CN1345301A - Improved compounds for protein binding - Google Patents

Improved compounds for protein binding Download PDF

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CN1345301A
CN1345301A CN00803375A CN00803375A CN1345301A CN 1345301 A CN1345301 A CN 1345301A CN 00803375 A CN00803375 A CN 00803375A CN 00803375 A CN00803375 A CN 00803375A CN 1345301 A CN1345301 A CN 1345301A
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compound
group
arbitrary
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nta
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殷萍
克里斯托弗·约翰·伯恩斯
马修·彼得·威尔金森
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Australian Membrane and Biotechnology Research Institute Ltd
University of Sydney
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Australian Membrane and Biotechnology Research Institute Ltd
University of Sydney
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/12—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

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

The compound that is used for protein bound improvement
Invention field
The present invention relates to new binding compounds, particularly the protein binding compound.This new compound is for being useful especially with protein binding to the surface that comprises film.In preferred form of the present invention, provide the biosensor of introducing these protein binding compounds.The invention still further relates to the midbody compound of use in binding compounds of the present invention synthetic.
Background of invention
Known ternary metal title complex in the document (1), it can be described to two the discrete metal-chelating groups and the coordination of metal.Usually the metal in ternary complex is Co 2+, Ni 2+, Cu 2+And Zn 2+Typical metal-complexing group is complexon I (NTA), iminodiethanoic acid (IDA), catechol and the heterogeneous ring compound that contains aromatic nitrogen, as imidazoles.By comprising that electromotive force calculates the several different methods sign ternary complex of (2) and X-radiocrystallography (3).The average stability constant (Ka) of the simple ternary complex that these researchs record is 10 3-10 4M -1(4).
Reported in 1975 in the process of separation and protein purification and used metal-IDA title complex, this process is called as immobilization metal affinity chromatography (IMAC) (5).Derivative with IDA in this process is connected in solid carrier such as sepharose, then sepharose is packed in the pillar.Metal ion is joined IDA part (passing through pillar by the dilute solution that makes metal ion), add protein mixture then.Except with metal-IDA species interacting proteins, the elute protein mixture is removed all proteins.By adding imidazoles solution, passing through to add strong metal sequestrant such as EDTA or EGTA, perhaps this or these protein are eluted from pillar then by reducing pH to 4.5-5.3 (6).
Hochuli in 1987 and co-worker thereof adopt the Ni-NTA title complex to come protein purification (7), particularly the protein (8) of 6 hyte propylhomoserin marks of reorganization design.The protein of histidine mark and the avidity that has between the solid carrier of NTA are about 10 13M -1(9).
Reported the method (10) on the hole of adopting the NTA derivative protein of histidine mark to be connected to polystyrene micropore plate in 1996.
1997, a plurality of uses that are combined in the NTA of quartzy slide surface were described to protein is connected to the method (11) on surface.Reported in 1998 by this technology the 5-hydroxyl look ammonia acceptor of histidine mark has been connected to quartz plate to study (12) by total internal reflection fluorescent.
Nineteen ninety-five, adopt bidentate, three teeth and four tooth metal-chelating group derivatize surfaces to be described to the method (13) of check and analysis thing.Reported that the protein with histidine mark was connected on the surface that has the mixing self assembled monolayer (SAM) on the golden side group NTA part to study (14) by the surface plasma laser resonant in 1996.
Reported in 1997 by adopt SPR as the commercial sensing device of measuring method with the protein bound of histidine mark to the surface (15) with NTA-nickel part.
Reported in 1997 on the surface of adopting the SAM that has a NTA part by use the Fab fragment to be attached to gold plating studying (16) by FTIR.Reported that also having NTA SAM partly by use detects metal ion (17).
Reported by use and had IDA-Cu 2+The unimolecular layer of part is bonded to unimolecular layer to study by X-radiocrystallography (18) and electron microscope method (19) with streptavidin.
Reported that the lipid that has a NTA group by use prepared the lipid film (20) of metal sensitivity in 1994.
Reported in 1996 to use to contain and had pyrene part and IDA-Cu on the surface of bilayer 2+The bilayer of the lipid of part is rich in the protein (21) of Histidine by the excimer fluoroscopic examination.
The material that is adopted in above-mentioned report has many shortcomings.The first, the stability of ternary complex, i.e. interaction between metallo-chelate metal and the protein for make protein be immobilized the sufficiently long time with observe and research too a little less than.The second, the metallo-chelate that is adopted and other unlabelled protein interact in nonspecific mode.In addition, in some analytical system, ternary complex may be destroyed in the presence of the chaff interference of certain unknown concentration.
Summary of the invention
The present inventor has developed the compound with the compound improved characteristics that has been disclosed in the literature than those.These compounds have a plurality of covalently bound metal-chelating groups.These compounds can be in order to be connected in protein on material and surface.
Therefore the present invention is the compound with general formula I
Y-(Z) n
Molecular formula I wherein, Y is a component, the polydentate ligand sequestrant of Z representative and metallic ion coordination; And n is at least 2 integer, preferably 2-9.
Z can be a polydentate ligand, its with as Co 2+, Ni 2+, Cu 2+, Zn 2+Metallic ion coordination.Donor atom Z can be σ-donor atom or π-donor atom.Donor atom can be selected from nitrogen, oxygen, sulphur, phosphorus and silicon.Donor atom is nitrogen preferably.Z can be bidentate, three teeth (as IDA) or four teeth (as NTA).Z is preferably except that cyclic or the polycyclic.Z is tetradentate ligands such as NTA preferably.
Y preferably provides at least three in order to directly or by optional covalently bound with the Z indirectly part of linking group.The main chain of the group Y of branch can be residue, oligopolymer or the polymkeric substance of compound.Linking group most preferably has linear backbone.
On the other hand, the invention provides compound as molecular formula II
X-Y-(Z) n
Molecular formula II wherein Y, Z and n as mentioned above, and X is a compartment.
X can be hydrophilic, hydrophobic or not only have hydrophilic area but also have hydrophobic region.X can be or comprise and replace or unsubstituted alkyl; randomly separated for example low polyoxyethylene glycol or other oligomeric alkyl glycol, aminoacid sequence, polypeptide or polymeric amide or oligoamide (as amino caproyl oligopolymer) by one or more heteroatomss (as oxygen, nitrogen, sulphur or their two or more combination).X can be or comprise lipid.This lipid can be to stride membrane lipid (MSL).Preferred X comprises hydrophilic area, for example polyalkylene oligopolymer, and hydrophobic region, and lipid for example, wherein X randomly links to each other with Y by spacer groups by hydrophobic region.
Another aspect the invention provides the compound as the molecule formula III
W-X-Y-(Z) n
The molecule formula III wherein, X, Y, Z and n as mentioned above, and W is in order to be connected with other molecule, perhaps to be connected with the surface, perhaps to be inlaid into the group in the film.
