-07-11 Mimétiques de glycosaminoglycane pénétrant des cellules Ceased WO2019013707A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024181456A1 (fr) * 2023-02-27 2024-09-06 富士フイルム株式会社 Composé et biomatériau marqué l'utilisant
WO2024181455A1 (fr) * 2023-02-27 2024-09-06 富士フイルム株式会社 Composé et biomatériau marqué l'utilisant
WO2024181454A1 (fr) * 2023-02-27 2024-09-06 富士フイルム株式会社 Composé et biomatériau marqué l'utilisant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014175838A1 (fr) * 2013-04-26 2014-10-30 Agency For Science, Technology And Research Modulation d'interactions polyvalentes de biopolymères présentant une chaîne principale de polyproline
WO2017082827A1 (fr) * 2015-11-12 2017-05-18 Agency For Science, Technology And Research Ingénierie de mimétiques des glycosaminoglycanes non saccharidiques structuralement définis, par l'intermédiaire d'un échafaudage polyproline

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014175838A1 (fr) * 2013-04-26 2014-10-30 Agency For Science, Technology And Research Modulation d'interactions polyvalentes de biopolymères présentant une chaîne principale de polyproline
WO2017082827A1 (fr) * 2015-11-12 2017-05-18 Agency For Science, Technology And Research Ingénierie de mimétiques des glycosaminoglycanes non saccharidiques structuralement définis, par l'intermédiaire d'un échafaudage polyproline

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU, P. ET AL.: "Tailored chondroitin sulfate glycomimetics via a tunable multivalent scaffold for potentiating NGF/TrkA-induced neurogenesis", CHEMICAL SCIENCE, vol. 6, no. 1, 15 October 2014 (2014-10-15), pages 450 - 456, XP055565944, [retrieved on 20180926] *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024181456A1 (fr) * 2023-02-27 2024-09-06 富士フイルム株式会社 Composé et biomatériau marqué l'utilisant
WO2024181455A1 (fr) * 2023-02-27 2024-09-06 富士フイルム株式会社 Composé et biomatériau marqué l'utilisant
WO2024181454A1 (fr) * 2023-02-27 2024-09-06 富士フイルム株式会社 Composé et biomatériau marqué l'utilisant

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