WO2000047548A1 - Improved compounds for protein binding - Google Patents

Improved compounds for protein binding Download PDF

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Publication number
WO2000047548A1
WO2000047548A1 PCT/AU2000/000075 AU0000075W WO0047548A1 WO 2000047548 A1 WO2000047548 A1 WO 2000047548A1 AU 0000075 W AU0000075 W AU 0000075W WO 0047548 A1 WO0047548 A1 WO 0047548A1
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compound according
group
membrane
substitute sheet
moiety
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PCT/AU2000/000075
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French (fr)
Inventor
Ping Yin
Christopher John Burns
Matthew Peter Wilkinson
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Australian Membrane And Biotechnology Research Institute
The University Of Sydney
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Application filed by Australian Membrane And Biotechnology Research Institute, The University Of Sydney filed Critical Australian Membrane And Biotechnology Research Institute
Priority to EP00904701A priority Critical patent/EP1150942A4/en
Priority to CA002341348A priority patent/CA2341348A1/en
Priority to AU26485/00A priority patent/AU2648500A/en
Priority to JP2000598469A priority patent/JP2002536428A/en
Publication of WO2000047548A1 publication Critical patent/WO2000047548A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/12—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

Definitions

  • the present invention relates to novel binding compounds, in particular protein binding compounds.
  • novel compounds are particularly useful for binding proteins to surface including membranes.
  • the present invention provides biosensors incorporating these protein binding compounds.
  • the present invention also extends to intermediate compounds for use in the synthesis of the binding compounds of the present invention.
  • Ternaiy metal complexes are well known in the literature (1) and can be described as the coordination of two discrete metal chelating groups to a metal.
  • the metals normally observed in ternary complexes are Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ .
  • Typical metal coordinating groups are nitrilotriacetic acid (NT A), iminodiacetic acid (IDA), catechols, and aromatic nitrogen containing heterocycles such as imidazole.
  • Ternary complexes have been characterised by numerous methods including potentiometric calculations (2) and X-ray crystallography (3). These studies have allowed the stability of simple ternary complexes to be determined with the average stability constant (Ka) being 10 3 -lu 4 M '1 (4).
  • IMAC Immobilised Metal Affinity Chromatography
  • the stability of the ternary complex i.e. the interaction between the metal chelate the metal and the protein, is often too weak for proteins to be immobilised for a sufficiently long period for observation and study.
  • the metal chelates employed interact in a non-specific manner with other non-tagged proteins.
  • the ternary complexes can be broken down in the presence of certain interferents at concentrations not unknown in certain assay systems.
  • the present inventors have developed compounds with improved characteristics to those compounds already disclosed in the literature. These compounds possess a plurality of metal-chelating groups covalently linked. The compounds can be used to attach proteins to materials and surfaces.
  • the present invention consists in a compound, the compound having the general Formula I
  • Y is a branching moiety and Z represents a polydentate ligand chelating agent that coordinates a metal ion; and n is an integer of at least 2, preferably from 2 to 9.
  • Z may be a polydentate ligand that coordinates a metal ion such as Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ .
  • the donor atoms of Z may be ⁇ - donor atoms or ⁇ - donor atoms.
  • the donor atoms may be selected from N, O, S, P and Si.
  • the donor atoms are N.
  • Z may be bidentate, tridentate (for example IDA) or quadradentate (for example NTA).
  • Z is other than a cyclic or polycyclic.
  • Z is a quadradentate ligand such as NTA.
  • Y provides at least three moieties for covalent attachment directly, or indirectly through an optional linking group, to Z.
  • the backbone of branching group Y may be a residue of a compound, an oligomer or a polymer.
  • the linking group has a linear backbone.
  • the present invention provides a compound of formula II
  • X may be hydrophilic, hydrophobic or have both hydrophobic and hydrophilic regions.
  • X may be or include a substituted or unsubstituted alkyl, optionally interrupted by one or more heteroatoms (eg 0, N, S or combinations of two or more thereof), for example oligoethylene glycol or other oligoalkylene
  • X may be, or include, a lipid.
  • the lipid may be a membrane spanning lipid (MSL).
  • MSL membrane spanning lipid
  • X includes an hydrophilic region, for example polyalkylene oligomer, and an hydrophobic region, for example a lipid, wherein X is attached to Y via an hydrophobic region optionally via a spacer.
  • the present invention provides a compound of Formula III
  • X, Y, Z and n are as described above and W is a group that allows for attachment to other molecules, or attachment to surfaces, or insertion into membrane(s).
  • W is a group which allows for attachment to other molecules including polymers such as Sepharose (such as an amine functional group, a carboxylic acid functional group, an alcohol functional group, a halide functional group) or attachment to surfaces (such as a thiol or disulfide for attachment to gold or other coinage metal surfaces, or such as a silane derivative for attachment to oxide surfaces) or insertion into membranes (such as a lipid group, or a membrane soluble protein, such as gramicidin). W may also be a group which enables non-covalent attachment such as biotin to streptavidin.
  • polymers such as Sepharose (such as an amine functional group, a carboxylic acid functional group, an alcohol functional group, a halide functional group) or attachment to surfaces (such as a thiol or disulfide for attachment to gold or other coinage metal surfaces, or such as a silane derivative for attachment to oxide surfaces) or insertion into membranes (such as a lipid group, or a membrane soluble
  • Y is a branching moiety that provides a plurality of moieties for covalent attachment of Z and a single moiety for covalent attachment of X.
  • Non-limiting illustrative examples of Y include: amino-polyols such as TRIS, bis-homotris
  • amino acids such as 3,5-diaminobenzoic acid, 5-aminoisophthalic acid,
  • Substitute Sheet peptides which possess multiple free acid and/or amine moieties, for example
  • Y is a branching moiety where there is a plurality of moieties for covalent attachment of Z and X.
  • Non-limiting illustrative examples of Y include:
  • polyamines such as spermjdine, spermine, pentaethylenehexamine
  • polyacids such as tartaric acid, trimesic acid, citric acid, Kemp's triacid
  • polyhydroxylated materials such as sugars
  • dendrons such as the commercially available “Starburst” compounds
  • nucleophiles such as halides, tosylates
  • groups to which nucleophiles readily add such as , ⁇ -unsaturated ketones
  • the compounds of the present invention may have a wide range of uses. In particular they are useful for the attachment of proteins and other biological macromolecules to surfaces. As will be understood this is a requirement of a multitude of sensing devices and assays.
  • Biosensors are well known in the art and are described in PCT/AU93/00620, PCT/AU96/00482, PCT/AU95/00763,
  • biosensors there is provided a membrane which includes ionophores and receptors. Binding of an analyte to the receptors causes a detectable change in the conductance or impedance of the membrane.
  • receptors directed against the analyte of interest are attached to the ionophore and to the membrane.
  • the receptors are proteins such as antibodies or antigen binding fragments thereof such as Fab'. It is believed that the binding compounds of the present invention will be useful in the attachment of such receptors to the ionophores and membrane of the biosensors.
  • FIG. 1 Binding of 6His.Rubisco to Jl.triNTA chip. 100, 200 and 400 nM binding curves shown (bottom to top). Binding curves are the responses from Fc2 (control, -Ni 2+ ) subtracted from Fcl.
  • Binding of 6His.CD40 to Jl.triNTA chip 100, 200, 400, 600 and 800 nM binding curves shown (bottom to top). Binding curves are the responses from Fc2 (control, -Ni 2+ ) subtracted from Fcl.
  • Binding of 6HIS.CD40 to triNTA has a 12 fold higher affinity than to NTA. This is mainly as a result of a 10-fold higher on rate (k .
  • the off rate (k for the NTA binding is likely to be exaggerated.
  • N6-carbobenzyloxy-L-lysine,2-tTimethylsilylethanol, l-(3-dimethylaminopropyl)-3- ethylcarbodiiuiide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N- hydroxysuccinamide (NHS) and 6-aminocaproic acid (X) were obtained from Sigma- Aldrich chemical company.
  • XXBoc was prepared by reacting XBoc NHS ester (itself obtained by protection of the amino group of X with BocON under standard conditions, and subsequent coupling with NHS using DCC) with X.
  • Dichloromethane was distilled over P 2 O 5 immediately prior to use.
  • Substitute Sheet 25 ml was added dropwise to the reaction mixture over 2 h. The solution was allowed to warm to room temperature and stirred at room temperature overnight. The reaction mixture was heated at 50°C for 2 h, then allowed to cool to room temperature. An aqueous solution of HC1 (IM, 40 ml) was added dropwise to the reaction mixture, and the resulting white precipitate was filtered and washed with HC1 (0.1M, 20 ml) and distilled water (2x20 ml). The resulting white solid was dried under high vacuum for several days to afford Z-LysNTA as a white solid (2.68 g, 95% yield).
  • Electrospray M/S m/z 397 (100%) (M+H + ), 398 (21%), 353 (30%), 792 (15%).
