CA2341348A1 - Improved compounds for protein binding - Google Patents
Improved compounds for protein binding Download PDFInfo
- Publication number
- CA2341348A1 CA2341348A1 CA002341348A CA2341348A CA2341348A1 CA 2341348 A1 CA2341348 A1 CA 2341348A1 CA 002341348 A CA002341348 A CA 002341348A CA 2341348 A CA2341348 A CA 2341348A CA 2341348 A1 CA2341348 A1 CA 2341348A1
- Authority
- CA
- Canada
- Prior art keywords
- compound according
- group
- membrane
- moiety
- attachment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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
Ternary metal complexes are well known in the literature (1) and can be described as the coordination of t~n~o discrete metal chelating groups to a metal.
The metals normally observed in ternary complexes are Coz+, Ni2+, Cuz+, Znz+.
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'-10~ 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 mhich 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. 101' M'' (9).
Substitute Sheet (Role 26) 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 SHT, 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-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-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 (Rule 26) RO/AU
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
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 t~n~o discrete metal chelating groups to a metal.
The metals normally observed in ternary complexes are Coz+, Ni2+, Cuz+, Znz+.
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'-10~ 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 mhich 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. 101' M'' (9).
Substitute Sheet (Role 26) 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 SHT, 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-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-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 (Rule 26) RO/AU
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+, Niz+, CuZ+, ZnZ+. The donor atoms of Z may be ~- donor atoms or n- 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 O, N, S or combinations of two or more thereof), for example oligoethylene glycol or other oligoalkylene Substitute Sheet (Rule 2F) ROlAU
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 Iipid may be a membrane spanning Iipid (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, COZH
COiH CO=H
i ~O H
H HzN N ~CO=H
HN
CO~H ~ ~CO~H ~ '-C02H
Substitute Sheep (Rule 26) RO/AU
WO 00/47548 _ PCT/AU00/00075 peptides which possess multiple free acid and/or amine moieties, for example N N~ NH=
HO H II H
HOiC HOiC HOG
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, ~H
HO O
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 oc,(i-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 (Rule ~6) RO/AU
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, -Niz+) 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 ~6) 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 (1S,). 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 (lcd) for the N'TA
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 yr 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)-
Z may be a polydentate ligand that coordinates a metal ion such as Co2+, Niz+, CuZ+, ZnZ+. The donor atoms of Z may be ~- donor atoms or n- 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 O, N, S or combinations of two or more thereof), for example oligoethylene glycol or other oligoalkylene Substitute Sheet (Rule 2F) ROlAU
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 Iipid may be a membrane spanning Iipid (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, COZH
COiH CO=H
i ~O H
H HzN N ~CO=H
HN
CO~H ~ ~CO~H ~ '-C02H
Substitute Sheep (Rule 26) RO/AU
WO 00/47548 _ PCT/AU00/00075 peptides which possess multiple free acid and/or amine moieties, for example N N~ NH=
HO H II H
HOiC HOiC HOG
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, ~H
HO O
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 oc,(i-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 (Rule ~6) RO/AU
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, -Niz+) 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 ~6) 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 (1S,). 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 (lcd) for the N'TA
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 yr 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 PROS immediately prior to use.
2. Synthesis of Z-LysNTA
p OH
O
~ OH
O' HN N
O
C t sHzaNzOs 396.39 396.153266 C54.5%H6.1%N7.1%032.3%
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°C. N'-Z-(L)-Lysine (2.0 g, 7.0 mmol) in aqueous NaOH (1.5 M, Substitute Sheet (Rule 2~) RO/AU
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 1H NMR (200 MHz, ds-DMSO) : S 7.42 (5H, m, -CsHs), 5.09 (2H, s, -CHZPh), 3.56 (4H, AB quartet, 2 x -NCHZCOZH), 3.41 (1H, overlapping dd, -NCHCOZH), 3.06 (2H, m, -NHCHi ), 1.75-1.25 (6H, overlapping multiplets, -NHCHZCHZCHZCHZ-) ppm.
'3C NMR (50 MHz, d~ MeOD) : 8 175.9 (2 x -C(O)O-), 175.8 (-C(O)O-), 158.9 (-C(O)NH), 138.4 (-CBHs), 129.4 (-C6Hs), 128.9 (-CeHs), 128.7 (-CeHs), 67.3 (-CHZFh), 66.7 (_ NCHC(O)-), 55.3 (2 x -NCHZC(O)-), 41.5 (-NHCHZ ), 30.7 (-NHCHZCHa-), 30.5 (-NHCHZCHZCHZCHz-), 24.6 (-NHCHZCHZCHZ-) 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.z 0~ /~~
O ° ~i(Cfhis ~ 0 O' HN N~ ~Si(CFi~s O
~Si(~s v CssHed'~~OeSis X7.10 696.366752 C 56.9% H B.7% N 4.0% O 18.4% SI 121~A
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 (Rule 2fi) RO/AU
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 flesh 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) 'H NMR (200 MHz, CDCI,) : E 7.34 (5H, m, -C6H5), 5.08 (2H, s, -CHZPh), 4.91 (1H, br t, -NH), 4.16 (6H, m, 3 x -OCHZ-), 3.60 (4H, s, 2 x -NCH2C(O)-), 3.39 (1H, t, -NCHC(O)-, 3JH-H = 7.5 Hz), 3.19 (2H, m, -NHCHZ ), 1.3-1.8 (6H, overlapping multiplets, -NHCHZCHZCHZCHz ), 0.93-1.04 (6H, overlapping multiplets, 3 x -CHZSi-), 0.04 (9H, s, 1 x -Si(CH3)3), 0.03 (18H, s, 2 x -Si(CH3)3) ppm.
'3C NMR (100 MHz, CDCl3) : 8 173.6 (-C(O)O-), 172.2 (2 x -C(O)O-), 157.1 (-OC(O)NH), 137.5 (-CsHS), 129.2 (-CsHs), 128.7 (-CBHS), 67.2 (-CHZPh), 65.4 (-NCHC(O)-}, 63.5 (1 x -OCHZ ), 63.3 (2 x -OCHZ-), 53.6 (2 x -NCHZC(O)-), 41.6 (-NHCHZ-), 30.8 (-NHCHZCHZ-), 30.1 (-NHCHzCHZCH2CH2-), 23.8 (-NHCH2CHZCHZ-), 18.3 (-CHZSi-), 18.1 (2 x -CHZSi-), -0.8 (3 x -Si(CH3)3) ppm.
Electrospray M/S : m/z 719.4 (100%) (M+Na+)
Dichloromethane was distilled over PROS immediately prior to use.
2. Synthesis of Z-LysNTA
p OH
O
~ OH
O' HN N
O
C t sHzaNzOs 396.39 396.153266 C54.5%H6.1%N7.1%032.3%
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°C. N'-Z-(L)-Lysine (2.0 g, 7.0 mmol) in aqueous NaOH (1.5 M, Substitute Sheet (Rule 2~) RO/AU
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 1H NMR (200 MHz, ds-DMSO) : S 7.42 (5H, m, -CsHs), 5.09 (2H, s, -CHZPh), 3.56 (4H, AB quartet, 2 x -NCHZCOZH), 3.41 (1H, overlapping dd, -NCHCOZH), 3.06 (2H, m, -NHCHi ), 1.75-1.25 (6H, overlapping multiplets, -NHCHZCHZCHZCHZ-) ppm.
