AU2648500A - Improved compounds for protein binding - Google Patents

Description

WO 00/47548 PCT/AUOO/00075 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 Ternary 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 Coz+, Ni 2 +, Cu 2 , Zn2+. Typical metal coordinating groups are nitrilotriacetic acid (NTA), 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 -10 4 M-' (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 His-tagged proteins for solid supports bearing NTA has been determined to be approx. 10" M 4 (9). Substitute Sheet WO 00/47548 PCT/AUOO/00075 2 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 5HT, 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 FTIR (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-Cuz+ 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-Cuz+ 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 WO 00/47548 3 PCT/AUOO/00075 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) ' 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 Coz+ Niz+, Cuz+, Zn 2 +. The donor atoms of Z may be a- donor atoms or 7c- donor atoms. The donor atoms may be selected from N, 0, 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)" 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 nihatiitt Sheet WO 00/47548 PCT/AUOO/00075 4 glycol, an amino acid sequence, a polypeptide or a poly- or oligoamide (for example an aminocaproyl oligomer). 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) 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,

CO

2 H

CO

2 H CO 2 H

H

2 N H 2 N N COH HN OH

CO

2 H

CO

2 H 0 2 H Substitute ShpAt WO 00/47548 PCT/AUOO/00075 5 peptides which possess multiple free acid and/or amine moieties, for example 0 0 0 H H HO N

NH

2 HOO NO H H 0 0

HO

2 C HO 2 C HO 2 C 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 spermidine, 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, OH 0-c HO0 OH 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 c,o-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, WO 00/47548 PCT/AUOO/00075 6 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 J1.triNTA chip. 100, 200 and 400 nM binding curves shown (bottom to top). Binding curves are the responses from Fc2 (control, -Niz+) subtracted from Fc1. Figure 2. Binding of 6His.CD40 to J1.triNTA chip. 100, 200, 400, 600 and 800 nM binding curves shown (bottom to top). Binding curves are the responses from Fc2 (control, -Niz+) 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 WO 00/47548 PCT/AUOO/00075 7 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 BlAcore NTA chip, compared to the triNTA on the gold chip (compare the absolute binding levels), the off rate (kd) 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-trimethylsilylethanol, 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide 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 01 immediately prior to use. 2. Synthesis of Z-LysNTA 0 OH 0 OH Q "O)HN ,N 0 OH 0

C

18

H

24

N

2 0 8 396.39 396.153266 C 54.5% H 6.1% N 7.1% 0 32.3% Z-LysNTA was synthesised following the procedure of Schmitt et al.' Bromoacetic acid (4.17 g, 30.0 mmol) was dissolved in aqueous NaOH (1.5 M, 15 ml) and cooled to 0 0 C. N'-Z-(L)-Lysine (2.0 g, 7.0 mmol) in aqueous NaOH (1.5 M, WO 00/47548 PCT/AU0O/00075 8 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 HCl (1M, 40 ml) was added dropwise to the reaction mixture, and the resulting white precipitate was filtered and washed with HCl (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 'H NMR (200 MHz, d-DMSO) : 5 7.42 (5H, m, -C 6 H,), 5.09 (2H, s, -CH 2 Ph), 3.56 (4H, AB quartet, 2 x -NCH 2

CO

2 H), 3.41 (1H, overlapping dd. -NCHCO 2 H), 3.06 (2H, m, NHCH 2 -), 1.75-1.25 (6H, overlapping multiplets, -NHCH2CH 2

CH

2

CH

2 -) ppm. 13 C NMR (50 MHz, d,-MeOD) :5 175.9 (2 x -C(O)O-), 175.8 (-C(O)O-), 158.9 (-C(O)NH), 138.4 (-C 6 H,), 129.4 (-CH,), 128.9 (-CbH,), 128.7 (-CH,), 67.3 (-CH 2 Ph), 66.7 ( NCHC(O)-), 55.3 (2 x -NCH 2 C(O)-), 41.5 (-NHCH 2 -), 30.7 (-NHCH 2

CH

2 -), 30.5 (

NHCH

2

CH

2

CH

2

CH

2 -), 24.6 (-NHCH 2

CH

2

CH

2 -) ppm. Electrospray M/S : m/z 397 (100%) (M+H+), 398 (21%), 353 (30%), 792 (15%). 3. Synthesis of ZLysNTA.TMSE 3 ZLysNTA.TMSE, was synthesised using a procedure outlined by Gao et al.