  • ZLysNTA.TMSE 3 was synthesised using a procedure outlined by Gao et al. 2
  • Substitute Sheet stirred at room temperature overnight. Distilled water (50 ml) was added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with dichloromethane (2x50 ml), and the organic extracts were combined and dried over anhydrous sodium carbonate. The mixture was filtered and all volatiles were removed under reduced pressure to afford a colourless oil. Purification by column chromatography on flash silica using a solvent gradient from dichloromethane to 5% methanol in dichloromethane afforded ZLysNTA.TMSE 3 as a colourless oil (0.75 g, 86%).
  • Electrospray M/S m/z 719.4 (100%) (M+Na + )
  • Substitute Sheet Rul 2 R ⁇ U multiplets 3.26-3.16 (10H, overlapping multiplets), 3.07 (IH, triplet), 1.85-1.30 (24H, overlapping multiplets, 12 x -CH 2 ), 0.97 (18H, overlapping multiplets, 9 x -CH 2 Si-), 0.05 (27H, s, 3 x -Si(CH 3 ) 3 ), 0.04 (54H, s, 6 x -Si(CH 3 ) 3 ) ppm.
  • Electrospray M/S m/z 2054.3 (100%) (M+Na + ), 1038.8 (20%), 613.3 (36%)
  • Electrospray M/S m/z 1151.4 (100%) (M+Na + ), 1129.2 (88%) (M+H + ), 1130.3 (56%) (M+2H + ), 1152.4 (53%) (M+Na + +H + ), 1165.3 (53%), 1143.3 (52%), 755.5 (50%), 907.2 (41%), 393.2 (26%).
  • Electrospray M/S m/z 2019.8 (35%), ( +Na + ), 1997.8 (100%) (M + ).
  • NTA TMSE 9 34 mg,0.0179 mmol was triturated with toluene, evaporated and dried under high vacuum.
  • Trifluoroacetic acid (1 ml) was added and stirred under nitrogen atmosphere 0-5° C for 2 hours, then at room temperature overnight. The trifluoroacetic acid was evaporated and the residue was triturated with toluene again, evaporated and dried to afford t ⁇ NTA (20 mg, 100%).
  • Gramicidin (75 mg, 0.0398 mmol) was dissolved in pyridine (0.5 ml) and succinic anhydride (20 mg, 0.200 mmol) was added. The mixture was stirred under nitrogen atmosphere at 50° C for 20 hours and evaporated. The crude product was passed down a sephadex LH-20 column in methanol, the eluate was evaporated and purified on a flash silica column using dichloromethane/methanol/water/acetic acid (400:50:4:1). The product was further purified by centrifuging with water. The water was decanted and the product was dried under high vacuum to give gramicidin succinate (53 mg, 67%).
  • Gramicidin succinate (21 mg, 0.0105 mmol), N-hydroxysuccinamide (12 mg, 0.1042 mmol) and 4-dimethylamino pyridine (2.5 mg, 0.0204 mmol) were combined with distilled tetrahydrofuran (10 ml). With stirring under nitrogen dicyclohexylcarbodiimide (22 mg, 0.1066 mmol) was added. The mixture was heated to reflux for one hour. The mixture was evaporated and passed down a sephadex LH- 20 column in methanol. Appropriate fractions were evaporated and dried to give gramicidin succinate NHS (22 mg, 100%).
  • Lysine tri NTA (20 mg, 0.0201 mmol) was dissolved in methanol (1 ml) and triethylamine (2 drops) was added to neutralise. Then gramicidin succinate NHS (15
  • Gramicidin lysine 2XBOC (12 mg,0.0052 mmol) was triturated with toluene, evaporated and dried under high vacuum. Trifluoroacetic acid (1 ml) was added, evaporated under nitrogen and dried. Again toluene was added, evaporated and dried under high vacuum. The crude amine was dissolved in pyridine (0.5 ml) and reacted with succinic anhydride (2.6 mg, 0.0259mmol). The reaction mixture was stirred at room temperature for 20 hours. Pyridine was removed under high vacuum and the residue was passed down the sephadex LH-20 column in methanol. The product was further purified on a flash silica column eluted with methanol to give gAlysine 2X Succinate (10 mg, 83%).
  • Substitute Sheet 0.0363mmol was added. The mixture was refluxed under nitrogen for 1 hour. The mixture was evaporated and purified on a sephadex LH-20 column in methanol. Appropriate fractions were evaporated and added to lysine tri NTA (8.5 mg, 0.0085 mmol). The mixture was stirred at room temperature for 18 hours. The reaction mixture was evaporated and purified on sephadex LH-20 in methanol (X2). Gramicidin lysine 2Xtri NTA (2.6 mg, 19%) was obtained (due to low solubility some compound was lost).
  • Electrospray M/S m/z 1173.9 (100%), 2146.4 (25%) (M+Na + ).
  • Trifluoroacetic acid (2 ml) was added to Biotin.triNTA.TMSE 9 (19 mg, 9 umol) at 0°C.
  • the reaction mixture was stirred at 0°C for 3 hour, after which time all volatiles were removed under reduced pressure.
  • Electrospray M/S m/z 1221.5 (100%) (M+H + ), 1243.4 (33%) (M+Na + ).
  • Electrospray M/S m/z 1486.6 (100%) (M+H + ), 1508.8 (62%) (M+Na + ).
  • the reaction mixture was added with chloroform (50 mL) and then washed successively with 40 mL portions of water (x2), 3% potassium carbonate (x2), water (x2), 5% HC1 (x2) and water (x2). (NB.
  • the phase separation was extremely tedious due to emulsion formation at each of the successive wash. In order to improve the process an addition of methanol was necessary.
  • the organic phases were combined and dried over sodium sulphate. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography. [Eluant; dichloromethane: acetone: methanol: ammonium hydroxide (8:5:5:3)] 122 mg of colourless waxy material was obtained.
  • Substitute Sheet ( Rule 26) RO/AU 2X-Boc (420mg) was treated with TFA (5mL) for 20 mins under nitrogen atmosphere. TFA was removed under reduduced pressure and dried under high vacuum. This residue was then dissolved in 9% aqueous sodium carbonate solution (6mL) and the solution cooled to 0°C. Fmoc-NMS ester (purchased from CALBIOCHEM) (420mg) dissolved in DMF(3mL) was added into the above stirring solution at 0°C and stirred at RT for 30 mins. Water (lOOmL) was added and aqueous solution extracted with ethyl acetate (2x50mL) and organic extracts were discarded. Aqueous layer acidified with concentrated hydrochloric acid (2-3mL) and cooled in an ice bath during which a precipitate appeared. This precipitate was filtered and dried to give white powder (210mg).
  • the Fmoc protected material (200mg) was dissolved in a solution of piperidine:DMF (20:80) stirred at room temperature for 10 minutes. DMF removed under reduced pressure and the crude material was purified by column chromatography (methanol :aqueous ammonia: DCm 10:2:88). Pure product was isolated as a colorless semi-solid (148mg).
  • the NHS ester of this material was prepared by reaction of the acid (50mg) with NHS (8mg) in the presence of DCC (15mg) and DMAP (2.6mg) in CH 2 C1 2 (3ml) for 4h and purified by passage through a Sephadex LH-20 column eluting with methanol.
  • SPR Surface Plasmon Resonance
  • This Jl.triNTA chip was docked into a BIAcore 2000 machine and the pumps flushed and experiments performed using HBS/EDTA running buffer (50 mM HEPES, 300 mM NaCl. 50 ⁇ M EDTA, pH 8 0) at a flow rate of 40 ⁇ l/min at 21 °C Flow cell 2 (Fc2) was used as the test cell relative to the control Flow cell 1 (Fcl)
  • running buffer containing 500 ⁇ M N ⁇ Cl 2 was injected through Fc2 only, which was then washed with running buffer for 5 min
  • the protein of interest 100-600 nM
  • Substitute Sheet 17 Liley, M., Keller, T.A., Duschl, C, Vogel, H., Langmuir, 1997, 13, 4190.

Abstract

Novel protein binding compounds of Formula (III): W-X-Y-(Z)n in which Y is a branching moiety, Z represents a polydentate ligand chelating agent that coordinates a metal ion; X is a spacer moiety; n is an integer of at least 2 and W is a group that allows for attachment to another molecules, attachment to surfaces, or insertion into membranes.

Description

Improved compounds for protein binding
FIELD OF THE INVENTION
The present invention relates to novel binding compounds, in particular protein binding compounds. The novel compounds are particularly useful for binding proteins to surface including membranes. In preferred forms the present invention provides biosensors incorporating these protein binding compounds. The present invention also extends to intermediate compounds for use in the synthesis of the binding compounds of the present invention.
BACKGROUND OF THE INVENTION
Ternaiy metal complexes are well known in the literature (1) and can be described as the coordination of two discrete metal chelating groups to a metal. The metals normally observed in ternary complexes are Co2+, Ni2+, Cu2+, Zn2+. Typical metal coordinating groups are nitrilotriacetic acid (NT A), iminodiacetic acid (IDA), catechols, and aromatic nitrogen containing heterocycles such as imidazole. Ternary complexes have been characterised by numerous methods including potentiometric calculations (2) and X-ray crystallography (3). These studies have allowed the stability of simple ternary complexes to be determined with the average stability constant (Ka) being 103-lu4 M'1 (4).