'3C NMR (50 MHz, d~ MeOD) : 8 175.9 (2 x -C(O)O-), 175.8 (-C(O)O-), 158.9 (-C(O)NH), 138.4 (-CBHs), 129.4 (-C6Hs), 128.9 (-CeHs), 128.7 (-CeHs), 67.3 (-CHZFh), 66.7 (_ NCHC(O)-), 55.3 (2 x -NCHZC(O)-), 41.5 (-NHCHZ ), 30.7 (-NHCHZCHa-), 30.5 (-NHCHZCHZCHZCHz-), 24.6 (-NHCHZCHZCHZ-) 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.z 0~ /~~
O ° ~i(Cfhis ~ 0 O' HN N~ ~Si(CFi~s O
~Si(~s v CssHed'~~OeSis X7.10 696.366752 C 56.9% H B.7% N 4.0% O 18.4% SI 121~A
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 (Rule 2fi) RO/AU
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 flesh 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) 'H NMR (200 MHz, CDCI,) : E 7.34 (5H, m, -C6H5), 5.08 (2H, s, -CHZPh), 4.91 (1H, br t, -NH), 4.16 (6H, m, 3 x -OCHZ-), 3.60 (4H, s, 2 x -NCH2C(O)-), 3.39 (1H, t, -NCHC(O)-, 3JH-H = 7.5 Hz), 3.19 (2H, m, -NHCHZ ), 1.3-1.8 (6H, overlapping multiplets, -NHCHZCHZCHZCHz ), 0.93-1.04 (6H, overlapping multiplets, 3 x -CHZSi-), 0.04 (9H, s, 1 x -Si(CH3)3), 0.03 (18H, s, 2 x -Si(CH3)3) ppm.
'3C NMR (100 MHz, CDCl3) : 8 173.6 (-C(O)O-), 172.2 (2 x -C(O)O-), 157.1 (-OC(O)NH), 137.5 (-CsHS), 129.2 (-CsHs), 128.7 (-CBHS), 67.2 (-CHZPh), 65.4 (-NCHC(O)-}, 63.5 (1 x -OCHZ ), 63.3 (2 x -OCHZ-), 53.6 (2 x -NCHZC(O)-), 41.6 (-NHCHZ-), 30.8 (-NHCHZCHZ-), 30.1 (-NHCHzCHZCH2CH2-), 23.8 (-NHCH2CHZCHZ-), 18.3 (-CHZSi-), 18.1 (2 x -CHZSi-), -0.8 (3 x -Si(CH3)3) ppm.
Electrospray M/S : m/z 719.4 (100%) (M+Na+)
4. Synthesis of LysNTA.TMSE3 o~si(C~)3 HEN N~ ~Si(CH3)3 ~O
~Si(CH~)~
O
CuHs,,Nz06Si3 562.97 562.328973 C 53.3% H 9.7% N S.0% O 17.1% Si 15.0%
Substitute Sheet (Rule 26) RO/E1U
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 HZ 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.TMSE3 as a colourless oil (0.127 g, quantitative yield).
Data 'H NMR (200 MHz, CDCl3) : 8 4.16 (6H, m, 3 x -OCHz-). 3.61 (4H, s. 2 x -NCHZC(O}-), 3.40 (1H, apparent triplet, -NCHC(O)-, ~JH_H = 7.5 Hz), 2.69 (2H, br t, NHZCHZ-, 'JH-H =
6.1 Hz), 1.3-1.8 (6H, overlapping multiplets, NHZCHZCHzCH2CHz ), 0.93-1.04 (6H, overlapping multiplets, 3 x -CHZSi-), 0.04 (9H, s, 1 x -Si(CH3)3), 0.03 (18H, s, Z x -Si(CH3),) PPm.
"C NMR (100 MHz, CDCl3) : 8 173.7 (-C(O)O-), 172.2 (2 x -C(O)O-), 65.6 (-NCHC(O}-), 63.5 (2 x -OCHZ ), 63.3 (1 x -OCH2 ), 53.6 (2 x -NCHZC(O)-), 42.7 (NHZCHZ ), 33.9 (NHZCHZCHZ-), 31.0 (NHaCHZCHZCHZCHi ), 23.9 (NHZCHZCHZCHZ-), 18.3 (-CHZSi-), 18.1 (2 x -CHZSi-), -0.8 (3 x -Si(CH,)3) ppm.
Electrospray M/S : m/z 563.3 (200%) (M+H+) Substitute Sheet (Rule ~) RO/AU
~Si(CH~)~
O
CuHs,,Nz06Si3 562.97 562.328973 C 53.3% H 9.7% N S.0% O 17.1% Si 15.0%
Substitute Sheet (Rule 26) RO/E1U
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 HZ 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.TMSE3 as a colourless oil (0.127 g, quantitative yield).
Data 'H NMR (200 MHz, CDCl3) : 8 4.16 (6H, m, 3 x -OCHz-). 3.61 (4H, s. 2 x -NCHZC(O}-), 3.40 (1H, apparent triplet, -NCHC(O)-, ~JH_H = 7.5 Hz), 2.69 (2H, br t, NHZCHZ-, 'JH-H =
6.1 Hz), 1.3-1.8 (6H, overlapping multiplets, NHZCHZCHzCH2CHz ), 0.93-1.04 (6H, overlapping multiplets, 3 x -CHZSi-), 0.04 (9H, s, 1 x -Si(CH3)3), 0.03 (18H, s, Z x -Si(CH3),) PPm.
"C NMR (100 MHz, CDCl3) : 8 173.7 (-C(O)O-), 172.2 (2 x -C(O)O-), 65.6 (-NCHC(O}-), 63.5 (2 x -OCHZ ), 63.3 (1 x -OCH2 ), 53.6 (2 x -NCHZC(O)-), 42.7 (NHZCHZ ), 33.9 (NHZCHZCHZ-), 31.0 (NHaCHZCHZCHZCHi ), 23.9 (NHZCHZCHZCHZ-), 18.3 (-CHZSi-), 18.1 (2 x -CHZSi-), -0.8 (3 x -Si(CH,)3) ppm.