In preferred embodiments, W is in order to comprise that with other molecule group (as amine functional group, carboxylic acid functional, alcohol functional group, halogen functional group) that polymkeric substance such as sepharose are connected or the group that is connected with the surface are (as mercaptan or the disulphide in order to be connected with gold or other coinage metallic surface, perhaps as silane derivative) in order to be connected with oxide surface, perhaps be inlaid into group in the film (as lipid groups, perhaps dissolve in the protein of film, as linear gramicidins).W also can be the group that can produce non-covalent connection, as non-covalent connection the between vitamin H and the streptavidin.
Preferred Y is a component, its provide a plurality of in order to the covalently bound part of Z and provide one in order to the covalently bound part of X.Nonrestrictive illustrative example to Y comprises:
Amino polyol, as TRIS, bis-homotris
Amino acid, as 3, the amino m-phthalic acid of 5-diaminobenzoic acid, 5-,
Figure A0080337500081
The peptide that contains a plurality of free acids and/or amine moiety, for example
Figure A0080337500091
In another embodiment, Y is a component, its contain a plurality of in order to Z and the covalently bound part of X.The nonrestrictive illustrative example of Y comprises:
Polyamine is as spermidine, spermine, penten;
Polyprotonic acid such as tartrate, 1,3,5-benzenetricarboxylic acid, citric acid, Kemp ' s three acid
Polyhydroxylated material such as sugar
Dendron, as commercial " Starburst " compound,
The compound that contains a plurality of epoxy moieties, perhaps contain easily by nucleophilic reagent (as halogen,
Tosylate or ester) group that replaces, perhaps nucleophilic reagent is easy to the group of addition
The compound of (as alpha, beta-unsaturated ketone), perhaps their combination.
Compound of the present invention can have purposes widely.Particularly they to be attached to the surface for the biomacromolecule with protein and other be useful.Be appreciated that this is the requirement of many sensing devices and analysis.
It is believed that compound of the present invention will be applied to biosensor especially.Biosensor is known in the art, and at PCT/AU93/00620, PCT/AU96/00482, PCT/AU95/00763, 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, among PCT/AU96/00369 and the PCT/AU94/00202 description is arranged, these documents are incorporated herein for referencial use.
These biosensors have the film that comprises ionophore and acceptor.Analyte and receptors bind cause the electricity of film to be led or the detectable variation of impedance.These biosensors provide the sensitive to special analyte to detect.
Usually, be oriented to the acceptor of interested analyte be connected in ionophore and film.Usually acceptor is protein such as antibody or its Fab such as Fab.It is believed that binding compounds of the present invention will be useful in ionophore that this acceptor is connected in biosensor and film.
Therefore in preferred embodiments, the invention reside in
In this manual, " comprising " this speech will be understood that to mean and comprise certain element, integer or step, perhaps element set, integer group or step group, rather than with any other element, integer or step, perhaps element set, integer group and step group foreclose.
Brief description of drawings
Fig. 1: 6 Histidine rubiscos are incorporated into J1. three NTA (triNTA) fragment.Show 100,200 and 400 nmoles/liter binding curve (from bottom to up).Binding curve is that Fc1 deducts Fc2 (contrast ,-Ni 2+) response.
Fig. 2: 6 Histidine CD40 are incorporated into J1. three NTA fragments.Show 100,200,400,600 and 800 nmoles/liter binding curve (from bottom to up).Binding curve is that Fc1 deducts Fc2 (contrast ,-Ni 2+) response.
Detailed Description Of The Invention
In order more to be expressly understood character of the present invention, preferred form of the present invention is described below with reference to embodiment.General introduction
6 Histidine CD40 are incorporated into three NTA to be had than it being incorporated into the high 12 times avidity of NTA.This mainly is the high 10 times result of association rate (Ka).But owing to compare (more absolute combination degree) with three NTA on the golden fragment, the density of NTA molecule on BLAcore NTA fragment is higher, so NTA bonded disengaging speed (offrate, K d) may be by exaggerative.
It will be understood to those of skill in the art that under the prerequisite that does not deviate from as broadly described the spirit and scope of the invention, can make multiple change and/or modification, as shown in the specific embodiment the present invention.Therefore these embodiments are illustrative and nonrestrictive.1. general process
N6-carbobenzoxy-(Cbz)-L-Methionin, 2-trimethyl silyl ethanol, hydrochloric acid 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), N-hydroxyl succinic diamide (NHS) and 6-aminocaprolc acid (X) are available from Sigma-Aldrich chemical company.XXBoc be by Xboc NHS ester (it self be by under standard conditions with the amino of BocON protection X, obtain with DCC and NHS coupling then) and X react and obtain.Face with preceding methylene dichloride P 2O 5Distillation.2.Z-Methionin NTA's is synthetic
Figure A0080337500111
C 18H 24N 2O 8
396.39
396.153266C54.5％ H6.1％ N7.1％ O32.3％
According to Schmitt etc. 1The synthetic Z-Methionin NTA of process.