2 O0 N Si(CHN) 0 c r ----- Si(CH3)3 C3 3 HeoN2OsSi 3 697.10 696.365752 C 56.9% H 8.7% N 4.0% 0 18.4% Si 12.1% 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 0 C was added EDC (1.25 g, 12.5 mmol). The reaction was stirred at 0 0 C for 1 hour, then allowed to warm to room temperature and WO 00/47548 PCT/AUOO/00075 9 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, as a colourless oil (0.75 g, 86%). Data Rf = 0.78 (5% methanol in dichloromethane) 'H NMR (200 MHz, CDCl 3 ) :5 7.34 (5H, m, -CH), 5.08 (2H, s, -CH 2 Ph), 4.91 (1H, br t, NH), 4.16 (6H, m, 3 x -OCH 2 -), 3.60 (4H, s, 2 x -NCH 2 C(O)-), 3.39 (1H, t, -NCHC(O)-, 'JH. H = 7.5 Hz), 3.19 (2H, m, -NHCH 2 -), 1.3-1.8 (6H, overlapping multiplets, NHCH 2

CH

2

CH

2

CH

2 -), 0.93-1.04 (6H, overlapping multiplets, 3 x -CH 2 Si-), 0.04 (9H, s, 1 x -Si(CH 3 ),), 0.03 (18H, s, 2 x -Si(CH 3

)

3 ) ppm.

'

3 C NMR (100 MHz, CDC1l) :8 173.6 (-C(O)O-), 172.2 (2 x -C(O)O-), 157.1 (-OC(O)NH), 137.5 (-CH,), 129.2 (-CH,), 128.7 (-CH,), 67.2 (-CH 2 Ph), 65.4 (-NCHC(O)-), 63.5 (1 x OCH 2 -), 63.3 (2 x -OCH 2 -), 53.6 (2 x -NCH 2 C(O)-), 41.6 (-NHCH 2 -), 30.8 (-NHCH 2

CH

2 -), 30.1 (-NHCH 2

CH

2

CH

2

CH

2 -), 23.8 (-NHCH 2

CH

2

CH

2 -), 18.3 (-CH 2 Si-), 18.1 (2 x -CH 2 Si-), 0.8 (3 x -Si(CH 3

)

3 ) ppm. Electrospray M/S : m/z 719.4 (100%) (M+Na*) 4. Synthesis of LysNTA.TMSE 3 0 o 5Si(CH3)3

H

2 N N ' Si(CH 3

)

3 0 0' Si(CH3)3 0 C25H 4

N

2 0Si 3 562.97 562.328973 C 53.3% H 9.7% N 5.0% 0 17.1% Si 15.0% Substitute Sheet WO 00/47548 PCT/AUOO/00075 10 ZLysNTA.TMSE, (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 H 2 gas. The reaction mixure was stirred at room temperature for 75 min, then filtered and all volatiles removed under reduced pressure. Analysis by 'H NMR showed complete hydrogenation had occurred, to afford LysNTA.TMSE, as a colourless oil (0.127 g, quantitative yield). Data 'H NMR (200 MHz, CDC1) : 5 4.16 (6H, m, 3 x -OCH 2 -), 3.61 (4H, s, 2 x -NCH 2 C(O)-), 3.40 (1H, apparent triplet, -NCHC(O)-, %JaH = 7.5 Hz), 2.69 (2H, br t, NH 2 CH,-, 'JH-H 6.1 Hz), 1.3-1.8 (6H, overlapping multiplets. NH 2

CH

2 CH2CHCH 2 -), 0.93-1.04 (6H, overlapping multiplets, 3 x -CH2Si-), 0.04 (9H, s, 1 x -Si(CH,),), 0.03 (18H, s, 2 x Si(CH),) ppm.