Electrospray M/S : m/z 563.3 (200%) (M+H+) Substitute Sheet (Rule ~) RO/AU
5. Synthesis of Ztn'NTA.TMSE9 ~~~saa~,b ~SNa4b CmHieot~s~s~9 2031.26 2029.108491 C55.0%H8.9%N5.5%018.1%Si12.4%
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 dichloromothane) 'H NMR (400 MHz. CDC13) : b 7.37 (2H, br t, NH). 7.34 (5H, m, -CBHS), 7.26 (1H, br t, NH), 5.15 (1H, br t, NH), 5.08 (2H, s, -CHZPh), 4.17 (18H, m, 9 x -OCHZ ), 3.60 (12H, two overlapping singlets in 2:2 ratio, 6 x -NCHZC(O)O-), 3.39-3.34 (5H, overlapping Substitute Sheet (Rule 2fi) ROIAU
multiplets), 3.26-3.16 (10H, overlapping multiplets), 3.07 (1H, triplet), 1.85-1.30 (24H, overlapping multiplets, 12 x -CHZ), 0.97 (18H, overlapping multiplets, 9 x -CHZSi-), 0.05 (27H, s, 3 x -Si(CH3)3), 0.04 (54H, s, 6 x -Si(CH3)3) ppm. -OC(O)NHCHZ
'3C NMR (100 MHz. CDCl3) : b 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-). 172.2 (2 x -C(O)O-), 157.2 (-OC(O)NH), 137.4 (-CeHs).
129.1 (-C6H5), 128.6 (-C6H5), 67.1 (-CHZPh). 66.4 (-NCHC(O)NH-), 65.5 (-NCHC(O)O-).
65.4 (2 x -NCHC(O)O-), 63.4 (6 x -OCHZCHZ-), 63.3 (3 x -OCHZCHZ ), 56.9 (Z x -NCHaC(O)NH-), 53.7 (4 x -NCHZC(O)O-), 53.4 (2 x -NCHZC(O)O-), 41.2 (-OC(O)NHCHZ ), 39.9 (3 x -C(O)NCHZ-), 30.7, 30.5, 30.2 (4 x -NHCHZCHZ ), 29.9, 29.5, 28.9 (4 x NHCHZCHZCHZCHZ ). 24.7, Z4.0, 23.8 (4 x -NHCH2CHZCH2-), 18.3 (3 x -CHZSi(CH3)3).
18.0 (6 x -CHZSi(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%)
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 dichloromothane) 'H NMR (400 MHz. CDC13) : b 7.37 (2H, br t, NH). 7.34 (5H, m, -CBHS), 7.26 (1H, br t, NH), 5.15 (1H, br t, NH), 5.08 (2H, s, -CHZPh), 4.17 (18H, m, 9 x -OCHZ ), 3.60 (12H, two overlapping singlets in 2:2 ratio, 6 x -NCHZC(O)O-), 3.39-3.34 (5H, overlapping Substitute Sheet (Rule 2fi) ROIAU
multiplets), 3.26-3.16 (10H, overlapping multiplets), 3.07 (1H, triplet), 1.85-1.30 (24H, overlapping multiplets, 12 x -CHZ), 0.97 (18H, overlapping multiplets, 9 x -CHZSi-), 0.05 (27H, s, 3 x -Si(CH3)3), 0.04 (54H, s, 6 x -Si(CH3)3) ppm. -OC(O)NHCHZ
'3C NMR (100 MHz. CDCl3) : b 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-). 172.2 (2 x -C(O)O-), 157.2 (-OC(O)NH), 137.4 (-CeHs).
129.1 (-C6H5), 128.6 (-C6H5), 67.1 (-CHZPh). 66.4 (-NCHC(O)NH-), 65.5 (-NCHC(O)O-).
65.4 (2 x -NCHC(O)O-), 63.4 (6 x -OCHZCHZ-), 63.3 (3 x -OCHZCHZ ), 56.9 (Z x -NCHaC(O)NH-), 53.7 (4 x -NCHZC(O)O-), 53.4 (2 x -NCHZC(O)O-), 41.2 (-OC(O)NHCHZ ), 39.9 (3 x -C(O)NCHZ-), 30.7, 30.5, 30.2 (4 x -NHCHZCHZ ), 29.9, 29.5, 28.9 (4 x NHCHZCHZCHZCHZ ). 24.7, Z4.0, 23.8 (4 x -NHCH2CHZCH2-), 18.3 (3 x -CHZSi(CH3)3).
18.0 (6 x -CHZSi(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 Ztr~'NTA
ZtnNTA.TMSE9 (0.12 g, 0.06mmo1) 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% HZO/0.05%
TFA to 50% acetonitrile/0.05%TFA in H20/0.05% 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 (Rule ~) RO/AU
Data 'H NMR (400 MHz, DIO) : 8 7.38 (5H, m, -CeH~), 5.08 (2H, s, -CHIPh), 4.06-3.78 (20H, m, 8 x -NCHzC(O)- + 4 x -NCHC(O)-), 3.23 (6H, apparent triplet, 3 x -C(O)NHCHI-), 3.08 (2H. apparent triplet, 1 x -OC(O)NHCHI-), 2.00-1.76 (8H, overlapping multiplets, 4 x -CHI), 1.60-1.40 (14H, overlapping multiplets, 7 x -CHI), 1.38-1.22 (2H, multiplet, 1 x -CHI) PPm.
"C NMR (100 MHz, DIO): 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%).
ZtnNTA.TMSE9 (0.12 g, 0.06mmo1) 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% HZO/0.05%
TFA to 50% acetonitrile/0.05%TFA in H20/0.05% 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 (Rule ~) RO/AU
Data 'H NMR (400 MHz, DIO) : 8 7.38 (5H, m, -CeH~), 5.08 (2H, s, -CHIPh), 4.06-3.78 (20H, m, 8 x -NCHzC(O)- + 4 x -NCHC(O)-), 3.23 (6H, apparent triplet, 3 x -C(O)NHCHI-), 3.08 (2H. apparent triplet, 1 x -OC(O)NHCHI-), 2.00-1.76 (8H, overlapping multiplets, 4 x -CHI), 1.60-1.40 (14H, overlapping multiplets, 7 x -CHI), 1.38-1.22 (2H, multiplet, 1 x -CHI) PPm.
"C NMR (100 MHz, DIO): 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 trJ NTA TMSE9 ° ,~ /p~sHeN~t, N 'P
O KtHab o ~-~"°'~
H ~ ~ ~N_ X ~ 31(CN,h N7Pi!~~'~~H~ V ~
O ~~i~ ~~N~
O~O
0' ~~~
G,NmN~ns4 N ~alrh Es~etMrr 1895.07 °
Mo4 Wt.: Id97.10 O
C. SLII: N. 9.IJ: N.5.91: D.17.~1: $i. t1.72 \
Z-tri NTA TMSE9 (38 mg, 0.0187 mmol) was dissolved in methanol (10 ml) end 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 (Rule 26) RO/AU
O KtHab o ~-~"°'~
H ~ ~ ~N_ X ~ 31(CN,h N7Pi!~~'~~H~ V ~
O ~~i~ ~~N~
O~O
0' ~~~
G,NmN~ns4 N ~alrh Es~etMrr 1895.07 °
Mo4 Wt.: Id97.10 O
C. SLII: N. 9.IJ: N.5.91: D.17.~1: $i. t1.72 \
Z-tri NTA TMSE9 (38 mg, 0.0187 mmol) was dissolved in methanol (10 ml) end 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 (Rule 26) RO/AU
8 PCT/AU00/00075 Data 'H n.m.r. (CDCl,) b 0.00 (81 H, m, Si(CH3)3), 0.96 (18H, m, CHZSi), 1.2-2.0 (24H, m), 3.1-3.7 (28H, m). 4.15 (18H, m. OCHZ) and 7.4 (2H, NHZ).