In 1975 the use of metal-IDA complexes in a procedure for the fractionation and purification proteins in a procedure known as Immobilised Metal Affinity Chromatography (IMAC) was reported (5). In this procedure a derivative of IDA was attached to a solid support such as Sepharose, and the Sepharose packed into a column. A metal ion was added to the IDA moiety (by passage of a dilute solution of the metal ion through the column) and a protein mixture was then added. Elution of the protein mixture through the column removes all proteins except those which interact with the metal-IDA species. This protein or proteins are then eluted from the column by addition of an imidazole solution, by addition of strong metal chelators such as EDTA or EGTA, or by lowering the pH to 4.5-5.3 (6).
In 1987 Hochuli and coworkers employed Ni-NTA complexes for use in the purification of proteins, (7) particularly recombinantly engineered 6-His tagged proteins (8). The affinity of Hϊs-tagged proteins for solid supports bearing NTA has been determined to be approx. 10" M"1 (9).
Substitute Sheet 6 RO/AU In 1996 a method of attaching His-tagged proteins to wells of polystyrene microwell plates was reported using a derivative of NTA (10).
In 1997 the use of multiple NTA's bound to a quartz microscope slide was described as a method of attaching proteins to surfaces (11). In 1998 the attachment of a His tagged 5HT3 receptor to a quartz slide by this technique for study by total internal reflection fluorescence was reported (12).
In 1995 the derivatisation of surfaces with bidentate, tridentate and quadradentate metal chelating groups was described as a method of detection of analytes (13). In 1996 the attachment of His-tagged proteins to the surface of mixed self-assembled monolayers (SAMs) on gold bearing pendant NTA moieties for study by surface plasmon resonance (SPR) was reported (14).
In 1997 the binding of His-tagged proteins to surfaces displaying NTA-nickel moieties by commercially available sensing devices employing SPR as the measurement protocol was reported (15).
In 1997 the use of SAM's presenting NTA moieties was reported for the binding of Fab fragments to a gold-coated surface for study by FTTR (16). The detection of metal ions through the use of SAM's presenting NTA moieties has also been reported (17).
The use of monolayers presenting IDA-Cu2+ moieties for the attachment of streptavidin to monolayers for study by X-ray crystallography (18) and electron microscopy was reported (19).
The preparation of metal sensitive lipid films through the use of lipids bearing NTA groups was reported in 1994 (20).
In 1996 the use of bilayer membranes containing lipids bearing a pyrene moiety and IDA-Cu2+ moieties presented at the bilayer surface for the detection of His-rich proteins by excimer fluorescence was reported (21).
There are a number of shortcomings with the materials employed in the reports discussed above. Firstly, the stability of the ternary complex, i.e. the interaction between the metal chelate the metal and the protein, is often too weak for proteins to be immobilised for a sufficiently long period for observation and study. Secondly, the metal chelates employed interact in a non-specific manner with other non-tagged proteins. Furthermore, the ternary complexes can be broken down in the presence of certain interferents at concentrations not unknown in certain assay systems.
Substitute Sheet R le 6 R A SUMMARY OF THE INVENTION
The present inventors have developed compounds with improved characteristics to those compounds already disclosed in the literature. These compounds possess a plurality of metal-chelating groups covalently linked. The compounds can be used to attach proteins to materials and surfaces.
Accordingly the present invention consists in a compound, the compound having the general Formula I
Y - (Z)n
Formula I
in which Y is a branching moiety and Z represents a polydentate ligand chelating agent that coordinates a metal ion; and n is an integer of at least 2, preferably from 2 to 9.
Z may be a polydentate ligand that coordinates a metal ion such as Co2+, Ni2+, Cu2+, Zn2+. The donor atoms of Z may be σ- donor atoms or π- donor atoms. The donor atoms may be selected from N, O, S, P and Si. Preferably the donor atoms are N. Z may be bidentate, tridentate (for example IDA) or quadradentate (for example NTA). Preferably Z is other than a cyclic or polycyclic. Preferably Z is a quadradentate ligand such as NTA.
Preferably Y provides at least three moieties for covalent attachment directly, or indirectly through an optional linking group, to Z. The backbone of branching group Y may be a residue of a compound, an oligomer or a polymer. Most preferably, the linking group has a linear backbone.
In a further aspect, the present invention provides a compound of formula II
X - Y - (Z)n Formula II in which Y, Z and n are as described above and X is a spacer moiety.
X may be hydrophilic, hydrophobic or have both hydrophobic and hydrophilic regions. X may be or include a substituted or unsubstituted alkyl, optionally interrupted by one or more heteroatoms (eg 0, N, S or combinations of two or more thereof), for example oligoethylene glycol or other oligoalkylene
Substitute Sheet glycol, an amino acid sequence, a polypeptide or a poly- or oligoamide (for example an aminocaproyl oligσmer). X may be, or include, a lipid. The lipid may be a membrane spanning lipid (MSL). In a preferred aspect, X includes an hydrophilic region, for example polyalkylene oligomer, and an hydrophobic region, for example a lipid, wherein X is attached to Y via an hydrophobic region optionally via a spacer.
In yet a further aspect, the present invention provides a compound of Formula III
W - X - Y - (Z)n
Formula III
in which X, Y, Z and n are as described above and W is a group that allows for attachment to other molecules, or attachment to surfaces, or insertion into membrane(s).
In a preferred embodiment W is a group which allows for attachment to other molecules including polymers such as Sepharose (such as an amine functional group, a carboxylic acid functional group, an alcohol functional group, a halide functional group) or attachment to surfaces (such as a thiol or disulfide for attachment to gold or other coinage metal surfaces, or such as a silane derivative for attachment to oxide surfaces) or insertion into membranes (such as a lipid group, or a membrane soluble protein, such as gramicidin). W may also be a group which enables non-covalent attachment such as biotin to streptavidin.
Preferably Y is a branching moiety that provides a plurality of moieties for covalent attachment of Z and a single moiety for covalent attachment of X. Non-limiting illustrative examples of Y include: amino-polyols such as TRIS, bis-homotris
amino acids such as 3,5-diaminobenzoic acid, 5-aminoisophthalic acid,
Figure imgf000006_0001
Substitute Sheet peptides which possess multiple free acid and/or amine moieties, for example
Figure imgf000007_0001
In an alternative embodiment Y is a branching moiety where there is a plurality of moieties for covalent attachment of Z and X. Non-limiting illustrative examples of Y include:
polyamines such as spermjdine, spermine, pentaethylenehexamine
polyacids such as tartaric acid, trimesic acid, citric acid, Kemp's triacid
polyhydroxylated materials such as sugars
dendrons such as the commercially available "Starburst" compounds,
Figure imgf000007_0002
compounds which possess multiple epoxide moieties, or compounds which possess groups readily displaced by nucleophiles (such as halides, tosylates) or groups to which nucleophiles readily add (such as ,β-unsaturated ketones) or a combination thereof.
The compounds of the present invention may have a wide range of uses. In particular they are useful for the attachment of proteins and other biological macromolecules to surfaces. As will be understood this is a requirement of a multitude of sensing devices and assays.
It is believed that the compounds of the present invention will find particular application in biosensors. Biosensors are well known in the art and are described in PCT/AU93/00620, PCT/AU96/00482, PCT/AU95/00763,
Substitute Sheet PCT/AU96/00368, PCT/AU97/00071, PCT/AU98/00423, PCT/AU98/00424, PCT/AU97/00294, PCT/AU93/00509, PCT/AU96/00304, PCT/AU89/00352, PCT/AU97/00014, PCT/AU92/00132, PCT/AU97/00316, PCT/AU90/00025, PCT/AU98/00417, PCT/AU96/00369, and PCT/AU94/00202, the disclosures of which are incorporated by reference.
In these biosensors there is provided a membrane which includes ionophores and receptors. Binding of an analyte to the receptors causes a detectable change in the conductance or impedance of the membrane. These biosensors provide sensitive detection of the presence of a particular analyte.
Typically, receptors directed against the analyte of interest are attached to the ionophore and to the membrane. Often the receptors are proteins such as antibodies or antigen binding fragments thereof such as Fab'. It is believed that the binding compounds of the present invention will be useful in the attachment of such receptors to the ionophores and membrane of the biosensors.
Accordingly in a preferred embodiment the present invention consists in
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Binding of 6His.Rubisco to Jl.triNTA chip. 100, 200 and 400 nM binding curves shown (bottom to top). Binding curves are the responses from Fc2 (control, -Ni2+) subtracted from Fcl.
Figure 2. Binding of 6His.CD40 to Jl.triNTA chip. 100, 200, 400, 600 and 800 nM binding curves shown (bottom to top). Binding curves are the responses from Fc2 (control, -Ni2+) subtracted from Fcl.
DETAILED DESCRIPTION
In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following examples.