Be dissolved in bromoacetic acid (4.17 gram, 30.0 mmoles) in the aqueous sodium hydroxide solution (1.5 mol, 15 milliliters) and be cooled to 0 ℃.In 2 hours, in reaction mixture, dropwise add N εThe aqueous sodium hydroxide solution (1.5 mol, 25 milliliters) of-Z-(L)-Methionin (2.0 grams, 7.0 mmoles).Solution is warmed to room temperature and at room temperature stirs and spend the night.Reaction mixture 50 ℃ of heating 2 hours, is chilled to room temperature then.Aqueous hydrochloric acid (1 mol, 40 milliliters) is dropwise added in the reaction mixture, then the white precipitate that obtains is filtered, and wash with hydrochloric acid (0.1 mol, 20 milliliters) and distilled water (2 * 20 milliliters).The white solid that obtains is dry a couple of days under high vacuum, obtains the white solid (2.68 grams, yield 95%) of Z-Methionin NTA.Data 1H NMR (200MHz, d 6-DMSO): δ 7.42 (5H, m ,-C 6H 5), 5.09 (2H, s ,-CH 2Ph), 3.56 (4H, the AB quartet, 2 *-NCH 2CO 2H), 3.41 (1H, overlapping dd ,-NCHCO 2H), 3.06 (2H, m ,-NHCH 2-), 1.75-1.25 (6H, overlapping multiplet ,-NHCH 2CH 2CH 2CH 2-) ppm. 13C?NMR(50MHz，d 4-MeOD)：δ175.9(2×-C(O)O-)，175.8(-C(O)O-)，158.9(-C(O)NH)，138.4(-C 6H 5)，129.4(-C 6H 5)，128.9(-C 6H 5)，128.7(-C 6H 5)，67.3(-CH 2Ph)，66.7(-NCHC(O)-)，55.3(2×-NCH 2C(O)-)，41.5(-NHCH 2)，30.7(-NHCH 2CH 2-)，30.5(-NHCH 2CH 2CH 2CH 2-)，24.6(-NHCH 2CH 2CH 2-)ppm。Electron spray(ES) M/S:m/z 397 (100%) (M+H +), 398 (21%), 353 (30%), 792 (15%).3.Z-Methionin NTA.TMSE 3Synthetic
According to Gao etc. 2The synthetic Z-Methionin NTA.TMSE of process 3
Figure A0080337500121
C 33H 60N 2O 8Si 3
697.10
696.365752C56.9％ H8.7％ N4.0％ O18.4％ Si12.1％
(1.25 restrain with EDC at 0 ℃, 12.5 mmole) add Z-Methionin NTA (0.50 gram, 1.25 2-trimethyl silyl ethanol (1.50 grams mmole),, 12.5 mmole) and 4-dimethylaminopyridine (0.50 gram, 4.15 mmoles) newly steam, solution in the dry methylene chloride (60 milliliters).Be reflected at 0 ℃ and stirred 1 hour down, be warmed to room temperature then and at room temperature stir and spend the night.Distilled water (50 milliliters) is added in the reaction mixture, separate organic layer.With methylene dichloride (2 * 50 milliliters) aqueous layer extracted, merge organic extract liquid, with dried over anhydrous sodium carbonate.Filtering mixt, all volatile matter are removed in decompression, obtain colorless oil.Employing from methylene dichloride to the dichloromethane solvent gradient that contains 5% methyl alcohol on flash distillation silicon-dioxide with the column chromatography purifying, obtain the Z-Methionin NTA.TMSE of colorless oil 3(0.75 gram, 86%).Data Rf=0.78 (methylene dichloride that contains 5% methyl alcohol) 1H NMR (200MHz, CDCl 3): δ 7.34 (5H, m ,-C 6H 5), 5.08 (2H, s ,-CH 2Ph), 4.91 (1H, br t ,-NH), 4.16 (6H, m, 3 *-OCH 2-), 3.60 (4H, s, 2 *-NCH 2C (O)-), 3.39 (1H, t ,-NCHC (O)-, 3J H-H=7.5Hz), 3.19 (2H, m ,-NHCH 2-), 1.3-1.8 (6H, overlapping multiplet ,-NHCH 2CH 2CH 2CH 2-), 0.93-1.04 (6H, overlapping multiplet, 3 *-CH 2Si-), 0.04 (9H, s, 1 *-Si (CH 3) 3), 0.03 (18H, s, 2 *-Si (CH 3) 3) ppm. 13CNMR(100MHz，CDCl 3)：δ173.6(-C(O)O-)，172.2(2×-C(O)O-)，157.1(-OC(O)NH)，137.5(-C 6H 5)，129.2(-C 6H 5)，128.7(-C 6H 5)，67.2(-CH 2Ph)，65.4(-NCHC(O)-)，63.5(1×-OCH 2-)，63.3(2×-OCH 2-)，53.6(2×-NCH 2C(O)-)，41.6(-NHCH 2-)，30.8(-NHCH 2CH 2-)，30.1(-NHCH 2CH 2CH 2CH 2-)，23.8(-NHCH 2CH 2CH 2-)，18.3(-CH 2Si-)，18.1(2×-CH 2Si-)，-0.8(3×-Si(CH 3) 3)ppm。Electron spray(ES) M/S:m/z 719.4 (100%) (M+Na +) 4. Methionin NTA.TMSE 3Synthetic
Figure A0080337500141
C 25H 54N 2O 6Si 3
562.97
562.328973C53.3％ H9.7％ N5.0％ O17.1％ Si15.0％
With Z-Methionin NTA.TMSE 3(0.167 gram, 0.24 mmole) is dissolved in the methyl alcohol (5 milliliters), adds the 10%Pd/C of the most advanced and sophisticated amount of spatula (spatule-tip), loads onto the air bag that contains hydrogen to reaction vessel.At room temperature stirred reaction mixture is 75 minutes, filters then, and all volatile matters are removed in decompression. 1H NMR analyzes and shows the complete hydrogenation of generation, obtains the Methionin NTA.TMSE of colorless oil 3(0.127 gram quantitative yield).Data 1H NMR (200MHz, CDCl 3): δ 4.16 (6H, m, 3 *-OCH 2-), 3.61 (4H, s, 2 *-NCH 2C (O)-), 3.40 (1H, apparent triplet ,-NCHC (O)-, 3J H-H=7.5Hz), 2.69 (2H, brt, NH 2CH 2-, 3J H-H=6.1Hz), 1.3-1.8 (6H, overlapping multiplet, NH 2CH 2CH 2CH 2CH 2-), 0.93-1.04 (6H, overlapping multiplet, 3 *-CH 2Si-), 0.04 (9H, s, 1 *-Si (CH 3) 3), 0.03 (18H, s, 2 *-Si (CH 3) 3) ppm. 13C?NMR(100MHz，CDCl 3)：δ173.7(-C(O)O-)，172.2(2×-C(O)O-)，65.6(-NCHC(O)-)，63.5(2×-OCH 2-)，63.3(1×-OCH 2-)，53.6(2×-NCH 2C(O)-)，42.7(NH 2CH 2-)，33.9(NH 2CH 2CH 2-)，31.0(NH 2CH 2CH 2CH 2CH 2-)，23.9(NH 2CH 2CH 2CH 2-)，18.3(-CH 2Si-)，18.1(2×-CH 2Si-)，-0.8(3×-Si(CH 3) 3)ppm。Electron spray(ES) M/S:m/z 563.3 (100%) (M+H +) 5.Z-three NTA.TMSE9 synthetic
Figure A0080337500151
C 93H 180N 8O 23Si 9
2031.26
2029.108491C55.0％ H8.9％ N5.5％ O18.1％ Si12.4％