8. Synthesis of trf NTA TMSE9 hemisuccinamide A solution of tri-NTA.TMSEg (0.5g), benzyl hemisuccinate (7lmg), 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 CHZC12 MeOH
(96:4) to separate 537mg of the benzyl ester intermediate. 500mg of this material was dissolved in methanol (60m1) 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. (CDCl3) 8 0.04 (81 H, m, Si(CH3)3), 0.96 (18H, m, CHzSi), 1.2-Z.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%), (M+Na'), 1997.8 (100%) (M+).
Substitute Sheet (Rule 26) RO/AU
°
°
~sHtulJ~Or$4 ~ ~O
Eaaat Mw. 1993.09 N~~Eilply Mel. Wt.: 199?.1'1 C. 37.31; H.1.9~ N.3.61: O. 19.27: f4 12.66 ~ O
O' _ O ~31(Glrh
8. Synthesis of trf NTA TMSE9 hemisuccinamide A solution of tri-NTA.TMSEg (0.5g), benzyl hemisuccinate (7lmg), 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 CHZC12 MeOH
(96:4) to separate 537mg of the benzyl ester intermediate. 500mg of this material was dissolved in methanol (60m1) 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. (CDCl3) 8 0.04 (81 H, m, Si(CH3)3), 0.96 (18H, m, CHzSi), 1.2-Z.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%), (M+Na'), 1997.8 (100%) (M+).
Substitute Sheet (Rule 26) RO/AU
°
°
~sHtulJ~Or$4 ~ ~O
Eaaat Mw. 1993.09 N~~Eilply Mel. Wt.: 199?.1'1 C. 37.31; H.1.9~ N.3.61: O. 19.27: f4 12.66 ~ O
O' _ O ~31(Glrh
9. Synthesis of tri NTA
0 off OH
N O
~OH
O O
~~-OH
O~ HN ~ O
HiN~N~N N v -OH
O' _OH
Cao~~n sa.cealau:994.a3 ~t~l. m: ~a.~
C, ag.28; H, 6.69: N. I i .26: O, 33.77 tri NTA TMSEg (34 mg,0.0179 mmol) was triturated with toluene, evaporated and dried under high vacuum. Triffuoroacetic 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. (CD30D) 8 1.2-2.0 (24H, m), 3.07 (6H, m, CH NHCO) and 3.1-3.7 (22H, m).
0 off OH
N O
~OH
O O
~~-OH
O~ HN ~ O
HiN~N~N N v -OH
O' _OH
Cao~~n sa.cealau:994.a3 ~t~l. m: ~a.~
C, ag.28; H, 6.69: N. I i .26: O, 33.77 tri NTA TMSEg (34 mg,0.0179 mmol) was triturated with toluene, evaporated and dried under high vacuum. Triffuoroacetic 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. (CD30D) 8 1.2-2.0 (24H, m), 3.07 (6H, m, CH 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%).
Substitute Sheet (Rule ~&) RO/AU
Data 'H n.m.r. (CD30D) b 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CHZCO), 2.9-3.4 (10H, m), 3.90 (2H, dd, CHI-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).
Substitute Sheet (Rule ~&) RO/AU
Data 'H n.m.r. (CD30D) b 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CHZCO), 2.9-3.4 (10H, m), 3.90 (2H, dd, CHI-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 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 'H n.m.r. (CD30D) S 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CHZCO), 2.82 (4H, s, NHS), 2.9-3.4 (10H, m), 3.90 (2H, dd, CHZ-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).
Data 'H n.m.r. (CD30D) S 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CHZCO), 2.82 (4H, s, NHS), 2.9-3.4 (10H, m), 3.90 (2H, dd, CHZ-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).
12. Preparation of Gramicidin succinate iri NTA
O OH
OOH
N~ O
OOH
O
HN ~OH
~~HN~N~(N~ Nf~OH
O ~p O-"OH
HN
~OH
O
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 (Rule 2fi) RO/E1U
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. (CD30D) 8 0.4-1.8 (90H, m), 2.0-2.2 (4H, m), 2.52 (4H. m. CHZCO), 3.0-3.8 (38H. m), 3.90 (2H, dd, CH2-gly), 4.0-4.8 (lsH, m), s.s-7.s (zOH, m) and s.la (iH, s, cHO).
MALDI m.s. 2963.4( (M+Na+)-HZO), 2982.9 (M+Na''), 2998 (M+K+)
O OH
OOH
N~ O
OOH
O
HN ~OH
~~HN~N~(N~ Nf~OH
O ~p O-"OH
HN
~OH
O
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 (Rule 2fi) RO/E1U
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. (CD30D) 8 0.4-1.8 (90H, m), 2.0-2.2 (4H, m), 2.52 (4H. m. CHZCO), 3.0-3.8 (38H. m), 3.90 (2H, dd, CH2-gly), 4.0-4.8 (lsH, m), s.s-7.s (zOH, m) and s.la (iH, s, cHO).
MALDI m.s. 2963.4( (M+Na+)-HZO), 2982.9 (M+Na''), 2998 (M+K+)
13. Preparation of gA lysine-XXBOC
OH
'I .BOC
g~H~~H~ ~H
O
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. (CD30D) b 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, CHZ gly), 4.0-4.8 (15H, m). 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).
substitute Sheet (Rule ~) RO/AU
OH
'I .BOC
g~H~~H~ ~H
O
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. (CD30D) b 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, CHZ gly), 4.0-4.8 (15H, m). 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).
substitute Sheet (Rule ~) RO/AU
14. Preparation of gAlysine XXBOC hemisuccinate OH
O
~~ N ~~ ~OH
H O H _ [~O
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. (CD30D) 8 0.4-1.8 (84H, m), 2.0-2.2 (8H, m), 2.58 (4H, dd, CHZCO), 2.9-3.4 (14H, m), 3.90 (2H, dd, CHi gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).
O
~~ N ~~ ~OH
H O H _ [~O
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. (CD30D) 8 0.4-1.8 (84H, m), 2.0-2.2 (8H, m), 2.58 (4H, dd, CHZCO), 2.9-3.4 (14H, m), 3.90 (2H, dd, CHi gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).
15. Synthesis of gAlysine2Xtr1 NTA
O OH
H
N~ O
OOH
O
OH O HN ~~H
6ANH N~N~N N N'~(N~Nf '~OH
H O H O ~p O~3.OH
HN O
~OH
N ~OH
~ O
~OH
O
Gramicidin lysine 2X succinate (8.5 mg, 0.037mmo1), N-hydroxy succinemide 4.2 mg, 0.0364mmol) and 4-dimethylamino pyridine (1 mg, 0.0081mmo1) were stirred in distilled tetrahydrofuran (5 ml) and dicyclohexylcarbadiimide (7.5 mg, Substitute Sheet (R.ule 26) RO/AU
0.0363mmo1) 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. (CD30D) b 0.4-1.8 (108H, m), 2.0-2.2 (8H, m), 2.62 (4H, dd, CH2C0), 3.0-3.8 (42H, m), 3.90 (ZH, dd, CHZ-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.OZ (M+K;).