'

3 C NMR (100 MHz, CDCl 3 ) :5 173.7 (-C(O)O-), 172.2 (2 x -C(O)O-), 65.6 (-NCHC(O)-), 63.5 (2 x -OCH 2 -), 63.3 (1 x -OCH-), 53.6 (2 x -NCH 2 C(O)-), 42.7 (NH 2

CH

2 -), 33.9

(NH

2 CH2CH 2 -), 31.0 (NH 2

CH

2

CH

2

CH

2

CH

2 -), 23.9 (NH 2

CH

2

CH

2

CH

2 -), 18.3 (-CH 2 Si-), 18.1 (2 x -CH 2 Si-), -0.8 (3 x -Si(CH,),) ppm. Electrospray M/S : m/z 563.3 (100%) (M+H+) Substitute Sheet /T-1-1 - n-/- TI\ A T T WO 00/47548 PCT/AUOO/00075 11 5. Synthesis of ZtriNTA.TMSE9 Si( b oC6) S(CH31b SH Si(C0h 0 0 0--Sl(CH 3 0 H 0 o i(CHsh

C

93 HsoNgsO23si 9 2031.26 2029.108491 C 55.0% H 8.9% N 5.5% 0 18.1% Si 12.4% ZLysNTA (60 mg, 0.15 mmol), 4-dimethylaminopyridine (60 mg, 0.51 mmol) and LysNTA.TMSE, (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.TMSE, as a colourless oil (0.235 g, 76%). Data Rf = 0.24 (5% methanol in dichloromethane) 'H NMR (400 MHz, CDCl 3 ) : 5 7.37 (2H, br t, NH), 7.34 (5H, m, -CH 5 ), 7.26 (1H, br t, NH), 5.15 (1H, br t, NH), 5.08 (2H, s, -CH 2 Ph), 4.17 (18H, m, 9 x -OCH 2 -), 3.60 (12H, two overlapping singlets in 2:1 ratio, 6 x -NCH 2 C(O)O-), 3.39-3.34 (5H, overlapping Substitute Sheet WO 00/47548 PCT/AUOO/00075 12 multiplets), 3.26-3.16 (10H, overlapping multiplets), 3.07 (1H, triplet), 1.85-1.30 (24H, overlapping multiplets, 12 x -CH2), 0.97 (18H, overlapping multiplets, 9 x -CH 2 Si-), 0.05 (27H, s, 3 x -Si(CH,),), 0.04 (54H, s, 6 x -Si(CH,),) ppm. -OC(O)NHCH 2 "C NMR (100 MHz. CDCl 3 ) :5 173.5 (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 (-CH,), 129.1 ( CGH,), 128.6 (-C 6 H,), 67.1 (-CH 2 Ph), 66.4 (-NCHC(O)NH-), 65.5 (-NCHC(O)O-), 65.4 (2 x NCHC(O)O-), 63.4 (6 x -OCH 2

CH

2 -), 63.3 (3 x -OCH 2

CH

2 -), 56.9 (2 x -NCH 2 C(O)NH-), 53.7 (4 x -NCH 2 C(O)O-), 53.4 (2 x -NCH 2 C(O)O-), 41.2 (-OC(O)NHCH 2 -), 39.9 (3 x C(O)NCH 2 -), 30.7, 30.5, 30.2 (4 x -NHCH 2

CH

2 -), 29.9, 29.5, 28.9 (4 x

NHCH

2

CH

2

CH

2

CH

2 -), 24.7, 24.0, 23.8 (4 x -NHCH 2

CH

2

CH

2 -), 18.3 (3 x -CH 2 Si(CH,),), 18.0 (6 x -CH 2 Si(CH 3 )3), -0.8 (9 x -Si(CH 3

)

3 ) ppm. Electrospray M/S : m/z 2054.3 (100%) (M+Na*), 1038.8 (20%), 613.3 (36%) 6. Synthesis of ZtriNTA o OH OH OH o O OoNN OH H HOH 00 o s . OH HN(! H 0 HO

C

4 8H 72

NE

5 O O O Exact Mass: 1128.47N rNiol. Wt.: 1129.13o C, 51.06; H, 6.43; N, 9.92; 0, 32.59 OH 0 ZtriNTA.TMSE, (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 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% H 2 0/0.05% TFA to 50% acetonitrile/0.05%TFA in H 2 0/0.05% 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).