With Z-Methionin NTA (60 milligrams, 0.15 mmole), 4-dimethylaminopyridine (60 milligrams, 0.51 mmole) and Methionin NTA.TMSE 3(0.37 gram, 0.66 mmole) is dissolved in the methylene dichloride (50 milliliters), and reaction mixture is chilled to 0 ℃.Add EDC (0.15 gram, 0.75 mmole),, be warmed to room temperature then, at room temperature stir and spend the night 0 ℃ of reaction stirred 1 hour.In reaction mixture, add distilled water (40 milliliters), separate organic layer.Water layer merges organic extract liquid, and uses dried over anhydrous sodium carbonate with methylene dichloride (2 * 40 milliliters) extraction.Filtering mixt, all volatile matters are removed in decompression, obtain colourless oily matter.The methylene dichloride of employing from 100% to the solvent gradient of the methylene dichloride that contains 5% methyl alcohol on flash distillation silicon-dioxide with the column chromatography purifying, obtain Z-three NTA.TMSE of colorless oil 9(0.235 gram, 76%).Data Rf=0.24 (methylene dichloride that contains 5% methyl alcohol) 1H NMR (400MHz, CDCl 3): δ 7.37 (2H, br t, NH), 7.34 (5H, m ,-C 6H 5), 7.26 (1H, br t, NH), 5.15 (1H, br t, NH), 5.08 (2H, s ,-CH 2Ph), 4.17 (18H, m, 9 *-OCH 2-), 3.60 (12H, two ratios are 2: 1 overlapping unimodal, 6 *-NCH 2C (O) O-), 3.39-3.34 (5H, overlapping multiplet), 3.26-3.16 (10H, overlapping multiplet), 3.07 (1H, triplets), 1.85-1.30 (24H, overlapping multiplet, 12 *-CH 2), 0.97 (18H, overlapping multiplet, 9 *-CH 2Si-), 0.05 (27H, s, 3 *-Si (CH 3) 3), 0.04 (54H, s, 6 *-Si (CH 3) 3) ppm.-OC(O)NHCH 2- 13C?NMR(100MHz，CDCl 3)：δ173.5(2×-C(O)O-)，173.5(-C(O)O-)，172.9(3×-C(O)NH-)，172.2(4×-C(O)O-)，172.1(2×-C(O)O-)，157.2(-OC(O)NH)，137.4(-C 6H 5)，129.1(-C 6H 5)，128.6(-C 6H 5)，67.1(-CH 2Ph)，66.4(-NCHC(O)NH-)，65.5(-NCHC(O)O-)，65.4(2×-NCHC(O)O-)，63.4(6×-OCH 2CH 2-)，63.3(3×-OCH 2CH 2-)，56.9(2×-NCH 2C(O)NH-)，53.7(4×-NCH 2C(O)O-)，53.4(2×-NCH 2C(O)O-)，41.2(-OC(O)NHCH 2-)，39.9(3×-C(O)NCH 2-)，30.7，30.5，30.2(4×-NHCH 2CH 2-)，29.9，29.5，28.9(4×NHCH 2CH 2CH 2CH 2-)，24.7，24.0，23.8(4×-NHCH 2CH 2CH 2-)，18.3(3×-CH 2Si(CH 3) 3-)，18.0(6×-CH 2Si(CH 3) 3-)，-0.8(9×-Si(CH 3) 3)ppm。Electron spray(ES) M/S:m/z 2054.3 (100%) (M+Na +), 1038.8 (20%), 613.3 (36%) 6.Z-three NTA's is synthetic
Figure A0080337500171
C 48H 72N 8O 23
Exact mass: 1128.47
Molecular weight: 1129.13C51.06; H6.43; N9.92; O32.59
With Z-three NTA.TMSE 9(0.12 gram, 0.06 mmole) is chilled to 0 ℃, adds trifluoroacetic acid (3 milliliters).0 ℃ of following stirred reaction mixture 3 hours, all volatile matters were removed in decompression then.With reversed-phase HPLC, in 40 minutes, adopt solvent gradient purification reaction mixture from 100% water/0.05%TFA to the water/0.05%TFA that contains 50% acetonitrile/0.05%TFA.The collection retention time is 21 minutes a HPLC stratographic main peak, obtains the white solid (20 milligrams, 30% yield) of pure Z-three NTA.Data 1H NMR (400MHz, D 2O): δ 7.38 (5H, m ,-C 6H 5), 5.08 (2H, s ,-CH 2Ph), 4.06-3.78 (20H, m, 8 *-NCH 2C (O)-+4 *-NCHC (O)-), 3.23 (6H, apparent triplet, 3 *-C (O) NHCH 2-), 3.08 (2H, apparent triplet, 1 *-OC (O) NHCH 2-), 2.00-1.76 (8H, overlapping multiplet, 4 *-CH 2), 1.60-1.40 (14H, overlapping multiplet, 7 *-CH 2), 1.38-1.22 (2H, multiplet, 1 *-CH 2) ppm. 13C?NMR(100MHz，D 2O)：172.3，171.2，170.8，168.6，159.0，137.5，129.5，129.0，128.3，118.0，115.5，67.8，67.5，56.0，55.5，41.0，39.6，39.0，28.5，27.4，23.7，23.0ppm。Electron spray(ES) 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. three NTA TMSE 9Synthetic
Figure A0080337500181
C 85H 174N 8O 21Si 9
Exact mass: 1895.07
Molecular weight: 1897.10C53.81; H9.24; N5.91; O17.71; Si13.32
With Z-three NTA TMSE 9(38 milligrams, 0.0187 mmole) are dissolved in the methyl alcohol (10 milliliters), add the palladium charcoal (10%) of catalytic amount.Under vacuum, wash mixture, and in nitrogen atmosphere, stirred 2 hours with hydrogen.Filter reaction mixture, evaporated filtrate obtains three NTA TMSE 9Clarifying oil (34 milligrams, 96%).Data 1H n.m.r. (CDCl 3) δ 0.00 (81H, m, Si (CH 3) 3), 0.96 (18H, m, CH 2Si), and 1.2-2.0 (24H, m), 3.1-3.7 (28H, m), 4.15 (18H, m, OCH 2) and 7.4 (2H, NH 2).8. three NTA TMSE 9Synthesizing of half succinic diamide
C 89H 178N 8O 24Si 9
Exact mass: 1995.09
Molecular weight: 1997.17C53.52; H8.98; N5.61; O19.23; Si12.66
Stir three NTA TMSE down at 0 ℃ 9The dichloromethane solution of (0.5 gram), hemisuccinic acid benzyl ester (71 milligrams), DMAP (64 milligrams).EDC (101 milligrams) is added in the reaction mixture, at room temperature continue to stir 16 hours.Removal of solvent under reduced pressure adopts methylene chloride-methanol (96: 4) to carry out the chromatographic separation residuum, isolates the intermediate of 537 milligrams benzyl ester.In methyl alcohol (60 milliliters), is catalyzer at normal temperatures hydrogenation 4 hour with 10%Pd/C (60 milligrams) with 500 milligrams of these substance dissolves.Obtain 474 milligrams of pure products.Data 1H n.m.r. (CDCl 3) δ 0.04 (81H, m, Si (CH 3) 3), 0.96 (18H, m, CH 2Si), 1.2-2.0 (24H, m), 2.5 (2H, m), 2.65 (2H, m), 3.2-3.45 (16H, m), 3.59 (s, 12), 4.16 (18H, m, OCH 2).Electron spray(ES) M/S:m/z 2019.8 (35%), (M+Na +), 1997.8 (100%) (M +).9. three NTA's is synthetic
Develop three NTA TMSE with toluene 9(34 milligrams, 0.0179 mmole), evaporation and dry under high vacuum condition.Add trifluoroacetic acid (1 milliliter), under 0-5 ℃, in nitrogen atmosphere, stirred 2 hours, at room temperature stir then and spend the night.Trifluoroacetic acid is removed in evaporation, with toluene development residuum, obtains three NTA (20 milligrams, 100%) after evaporation and the drying once more.Data 1H n.m.r. (CD 3OD) δ 1.2-2.0 (24H, m), 3.07 (6H, m, CH 2NHCO) and 3.1-3.7 (22H, m).10. the preparation of linear gramicidins succinate