O OH
H
N~ O
OOH
O
OH O HN ~~H
6ANH N~N~N N N'~(N~Nf '~OH
H O H O ~p O~3.OH
HN O
~OH
N ~OH
~ O
~OH
O
Gramicidin lysine 2X succinate (8.5 mg, 0.037mmo1), N-hydroxy succinemide 4.2 mg, 0.0364mmol) and 4-dimethylamino pyridine (1 mg, 0.0081mmo1) were stirred in distilled tetrahydrofuran (5 ml) and dicyclohexylcarbadiimide (7.5 mg, Substitute Sheet (R.ule 26) RO/AU
0.0363mmo1) 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. (CD30D) b 0.4-1.8 (108H, m), 2.0-2.2 (8H, m), 2.62 (4H, dd, CH2C0), 3.0-3.8 (42H, m), 3.90 (ZH, dd, CHZ-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.OZ (M+K;).
16. Synthesis of tttNTA.TMSEs aro~~b CesHmNa~a s~s 1897.12 1895.071711 C 53.8% H 9.2°.6 N 5.9°.6 O 17.7% Si 13.3°.6 A solution of ZtriNTA.TMSEg (0.1 g, 50 umol) was dissolved in methanol (10 ml). Palladium on carbon (10%) was added (ca. 10 mg) and a balloon containing HZ
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 (Rule ~6) RO/AU
ZO
removed from the reaction micxture under reduced pressure to afford trisNTA.TMSE9 as a colourless oil (0.09 g, quantitative).
Data Electrospray 1~1/S : m/z 1898.3 (100%) (M+H;)
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 (Rule ~6) RO/AU
ZO
removed from the reaction micxture under reduced pressure to afford trisNTA.TMSE9 as a colourless oil (0.09 g, quantitative).
Data Electrospray 1~1/S : m/z 1898.3 (100%) (M+H;)
17. Synthesis of 8iotin.t~iNTA.TMSE9 ~si(Cfbh si(ci-bh ~si(aih ~si(Cl~,h 'o ~si(cfbh u(a~,h o/
i(CHsh ~Si(Chhh C95H188N10023SSIg 2123.42 2121.149311 C 53.7% H 8.9% N 6.6% O 17.3% S 1.5% SI 11.9%
Biotin (20 mg, 0.08 mmol), 4-dimethylaminopyridine (12 mg, 0.1 mmol) and trihlTA.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 2 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 ~) RO/AU
Data Electrospray M/S : m/z 1173.9 (100%), 2146.4 (25%) (M+Na+).
i(CHsh ~Si(Chhh C95H188N10023SSIg 2123.42 2121.149311 C 53.7% H 8.9% N 6.6% O 17.3% S 1.5% SI 11.9%
Biotin (20 mg, 0.08 mmol), 4-dimethylaminopyridine (12 mg, 0.1 mmol) and trihlTA.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 2 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 ~) RO/AU
Data Electrospray M/S : m/z 1173.9 (100%), 2146.4 (25%) (M+Na+).
18. Synthesis of Biotin.triNTA
off off ~o a~
\\o C5°Ha°N~°02~S
1221.29 1220.511851 C49.2%H6.6YeN11.5%030.tYeS2.6%
Trifluoroacetic acid (2 ml) was added to Biotin.triNTA.TMSEg (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 = 24.2 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 2221.5 (100%) (M+H''), 1243.4 (33%) (M+Na+) Substitute Sheet (R,ule 26) RO/AL:
off off ~o a~
\\o C5°Ha°N~°02~S
1221.29 1220.511851 C49.2%H6.6YeN11.5%030.tYeS2.6%
Trifluoroacetic acid (2 ml) was added to Biotin.triNTA.TMSEg (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 = 24.2 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 2221.5 (100%) (M+H''), 1243.4 (33%) (M+Na+) Substitute Sheet (R,ule 26) RO/AL:
19. Synthesis of Lipid-triNTA
OH ~OH
O /- 'O
N OH
HN y O
O
OH
O
C~H~27NgOy3 1488.84 1485.904482 c58.2%H8.6%N8.5%024.7%
A solution of ditetradecylamine (100mg) in CHzCl2 (5m1} was added to a solution of succinic anhydride (120mg) and triethylamine (40mg) in CHZCIZ
(5m1) at room temperature. The reaction was stirred overnight, the solvents removed in vacuo and the residue chromatographed using CHZCIa-MeOH (95:5 4 85:5) to give 60mg of ditetradecylamine hemisuccinamide. This material was added to a solution of tn'NTA.TMSE9 in CHzCIz (10m1) followed by DMAP (l4mg). 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.
l0mg of this material was dissolved in TFA (1m1) and stirred under nitrogen for 3h.
The solvent was removed in vacuo and the solid remaining washed with water (1m1) 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 ,26) RO/AU
OH ~OH
O /- 'O
N OH
HN y O
O
OH
O
C~H~27NgOy3 1488.84 1485.904482 c58.2%H8.6%N8.5%024.7%
A solution of ditetradecylamine (100mg) in CHzCl2 (5m1} was added to a solution of succinic anhydride (120mg) and triethylamine (40mg) in CHZCIZ
(5m1) at room temperature. The reaction was stirred overnight, the solvents removed in vacuo and the residue chromatographed using CHZCIa-MeOH (95:5 4 85:5) to give 60mg of ditetradecylamine hemisuccinamide. This material was added to a solution of tn'NTA.TMSE9 in CHzCIz (10m1) followed by DMAP (l4mg). 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.
l0mg of this material was dissolved in TFA (1m1) and stirred under nitrogen for 3h.
The solvent was removed in vacuo and the solid remaining washed with water (1m1) 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 ,26) RO/AU
20. Synthesis of Membrane-spanning lipid phosphatyl choline (MSL-PC) NMe3 Ct24~"~22oN~24PS2 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-OH' (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 choline 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% 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. 8(CDC13:CD30D (3:1)) 0.82-0.92 (m, 30H, phytanyl CH3's), 0.92-1.80, m, 100H, phytanyl CHz's and CH's), 2.61 (t, 2H, CHZCHZS), 2.65 (s, 8H, succinate H's), 3.20 (s, 9H, -N(CH )3), 3.35-3.74 (m, 44H, -OCI-iZ, -OCH, CHZN), 3.85 (s, 2H, SCHZPh), 3.92 (t, 2H, CH OP), 4.10 (m, 2H, POCH~CHZ), 4.20 (m, 8H, ester H's), 6.90 & 7.43 (d, 8H, biphenyl), 7.30 (s, 5H, PhCHz).