Substitute Sheet (Rule 26) RO/AU Summary
Binding of 6HIS.CD40 to triNTA has a 12 fold higher affinity than to NTA. This is mainly as a result of a 10-fold higher on rate (k . However, due to the higher density of NTA molecules on the BIAcore NTA chip, compared to the triNTA on the gold chip (compare the absolute binding levels), the off rate (k for the NTA binding is likely to be exaggerated.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
1. General Procedures
N6-carbobenzyloxy-L-lysine,2-tTimethylsilylethanol, l-(3-dimethylaminopropyl)-3- ethylcarbodiiuiide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), N- hydroxysuccinamide (NHS) and 6-aminocaproic acid (X) were obtained from Sigma- Aldrich chemical company. XXBoc was prepared by reacting XBoc NHS ester (itself obtained by protection of the amino group of X with BocON under standard conditions, and subsequent coupling with NHS using DCC) with X. Dichloromethane was distilled over P2O5 immediately prior to use.
2. Synthesis of Z-LysNTA
Figure imgf000009_0001
C18H24N2θ8
396.39
396.153266
C 54.5% H 6.1% N 7.1 % O 32.3%
Z-LysNTA was synthesised following the procedure of Schmitt et al.1
Bromoacetic acid (4.17 g, 30.0 mmol) was dissolved in aqueous NaOH (1.5 M, 15 ml) and cooled to 0°C. Nε-Z-(L)-Lysine (2.0 g, 7.0 mmol) in aqueous NaOH (1.5 M,
Substitute Sheet 25 ml) was added dropwise to the reaction mixture over 2 h. The solution was allowed to warm to room temperature and stirred at room temperature overnight. The reaction mixture was heated at 50°C for 2 h, then allowed to cool to room temperature. An aqueous solution of HC1 (IM, 40 ml) was added dropwise to the reaction mixture, and the resulting white precipitate was filtered and washed with HC1 (0.1M, 20 ml) and distilled water (2x20 ml). The resulting white solid was dried under high vacuum for several days to afford Z-LysNTA as a white solid (2.68 g, 95% yield).
Data
Η NMR (200 MHz, rJ6-DMSO) : δ 7.42 (5H, m, -C6H5), 5.09 (2H, s, -CH2Ph), 3.56 (4H, AB quartet, 2 x -NCH2CO2H), 3.41 (1H, overlapping dd. -NCHCO2H), 3.06 (2H. m, - NHCH2-), 1.75-1.25 (6H, overlapping multiplets, -NHCH2CH2CH2CH2-) ppm.
13C NMR (50 MHz, d4-MeOD) : δ 175.9 (2 x -C(O)O-), 175.8 (-C(O)O-), 158.9 (-C(O)NH), 138.4 (-C6H5), 129.4 (-C6H5), 128.9 (-CbH5), 128.7 (-CβH5), 67.3 (-CH2Ph), 66.7 (- NCHC(O)-), 55.3 (2 x -NCH2C(O)-), 41.5 (-NHCH2-), 30.7 (-NHCH2CH2-), 30.5 (- NHCH2CH2CH2CH2-), 24.6 (-NHCH2CH2CH2-) ppm.
Electrospray M/S : m/z 397 (100%) (M+H+), 398 (21%), 353 (30%), 792 (15%).
3. Synthesis of ZLysNTA.TMSE3
ZLysNTA.TMSE3 was synthesised using a procedure outlined by Gao et al.2
Figure imgf000010_0001
To a solution of Z-LysNTA (0.50 g, 1.25 mmol), 2-trimethylsilylethanol (1.50 g, 12.5 mmol) and 4-dimethylaminopyridine (0.50 g, 4.15 mmol) in freshly distilled, dried dichloromethane (60 ml) at 0°C was added EDC (1.25 g, 12.5 mmol). The reaction was stirred at 0°C for 1 hour, then allowed to warm to room temperature and
Substitute Sheet stirred at room temperature overnight. Distilled water (50 ml) was added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with dichloromethane (2x50 ml), and the organic extracts were combined and dried over anhydrous sodium carbonate. The mixture was filtered and all volatiles were removed under reduced pressure to afford a colourless oil. Purification by column chromatography on flash silica using a solvent gradient from dichloromethane to 5% methanol in dichloromethane afforded ZLysNTA.TMSE3 as a colourless oil (0.75 g, 86%).
Data
Rf = 0.78 (5% methanol in dichloromethane)
Η NMR (200 MHz, CDC13) : δ 7.34 (5H, , -CβHs), 5.08 (2H, s, -CH2Ph), 4.91 (IH, br t, - NH), 4.16 (6H, m, 3 x -OCH2-), 3.60 (4H. s, 2 x -NCH2C(O)-), 3.39 (IH, t, -NCHC(O)-, 3JH. H = 7.5 Hz), 3.19 (2H, m, -NHCH2-), 1.3-1.8 (6H, overlapping multiplets, - NHCH2CH2CH2CH2-), 0.93-1.04 (6H, overlapping multiplets, 3 x -CH2Si-), 0.04 (9H, s, 1 x -Si(CH3)3), 0.03 (18H, s, 2 x -Si(CH3)3) ppm.
13C NMR (100 MHz, CDC13) : δ 173.6 (-C(O)O-), 172.2 (2 x -C(O)O-), 157.1 (-OC(O)NH), 137.5 (-CβHs), 129.2 (-CβH5), 128.7 (-C6H5), 67.2 (-CH2Ph), 65.4 (-NCHC(O)-), 63.5 (1 x - OCE-2-), 63.3 (2 x -OCH2-), 53.6 (2 x -NCH2C(0)-), 41.6 (-NHCH2-), 30.8 (-NHCH2CH2-), 30.1 (-NHCH2CH2CH2CH2-), 23.8 (-NHCH2CH2CH2-), 18.3 (-CH2Si-), 18.1 (2 x -CH2Si-), - 0.8 (3 x -Si(CH3)3) ppm.
Electrospray M/S : m/z 719.4 (100%) (M+Na+)
4. Synthesis of LysNTA.TMSE3
Figure imgf000011_0001
C25H5 N206Si3
562.97
562.328973
C 53.3% H 9.7% N 5.0% O 17.1% Si 15.0%
Substitute Sheet Rule 26 ZLysNTA.TMSE3 (0.167 g, 0.24 mmol) was dissolved in methanol (5 ml), a spatula- tip of 10% Pd/C was added and the reaction vessel was fitted with a balloon containing H2 gas. The reaction mixure was stirred at room temperature for 75 min, then filtered and all volatiles removed under reduced pressure. Analysis by Η NMR showed complete hydrogenation had occurred, to afford LysNTA.TMSE3 as a colourless oil (0.127 g, quantitative yield).
Data
Η NMR (200 MHz, CDC13) : δ 4.16 (6H, m, 3 x -OCH2-), 3.61 (4H, s, 2 x -NCH2C(O)-), 3.40 (IH, apparent triplet, -NCHC(O)-, 3JH.H = 7.5 Hz), 2.69 (2H, br t, NH2CH2-, 3JH.H = 6.1 Hz), 1.3-1.8 (6H, overlapping multiplets. NH2CH2CH2CH2CH2-), 0.93-1.04 (6H, overlapping multiplets, 3 x -CH2Si-), 0.04 (9H, s, 1 x -Si(CH3)3), 0.03 (18H, s, 2 x - Si(CH3)3) ppm.
13C NMR (100 MHz, CDC13) : δ 173.7 (-C(O)O-), 172.2 (2 x -C(O)O-), 65.6 (-NCHC(O)-), 63.5 (2 x -OCH2-), 63.3 (1 x -OCH2-), 53.6 (2 x -NCH2C(O)-), 42.7 (NH2CH2-), 33.9 (NH2CH2CH2-), 31.0 (NH2CH2CH2CH2CH2-), 23.9 (NH2CH2CH2CH2-), 18.3 (-CH2Si-), 18.1 (2 x -CH2Si-), -0.8 (3 x -Si(CH3)3) ppm.
Electrospray M/S : m/z 563.3 (100%) (M+H+)
Substitute Sheet (Rule 6) RO/AU 5. Synthesis of Zfr/NTA.TMSE9
Figure imgf000013_0001
C93H|8oN8θ23Si9
2031.26
2029.108491
C 55.0% H 8.9% N 5.5% O 18.1% Si 12.4%
ZLysNTA (60 mg, 0.15 mmol), 4-dimethylaminopyridine (60 mg, 0.51 mmol) and LysNTA.TMSE3 (0.37 g, 0.66 mmol) were dissolved in dichloromethane (50 ml) and the reaction mixture was cooled to 0°C. EDC (0.15 g, 0.75 mmol) was added, and the reaction was stirred at 0°C for 1 hour, then allowed to warm to room temperature and stirred at room temperature overnight. Distilled water (40 ml) was added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with dichloromethane (2x40 ml), and the organic extracts were combined and dried over anhydrous sodium carbonate. The mixture was filtered and all volatiles were removed under reduced pressure to afford a colourless oil. Purification by column chromatography on flash silica using a solvent gradient from 100 % dichloromethane to 5% methanol in dichloromethane afforded ZtriNTA.TMSE9 as a colourless oil (0.235 g, 76%).