Linear gramicidins (75 milligrams, 0.0398 mmole) is dissolved in the pyridine (0.5 milliliter), adds succinyl oxide (20 milligrams, 0.200 mmole).Mixture stirred 20 hours in nitrogen atmosphere under 50 ℃, then evaporation.With the methanol solution of crude product by the sephadexLH-20 post, evaporation of eluate, and with the flash distillation silica column with methylene chloride/water/acetate (400: 50: 4: 1) purifying.The product water is centrifugal to carry out further purifying.Behind the decantation water, desciccate under high vacuum obtains linear gramicidins succinate (53 milligrams, 67%).Data 1H n.m.r. (CD 3OD) δ 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CH 2CO), and 2.9-3.4 (10H, m), 3.90 (2H, dd, CH 2-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).11. the preparation of linear gramicidins succsinic acid NHS ester
Linear gramicidins succinate (21 milligrams, 0.0105 mmole), N-hydroxyl succinic diamide (12 milligrams, 0.1042 mmole) and 4-Dimethylamino pyridine (2.5 milligrams, 0.0204 mmole) and distillatory tetrahydrofuran (THF) (10 milliliters) are merged.Add dicyclohexylcarbodiimide (22 milligrams, 0.1066 mmole) under in nitrogen, stirring.Mixture heating up refluxed 1 hour.Evaporating mixture makes mixture pass through Sephadex LH-20 post in methyl alcohol.With suitable part evaporation and dry, obtain linear gramicidins succsinic acid NHS ester (22 milligrams, 100%).Data 1H n.m.r. (CD 3OD) δ 0.4-1.8 (66H, m), 2.0-2.2 (4H, m), 2.56 (4H, s, CH 2CO), 2.82 (4H, s, NHS) 2.9-3.4 (10H, m), 3.90 (2H, dd, CH 2-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).12. the preparation of linear gramicidins succinate three NTA
Methionin three NTA (20 milligrams, 0.0201 mmole) are dissolved in the methyl alcohol (1 milliliter), add triethylamine (2) neutralization.Methyl alcohol (2 milliliters) solution that adds linear gramicidins succinate NHS (15 milligrams, 0.0072 mmole) then.Reaction mixture is used Sephadex LH-20 column purification then 50 ℃ of heating 24 hours in methyl alcohol.Obtain linear gramicidins succinate Methionin three NTA (14 milligrams, 65%).Data 1H n.m.r. (CD 3OD) δ 0.4-1.8 (90H, m), 2.0-2.2 (4H, m), 2.52 (4H, m, CH 2CO), and 3.0-3.8 (38H, m), 3.90 (2H, dd, CH 2-gly), 4.0-4.8 (16H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).MALDI?m.s.2963.4((M+Na +)-H 2O)，2982.9(M+Na +)，2998(M+K +)。13.gA the preparation of Methionin-XXBOC GA Methionin XXBOC
With (15 milligrams of linear gramicidins Methionins, 0.0076 mmole) be dissolved in the methyl alcohol (2 milliliters), and adding monovalent triethylamine, with the N-hydroxyl succinic diamide ester (10 milligrams, 0.0226 mmole) of itself and XXBOC (by in the exsiccant methylene dichloride, XXBOC and 1 equivalent DCC and NHS and 1 equivalent DMAP reaction being prepared) reaction.Reaction mixture at room temperature stirred 18 hours.Evaporation reaction mixture, and in methyl alcohol, pass through Sephadex LH-20 post.To contain the elutriant evaporation of suitable part, and (400: 40: 4: 1) purifying obtained gA Methionin 2XBOC (12 milligrams, 68%) with methylene chloride/water/acetate on the flash distillation silica column.Data 1H n.m.r. (CD 3OD) δ 0.4-1.8 (84H, m), 1.41 (9H, s, BOC), 2.0-2.2 (4H, m), 2.9-3.4 (14H, m), 3.90 (2H, dd, CH 2-gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).14.gA the preparation of Methionin XXBOC hemisuccinic acid ester
Linear gramicidins Methionin 2XBOC (12 milligrams, 0.0052 mmole) is developed with toluene, and vaporising under vacuum is also dry.Add trifluoroacetic acid (1 milliliter), evaporation and dry in nitrogen.Add toluene once more, in a vacuum evaporation and dry.Thick amine solvent in pyridine (0.5 milliliter), and is reacted with succinyl oxide (2.6 milligrams, 0.0259 mmole).Reaction mixture was at room temperature stirred 20 hours.Remove pyridine under the high vacuum, residuum passes through Sephadex LH-20 post in methyl alcohol.Product is further purified with methanol-eluted fractions on the flash distillation silica column, obtains gA Methionin 2X succinate (10 milligrams, 83%).Data 1H n.m.r. (CD 3OD) δ 0.4-1.8 (84H, m), 2.0-2.2 (8H, m), 2.58 (4H, dd, CH 2-CO), and 2.9-3.4 (14H, m), 3.90 (2H, dd, CH 2-gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).15.gA Methionin 2X three NTA's is synthetic
With (8.5 milligrams of linear gramicidins Methionin 2X succinates, 0037 mmole), N-hydroxyl succinic diamide is (4.2 milligrams, 0.0364 mmole) and (1 milligram of 4-dimethylaminopyridine, 0.0081 mmole) in distilled tetrahydrofuran (THF) (5 milliliters), stir, and adding dicyclohexylcarbodiimide (7.5 milligrams, 0.0363 mmole).Mixture refluxed in nitrogen 1 hour.Evaporating mixture, and in methyl alcohol, pass through sephadex LH-20 column purification.Evaporate suitable part, add Methionin three NTA (8.5 milligrams, 0.0085 mmole).Mixture at room temperature stirred 18 hours.Evaporation reaction mixture passes through sephadex LH-20 column purification in methyl alcohol (X2).Obtain gA Methionin 2X three NTA (2.6 milligrams, 19%) (because solubleness is low, so lose some compounds).Data 1H n.m.r. (CD 3OD) δ 0.4-1.8 (108H, m), 2.0-2.2 (8H, m), 2.62 (4H, dd, CH 2CO), and 3.0-3.8 (42H, m), 3.90 (2H, dd, CH 2-gly), 4.0-4.8 (15H, m), 6.8-7.6 (20H, m) and 8.18 (1H, s, CHO).MALDI?m.s.?3291.17(M+Na +)，3309.02(M+K +)。16. three NTA.TMSE 9Synthetic
C 85H 174N 8O 21Si 9
1897.12
1895.071711C53.8％ H9.2％ N5.9％ O17.7％ Si13.3％
With Z-three NTA.TMSE 9(0.1 gram, 50 micromoles) are dissolved in the methyl alcohol (10 milliliters).Add palladium charcoal (10%) (about 10 milligrams) and load onto the air bag that contains hydrogen.At room temperature stirred reaction mixture is 90 minutes, and TLC analyzes the completely dissolve of demonstration initial substance afterwards during this period of time.Reaction mixture filters by the short plug of iron calcium acid four calcium stones, and all volatile matters are removed in decompression from reaction mixture, obtain three NTA.TMSE 9Colorless oil (0.09 gram, quantitatively).Data electron spray(ES) M/S:m/z 1898.3 (100%) (M+H +) 17. vitamin Hs, three NTA.TMSE9 synthetic