Substitute Sheet (Bole 26) RO/AU
Electrospray MS m/z: 2206 (M+2), 1112, 587, 488
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. 8(CDC13:CD30D (3:1)) 0.82-0.92 (m, 30H, phytanyl CH3's), 0.92-1.80, m, 100H, phytanyl CHz's and CH's), 2.61 (t, 2H, CHZCHZS), 2.65 (s, 8H, succinate H's), 3.20 (s, 9H, -N(CH )3), 3.35-3.74 (m, 44H, -OCI-iZ, -OCH, CHZN), 3.85 (s, 2H, SCHZPh), 3.92 (t, 2H, CH OP), 4.10 (m, 2H, POCH~CHZ), 4.20 (m, 8H, ester H's), 6.90 & 7.43 (d, 8H, biphenyl), 7.30 (s, 5H, PhCHz).
Substitute Sheet (Bole 26) RO/AU
Electrospray MS m/z: 2206 (M+2), 1112, 587, 488
21. Synthesis of bis(N-methyl)-C38 diamine M~HN-CyoH~eeNZOa Exact Mass: 1373.29 Mol. Wt.: 1374.31 C, 78.66; H, 12.32: N, 2.04; O, 8.99 To the bola-amphiphile C38-diol' (300mg) in THF (10m1) cooled to 0° was added triethylamine (921) and methanesulphonyl chloride (511). The solution was stirred overnight at room temperature and diluted with ether (30m1). The organic phase was washed with saturated NaHC03 (2 X 30mL) and water (2 X 60m1), dried (NaZSO,) and concentrated in vacuo. The materiel obtained (315mg) was added to a pressure tube, cooled to -80°, and chilled methylamine (12m1) 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 CHZCIz and stirred with KZC03. The solid was removed by filtration and the filtrate dried in vacuo. The residue was chromatographed using CHZCl2-MeOH-NH3 (96:4:0.1 a 92:8:0.1) to separate 242 mg pure product.
Data 1H n.m.r. (CDCI3) 8 0.80-0.95 (30H, m, Phvtanyl 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-O and CHZ-O); 3.98 (4H, t. CH OAr); 6.93, 7.43 (8H, AA'XX' multiplets, atom.-H
After standing at room temperature overnight excess methylamine was allowed to evaporate, and the residue dissolved in CHZCIz and stirred with KZC03. The solid was removed by filtration and the filtrate dried in vacuo. The residue was chromatographed using CHZCl2-MeOH-NH3 (96:4:0.1 a 92:8:0.1) to separate 242 mg pure product.
Data 1H n.m.r. (CDCI3) 8 0.80-0.95 (30H, m, Phvtanyl 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-O and CHZ-O); 3.98 (4H, t. CH OAr); 6.93, 7.43 (8H, AA'XX' multiplets, atom.-H
22. Synthesis of XXFmoc / O~N N OH
H
O O
C~~Hs.~N20s Exact Mass: 466.25 Mol. Wt.: 466.57 C, 69.50; H, 7.35; N, 6.00; O, 17.15 substitute Sheet (Rule 2fi) RO/AU
2X-Hoc (420mg) was treated with TFA (5mL) for 20 rains 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 DI~IF(3mL) was added into the above stirring solution at 0°C and stirred at RT for 30 rains. Water (100mL) 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).
Date NMR (CDC13) 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*)
H
O O
C~~Hs.~N20s Exact Mass: 466.25 Mol. Wt.: 466.57 C, 69.50; H, 7.35; N, 6.00; O, 17.15 substitute Sheet (Rule 2fi) RO/AU
2X-Hoc (420mg) was treated with TFA (5mL) for 20 rains 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 DI~IF(3mL) was added into the above stirring solution at 0°C and stirred at RT for 30 rains. Water (100mL) 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).
Date NMR (CDC13) 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 (NCH3)2 mono BOC
C9sHnsNna Fxret Afasa: LtT3.34 hl°4 ~Vt.: ld7d.42 C, 77.39; I-I, 12.03; N, 1.90; O, 8.G9 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 She:
(Rule 26) ROIAU
Data NMR (CDCl,) 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 (h4+, 100%)
C9sHnsNna Fxret Afasa: LtT3.34 hl°4 ~Vt.: ld7d.42 C, 77.39; I-I, 12.03; N, 1.90; O, 8.G9 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 She:
(Rule 26) ROIAU
Data NMR (CDCl,) 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 (h4+, 100%)
24. Synthesis of i3oc-C38-2X-Fmoc o ~N
O~HN
N
H O
C~:xHnrW Oi:
E~ut Mass: 1921.58 Mol. Wt.: 1922.98 C. 76.20: H. 10.90: N, 2.91: O. 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 (S~o methanol in dichloromethane) to give pure product (ZOOmg).
Data Nmr (CDCl3) 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 (Rule 2G) RO/AU
O~HN
N
H O
C~:xHnrW Oi:
E~ut Mass: 1921.58 Mol. Wt.: 1922.98 C. 76.20: H. 10.90: N, 2.91: O. 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 (S~o methanol in dichloromethane) to give pure product (ZOOmg).
Data Nmr (CDCl3) 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 (Rule 2G) RO/AU
25. Synthesis of MSL-tri-NTA
o H~ .--.
N N
H MHO O NH Ct~9H3oeNu0usz O Exact Mass: 3394.17 HO~ Mot. lNt.: 3396.55 C, B3.30; H, 9.14; N, 4.95; O, 20.73; S. 1.89 HON
~~O
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.' 'I
The NHS ester of this material was prepared by reaction of the acid (50mg) with NHS
(8mg) in the presence of DCC (l5mg) and DMAP (2.6mg) in CHZC12 (3m1) for 4h and purified by passage through a Sephadex LH-20 column eluting with methanol.
Substitute Sheet ~,ule 26) R,0/AU
This active ester was reacted in methanol with the fully deprotected C38diamine-trllVTA 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-CHZCI2with 0.05% TFA).
o H~ .--.
N N
H MHO O NH Ct~9H3oeNu0usz O Exact Mass: 3394.17 HO~ Mot. lNt.: 3396.55 C, B3.30; H, 9.14; N, 4.95; O, 20.73; S. 1.89 HON
~~O
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.' 'I
The NHS ester of this material was prepared by reaction of the acid (50mg) with NHS
(8mg) in the presence of DCC (l5mg) and DMAP (2.6mg) in CHZC12 (3m1) for 4h and purified by passage through a Sephadex LH-20 column eluting with methanol.
Substitute Sheet ~,ule 26) R,0/AU
This active ester was reacted in methanol with the fully deprotected C38diamine-trllVTA 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-CHZCI2with 0.05% TFA).
26. Kinetic analysis of 6His-tagged Frotein 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 ~L) containing 1.2 ~M
MSLtriNTA, 24 N,M 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 /CL across the surface and air dried in the laminar flow hood. The chip was then re-sheathed and sealed with plastic film (Parafilm).