Data
Rf = 0.24 (5% methanol in dichloromethane)
Η NMR (400 MHz, CDC13) : δ 7.37 (2H, br t, NH), 7.34 (5H, m, -CeHs), 7.26 (IH, br t, NH), 5.15 (IH, br t, NH), 5.08 (2H, s, -CH2Ph), 4.17 (18H, , 9 x -OCH2-), 3.60 (12H, two overlapping singlets in 2:1 ratio, 6 x -NCH2C(0)0-), 3.39-3.34 (5H, overlapping
Substitute Sheet Rul 2 R ΔU multiplets), 3.26-3.16 (10H, overlapping multiplets), 3.07 (IH, triplet), 1.85-1.30 (24H, overlapping multiplets, 12 x -CH2), 0.97 (18H, overlapping multiplets, 9 x -CH2Si-), 0.05 (27H, s, 3 x -Si(CH3)3), 0.04 (54H, s, 6 x -Si(CH3)3) ppm. -OC(O)NHCH2-
"C NMR (100 MHz. CDC13) : δ 173.5 (2 x -C(O)O-), 173.5 (-C(O)O-), 172.9 (3 x -C(O)NH- ), 172.2 (4 x -C(O)O-), 172.1 (2 x -C(O)O-), 157.2 (-OC(O)NH), 137.4 (-C6H5), 129.1 (- C6H5), 128.6 (-C6H5), 67.1 (-CH2Ph), 66.4 (-NCHC(O)NH-), 65.5 (-NCHC(O)O-), 65.4 (2 x - NCHC(O)O-), 63.4 (6 x -OCH2CH2-), 63.3 (3 x -OCH2CH2-), 56.9 (2 x -NCH2C(O)NH-), 53.7 (4 x -NCH2C(O)0-), 53.4 (2 x -NCH2C(O)O-), 41.2 (-OC(0)NHCH2-), 39.9 (3 x - C(O)NCH2-), 30.7, 30.5, 30.2 (4 x -NHCH2CH2-), 29.9, 29.5, 28.9 (4 x NHCH2CH2CH2CH2-), 24.7, 24.0, 23.8 (4 x -NHCH2CH2CH2-), 18.3 (3 x -CH2Si(CH3)3), 18.0 (6 x -CH2Si(CH3)3), -0.8 (9 x -Si(CH3)3) ppm.
Electrospray M/S : m/z 2054.3 (100%) (M+Na+), 1038.8 (20%), 613.3 (36%)
6. Synthesis of Zfr/NTA
Figure imgf000014_0001
Z.r NTA.TMSE9 (0.12 g, 0.06mmol) was cooled to 0°C and trifluoroacetic acid (3 ml) was added. The reaction mixture was allowed to stir at 0°C for 3 h, after which time all volatiles were removed under reduced pressure. The reaction mixture was purified by reverse phase HPLC using a solvent gradient from 100% H2O/0.05% TFA to 50% acetonitrile/0.05%TFA in H2O/0.05% TFA over 40 min. Collection of the main peak in the HPLC chromatogram, with retention time 21 min, afforded pure ZtriNTA as a white solid (20 mg. 30% yield).
Substitute Sheet Data
Η NMR (400 MHz, D2O) : δ 7.38 (5H, , -CβHs), 5.08 (2H, s, -CH2Ph), 4.06-3.78 (20H, m, 8 x -NCH2C(O)- + 4 x -NCHC(O)-), 3.23 (6H, apparent triplet, 3 x -C(O)NHCH2-), 3.08 (2H, apparent triplet, 1 x -OC(O)NHCH2-), 2.00-1.76 (8H. overlapping multiplets, 4 x -CH2), 1.60-1.40 (14H, overlapping multiplets, 7 x -CH2), 1.38-1.22 (2H, multiplet, 1 x -CH2) ppm.
13C NMR (100 MHz, D2O): 172.3, 171.2, 170.8, 168.6, 159.0, 137.5, 129.5, 129.0, 128.3, 118.0, 115.5, 67.8, 67.5, 56.0, 55.5, 41.0, 39.6, 39.0, 28.5, 27.4, 23.7, 23.0 ppm
Electrospray M/S : m/z 1151.4 (100%) (M+Na+), 1129.2 (88%) (M+H+), 1130.3 (56%) (M+2H+), 1152.4 (53%) (M+Na++H+), 1165.3 (53%), 1143.3 (52%), 755.5 (50%), 907.2 (41%), 393.2 (26%).
7. Synthesis of
Figure imgf000015_0001
Z-t NTA TMSE9 (38 mg, 0.0187 mmol) was dissolved in methanol (10 ml) and a catalytic amount of palladium on charcoal (10%) was added. The mixture was flushed with hydrogen under vacuum and stirred under an atmosphere of hydrogen gas for 2 hours. The reaction mixture was filtered and the filtrate was evaporated to give a clear oil of tri NTA TMSE9 (34 mg, 96%).
Substitute Sheet Data
Η n.m.r. (CDC13) δ 0.00 (81 H, m, Si(CH3)3), 0.96 (18H, m, CH2Si), 1.2-2.0 (24H, m), 3.1-3.7 (28H, m). 4.15 (18H, m. OCH2) and 7.4 (2H, NH2).
8. Synthesis of tri NTA TMSE9 hemisuccinamide
Figure imgf000016_0001
A solution of tri-NTA.TMSE9 (0.5 ), benzyl hemisuccinate (71mg), DMAP (64mg) in dichloromethane was stirred at 0°. EDC (lOlmg) was added to the reaction mixture and stirring continued at room temperature for 16h. The solvent was removed under reduced pressure and the residue chromatographed using CH2Cl2-MeOH (96:4) to separate 537mg of the benzyl ester intermediate. 500mg of this material was dissolved in methanol (60ml) and hydrogenolysed at atmospheric pressure using 10% Pd/C (60mg) as catalyst for 4h. The pure product was obtained in 474mg.
Data
Η n.m.r. (CDC13) δ 0.04 (81 H, m, Si(CH3)3), 0.96 (18H, m, CH2Si), 1.2-2.0 (24H, m), 2.5 (2H, m). 2.65 (2H, m), 3.2-3.45 (16H, m), 3.59 (s, 12), 4.16 (18H, m, OCH2).
Electrospray M/S : m/z 2019.8 (35%), ( +Na+), 1997.8 (100%) (M+).
Substitute Sheet 9. Synthesis of tri NTA
Figure imgf000017_0001
tri NTA TMSE9 (34 mg,0.0179 mmol) was triturated with toluene, evaporated and dried under high vacuum. Trifluoroacetic acid (1 ml) was added and stirred under nitrogen atmosphere 0-5° C for 2 hours, then at room temperature overnight. The trifluoroacetic acid was evaporated and the residue was triturated with toluene again, evaporated and dried to afford tή NTA (20 mg, 100%).
Data
Η n.m.r. (CD3OD) δ 1.2-2.0 (24H, m), 3.07 (6H, m, CH2NHCO) and 3.1-3.7 (22H, m).
10. Preparation of Gramicidin succinate
Gramicidin (75 mg, 0.0398 mmol) was dissolved in pyridine (0.5 ml) and succinic anhydride (20 mg, 0.200 mmol) was added. The mixture was stirred under nitrogen atmosphere at 50° C for 20 hours and evaporated. The crude product was passed down a sephadex LH-20 column in methanol, the eluate was evaporated and purified on a flash silica column using dichloromethane/methanol/water/acetic acid (400:50:4:1). The product was further purified by centrifuging with water. The water was decanted and the product was dried under high vacuum to give gramicidin succinate (53 mg, 67%).
Substitute Sheet Data
Η n.m.r. (CD3OD) δ 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CH2CO), 2.9-3.4 (10H. m). 3.90 (2H, dd, CH2-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (IH, s, CHO).
11. Preparation of gramicidin succinate NHS ester
Gramicidin succinate (21 mg, 0.0105 mmol), N-hydroxysuccinamide (12 mg, 0.1042 mmol) and 4-dimethylamino pyridine (2.5 mg, 0.0204 mmol) were combined with distilled tetrahydrofuran (10 ml). With stirring under nitrogen dicyclohexylcarbodiimide (22 mg, 0.1066 mmol) was added. The mixture was heated to reflux for one hour. The mixture was evaporated and passed down a sephadex LH- 20 column in methanol. Appropriate fractions were evaporated and dried to give gramicidin succinate NHS (22 mg, 100%).
Data
Η n.m.r. (CD3OD) δ 0.4-1.8 (66H, in), 2.0-2.2 (4H, m), 2.56 (4H, s, CH2CO), 2.82 (4H, s, NHS), 2.9-3.4 (10H. m), 3.90 (2H, dd. CH2-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (IH, s, CHO).
12. Preparation of Gramicidin succinate tri NTA
Figure imgf000018_0001
Lysine tri NTA (20 mg, 0.0201 mmol) was dissolved in methanol (1 ml) and triethylamine (2 drops) was added to neutralise. Then gramicidin succinate NHS (15
Substitute Sheet mg, 0.0072 mmol) was added in methanol (2 ml). The reaction mixture was heated at 50° C for 24 hours, then purified on a Sephadex LH-20 column in methanol. Gramicidin succinate lysine tri NTA (14 mg, 65%) was obtained.