C 95H 188N 10O 23SSi 9
2123.42
2121.149311C53.7％ H8.9％ N6.6％ O17.3％ S1.5％ Si11.9％
With vitamin H (20 milligrams, 0.08 mmole), 4-Dimethylamino pyridine (12 milligrams, 0.1 mmole) and three NTA.TMSE 9(0.10 gram, 0.1 mmole) joins in drying, the new methylene dichloride (5 milliliters) that steams, and reaction mixture is chilled to 0 ℃.Add EDC (40 milligrams, 0.2 mmole), be reflected at 0 ℃ and stirred 1 hour, be warmed to room temperature then and also at room temperature stirred 3 days.All volatile matters are removed in decompression from reaction mixture, mixture is the elutriant purifying with methyl alcohol with Sephadex LH-20 post.Merge the part that contains the spot of Rf=0.1 in 5% ethanol/methylene.Obtain vitamin H three NTA.TMSE 9Colourless oily matter (73 milligrams, 0.03 mmole, 34%).Data electron spray(ES) M/S:m/z 1173.9 (100%), 2146.4 (25%) (M+Na +) 18. vitamin Hs, three NTA synthetic
Figure A0080337500271
C 50H 80N 10O 23S
1221.29
1220.511851C49.2％ H6.6％ N11.5％ O30.1％ S2.6％
Under 0 ℃, trifluoroacetic acid (2 milliliters) is added vitamin H three NTA TMSE 9In (19 milligrams, 9 micromoles).0 ℃ of following stirred reaction mixture 3 hours, all volatile matters were removed in decompression then.Adopt in 40 minutes from 100% solvent orange 2 A to the solvent orange 2 A gradient (solvent orange 2 A: water/0.05%TFA that contains 50% solvent B by the reversed-phase HPLC on Vydac C18 post; Solvent B: the analyze reaction mixture of acetonitrile/0.05%TFA) shows a main peak of Rt=24.1 minute of retention time.With anti-phase preparation HPLC separate reacted mixture, obtain the white solid (1.0 milligrams, 0.8 micromole, 9%) of vitamin H three NTA.Data electron spray(ES) M/S:m/z 1221.5 (100%) (M+H +), 1243.4 (33%) (M+Na +) 19. lipids-three NTA synthetic
Figure A0080337500281
C 72H 127N 9O 23
1486.84
1485.904482C58.2％ H8.6％ N8.5％ O24.7％
At room temperature methylene dichloride (5 milliliters) solution with two tetradecy lamines (100 milligrams) joins in methylene dichloride (5 milliliters) solution of succinyl oxide (120 milligrams) and triethylamine (40 milligrams).Reaction is stirred and is spent the night, solvent removed in vacuo, the residuum methylene chloride-methanol (
Figure A0080337500282
) carry out chromatographic separation, obtain 60 milligram of two tetradecy lamine half succinic diamide.This material is added three NTA TMSE 9Methylene dichloride (10 milliliters) solution in, add DMAP (14 milligrams) subsequently.Reaction mixture is chilled to 0 ℃, adds EDC (22 milligrams), and mixture at room temperature stirred 3 days.Remove then and desolvate, residuum SephadexLH-20 column purification is used methanol-eluted fractions, obtains 97 milligrams of pure substances.10 milligrams of these substance dissolves in TFA (1 milliliter), were stirred 3 hours in nitrogen.Solvent removed in vacuo, water (1 milliliter) washs remaining solid, vacuum-drying.This material separates obtaining expecting product (4 milligrams) by preparation HPLC (C18 Alltime post contains the acetonitrile of 1%TFA) purifying.Data electron spray(ES) M/S:m/z 1486.6 (100%) (M+H +), 1508.8 (62%) (M+Na +) .20. strides the synthetic of film phosphorylcholine fat (MSL-PC)
C 124H 220NO 24PS 2
Exact mass: 2202.52
Molecular weight: 2204.17C67.57; H10.06; N0.64; O17.42; P1.41; S2.91
With MSL-OH 3(895 milligrams, 0.44 mmole) be dissolved in contain quinoline (0.12 milliliter, 2.2 equivalents are used the sodium phosphate drying, with preceding with zinc dust distillation) chloroform (6 milliliters) in.Under the room temperature it is slowly added the new POCl that steams 3In (0.09 milliliter, 2.2 equivalents).Mixture stirred 30 minutes down at 45 ℃.The cooling back adds the toluenesulphonic acids choline (363 milligrams, 1.3 mmoles) that is dissolved in the exsiccant pyridine (1 milliliter), and reaction mixture at room temperature stirs and spends the night.Add entry (0.5 milliliter), mixture stirred 1 hour in addition.In reaction mixture, add chloroform (50 milliliters), use water (* 2), 3% salt of wormwood (* 2), water (* 2), 5% hydrochloric acid (* 2) and the water (* 2) of 40 milliliters of every parts to wash successively then.(note: because each washing all can form emulsion, so phase separation is quite time-consuming.In order to improve this process, it is necessary adding methyl alcohol.) merge organic phase, and use the sodium phosphate drying.Removal of solvent under reduced pressure, [(elutriant: methylene dichloride: acetone: methyl alcohol: aqua ammonia (8: 5: 5: 3)) obtains 122 milligrams of colourless waxy materials to residuum by silica gel chromatography.Data 1H n.m.r. δ (CDCl 3: CD 3OD (3: 1)) 0.82-0.92 (m, 30H, phytane base CH 3), 0.92-1.80 (m, 100H, phytane base CH 2And CH), 2.61 (t, 2H, CH 2CH 2S), 2.65 (s, 8H, succsinic acid H), 3.20 (s, 9H ,-N (CH 3) 3), 3.35-3.74 (m, 44H ,-OCH 2,-OCH, CH 2N), 3.85 (s, 2H, SCH 2Ph), 3.92 (t, 2H, CH 2OP), 4.10 (m, 2H, POCH 2CH 2), 4.20 (m, 8H, the H of ester), 6.90 and 7.43 (d, 8H, xenyls), 7.30 (s, 5H, PhCH 2).Electron spray(ES) M/S:m/z 2206 (M+2), 1112,587,48821. 2 (N-methyl)-C38 diamines synthetic
Figure A0080337500301
Exact mass: 1373.29
Molecular weight: 1374.31C78.66; H12.32; N2.04; O6.99
Suddenly draw amphiphile C38 glycol (bola-amphiphile C38-diol to being cooled to 0 ℃ be dissolved among the THF (10 milliliters) 3) add triethylamine (92 microlitre) and methane sulfonyl chloride (51 microlitre) in THF (10 milliliters) solution of (300 milligrams).Solution at room temperature stirs and spends the night, and dilutes with ether (30 milliliters).Organic phase is washed with saturated sodium bicarbonate (2 * 20 milliliters) and water (2 * 60 milliliters), dry (sodium sulfate) and vacuum concentration.The material (315 milligrams) that obtains is added in the penstock, be cooled to-80 ℃, add the methylamine (12 milliliters) of chilling.Seal up pressure piping, and mixture is warmed to room temperature.After at room temperature placement is spent the night, evaporate excessive methylamine, residuum is dissolved in the methylene dichloride, stir with salt of wormwood.Solids removed by filtration, filtrate vacuum-drying.Residuum methylene chloride-methanol-ammonia (
Figure A0080337500302
) carry out chromatographic separation, obtain 242 milligrams of pure products.Data 1H n.m.r. (CDCl 3) δ 0.80-0.95 (30H, m, phytane base CH 3), 0.95-1.70 (100H, m, phytane base CH 2And CH), 2.45 (6H, s), 2.69 (4H, m), 3.40-3.80 (14H, m, CH-O and CH 2-O), 3.98 (4H, t, CH 2OAr), 6.93,7.43 (8H, AA ' XX ' multiplet, fragrance-H).22.XXFmoc synthetic
Figure A0080337500311
C 27H 34N 2O 5
Exact mass: 466.25
Molecular weight: 466.57C69.50; H7.35; N6.00; O17.15
In nitrogen, handled 2X-Boc (420 milligrams) 20 minutes with TFA (5 milliliters).TFA is removed in decompression, and dry under high vacuum.Then this residuum is dissolved in 9% the aqueous sodium carbonate (6 milliliters), and solution is cooled to 0 ℃.To be dissolved in Fmoc-NMS ester (available from CALBIOCHEM) (420 milligrams) among the DMF (3 milliliters) and join toply in the solution of 0 ℃ of stirring, and at room temperature stir 30 minutes.Add entry (100 milliliters),, discard organic extract liquid with ethyl acetate (2 * 50 milliliters) extraction water solution.Water layer concentrated hydrochloric acid (2-3 milliliter) acidifying, and in ice bath, cool off, precipitation appears therebetween.Filter this throw out and dry, obtain white powder (210 milligrams).Data NMR (CDCl 3) 1.2-1.8 (m, 12H), 2.20 (t, 2H), 2.35 (t, 2H), 3.25 (m, 4H), 4.259m, 1H), 4.40 (m, 1H), 5.05 (m, 1H), 5.53 (m, 1H), 6.65 (m, 1H), 7.3-7.5 (m, 4H), 7.6 (d, 2H), 7.25 (d, 2H) EI mass spectrum 483.9 (M+Na +) 23.C38 (NCH 3) 2Single BOC's is synthetic
The suspension of C38 diamines (320 milligrams), BOC-ON (63 milligrams), triethylamine (37 milligrams), THF (3 milliliters) and water (2 milliliters) at room temperature stirred spend the night.Reaction mixture water (50 milliliters) dilutes, and extracts with methylene dichloride (2 * 80 milliliters).Merge organic extract liquid,, use dried over mgso with salt solution (50 milliliters) washing, and removal of solvent under reduced pressure.Crude product is with chromatogram purification (methylene dichloride that contains 5-7% methyl alcohol).Obtain the pure substance (150 milligrams) of thick liquid nano.Data NMR (CDCl 3) 0.7-0.9 (m, 30H), 1.0-1.9 (m, 109H), 2.51 (s, 3H), 2.7 (m, 2H), 2.91 (s, 3H), 3.1-3.7 (m, 16H), 4.0 (t, 4H), 6.9 (d, 4H), 7.4 (d, 4H) ES mass spectrum 1474.9 (M +, 100%) and 24.Boc-C38-2X-Fmoc synthetic
Figure A0080337500322
C 122H 208N 4O 12
Exact mass: 1921.58
Molecular weight: 1922.98C76.20; H10.90; N2.91; O9.98
The mixture of 2X-Fmoc (47 milligrams), DCC (35 milligrams), the single Boc (150 milligrams) of DMAP (3 milligrams), C38-and DCM (10 milliliters) was at room temperature stirred 24 hours.Removal of solvent under reduced pressure, crude product obtains pure products (200 milligrams) with column chromatography (methylene dichloride that contains 5% methyl alcohol) purifying.Data NMR (CDCl 3) 0.8-0.9 (m, 30H), 0.95-2.5 (m, 125H), 2.9,2.95,3.05 (3xs, 6H), 3.1-3.75 (m, m, 22H), 4.0 (t, 4H), 4.1-4.4 (m, 3H), 6.9 (d, 4H), 7.3-7.4 (m, 4H), 7.5 (d, 4H), 7.6 (d, 2H), 7.75 (d, 2H) ES mass spectrum 1923.3 (M +), 1945.1 (M+Na +) 25.MSL-three-NTA synthetic
The material (200 milligrams) of Fmoc protection is dissolved in the solution of piperidines: DMF (20: 80), at room temperature stirred 10 minutes.DMF is removed in decompression, with column chromatography (methyl alcohol: ammoniacal liquor: DCm 10: 2: 88) purifying crude product.Separate the pure products (148 milligrams) that obtains colourless semi-solid.
This C38 derivative of a part (210 milligrams), three NTA derivatives (135 milligrams), DCC (33 milligrams) and DMAP (3 milligrams) are dissolved in the exsiccant methylene dichloride (3 milliliters), at room temperature placed 18 hours.Removal of solvent under reduced pressure with column chromatography purifying crude product, obtains the pure substance (179 milligrams) of colourless viscous liquid shape.
The material (100 milligrams) of Boc and TMSE protection is dissolved among the TFA (2 milliliters), at room temperature stirred 4 hours.The decompression remove TFA, and under vacuum dry sample.
The synthetic of the disulphide acid that molecular formula is following reported in other place 4.
By in the presence of DCC (15 milligrams) and DMAP (2.6 milligrams), acid (50 milligrams) was reacted 4 hours in methylene dichloride (3 milliliters) with NHS (8 milligrams), and be elutriant Sephadex LH-20 column purification with methyl alcohol, prepare the NHS ester of this material.
With this active ester in methyl alcohol with substance C 38 diamines-three NTA (100 milligrams) reaction of the complete deprotection of above preparation.Stir under the room temperature after 16 hours, remove and desolvate, thick material is that elutriant uses Sephadex LH-20 post by partial purification with methyl alcohol.The material that obtained is further purified with preparation HPLC (C18 Alltima post contains methyl alcohol-methylene dichloride of 0.05%TFA).26.6 the albumen of hyte propylhomoserin mark and three NTA bonded dynamic analyses
Adopt the BIAcore technology to carry out bonded surface plasma laser resonant (SPR) analysis.Commercial J1 chip (BIAcore, Uppsala, Sweden) is modified by applying lipid layer.In a ultra-clean shield cap, BIAcore J1 chip is extracted with the exposed gold surface.The ethanolic soln (100 microlitre) that will contain 1.2 micromoles per liter MSL, three NTA, 24 micromoles per liter MSLPC directly is dispersed on the gold.During insulation 1 hour, added ethanol every 10-15 minute and evaporate fully to prevent alcoholic acid.Measure the washing with alcohol gold surface of 10 * 100 microlitres then by transfer pipet, and air-dry in the ultra-clean shield cap.Then chip is put again and seal with plastics film (Parafilm).
These J1. three NTA chips are connected into the BIAcore2000 machine, 21 ℃ with pump flushing, flow velocity is flow damping fluid (50 mmoles/rise HEPES, 300 mmoles/a rise sodium-chlor of the HBS/EDTA of 40 mul/min, 50 micromoles per liter EDTA pH8.0) test.With respect to contrast flow-through cell 1 (Fc1), flow-through cell 2 (Fc2) is as test tank.Therefore, the mobile damping fluid that contains 500 micromoles per liter nickelous chlorides only injects by Fc2, then with damping fluid washing Fc25 minute.Then protein of interest matter (100-600 nmole/liter) is injected (using the KINJECT function), by Fc1 and Fc2 3-7 minute, then with dissociating phase 7-15 minute.Then the surface with the mobile damping fluid regeneration that contains 350 mmoles/rise EDTA removing nickelous chloride, thereby the protein of 6 hyte propylhomoserin marks is discharged from three NTA molecules.To each protein, shown in concentration (Fig. 1, repeating step 2-4 in scope Fig. 2).The negative control that adopts is the protein with 6 hyte propylhomoserin marks.The protein that the result shows three NTA and 6 hyte propylhomoserin marks with high-affinity combine (Fig. 1, Fig. 2).6 Histidine rubiscos and 6 Histidine CD40 and the three NTA bonded equilibrium constants be respectively 300 picomole/liter and 2.9 nmoles/liter.There is not Ni 2+Existence under these protein do not combine with three NTA, and the protein of 6 hyte propylhomoserin marks of no use does not combine with three NTA yet.
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It will be understood to those of skill in the art that under the prerequisite that does not deviate from as broadly described the spirit and scope of the invention, can make multiple variation or modification the present invention shown in specific embodiments.Therefore where face in office, these embodiments all are illustrative and nonrestrictive.

Claims (28)

1. the compound of general formula I
Y-(Z) n
I wherein Y is a component, the polydentate ligand sequestrant of Z representative and metallic ion coordination; And n is at least 2 integer.
2. compound as claimed in claim 1, wherein n is 2 to 9 integer.
3. compound as claimed in claim 2, wherein n is 3 at least.
4. the described compound of arbitrary as described above claim, wherein n is 4.
5. the described compound of arbitrary as described above claim, wherein the donor atom of Z is a nitrogen.
6. compound as claimed in claim 5, wherein Z is a tetradentate ligands.
7. compound as claimed in claim 7, wherein Z is the NTA residue.
8. the described compound of arbitrary as described above claim, wherein Y provides at least three parts, and each part directly is covalently attached to Z, perhaps is connected in Z indirectly by optional linking group.
9. the described compound of arbitrary as described above claim, wherein Y has the main chain from oligopolymer or polymer formation.
10. compound as claimed in claim 9, main chain wherein is a straight chain.
11. as the described compound of arbitrary claim among the claim 1-8, wherein Y is selected from the group of being made up of amino polyol, amino acid, amino polycarboxylic acid, polyamine, polyprotonic acid and polyol.
12. compound as claimed in claim 9, wherein Z is selected from the group of being made up of peptide and dendron.
13. the described compound of arbitrary as described above claim has molecular formula II
X-Y-(Z) n
II is Y, Z and n such as preceding definition wherein, and X is a partitioned portion.
14. compound as claimed in claim 13, wherein X is hydrophilic, hydrophobic or has hydrophobic region and hydrophilic area.
15. compound as claimed in claim 14, wherein X is or comprises the part that is selected from the group of being made up of replacement or unsubstituted alkyl, is randomly separated by one or more heteroatomss, low polyalkylene oxide, aminoacid sequence, polypeptide, oligoamide, polymeric amide and lipid.
16. compound as claimed in claim 15, wherein X is selected from by low polyoxyethylene glycol, amino hexanoyl oligopolymer and strides the group that membrane lipid (MSL) is formed.
17. as the described compound of arbitrary claim among the claim 13-16, wherein X comprises hydrophilic area, hydrophobic region.
18. as the described compound of arbitrary claim among the claim 13-17, wherein Y is a component, it provide a plurality of in order to the part that is covalently attached to Z and one in order to be covalently attached to the part of X.
19. compound as claimed in claim 18, wherein Y is selected from by amino polyol, amino acid, the peptide that contains a plurality of free acids and/or amine moiety, polyhydroxylated material and contains and is easy to the group that replaced by nucleophilic reagent or nucleophilic reagent and is easy to the compound of the group of addition on it or the group that their combination is formed.
20. compound as claimed in claim 19, wherein Y is selected from by TRIS, bis-homotris, 3, the amino m-phthalic acid of 5-diaminobenzoic acid, 5-, The group that sugar, dendron and alpha, beta-unsaturated ketone are formed.
21., have the molecule formula III as the described compound of arbitrary claim among the claim 13-20
W-X-Y-(Z) n
III is X, Y, Z and n such as preceding definition wherein, and W allows to be connected in other molecule, is connected in the surface or is inlaid into group in the film.
22. compound as claimed in claim 21, wherein W comprises the protein that is selected from amine functional group, carboxylic acid functional, alcohol functional group, halogen functional group, mercaptan, disulphide, silane derivative, dissolves in film, the one or more functional groups that allow group, ionophore or the lipid groups of non-covalent connection.
23. compound as claimed in claim 22, wherein W is linear gramicidins or vitamin H.
24. comprise the biosensor of self-assembled film, described film comprises a plurality of binding compounds as the described molecule formula III of arbitrary claim among the claim 13-23, wherein W is the ionophore that embeds in the film.
25. biosensor as claimed in claim 24, ionophore wherein is a linear gramicidins.
26. as claim 24 or 25 described biosensors, film wherein comprises the binding compounds of second kind of a plurality of molecule formula III, W wherein is the parents' thing that embeds in the film.
27. as the purposes of the described compound of arbitrary claim in the metal affinity chromatography among the claim 1-23.
28. comprise metal affinity chromatographic column as the described compound of arbitrary claim among the claim 1-23.
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