This J 1.triNTA chip was docked into a Bl:Acore 2000 machine and the pumps flushed and experiments performed using HBS/EDTA running buffer (50 mM HEPES, 300 mM NaCh ~0 ~tM
EDTA, pH 8.0) at a flow race of 40 ~I/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 ~M NiCIZ was injected through Fc2 only, which was then washed with running buffer for ~
min. The protein of interest ( 100-600 nM) was then injected (using KINJECT function) through Fc 1 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 NiCl2 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. I, 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 Ni2+ and proteins without the 6His tag also did not bind triNTA.
Substitute Sheet (Rule 26) RO/AU
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 ~L) containing 1.2 ~M
MSLtriNTA, 24 N,M 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 /CL across the surface and air dried in the laminar flow hood. The chip was then re-sheathed and sealed with plastic film (Parafilm).
This J 1.triNTA chip was docked into a Bl:Acore 2000 machine and the pumps flushed and experiments performed using HBS/EDTA running buffer (50 mM HEPES, 300 mM NaCh ~0 ~tM
EDTA, pH 8.0) at a flow race of 40 ~I/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 ~M NiCIZ was injected through Fc2 only, which was then washed with running buffer for ~
min. The protein of interest ( 100-600 nM) was then injected (using KINJECT function) through Fc 1 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 NiCl2 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. I, 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 Ni2+ and proteins without the 6His tag also did not bind triNTA.
Substitute Sheet (Rule 26) RO/AU
27. References 1. Schmitt, L.; Dietrich, C.; Tampe R. JACS, 1994, 116, 8485.
2. Geo. C.; Lin C.-H.; Lo, C.-H.L.; Mao. S.; Wirsching, P.: Lerner, R.A.;
Janda, K.D.
PNlIS, 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.
1. Sigel, H., Angew. Chem. Int. Ed. Engl., 1975, 14, 394.
2. Martin, R.P., Petit-Ramel, hi.M., Scharff, J.P., in "Metal Ions in Biological Systems" 1976 vol 2 p 1 (Iviarcel Dekker) 3. Burns, C.j., Field, L.D., Hambley, T., Lin, T., Ridley, D.D., Turner, P., Wilkinson,1~LP., manuscript in preparation;
Bocarsly, J.R., Chiang, IvLY., 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, L, 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., Stiiber, 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.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.
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., Pliickthun, A., Anal. Biochem., 1997, 252, 217.
Substitute Sheet (Rule 26) RO/AU
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., Tampe, R., J. Am. Chem. Soc., 1994, 116, 8485.
22. blaloney, 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 (Rule 2fi) RO/~1U
2. Geo. C.; Lin C.-H.; Lo, C.-H.L.; Mao. S.; Wirsching, P.: Lerner, R.A.;
Janda, K.D.
PNlIS, 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.
1. Sigel, H., Angew. Chem. Int. Ed. Engl., 1975, 14, 394.
2. Martin, R.P., Petit-Ramel, hi.M., Scharff, J.P., in "Metal Ions in Biological Systems" 1976 vol 2 p 1 (Iviarcel Dekker) 3. Burns, C.j., Field, L.D., Hambley, T., Lin, T., Ridley, D.D., Turner, P., Wilkinson,1~LP., manuscript in preparation;
Bocarsly, J.R., Chiang, IvLY., 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, L, 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., Stiiber, 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.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.
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., Pliickthun, A., Anal. Biochem., 1997, 252, 217.
Substitute Sheet (Rule 26) RO/AU
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., Tampe, R., J. Am. Chem. Soc., 1994, 116, 8485.
22. blaloney, 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 (Rule 2fi) RO/~1U
Claims (28)
1. A compound having the general Formula 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.
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.
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 covalently attached directly, or indirectly through an optional linking group, to Z.
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 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.
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
in which Y, Z and n are as defined above and X is a spacer moiety.
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.
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.
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, sugars, dendrons and .alpha.,.beta.-unsaturated ketones.
21. A compound according to any one of claims 13 to 20 of Formula 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.
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
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.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPP8563 | 1999-02-08 | ||
| AUPP8563A AUPP856399A0 (en) | 1999-02-08 | 1999-02-08 | Improved compounds for protein binding |
| PCT/AU2000/000075 WO2000047548A1 (en) | 1999-02-08 | 2000-02-08 | Improved compounds for protein binding |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2341348A1 true CA2341348A1 (en) | 2000-08-17 |
Family
ID=3812765
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002341348A Abandoned CA2341348A1 (en) | 1999-02-08 | 2000-02-08 | Improved compounds for protein binding |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1150942A4 (en) |
| JP (1) | JP2002536428A (en) |
| CN (1) | CN1345301A (en) |
| AU (1) | AUPP856399A0 (en) |
| CA (1) | CA2341348A1 (en) |
| WO (1) | WO2000047548A1 (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1506402A2 (en) * | 2002-03-28 | 2005-02-16 | Rutgers, The State University of New Jersey | Bis-transition-metal-chelate-probes |
| US6919333B2 (en) * | 2002-11-12 | 2005-07-19 | Rutgers, The State University Of New Jersey | Bis-transition-metal-chelate probes |
| DE10227599B4 (en) * | 2002-06-20 | 2005-04-28 | Proteome Factory Ag | Method and reagent for the specific identification and quantification of one or more proteins in a sample |
| AU2004254295B2 (en) * | 2003-07-08 | 2010-05-27 | Tacnia Pty Ltd | Improvements in sensor chips |
| AU2003903504A0 (en) | 2003-07-08 | 2003-07-24 | Johnson, Daniel | Improvements in sensor chips |
| JP4970035B2 (en) * | 2003-08-21 | 2012-07-04 | リポテック・プロプライエタリー・リミテッド | Targeting dendritic cells in vivo |
| US7371585B2 (en) | 2003-12-31 | 2008-05-13 | Genencor International, Inc. | Membranes incorporating recognition moieties |
| JP2008508333A (en) * | 2004-08-05 | 2008-03-21 | ヨハン ウォルフガング ゲーテ−ウニベルジテート フランクフルト アム マイン | Multivalent chelators for modification and organization of target molecules |
| DE102004038134B4 (en) * | 2004-08-05 | 2013-07-25 | Johann Wolfgang Goethe-Universität Frankfurt am Main | Multivalent chelators for modifying and organizing target molecules, methods for their preparation and their use |