WO 00/47548 PCT/AUOO/00075 13 Data 'H NMR (400 MHz, D 2 0) :5 7.38 (5H, m, -CHL), 5.08 (2H, s, -CH 2 Ph), 4.06-3.78 (20H, m, 8 x -NCH 2 C(O)- + 4 x -NCHC(O)-), 3.23 (6H, apparent triplet, 3 x -C(O)NHCH 2 -), 3.08 (2H, apparent triplet, 1 x -OC(O)NHCH 2 -), 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

-CH

2 ) ppm. "C NMR (100 MHz, D 2 O): 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 tri NTA TMSE9 S_/CH (H3) o S0(C (Cb N O o Oo 0 H"'>N N |C 0 0 0 HNN 0 Si(C b 0" S KCH Z-tri NTA TMSE, (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 TMSE, (34 mg, 96%).

WO 00/47548 PCT/AUOO/00075 14 Data 'H n.m.r. (CDCl,) 5 0.00 (81 H, m, Si(CH,),), 0.96 (18H, m, CH 2 Si), 1.2-2.0 (24H, m), 3.1-3.7 (28H, m), 4.15 (18H, m. OCH 2 ) and 7.4 (2H, NH 2 ). 8. Synthesis of tri NTA TMSE, hemisuccinamide 0 O Si0CH 0 0O 00 HO N N NNSi(C~i NN Ect Mw: 1995.09 N SKCHA Mol. Wt.: 1997.17N C, 53.52; H, 8.98; N 5.61; 0. 19.23; Si, 12.66 0 O0 SRCHA A solution of tri-NTA.TMSE, (0.5g), benzyl hemisuccinate (71mg), DMAP (64mg) in dichloromethane was stirred at 0*. EDC (101mg) 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 CH 2 Cl-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 'H n.m.r. (CDCl,) 5 0.04 (81 H, m, Si(CH 3

)

3 ), 0.96 (18H, m, CH 2 Si), 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, OCH 2 ). Electrospray M/S : m/z 2019.8 (35%), (M+Na*), 1997.8 (100%) (M*).

WO 00/47548 PCT/AUOO/00075 15 9. Synthesis of tri NTA o /OH OH 0 H O 0 O ,HN OH NN

H

2 N N OH 0 o4H6B2 0H OH HN OH ECetH 3 N 2 OH Exact Was: 994.439 C, 48.28; H, 6.69; N, 11.26; 0, 33.77 OH tri NTA TMSE, (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 tri NTA (20 mg, 100%). Data 'H n.m.r. (CD,OD) 5 1.2-2.0 (24H, m), 3.07 (6H, m, CH 2 NHCO) 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%).

WO 00/47548 PCT/AUOO/00075 16 Data 'H n.m.r. (CDOD) 5 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CH 2 CO), 2.9-3.4 (10H. m). 3.90 (2H, dd, CH 2 -gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, 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 mnmol) 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 'H n.m.r. (CDOD) 5 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CH 2 CO), 2.82 (4H, s, NHS), 2.9-3.4 (10H. m), 3.90 (2H, dd. CH 2 -gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO). 12. Preparation of Gramicidin succinate tri NTA O OH OH N O OH NYOH 0 0H O HN H 0 OH HN 0 N O N>OH 0 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 WO 00/47548 PCT/AUOO/00075 17 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 'H n.m.r. (CD2OD) 8 0.4-1.8 (90H, m), 2.0-2.2 (4H, m), 2.52 (4H. m. CH 2 CO), 3.0-3.8 (38H. m), 3.90 (2H, dd, CH 2 -gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO). MALDI m.s. 2963.4( (M+Na*)-H 2 O), 2982.9 (M+Na*), 2998 (M+K+). 13. Preparation of gA lysine-XXBOC OH 0 H gANH5- N .. sN OC H ~ 0H gAlysXXBOC 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 'H n.m.r. (CD2OD) 5 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, CH 2 -gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO). Substitute Sheet WO 00/47548 PCT/AUOO/00075 18 14. Preparation of gAlysine XXBOC hemisuccinate OH 00 H0 gA IN OH HO So 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 'H n.m.r. (CDOD) 8 0.4-1.8 (84H, m), 2.0-2.2 (8H, m), 2.58 (4H, dd, CH 2 CO), 2.9-3.4 (14H, m), 3.90 (2H, dd, CH 2 -gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO). 15. Synthesis of gAlysine2Xtri NTA O OH OH 00Q OH 0 HN -OH gAI ' N OH OH H 0 H Q0~ 0 OH HN 0 OH N oH 0 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, WO 00/47548 PCT/AUOO/00075 19 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 'H n.m.r. (CDOD) 5 0.4-1.8 (108H, m), 2.0-2.2 (8H, m), 2.62 (4H, dd, CH2CO), 3.0-3.8 (42H, m), 3.90 (2H, dd, CH,-gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO). MALDI m.s. 3291.17 (M+Na+), 3309.02 (M+K*). 16. Synthesis of triNTA.TMSE 9 H2N N O3 0 0 1 S(CHb3 CH0 3 )3 SI(CH3h casHllJiNsO21si9 1897.12 1895.071711 C 53.8% H 9.2% N 5.9% 0 17.7% Si 13.3% A solution of ZtriNTA.TMSE, (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 WO 00/47548 PCT/AUOO/00075 20 removed from the reaction micxture under reduced pressure to afford trisNTA.TMSE, 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 N H H N-- ' Si (ck-b H- 0 00 H N , H 0 0 0 si(cHa)b 0 si(CH ) si(CHa)3

C

95

H

188

N

10 0 23 SSig 2123.42 2121.149311 C 53.7% H 8.9% N 6.6% 0 17.3% S 1.5% Si 11.9% Biotin (20 mg, 0.08 mmol), 4-dimethylaminopyridine (12 mg, 0.1 mmol) and triNTA.TMSE, (0.10 g, 0.1 mmol) were added to dry, freshly distilled dichloromethane (5 ml) and the reaction mixture was cooled to OC. EDC (40 mg, 0.2 mmol) was added, and the reaction was stirred at 0 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.MTSE, was obtained as a colourless oil (73 mg, 0.03 mmol, 34%). Substitute Sheet WO 00/47548 PCT/AU0O/00075 21 Data Electrospray M/S : m/z 1173.9 (100%), 2146.4 (25%) (M+Na*). 18. Synthesis of Biotin.triNTA OH OH 0 OH HNy NH H OH 0 CH CSoHSON10023S 1221.29 1220.511851 C 49.2% H 6.6% N 11.5% N 30.1% S 2.6% Trifluoroacetic acid (2 ml) was added to Biotin.triNTA.TMSE ' (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 : H20 / 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 WO 00/47548 PCT/AUOO/00075 22 19. Synthesis of Lipid-triNTA OH OH N OH O HN 0 OH N ON N HN N OH H 0 0 K : OH 0

C

72

H

12 7N 9 0 2 3 1486.84 1485.904482 C 58.2% H 8.6% N 8.5% 0 24.7% A solution of ditetradecylamine (100mg) in CH 2 Cl 2 (5ml) was added to a solution of succinic anhydride (120mg) and triethylamine (40mg) in CH 2 C1 2 (5ml) at room temperature. The reaction was stirred overnight, the solvents removed in vacuo and the residue chromatographed using CH 2 Cl 2 -MeOH (95:5 <H] 85:5) to give 60mg of ditetradecylamine hemisuccinamide. This material was added to a solution of triNTA.TMSE, in CH 2 Cl 2 (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. 10mg of this material was dissolved in TFA (iml) 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 CHCN) to separate the desired product (4mg). Data Electrospray M/S : m/z 1486.6 (100%) (M+H*), 1508.8 (62%) (M+Na*). Substitute Sheet WO 00/47548 PCT/AUOO/00075 23 20. Synthesis of Membrane-spanning lipid phosphatyl choline (MSL-PC) 0 0 || + o ~ ~~~ o0s m owo g s- e/ e NMe3 00 -0

C

124

H

220

NO

24

PS

2 Exact Mass: 2202.52 Mol. Wt.: 2204.17 C, 67.57; H, 10.06; N, 0.64; 0, 17.42; P, 1.41; S, 2.91

MSL-OH

3 (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 POCl, (0.09 mL, 2.2 equiv.) at room temperature. The mixture was stirred at 45*C for 30 min. After cooling choline tosylate (363 mg, 1.3 inmol) 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% HCl (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 'H n.m.r. 6(CDCl,:CDOD (3:1)) 0.82-0.92 (m, 30H, phytanyl CH,'s), 0.92-1.80, m, 100H, phytanyl CH 2 's and CH's), 2.61 (t, 2H, CH 2

CH

2 S), 2.65 (s, 8H, succinate H's), 3.20 (s, 9H, -N(CH 3 ):), 3.35-3.74 (m, 44H, -OCH 2 , -OCH, CH 2 N), 3.85 (s, 2H, SCH 2 Ph), 3.92 (t, 2H, CH 2 OP), 4.10 (m, 2H, POCHCH 2 ), 4.20 (m, 8H, ester H's), 6.90 & 7.43 (d, 8H, biphenyl), 7.30 (s, 5H, PhCH 2 ). Substitun sha WO 00/47548 PCT/AUOO/00075 24 Electrospray MS m/z: 2206 (M+2), 1112, 587, 488 21. Synthesis of bis(N-methyl)-C38 diamine o 0 MeHN C90HI 68

N

2 0 6 NHMe Exact Mass: 1373.29 Mol. Wt.: 1374.31 C, 78.66; H, 12.32; N, 2.04; 0, 6.99 To the bola-amphiphile C38-diol 3 (300mg) in THF (10ml) cooled to 00 was added triethylamine (92 1) and methanesulphonyl chloride (5 1 1 ). The solution was stirred overnight at room temperature and diluted with ether (30ml). The organic phase was washed with saturated NaHCO, (2 X 3OmL) and water (2 X 60ml), dried (Na 2

SO

4 ) and concentrated in vacuo. The material obtained (315mg) was added to a pressure tube, cooled to -800, 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 CH 2 Cl 2 and stirred with K 2 CO. The solid was removed by filtration and the filtrate dried in vacuo. The residue was chromatographed using

CH

2 Cl 2 -MeOH-NH, (96:4:0.1 <R] 92:8:0.1) to separate 242 mg pure product. Data 'H n.m.r. (CDCl 3 ) 5 0.80-0.95 (30H, m, Phytanyl CH,'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-O and CH 2 -O); 3.98 (4H, t. CH 2 0Ar); 6.93, 7.43 (8H, AA'XX' multiplets, arom.-H 22. Synthesis of XXFmoc - 0 HOH H / 0

C

27

H

34

N

2 0 5 Exact Mass: 466.25 Mol. Wt.: 466.57 C, 69.50; H, 7.35; N, 6.00; 0, 17.15 Substitute Sheet WO 00/47548 PCT/AUOO/00075 25 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 (100mL) was added and aqueous solution extracted with ethyl acetate (2x5OmL) 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 (CDCl 3 ) 1.2-1.8 (m, 12H), 2.20(t, 2H), 2.35(t,2H), 3.25(m, 4H), 4.259m, 1H), 4.40(m, 1H), 5.05(m, 1H), 5.53(m, 1H), 6.65(m, 1H), 7.3-7.5(m, 4H), 7.6(d, 2H), 7.25(d, 2H) Mass Spec EI 483.9(M+Na*) 23. Synthesis of C38 (NCH 3

)

2 mono BOC -o

~

Me' N

C

95 H-1 76

N

2 0 8 eN- Me N O Exact Mass: 1473.34 MeHN 0 Mol. Wt.: 1474.42 C, 77.39; L 12.03; N, 1.90; 0, 8.68 A suspension of C38diamine (320mg), BOC-ON (63mg), triethylamine (37mg), THF(3mLO and water (2mL) was stirred at room temperature over night. Reaction mixture diluted with water (50mL) and extracted with dichloromethane (2x8OmL). Combined organic extracts were washed with brine (5OmL) 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) Riihej-ditntp. Sheu WO 00/47548 PCT/AUOO/00075 26 Data NMR (CDCl 3 ) 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 O MO HN 0 O O - 0 0 "' / 22

H

2

N

4 OI, E.xat Mass: 1921.58 Mol. Wt.: 1922.98 C, 76.20; H, 10.90. N, 2.91; 0. 9.98 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 (CDCl 3 ) 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*) Substiin~pin hn+- WO 00/47548 PCT/AUOO/00075 27 25. Synthesis of MSL-tri-NTA -O O HO O NMe 0Oe- 0 HOy N 0 N N HO HO", N _CN HOH

C

179 H HO NExact Mass: 3394.17 Mol. Wt.: 3396.55 HO C, 63.30; H, 9.14; N, 4.95; 0, 20.73; S, 1.89

HO.

7 (N HO 0 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 (100mg) 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 O O 0 a 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. Substitute Sheei WO 00/47548 PCT/AUOO/00075 28 This active ester was reacted in methanol with the fully deprotected C38diamine triNTA material prepared above (100mg). 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-CH 2 Cl 2 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 J1 chip (BIAcore, Uppsala. Sweden) was modified by the coating of a lipid layer. In a laminar flow hood, the BIAcore J1 chip was unsheathed to expose the gold surface. A solution (100 UL) containing 1.2 4M MSLtriNTA, 24 pM 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 uL 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 BlAcore 2000 machine and the pumps flushed and experiments performed using HBS/EDTA running buffer (50 mM HEPES, 300 mM NaCl. 50 pM EDTA, pH 8.0) at a flow rate of 40 p1/min at 21 0 C. Flow cell 2 (Fc2) was used as the test cell relative to the control Flow cell 1 (Fcl). Hence, running buffer containing 500 pM NiCl 2 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 NiCl 2 and hence released 6His-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 6His tag absent. The results indicated that triNTA binds 6His-tagged proteins with high affinity (Fig. 1, 2); 6His.Rubisco and 6His.CD40 bound triNTA with equilibrium constants of 300 pM and 2.9 nM, respectively. These proteins did not bind triNTA in the absence of Ni 2 ' and proteins without the 6His tag also did not bind triNTA. AnhRtitnit Sheet WO 00/47548 PCT/AUOO/00075 29 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. 5 PNAS, 1997, 94, 11777. 3. Raguse, B., Culshaw, P.N., Prashar,J.K., Raval, K., Tetrahedron 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. 10 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; 15 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. 20 8. Hochuli, E., Dobeli, H., Schacher, A., J. Chromatog., 1987, 411, 177 9. Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R., Stfiber, 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., 25 1996,234,60. 12. Schmid, E.L., Keller, T.A., Dienes, Z., Vogel, H., Anal. Chem., 1997, 69, 1979. 13. Schmid, E.L., Tairi, A.-P., Hovius, R., Vogel, H., Anal. Chem., 1998, 70, 133. 30 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., Hsmiliinen, M., Edebratt, F., Hansson, A., Lidholm, J., Magnusson, K., Karlsson, A.F., Plilckthun, 35 A., Anal. Biochem., 1997, 252, 217.

WO 00/47548 PCT/AUOO/00075 30 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., 5 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., Tamp6, R., J. Am. Chem. Soc., 1994, 116, 8485. 22. Maloney, K., Shnek, D., Sasaki, D., Arnold, F., Chem. Biol. 1996, 3, 185. 10 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 15 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

Claims (28)

1. A compound having the general Formula I 5 Y - (Z) I in which Y is a branching moiety, Z represents a polydentate ligand chelating 10 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. 15

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. 20

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. 25

8. A compound according to any one of the preceding claims wherein Y provides at least three moieties each moeity being covalently 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 30 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 35 from the group consisting of amino-polyols, amino acids, amino polycarboxylic acids, polyamines, polyacids and polyhydroxylated compounds. WO 00/47548 PCT/AUO0/00075 32

12. A compound according to claim 9 wherein Z is selected from the group consisting of peptides and dendrons. 5

13. A compound according to any one of the preceding claims of formula II X - Y - (Z), II in which Y, Z and n are as defined above and X is a spacer moiety. 10

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 15 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 20 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. 25

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. 30

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. 35 Substitute Sheet m..s- Pci rIATT WO 00/47548 PCT/AUOO/00075 33

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, CO 2 H CO 2 H CO 2 H H 2 N H 2 N N CO 2 H H, 1 -CO 2 H K ~HN- O 2 5 CO 2 H CO 2 H 02H 0 0 0 H H HO N N NH 2 HOO NO H H 0 0 HO 2 C HO 2 C HO 2 C sugars, dendrons and axp-unsaturated ketones. 10

21. A compound according to any one of claims 13 to 20 of Formula III W - X - Y - (Z). III 15 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. 20

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. 25

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 30 membrane Substitute Sheet I n__1 n ^ /" A TT WO 00/47548 PCT/AUOO/00075 34

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 5 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. 10

28. A metal affinity chromatographycolumn comprising a compound in accordance with any one of claims 1 to 23. 15 Substitute Sheet