Data
Η n.m.r. (CD3OD) δ 0.4-1.8 (90H, m), 2.0-2.2 (4H, m), 2.52 (4H. m. CH2CO), 3.0-3.8 (38H. m), 3.90 (2H, dd, CH2-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (IH, s, CHO).
MALDI m.s. 2963.4( (M+Na+)-H2O), 2982.9 (M+Na+), 2998 (M+K+).
13. Preparation of gA lysine-XXBOC
Figure imgf000019_0001
Gramicidin lysine (15 mg, 0.0076mmol) was dissolved in methanol (2 ml) and one equivalent of triethylamine was added, this was reacted with the N- hydroxysuccinimide ester of XXBOC (10 mg, 0.0226 mmol) (prepared by reacting XXBOC with 1 equiv. of DCC and NHS and 0.1 equiv. of DMAP in dry dichloromethane). The reaction mixture was stirred at room temperature for 18 hours. The mixture was evaporated and passed down a Sephadex-LH-20 column in methanol. The eluate containing the appropriate fractions were evaporated and purified on a flash silica column using dichloromethane/methanol/water/acetic acid (400:40:4:1) to afford gAlysine 2XBOC (12 mg, 68%).
Data
Η n.m.r. (CD3OD) δ 0.4-1.8 (84H, m), 1.41 (9H, s. BOC), 2.0-2.2 (4H, m), 2.9-3.4 (14H, m), 3.90 (2H, dd, CH2-gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (IH, s, CHO).
Substitute Sheet (Rule 26) RO/AU 14. Preparation of gAlysine XXBOC hemisuccinate
Figure imgf000020_0001
Gramicidin lysine 2XBOC (12 mg,0.0052 mmol) was triturated with toluene, evaporated and dried under high vacuum. Trifluoroacetic acid (1 ml) was added, evaporated under nitrogen and dried. Again toluene was added, evaporated and dried under high vacuum. The crude amine was dissolved in pyridine (0.5 ml) and reacted with succinic anhydride (2.6 mg, 0.0259mmol). The reaction mixture was stirred at room temperature for 20 hours. Pyridine was removed under high vacuum and the residue was passed down the sephadex LH-20 column in methanol. The product was further purified on a flash silica column eluted with methanol to give gAlysine 2X Succinate (10 mg, 83%).
Data
Η n.m.r. (CD3OD) δ 0.4-1.8 (84H, m), 2.0-2.2 (8H, m), 2.58
(4H, dd, CH2CO), 2.9-3.4 (14H, m), 3.90 (2H, dd, CH2-gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (IH, s, CHO).
15. Synthesis of gAlysine2Xfrv NTA
Figure imgf000020_0002
Gramicidin lysine 2X succinate (8.5 mg, 0.037mmol), N-hydroxy succinamide ( 4.2 mg, 0.0364mmol) and 4-dimethylamino pyridine (1 mg, 0.0081mmol) were stirred in distilled tetrahydrofuran (5 ml) and dicyclohexylcarbodiimide (7.5 mg,
Substitute Sheet 0.0363mmol) was added. The mixture was refluxed under nitrogen for 1 hour. The mixture was evaporated and purified on a sephadex LH-20 column in methanol. Appropriate fractions were evaporated and added to lysine tri NTA (8.5 mg, 0.0085 mmol). The mixture was stirred at room temperature for 18 hours. The reaction mixture was evaporated and purified on sephadex LH-20 in methanol (X2). Gramicidin lysine 2Xtri NTA (2.6 mg, 19%) was obtained (due to low solubility some compound was lost).
Data
Η n.m.r. (CD3OD) δ 0.4-1.8 (108H, m), 2.0-2.2 (8H, m), 2.62 (4H, dd, CH2CO), 3.0-3.8 (42H, ), 3.90 (2H, dd, CH2-gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (IH, s, CHO).
MALDI m.s. 3291.17 (M+Na+), 3309.02 (M+K+).
16. Synthesis of triNTA.TMSE9
Figure imgf000021_0001
CβjHmNgOjiSi,
1897.12
1895.071711
C 53.8% H 9.2% N 5.9% O 17.7% Si 13.3%
A solution of ZtiϊNTA.TMSE9 (0.1 g, 50 umol) was dissolved in methanol (10 ml). Palladium on carbon (10%) was added (ca. 10 mg) and a balloon containing H2 gas was fitted. The reaction mixture was stirred at room temperature for 90 min, after which time TLC analysis showed the complete disappearance of the starting material. The reaction mixture was filtered through a short plug of Celite and all volatiles were
Substitute Sheet Rul removed from the reaction micxture under reduced pressure to afford trisNTA.TMSE9 as a colourless oil (0.09 g, quantitative).
Data
Electrospray M/S : m/z 1898.3 (100%) (M+H
17. Synthesis of Biotin.triNTA.TMSE9
Figure imgf000022_0001
2121.149311
C 53.7% H 8.9% N 6.6% O 17.3% S 1.5% Si 11.9%
Biotin (20 g, 0.08 mmol), 4-dimethylaminopyτidine (12 mg, 0.1 mmol) and triNTA.TMSE9 (0.10 g, 0.1 mmol) were added to dry, freshly distilled dichloromethane (5 ml) and the reaction mixture was cooled to 0°C. EDC (40 mg, 0.2 mmol) was added, and the reaction was stirred at 0°C for 1 hour, then allowed to warm to room temperature and stirred at room temperature for 3 days. All volatiles were removed from the reaction mixture under reduced pressure, and the mixture was purified using a Sephadex LH20 column using MeOH as eluant. Fractions containing spots with Rf = 0.1 in 5% methanol/dichloromethane were combined. Biotin.triNTA.MTSE9 was obtained as a colourless oil (73 mg, 0.03 mmol, 34%).
Substitute Sheet Rule 26 RO AU Data
Electrospray M/S : m/z 1173.9 (100%), 2146.4 (25%) (M+Na+).
18. Synthesis of Biotin.triNTA
Figure imgf000023_0001
CS0H80N10O23S
1221.29
1220.511851
C 49.2% H 6.6% N 11.5% O 30.1% S 2.6%
Trifluoroacetic acid (2 ml) was added to Biotin.triNTA.TMSE9 (19 mg, 9 umol) at 0°C. The reaction mixture was stirred at 0°C for 3 hour, after which time all volatiles were removed under reduced pressure. Analysis of the reaction mixture by reverse phase HPLC on a Vydac C18 column using a gradient from 100% solvent A to 50% solvent B in solvent A over 40 min (solvent A : H2O / 0.05%TFA ; solvent B : acetonitrile / 0.05% TFA) showed one main peak with a retention time Rt = 24.1 min. Separation of the reaction mixture by preparative reverse phase HPLC afforded Biotin.triNTA as a white solid (1.0 mg, 0.8 umol, 9%).
Data
Electrospray M/S : m/z 1221.5 (100%) (M+H+), 1243.4 (33%) (M+Na+).
Substitute Sheet 26 RO/AU 19. Synthesis of Lipid-triNTA
Figure imgf000024_0001
C72H127N9023
14S6.S4
1485.904482
C 58.2% H 8.6% N 8.5% O 24.7%
A solution of ditetradecylamine (lOOmg) in CH2C12 (5ml) was added to a solution of succinic anhydride (120mg) and triethylamine (40mg) in CH2C12 (5ml) at room temperature. The reaction was stirred overnight, the solvents removed in vacuo and the residue chromatographed using CH2Cl2-MeOH (95:5 O 85:5) to give 60mg of ditetradecylamine hemisuccinamide. This material was added to a solution of triNTA.TMSE9 in CH2C12 (10ml) followed by DMAP (14mg). The reaction mixture was cooled to 0°, EDC (22mg) was added, and the mixture stirred for 3 days at room temperature. The solvent was then removed, and the residue purified by passage through Sephadex LH-20, eluting with methanol, to give 97mg pure material. lOmg of this material was dissolved in TFA (1ml) and stirred under nitrogen for 3h. The solvent was removed in vacuo and the solid remaining washed with water (1ml) and dried in vacuo. This material was purified by preparative HPLC (C18 Alltima column, 1% TFA in CH3CN) to separate the desired product (4mg).
Data
Electrospray M/S : m/z 1486.6 (100%) (M+H+), 1508.8 (62%) (M+Na+).
Substitute Sheet (Rule 6) RO/AT 20. Synthesis of Membrane-spanning lipid phosphatyl choiine (MSL-PC)
Figure imgf000025_0001
C124H220NO24PS2
Exact Mass: 2202.52
Mol. Wt.: 2204.17
C, 67.57; H, 10.06; N, 0.64; O, 17.42; P, 1.41; S, 2.91
MSL-OH3 (895 mg, 0.44 mmol) was dissolved in chloroform (6 mL) containing quinoline (0.12 mL, 2.2 equiv., dried with sodium sulphate and then distilled over zinc powder prior to use). This was slowly added to freshly distilled POCl3 (0.09 mL, 2.2 equiv.) at room temperature. The mixture was stirred at 45°C for 30 min. After cooling choiine tosylate (363 mg, 1.3 mmol) dissolved in dry pyridine (1 mL) was added and the reaction mixture was stirred at room temperature overnight. Water (0.5 mL) was added and the mixture was further stirred for 1 hr. The reaction mixture was added with chloroform (50 mL) and then washed successively with 40 mL portions of water (x2), 3% potassium carbonate (x2), water (x2), 5% HC1 (x2) and water (x2). (NB. The phase separation was extremely tedious due to emulsion formation at each of the successive wash. In order to improve the process an addition of methanol was necessary.) The organic phases were combined and dried over sodium sulphate. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography. [Eluant; dichloromethane: acetone: methanol: ammonium hydroxide (8:5:5:3)] 122 mg of colourless waxy material was obtained.
Data
Η n.m.r. J(CDCl3:CD3OD (3:1)) 0.82-0.92 (m, 30H, phytanyl CH3's), 0.92-1.80, m, 100H, phytanyl CH2's and CH's), 2.61 (t, 2H, CH2CH2S), 2.65 (s, 8H, succinate H's), 3.20 (s, 9H, -N(CH3)3), 3.35-3.74 (m, 44H, -OCH2, -OCH, CH2N), 3.85 (s, 2H. SCH2Ph), 3.92 (t, 2H, CH2OP), 4.10 (m, 2H, POCH2CH2), 4.20 (m, 8H, ester H's), 6.90 & 7.43 (d, 8H, biphenyl), 7.30 (s, 5H, PhCH2).
Substitute Sheet Rul Electrospray MS m z: 2206 (M+2), 1112, 587, 488
21. Synthesis of bis(N-methyl)-C38 diamine
Figure imgf000026_0001
To the bola-amphiphile C38-diol3 (300mg) in THF (10ml) cooled to 0° was added triethylamine (92μl) and methanesulphonyl chloride (51μl). The solution was stirred overnight at room temperature and diluted with ether (30ml). The organic phase was washed with saturated NaHCO3 (2 X 30mL) and water (2 X 60ml), dried (Na2SO4) and concentrated in vacuo. The material obtained (315mg) was added to a pressure tube, cooled to -80°, and chilled methylamine (12ml) was added. The pressure tube was sealed and the mixture allowed to warm to room temperature. After standing at room temperature overnight excess methylamine was allowed to evaporate, and the residue dissolved in CH2C12 and stirred with K2C03. The solid was removed by filtration and the filtrate dried in vacuo. The residue was chromatographed using CH2Cl2-MeOH-NH3 (96:4:0.1 O 92:8:0.1) to separate 242 mg pure product.
Data
Η n.m.r. (CDC13) δ 0.80-0.95 (30H, m, Phytanyl CH3's); 0.95-1.70 (100H, m, Phytanyl CH/s and CH's); 2.45 (6H, s), 2.69 (4H, m), 3.40-3.80 (14H, m, CH-0 and CH2-O); 3.98 (4H, t. CH2OAr); 6.93, 7.43 (8H, AA'XX' multiplets, arom.-H
22. Synthesis of XXFmoc
Figure imgf000026_0002
Substitute Sheet (Rule 26) RO/AU 2X-Boc (420mg) was treated with TFA (5mL) for 20 mins under nitrogen atmosphere. TFA was removed under reduduced pressure and dried under high vacuum. This residue was then dissolved in 9% aqueous sodium carbonate solution (6mL) and the solution cooled to 0°C. Fmoc-NMS ester (purchased from CALBIOCHEM) (420mg) dissolved in DMF(3mL) was added into the above stirring solution at 0°C and stirred at RT for 30 mins. Water (lOOmL) was added and aqueous solution extracted with ethyl acetate (2x50mL) and organic extracts were discarded. Aqueous layer acidified with concentrated hydrochloric acid (2-3mL) and cooled in an ice bath during which a precipitate appeared. This precipitate was filtered and dried to give white powder (210mg).
Data
NMR (CDCI3) 1.2-1.8 (m, 12H), 2.20(t, 2H), 2.35(t,2H), 3.25(m, 4H), 4.259m, IH), 4.40(m, IH), 5.05(m, IH), 5.53(m, IH), 6.65(m, IH), 7.3-7.5(m, 4H), 7.6(d, 2H), 7.25(d, 2H)
Mass Spec El 483.9(M+Na+)
23. Synthesis of C38 (NCH3)2 mono BOC
Figure imgf000027_0001
A suspension of C38diamine (320mg), BOC-ON (63mg), triethylamine (37mg), THF(3mL0 and water (2mL) was stirred at room temperature over night. Reaction mixture diluted with water (50mL) and extracted with dichloromethane (2x80mL). Combined organic extracts were washed with brine (50mL) dried with magnesium sulphate and solvent removed under reduced pressure. Crude material was purified by chromatography (5-7%methanol in dichloromethane). To give pure material as a viscous liquid (150mg)
Substitute Sheei Data
NMR (CDCI3) 0.7-0.9 (m, 30H), 1.0-1.9 (m, 109H), 2.51(s, 3H), 2.7(m, 2H), 2.91(s, 3H), 3.1-3.7(m, 16H), 4.0(t, 4H), 6.9(d, 4H), 7.4(d, 4H)
Mass ES 1474.9 (M+, 100%)
24. Synthesis of Boc-C38-2X-Fmoc
Figure imgf000028_0001
A mixture of 2X-Fmoc (47mg), DCC(35mg), DMAP(3mg), C38-monoBoc (150mg) and DCM (10 mL) was stirred at room temperature for 24 hours. Solvent removed under reduced pressure and the crude material purified by column chromatography (5% methanol in dichloromethane) to give pure product (200mg).
Data
Nmr (CDC13) 0.8-0.9(m, 30H), 0.95-2.5 (m, 125H), 2.9, 2.95, 3.05 (3xs, 6H), 3.1-3.75(m, m, 22H), 4.0(t, 4H), 4.1-4.4(m, 3H), 6.9 (d, 4H), 7.3-7.4(m, 4H), 7.5(d, 4H), 7.6(d, 2H), 7.75 (d, 2H)
Mass ES 1923.3(M+), 1945.1(M+Na+)
Substitute Sheet 25. Synthesis of MSL-tri-NTA
Figure imgf000029_0001
The Fmoc protected material (200mg) was dissolved in a solution of piperidine:DMF (20:80) stirred at room temperature for 10 minutes. DMF removed under reduced pressure and the crude material was purified by column chromatography (methanol :aqueous ammonia: DCm 10:2:88). Pure product was isolated as a colorless semi-solid (148mg).
A portion of this C38-derivative (210mg), triNTA derivative (135mg), DCC (33mg) and DMAP (3mg) was dissolved in dry dichloromethane (3mL) and allowed to stand at RT for 18h. The solvent was removed under reduced pressure and the crude material purified by column chromatography to give the pure material as a colorless thick liquid (179mg).
The Boc and TMSE protected material (lOOmg) was dissolved TFA (2mL) and stirred for 4h at room temperature. TFA was removed under reduced pressure and the sample dried under high vacuum. The synthesis of the disulfide acid shown below has been reported elsewhere.4
Figure imgf000029_0002
The NHS ester of this material was prepared by reaction of the acid (50mg) with NHS (8mg) in the presence of DCC (15mg) and DMAP (2.6mg) in CH2C12 (3ml) for 4h and purified by passage through a Sephadex LH-20 column eluting with methanol.
Substitute Sheei Rule26 RO/AU This active ester was reacted in methanol with the fully deprotected C38diamine- tr/NTA material prepared above (lOOmg). After stirring at room temperature for 16h the solvent was removed and the crude material partially purified by passage through a Sephadex LH-20 column eluting with methanol. The material obtained was further purified by preparative HPLC (C18 Alltima column, MeOH-CH2Cl2 with 0.05% TFA).
26. Kinetic analysis of 6His-tagged protein binding to triNTA
Surface Plasmon Resonance (SPR) analysis of binding was performed using BIAcore technology. The commercially available Jl chip (BIAcore, Uppsala. Sweden) was modified by the coating of a lipid layer. In a laminar flow hood, the BIAcore Jl chip was unsheathed to expose the gold surface. A solution (100 μL) containing 1.2 μM MSLtiiNTA, 24 μM MSLPC in ethanol, was dispensed directly on to the gold. During incubation for 1 hour, ethanol was added every 10-15 minutes to prevent the complete evaporation of ethanol. The gold surface was then washed with ethanol by pipetting 10 x 100 μL across the surface and air dried in the laminar flow hood. The chip was then re- sheathed and sealed with plastic film (Parafilm).
This Jl.triNTA chip was docked into a BIAcore 2000 machine and the pumps flushed and experiments performed using HBS/EDTA running buffer (50 mM HEPES, 300 mM NaCl. 50 μM EDTA, pH 8 0) at a flow rate of 40 μl/min at 21 °C Flow cell 2 (Fc2) was used as the test cell relative to the control Flow cell 1 (Fcl) Hence, running buffer containing 500 μM NιCl2 was injected through Fc2 only, which was then washed with running buffer for 5 min The protein of interest (100-600 nM) was then injected (using KINJECT function) through Fcl and Fc2 for 3-7 min. followed by a dissociation phase of 7-15 min The surface was then regenerated using running buffer containing 350 mM ETDA. which removed NιCl2 and hence released 6Hιs-tagged protein from the triNTA molecule Steps 2-4 were repeated for each protein at the range of conentrations indicated (Fig 1, 2) Negative controls used were identical proteins with the 6Hιs tag absent The results indicated that triNTA binds 6Hιs-tagged proteins with high affinity (Fig 1, 2). 6Hιs Rubisco and 6Hιs CD40 bound tπNTA with equilibrium constants of 300 pM and 2 9 nM. respectively These proteins did not bind triNTA in the absence of Nι2+ and proteins without the 6Hιs tag also did not bind tπNTA
Substitute Sheet 27. References
1. Schmitt, L.; Dietrich, C; Tampe R. JACS. 1994, 116, 8485.
2. Gao. C; Lin C.-H; Lo, C.-H.L.; Mao. S.: Wirsching. P.: Lerner, R.A.; Janda, K.D. PNΛS, 1997. 94, 11777.
3. Raguse, B., Culshaw, P.N., Prashar, J.K., Raval, K., Tetraliedron Lett., in press.
4. Raguse, B., Braach-Maksvytis, V.L.B., Cornell, B.A., King, L.G., Osman, P.D.J., Pace, R.J, Wieczorek, L., Langmuir. 1998, 14. 648.
1. Sigel, H., Angew. Chem. Int. Ed. Engl, 1975, 14, 394.
2. Martin, R.P., Petit-Ramel, M.M., Scharff, J.P., in "Metal Ions in Biological Systems" 1976 vol 2 p 1 (Marcel Dekker)
3. Burns, C.J., Field, L.D., Hambley, T., Lin, T., Ridley, D.D., Turner, P., Wilkinson, M.P., manuscript in preparation; Bocarsly, J.R., Chiang, M.Y., Bryant, L., Barton, J.K. Inorg. Chem., 1990,
4. 29, 4898.
5. Anderegg, G., Pure Appl. Chem., 1982, 54, 2693.
6. Porath, J., Carlsson, J., Olsson, I., Belfrage, G., Nature 1975, 258, 598.
7. Sulkowski, E., Trends Biotechnol., 1985, 3, 1. 8. Hochuli, E., Dobeli, H., Schacher, A., J. Chromatog., 1987, 411, 177
9. Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R., Stύber, D., Bio/Technology., 1988, 6, 1321.
10. 'The QIAexpressionist', 2nd Edition, QIAGEN, 1995.??
11. Paborsky, L.R., Dunn, K.E., Gibbs, C.S., Dougherty, J.P., Anal. Biochem., 1996, 234, 60.
12. Schmid, E ., Keller, T.A., Dienes, Z., Vogel, H., Anal. Chem., 1997, 69, 1979.
13. Schmid, E ., Tairi, A. -P., Hovius, R., Vogel, H., Anal. Chem., 1998, 70, 133. 14. US Patent 5,620,859
15. Sigal, G.B., Bamdad. C, Barberis, A., Strominger, J., Whitesides, G.M., Anal. Chem, 1996, 68, 490.
16. Nieba, L., Nieba-Axmann, S.E., Persson, A., Hamalainen, M., Edebratt, F., Hansson, A.. Lidholm, J., Magnusson, K., Karlsson, A.F., Plύckthun, A., Anal. Biochem., 1997, 252, 217.
Substitute Sheet 17. Liley, M., Keller, T.A., Duschl, C, Vogel, H., Langmuir, 1997, 13, 4190.
18. Stora, T., Hovius, R., Dienes, Z., Pachoud, M., Vogel, H., Langmuir, 1997, 13, 5211..
19. Frey, W., Schief, W.R., Pack, D.W., Chen, C.T., Chilkoti, A., Stayton, P., Vogel, V., Arnold, F.H., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 4937.
20. Frey, W., Brink, J., Schief, W.R., Chiu, W., Vogel, V., Biophys J., 1998, 74, 2674.
21. Schmitt, L., Dietrich, C, Tampe, R., J. Am. Chem. Soc, 1994, 116, 8485.
22. Maloney, K., Shnek, D., Sasaki, D., Arnold, F., Chem. Biol. 1996, 3, 185. 23. Gao, C, Lin, C.-H., Lo, C.-H.L., Mao, S., Wirsching, P., Lerner, R.A.,
Janda, K.D., Proc. Natl. Acad. Sci., 1997, 94, 11777.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Substitute Sheet (Rule 26) RO/AU

Claims

CLAIMS:
1. A compound having the general Formula I
Y - (Z)„ I
in which Y is a branching moiety, Z represents a polydentate ligand chelating agent that coordinates a metal ion; and n is an integer of at least 2.
2. A compound according to claim 1 wherein n is an integer of from 2 to 9.
3. A compound according to claim 2 wherein n is at least 3.
4. A compound according to any one of the preceding claims wherein n is 4.
5. A compound according to any one of the preceding groups wherein the donor atom of Z is N.
6. A compound according to claim 5 where Z is a quadradentate ligand.
7. A compound according to claim 7 wherein Z is an NTA residue.
8. A compound according to any one of the preceding claims wherein Y provides at least three moieties each moeity being covaiently attached directly, or indirectly through an optional linking group, to Z.
9. A compound according to any one of the preceding claims wherein Y has a backbone formed from an oligomer or a polymer.
10. A compound according to claim 9 wherein the backbone is linear.
11. A compound according to any one of claims 1 to 8 wherein Y is selected from the group consisting of amino-polyols, amino acids, amino polycarboxylic acids, polyamines, polyacids and polyhydroxylated compounds.
Substitute Sheet
12. A compound according to claim 9 wherein Z is selected from the group consisting of peptides and dendrons.
13. A compound according to any one of the preceding claims of formula II
X - Y - (Z)n II in which Y, Z and n are as defined above and X is a spacer moiety.
14. A compound according to claim 13 wherein X is hydrophilic, hydrophobic or has hydrophobic and hydrophilic regions.
15. A compound according to claim 14 wherein X is or includes a moiety selected from the group consisting of substituted or unsubstituted alkyl, optionally interrupted by one or more heteroatoms, oligoalkylene oxides, amino acid sequences, polypeptides, oligoamides, polyamides and lipids.
16. A compound according to claim 15 wherein X is selected from the group consisting of oligoethylene glycol, an aminocaproyl oligomer and a membrane spanning lipid (MSL).
17. A compound according to any one of claims 13 to 16 wherein X includes an hydrophilic region, an hydrophobic region.
18. A compound according to any one of claims 13 to 17 wherein Y is a branching moiety that provides a plurality of moieties for covalent attachment of Z and a single moiety for covalent attachment to X.
19. A compound according to claim 18 wherein Y is selected from the group consisting of aminopolyols, amino acids, peptides possessing multiple free acid and/or amine moieties, polyhydroxylated materials and compounds possessing groups readily displaced by nucleophiles or groups to which nucleophiles readily add, or a combination thereof.
Substitute Sheet (Rule 26) RO/AU
20. A compound according to claim 19 wherein Y is selected from the group consisting of TRIS, bis-homotris, 3,5-diaminobenzoic acid, 5-aminoisophthalic acid,
Figure imgf000035_0001
, sugars, dendrons and α,β-unsaturated ketones.
21. A compound according to any one of claims 13 to 20 of Formula III
W - X - Y - (Z).
III
in which X, Y, Z and n are as defined above and W is a group that allows for attachment to another molecules, attachment to surfaces, or insertion into membranes.
22. A compound according to claim 21 wherein W includes a functional group selected from one or more of an amine functional group, a carboxylic acid functional group, an alcohol functional group, a halide functional group, a thiol, a disulfide, a silane derivative, a membrane soluble protein, a group allowing non-covalent attachment, an ionophore or a lipid group.
23. A compound according to claim 22 wherein W is gramicidin or biotin.
24. A biosensor comprising a self-assembled membrane, the membrane comprising a plurality of binding compounds of Formula III according to any one of claims 13 to 23 in which W is an ionophore which is embedded in the membrane
Substitute Sheet (Rule 26) RO/ATJ
25. A biosensor according to claim 24 wherein the ionophore is gramicidin.
26. A biosensor according to claim 24 or claim 25 wherein the membrane includes a second plurality of binding compounds of Formula III in which W is an amphiphile that is embedded in the membrane.
27. Use of a compound according to any one of claims 1 to 23 in metal affinity chromatography.
28. A metal affinity chromatographycolumn comprising a compound in accordance with any one of claims 1 to 23.
Substitute Sheet (Rule 26) RO/ATJ
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EP1150942A1 (en) 2001-11-07
CA2341348A1 (en) 2000-08-17
EP1150942A4 (en) 2003-02-12
JP2002536428A (en) 2002-10-29
AUPP856399A0 (en) 1999-03-04

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