| EP2015071A1 (en) | 2007-07-13 | 2009-01-14 | FUJIFILM Corporation | Carrier, process for producing same, bioreactor, and chip for surface plasmon resonance analysis |
| US8137733B2 (en) | 2007-11-22 | 2012-03-20 | Fujifilm Corporation | Process for producing a carrier |
| JP2010190891A (en) * | 2009-01-22 | 2010-09-02 | Fujifilm Corp | Carrier, method of manufacturing the same, and extraction operation instrument |
| DE102010008417A1 (en) * | 2010-02-18 | 2011-08-18 | Johann Wolfgang Goethe-Universität Frankfurt am Main, 60323 | High affinity multivalent chelator compounds (MCHs) and their use for the structural and functional analysis of target molecules |
| EP2841446A2 (en) * | 2012-04-26 | 2015-03-04 | IBA GmbH | Adapter molecule capable of reversibly equipping a fusion protein carrying an oligohistidine affinity tag with a further affinity tag and methods of using the same |
| US10557852B2 (en) * | 2014-04-29 | 2020-02-11 | Yeda Research And Development Co. Ltd. | Fluorescent molecular sensor for targeting changes in protein surfaces, and methods of use thereof |
| US11639929B2 (en) * | 2014-04-29 | 2023-05-02 | Yeda Research And Development Co. Ltd. | Universal histidine-tag binding compounds and methods of use thereof as fluorescent probes and sensors |
| CN110183672B (en) | 2019-05-31 | 2021-07-09 | 天津大学 | PETX polymer, preparation method and three-dimensional thorn-shaped sensor interface |
| CN113999279A (en) * | 2020-11-04 | 2022-02-01 | 中国药科大学 | Dumbbell type amphiphilic peptide dendrimer, synthesis and application of dendrimer as drug delivery system |
Family Cites Families (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3158635A (en) * | 1959-03-18 | 1964-11-24 | Stauffer Chemical Co | Bis-adduction products and methods of preparing same |
| US3700671A (en) * | 1967-05-15 | 1972-10-24 | Monacelli Walter J | Chelating compositions |
| GB8610551D0 (en) * | 1986-04-30 | 1986-06-04 | Hoffmann La Roche | Polypeptide & protein derivatives |
| US5342604A (en) * | 1988-10-31 | 1994-08-30 | The Dow Chemical Company | Complexes possessing ortho ligating functionality |
| SE462454B (en) * | 1988-11-10 | 1990-06-25 | Pharmacia Ab | METHOD FOR USE IN BIOSENSORS |
| US5096946A (en) * | 1989-08-18 | 1992-03-17 | Rainer Norman B | Polymer product for the selective absorption of dissolved ions |
| US5800802A (en) * | 1989-10-11 | 1998-09-01 | Subramanian; Ramaswamy | Chelator IDAC-2 |
| US5580527A (en) * | 1992-05-18 | 1996-12-03 | Moltech Corporation | Polymeric luminophores for sensing of oxygen |
| DE4430023A1 (en) * | 1994-08-24 | 1996-02-29 | Boehringer Mannheim Gmbh | Electrochemical sensor |
| DE4433980C2 (en) * | 1994-09-23 | 1996-08-22 | Boehringer Ingelheim Int | Process and biosensor hit for investigating the interaction of biomolecules by means of surface plasma resonance |
| US5620850A (en) * | 1994-09-26 | 1997-04-15 | President And Fellows Of Harvard College | Molecular recognition at surfaces derivatized with self-assembled monolayers |
| DE19505960A1 (en) * | 1995-02-21 | 1996-08-22 | Deutsches Krebsforsch | Conjugate for the individual dosage of drugs |
| ATE198079T1 (en) * | 1995-04-07 | 2000-12-15 | Mogens Havsteen Jakobsen | METHOD FOR PHOTOCHEMICAL IMMOBILIZATION OF LIGANDS USING QUINONES |
| DE19518421A1 (en) * | 1995-05-19 | 1996-11-21 | Basf Ag | Aspartic acid oligomers and derivs. with high chelating capacity |
| DE19607279A1 (en) * | 1996-02-27 | 1997-08-28 | Bayer Ag | Membrane biosensors supported by solid bodies |
| JP2000514799A (en) * | 1996-07-03 | 2000-11-07 | プレジデント・アンド・フェローズ・オブ・ハーバード・カレッジ | Techniques using oligonucleotide linkers and immobilized and linked oligonucleotides |
| GB9813776D0 (en) * | 1998-06-25 | 1998-08-26 | Smithkline Beecham Plc | Novel compounds |
-
1999
- 1999-02-08 AU AUPP8563A patent/AUPP856399A0/en not_active Abandoned
-
2000
- 2000-02-08 WO PCT/AU2000/000075 patent/WO2000047548A1/en not_active Application Discontinuation
- 2000-02-08 CA CA002341348A patent/CA2341348A1/en not_active Abandoned
- 2000-02-08 EP EP00904701A patent/EP1150942A4/en not_active Withdrawn
- 2000-02-08 JP JP2000598469A patent/JP2002536428A/en active Pending
- 2000-02-08 CN CN00803375A patent/CN1345301A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002536428A (en) | 2002-10-29 |
| WO2000047548A1 (en) | 2000-08-17 |
| CN1345301A (en) | 2002-04-17 |
| EP1150942A1 (en) | 2001-11-07 |
| AUPP856399A0 (en) | 1999-03-04 |
| EP1150942A4 (en) | 2003-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2341348A1 (en) | Improved compounds for protein binding | |
| JP5339310B2 (en) | Glycoligand complex and protein analysis method using the ligand complex | |
| JPH07505631A (en) | Novel amphetamine derivatives, protein and polypeptide amphetamine-derived conjugates and labels | |
| US7320867B2 (en) | Linker compound, ligand, and producing method thereof | |
| EP0232693A2 (en) | Conjugates of vinblastine and its derivatives, process for their preparation and pharmaceutical compositions containing them | |
| KR20210125484A (en) | Method for preparing drug-linker MC-MMAF used in antibody drug conjugate and intermediates thereof | |
| Brettreich et al. | Convergent Synthesis of 1→ 3 C-Branched Polyamide Dendrons | |
| CN113365969B (en) | Connector containing phthalic aldehyde and application thereof in preparation of antibody-drug conjugate | |
| AU2648500A (en) | Improved compounds for protein binding | |
| AU2004203380A1 (en) | Improved compounds for protein binding | |
| US7238339B2 (en) | Bifunctional chelating compounds containing hydroxamic acid residues | |
| JP3565350B2 (en) | Protecting or anchoring groups and their use | |
| WO2006070737A1 (en) | β-BENZYLOXYASPARTIC ACID DERIVATIVE HAVING TWO SUBSTITUENTS ON BENZENE RING | |
| Paprica et al. | Preparation of novel cyclosporin A derivatives | |
| WO1992012122A1 (en) | N-protected aminoalkylaldehydes | |
| US9968683B2 (en) | Method for conjugating molecules | |
| FR2817259A1 (en) | RADIOPHARMACEUTICAL METAL CHELATING COMPOUND, PROCESS FOR PRODUCTION THEREOF, AND DIAGNOSTIC KIT | |
| US5432288A (en) | Derivatives of ryanodine and dehydroryanodine | |
| JP2764560B2 (en) | Ruthenium tertiary polyamine complex capable of immobilizing nitrogen | |
| JP2001521905A (en) | Hypsin reagent for peptide synthesis | |
| WO2002040507A2 (en) | Novel compounds for uses in radioimmunoscintigraphy and/or radioimmunotherapy of cancers | |
| US5510500A (en) | Derivatives of ryanodine and dehydroryanodine | |
| US5679701A (en) | Derivatives of ryanodine and dehydroryanodine | |
| WO1992018499A1 (en) | Novel ester derivatives of ryanodine and dehydroryanodine | |
| CN100398554C (en) | Linker compound, ligand, and producing method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |