CN1563116A - New type ramification of resin containing iodine, synthetic method and application in researching protein group based on mass spectrum - Google Patents

New type ramification of resin containing iodine, synthetic method and application in researching protein group based on mass spectrum Download PDF

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CN1563116A
CN1563116A CN 200410017662 CN200410017662A CN1563116A CN 1563116 A CN1563116 A CN 1563116A CN 200410017662 CN200410017662 CN 200410017662 CN 200410017662 A CN200410017662 A CN 200410017662A CN 1563116 A CN1563116 A CN 1563116A
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solid
resin
group
derivative
iodine
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CN1288177C (en
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郭寅龙
张立
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Shanghai Institute of Organic Chemistry of CAS
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Shanghai Institute of Organic Chemistry of CAS
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Abstract

The invention relates to synthesis of a kind of raw derivative containing iodine and isotope label and its application in the research of proteomics based on mass spectrography. The structure of the reagent described by said invention is Solid-Link-R'(H/D)-COCH2I, in which solid portion is resin with benzyl aminyl as reaction group i link portion is connecting portion which can be used for making solid-phase synthesis, in which C-N bond which is unstable for acid is contained; R'(H/D) portion is amino acid containig8-10 hydrogen/deuterium substituted chains or branch chains (for example leucine, isoleucine, valine and methionine, etc.), and the iodacetyl is selective reaction activity portion of mercapto-group.

Description

A kind ofly novelly contain iodine resin derivative, synthetic method and based on the application in the mass spectral protein science research
Technical field
The present invention relates to the solid derivative of a class formation novelty, relate to the especially application prospect in protein science research of the design, synthetic method of this compounds and the application aspect biochemical thereof, particularly relate to the novel iodine resin derivative that contains of a class, its synthetic method and the application aspect biochemical thereof be the application prospect in protein science research especially.
Technical background
At present, the research of genomics has entered a metastable stage, and simultaneously, the research of gene product (comprising RNA and protein) is just attracting increasing sight.Wherein particularly proteinic research just with the form of protein science research, has been subjected to attention day by day.Protein, as active substance important in the organism, the complicacy of its research is considerably beyond gene and RNA.And the multiple information that protein showed: degree of modification, the variation of amount, the variation of structure and distribution is all directly relevant with the variation of physiological function, so about proteinic research is the most effective research aspect in the life research, also be one of the most challenging research field (referring to J.Bio.Chem.2001; 49:45497-45500).
For the protein system of complexity, what at present general and effective research process used is after two-dimensional gel electrophoresis separates, and carries out mass spectrometric detection again, finishes qualitative, quantitative, diversity ratio method.This method is that present range of application is the widest, develop the most perfect protein science aspect research method (referring to Anal.Chem.1994,24:4390-4399; Proteomics 2001 1:3-12).But, its inherent shortcoming is arranged as the two-dimensional gel electrophoresis of separation means.Such as for separating strongly-acid and strong basicity albumen, the albumen that content is low, and some membranin, the result can not be satisfactory.And the level of automation of this method is difficult to improve, add its carry out aspect the albumen comparative analysis limitation (referring to Proc.Natl.Acad.Sci.U.S.A.2000,17:9390-9395; Trends in analyticai chemistry 2003 22:273-281), has impelled the development and the application of novel method.
In recent years, development along with mass-spectrometric technique, soft ionization technology (electron spray ionisation ESI technology particularly, substance assistant laser desorpted ionized MAIDI technology) appearance and development, the analysis and research means that make mass spectrum become to become more and more important in the protein science research, the research method that relies on the protein science that mass spectrum sets up has also caused increasing attention and development.Wherein, the isotopic labeling method that is suitable for mass spectrometric detection attracts people's attention very much.This isotopic labeling method comprises in the body and two kinds of external marks.The body internal labeling be in nutrient solution, introduce the isotopic labeling part (referring to Proc.Natl.Acad.Sci.U.S.A.1999,96:6591-6596; J.Am.Chem.Soc.1999,121:7949-7950), it is of limited application; And in the external mark, isotopic labeling partly is to introduce in analytic process, because of it uses suffered less-restrictive, so come into one's own in recent years.Wherein, stable isotope affinity labelling (ICAT) technology is to develop comparatively perfect method (referring to Nat.Biotechnol.1999,10:994-999).In the ICAT method, proteic mark, separation can rely on ICAT reagent and realize, detailed process is that this reagent is in selected marker sulfydryl part, biotin moiety in the reagent can be used for carrying out the affine separation of mark peptide again, greatly simplify the complexity of analyte, improved the efficient of analyzing.This technology has had commercial prod to occur at present, and corresponding software has also carried out development and application.But along with the application of ICAT reagent, increasing problem occurred, the separation efficiency problem that wherein depends on the biotin moiety in this reagent is a very important aspect.
Also occurred at present including the isotope-labeled reagent of solid (referring to Anal.Chem.2002,19:4969-4979; Nat.Biotechnol.2002,5:512-515), the advantage of this type of reagent tentatively shows.
Summary of the invention
The problem that will solve of the present invention provide a class brand-new contain the isotope-labeled reagent of solid.
Another problem that the present invention will solve provides the method for synthetic mentioned reagent.
The problem that also will solve of the present invention provides the application of mentioned reagent in protein science research.
The invention provides a kind of new reagent, is a kind of iodine resin derivative that contains, and it has following structure:
Solid-Link-R’(H/D)-COCH 2I
Wherein, Solid is the solid phase part, is to have the resin of methylamino-as reactive group, and the described methylamino-that has is preferably methylamino acrylic resin (Aminomethyl polystyreneresin) as the resin of reactive group;
Link is the connection portion, is the connection portion that can be used for solid phase synthesis, wherein contains the unsettled C-N key to acid;
R ' is a mark part (H/D), is to contain chain that 8-12 hydrogen/deuterium replaces or a catenate amino acid (chain that preferred 8-10 hydrogen/deuterium replaces or prop up catenate amino acid), and described amino acid is preferably as leucine, Isoleucine, Xie Ansuan, methionine(Met) etc.;
Iodacetyl is the selective reaction active part of sulfydryl.
Iodine resin derivative, the wherein b of containing provided by the invention), c) part provides amino, carboxylic group, so that solid phase connects, mark and activity etc. partly interconnects.This connection portion that contains the iodine resin derivative provides and can be connected with mark part and at acidic conditions generation cracked C-N key.This contains in the iodine resin derivative and exists electron-donating group to help mass spectroscopy.This contains a chain or a catenate amino acid that iodine resin derivative mark part comprises that 8-12 hydrogen/deuterium replaces, contains the locating information of limiting analysis thing residue.
The iodine resin derivative that contains provided by the invention, described connection portion preferred construction general formula is as follows, wherein contains the unsettled C-N key to acid,
Figure A20041001766200071
Substituting group on the described substituted-phenyl is single the replacement or polysubstituted electron-donating group.Described electron-donating group is preferably C 1～C 3Alkoxyl group, OH, Me 2NCH 2CH 2O, Et 2NCH 2CH 2O, NH 2, C 1～C 4Alkyl.
Further preferably have following general structure:
Figure A20041001766200081
The iodine resin derivative that contains provided by the invention further preferably has following general structure:
Resin is foregoing resin in the formula.
The connection that contains each several part in the iodine resin derivative of the present invention can be that deprotection under alkaline condition carries out condensation reaction with carboxyl again by the amino of fluorenylmethyloxycarbonyl (Fmoc) protection, and the amino circulation of deprotection is again finished.
Specifically, the iodine resin derivative that contains of the present invention, can be synthetic by following method:
In non-protonic solvent, the solid support resin and the connection portion that have the aminotoluene base, condensing agent BOP/DCC/DIC, the mol ratio of condensation catalyst HOBt is preferably 1: 1～and 5: 1～5: 1～5, more preferably 1: 3: 3: 3, at 0 ℃-50, be preferably 10 ℃-50 ℃, reaction times is preferably 0.5～5 hour, and more preferably 1-3 hour, suction filtration, solid part can be used non-protonic solvent, protic solvent washs respectively, and drying is weighed, calculate loaded value (loading value), be the mole number of the contained avtive spot of 1 gram solid, unit is mmole/gram, is applied to the calculating that feeds intake of reacting below.(wherein BOP is benzotriazole-1-oxygen-three-(dimethylin) phosphine hexafluorophosphate, and DCC is a dicyclohexylcarbodiimide, and DIC is a DIC, and HOBt is the hydroxyl benzotriazole).
Above-mentioned solid is carried out end socket (capping) reaction with aceticanhydride in non-protonic solvent, the unreacted amino of acetylize, 10-50 ℃ was reacted 0.5～3 hour; more preferably 1 hour, suction filtration, solid can be used protic solvent; protic solvent washs respectively; drying under alkaline condition, is taken off the Fmoc protection; enter the condensation of next round after the washing drying; washing, the deprotection process also is connected to the active group iodo-acid amide at last and gets destination agent on the solid.
Be the synthetic route that example illustrates reagent of the present invention with following agent structure below:
Figure A20041001766200091
Synthetic route:
(1) in aprotic solvent, under the 0-50 ℃ of condition, methylamino acrylic resin (AminomethylPolystyrene Resin), Rink Amide connecting arm (Rink Amide Linker, 4-[(2,4-dimethoxyphenyl) (Fmoc-amino) methyl] phenoxyacetic acid), condensing agent, condensation catalyst hydroxyl benzotriazole (HOBt) hybrid reaction 2-5 hour
The mol ratio of each component is 1: 3: 3: 3.Suction filtration washs the dry compound 1 that gets.Weighing to calculate loads (loading) value, is the mole number of the contained avtive spot of 1 gram solid, and unit is mmole/gram.
IR：3433，1720，1686.
The gained compound reacted the unreacted amino of acetylize with aceticanhydride 1 hour at 10-50 ℃ again in non-protonic solvent.Wherein used condensing agent can be BOP, benzotriazole-1-oxygen-three-(dimethylin) phosphine hexafluorophosphate (benzotriazol-l-yloxy) tris (dimethylamino) phosphonium hexafluoro-phosphate), dicyclohexylcarbodiimide (DCC), DIC (DIC) etc., aprotic solvent can be N, dinethylformamide (DMF), methylene dichloride (CH 2Cl 2), trichloromethane (CHCl 3) etc.;
The washing solvent for use is followed successively by N, N-dimethylformamide (DMF), methylene dichloride (CH 2Cl 2), methyl alcohol (CH 3OH); The amino used condition of protective reaction is benzene-aceticanhydride (1: 1).
(2) under alkaline condition, compound 1 takes off the Fmoc protection, washs the dry compound 2 that gets.Wherein used alkaline condition is piperidines/DMF (20-40%).The washing solvent for use is the same.
IR：3433，3428，1665.
(3) in aprotic solvent and under 0-50 ℃ of condition, compound 2, Fmoc-leu (10H/D), condensing agent, condensation catalyst HOBt hybrid reaction 1-5 hour, suction filtration, wash dry must compound 3.The mol ratio of each component, condensing agent, aprotic solvent, the washing solvent for use is the same.
IR：3430，3328，1676，1610.
Fmoc-leu (H/D) is by leu (H/D) preparation, and the operation reference literature carries out (referring to Int.J.Pept.Protein Res., 27 (4), 398-400,1986).
(4) under alkaline condition, compound 3 takes off the Fmoc protection, washs the dry compound 4 that gets.
IR：3381，3375，1669，1610.
(5) in aprotic solvent and under 0-50 ℃ of condition, compound 4, iodoacetic acid, condensing agent, condensation catalyst HOBt hybrid reaction 1-5 hour, the mol ratio of each component is 1: 6: 3: 3.Get compound 5 after the washing drying.Other condition is the same.
IR：3427，3301，1670，1610，538.
Amount of iodine is analyzed: I (amount of iodine) 5.60%.
Calculating can get this, and to contain iodine solid-phase reagent active group number be 0.44mmol/g.
In the synthetic method of the present invention, whether each step condensation reaction can detect remaining amino judgement reaction in order to sensitive ninhydrin reaction complete.If find not exclusively, repeat condensation step to reacting completely.Wash used solvent and be preferably DMF, CH 2Cl 2, CH 3OH etc.Take off the used alkaline reagents of Fmoc protection and be preferably piperidines/DMF (20-40%).Used device preferably has the vierics of the stop,threeway of polypropylene material, reacts on vibrator.
The iodine resin derivative that contains provided by the invention can be used as isotope-labeled reagent.In particular for halfcystine, contain the peptide of halfcystine, proteinic qualitative, quantitatively and aspect such as comparative analysis.
Description of drawings
Fig. 1 is the experimental procedure of extraction and analysis that contains the peptide section of halfcystine among the embodiment 1
Fig. 2 is the experimental procedure of the comparative analysis of egg white mixture among the embodiment 2
Specific implementation method
To help to understand the present invention by specific implementation method once, but not limit content of the present invention.
1. reagent composite part
Synthesizing of embodiment 1:Rink Amide-AM resin (Rink Amide-AM Resin) (1)
With 200mg (0.22mmol, 1.1mmol/g) methylamino acrylic resin resin, 360mg (0.66mmol) Rink Amide connecting arm, 300mg (0.66mmol) BOP, 100mg (0.66mmol) HOBt places reactor, adds 5ml N, N-dimethylformamide (DMF), at room temperature, reactor is placed oscillator reaction 1 hour.Suction filtration, solid DMF, CH 3OH, CH 2Cl 2Wash respectively three times, drying weigh the 296mg faint yellow solid, calculate to load (loading) value and be 0.89mmol/g.This solid is placed reactor, adding the 5ml aceticanhydride, 5ml benzene, the concussion reaction is 1 hour under the room temperature, behind the suction filtration, solid DMF, CH 3OH, CH 2Cl 2After washing three times respectively, drying gets little yellow solid, compound 1.
IR：1750
Embodiment 2: Rink Amide-AM resin (Rink Amide-AMResin) (2) synthetic that takes off Fmoc protection
100mg compound 1 is placed reactor, add the DMF solution of 10ml 20% piperidines, the concussion reaction is 5 minutes under the room temperature, suction filtration, solid DMF, CH 3OH, CH 2Cl 2After washing three times respectively, add the DMF solution of 10ml 20% piperidines again, the concussion reaction is 15 minutes under the room temperature, suction filtration, solid DMF, CH 3OH, CH 2Cl 2Wash respectively three times, drying gets little yellow solid, compound 2.
The RinkAmide-AM resin (N-(Fmoc-Leu (10H/D)) Rink Amide-AM Resin) (3) that the leucine of embodiment 3:Fmoc protection (10 hydrogen or deuterium replace) replaces synthetic
86mg (0.077mmol) compound 2 is placed reactor, add 102mg (0.231mmol) BOP, 31mg (0.231mmol) HOBt, and 81mg (0.231mmol) Fmoc-leu-(D 10) place reactor, add 5ml DMF.Room temperature reaction 1 hour, suction filtration, solid DMF, CH 3OH, CH 2Cl 2Wash respectively three times, drying detects remaining amino with sensitive ninhydrin reaction (Kaiser ' s color reaction), judge reaction whether fully (referring to Anal.Biochem.1970,34:595-598), if incomplete, repeat above-mentioned reaction to reacting completely, get little yellow solid, compound 3.
Embodiment 4: the Rink Amide-AM resin (N-Leu (10H/D)-Rink Amide-AM Resin) (4) that leucine (10 hydrogen or deuterium replace) replaces synthetic
86mg (0.077mmol) compound 3 is placed reactor, take off the Fmoc protection, operation is with embodiment 2, and washing is dry, gets little yellow solid, compound 4.
Embodiment 5: synthetic (5) that contain the iodine resin derivative
With 50mg (0.045mmol) compound 4,62mg (0.135mmol) BOP, 22mg (0.135mmol) HOBt, and 50mg (0.27mmol) iodoacetic acid places reactor, adding 5ml N, N-dimethylformamide (DMF), at room temperature, reactor is placed oscillator reaction 1 hour.Suction filtration, solid DMF, CH 3OH, CH 2Cl 2Wash respectively three times, drying gets faint yellow solid, compound 5.
2. reagent applying portion
Principle as shown in the formula:
Figure A20041001766200141
Embodiment 1: the extraction and analysis that contains the peptide section of halfcystine
Experimental procedure is as shown in Figure 1:
Operation steps is as follows:
1. with peptide mixt 1: contain 10nmol LT-9 (LLGTGSFCT), 10nmol MN-8 (MGNWLMGN), 20nmol GS-10 (GTLFQKMNGS), 20nmol SF-9 (SLRGKPHSF), peptide mixt 2: contain 20nmol LT-9 (LLGTGSFCT), 20nmol MN-8 (MGNWLMGN), 10nmol GS-10 (GTLFQKMNGS), 10nmol SF-9 (SLRGKPHSF), be dissolved in respectively in the aqueous solution (pH8.0-8.2) of ammonium hydrogencarbonate of 500 μ L of 0.03M, the tributylphosphine (TBP) that adds 2 μ mol, concussion reaction 30 minutes.
2. what add in the reaction solution that contains peptide mixt 1 that 10 hydrogen of 5mg replace contains iodine resin (10H), what add in the reaction solution that contains peptide mixt 2 that 10 deuteriums of 5mg replace contains iodine resin (10D), concussion reaction 1 hour, the beta-mercaptoethanol quencher reaction that in two reaction solutions, adds 2 μ l respectively, suction filtration, collect filtrate, this filtrate is also carried out μLC-MS analyzes, and is labeled as elutriant, and solid water, methyl alcohol, methylene dichloride wash respectively three times, drying.
3. above-mentioned solid is placed acidic solution, the composition of acidic solution is: trifluoroacetic acid-dithioglycol-thioanisole-phenol-water=81.5: 2.5: 5: 5: 6, and concussion reaction 2.5 hours.Suction filtration, filtrate is concentrated into dried under nitrogen gas stream, add the 5ml ether, can see that the adularescent decorating film produces, and adds the aqueous solution (PH8.0-8.2) of the ammonium hydrogencarbonate of 100 μ l 0.03M again, the solution layering of concussion back, after removing most of ether layer, centrifugal, after the water intaking layer concentrates, carry out μ LC-MS and analyze, be labeled as recovery liquid.
4. μIt is the A phase that LC-MS analyzes used condition: the aqueous solution that contains 0.1% formic acid; B phase: the acetonitrile solution that contains 0.1% formic acid.Gradient elution, B phase 3-65% in 60 minutes.
μAfter LC-MS analyzed, the result was as follows:
1. do not have in the elutriant to find to contain the signal of halfcystine peptide section LT-9 (LLGTGSFCT), but other three the peptide section MN-8 (MGNWLMGN) that do not contain halfcystine, GS-10 (GTLFQKMNGS), SF-9 (SLRGKPHSF) are arranged.This explanation contains the effect that iodine resin has selective binding to the peptide section that contains halfcystine.
2. only find to contain the signal of halfcystine peptide section LT-9 (LLGTGSFCT)+mark part (170/180Da) in the recovery liquid.There are not other three signals that do not contain halfcystine peptide section.This explanation not with contain iodine resin bonded peptide section and can remove by washing.
3. in the recovery liquid, the signal to noise ratio of LT-9+ mark part (170/180Da) obviously is better than the signal to noise ratio of the LT-9 of mixed peptide.This illustrates that this contains iodine resin all is very high to extraction and the organic efficiency that contains halfcystine peptide section.
4. the strength of signal that contains the LT-9 that contains the iodine resin mark that 10H replaces in the recovery liquid is 1/2 of the LT-9 strength of signal that contains the iodine resin mark that replaces of 10D, and the content of LT-9 is proportional in this and two peptide mixts.This illustrates that this kind method can be used for carrying out the comparative analysis of different samples.
Embodiment 2: the comparative analysis of egg white mixture
Experimental procedure is as shown in Figure 2:
1. with egg white mixture 1: N,O-Diacetylmuramidase (30nmol), catalase (50nmol), cytochrome C (50nmol), ovalbumin (30nmol) and bovine serum albumin (50nmol), egg white mixture 2: N,O-Diacetylmuramidase (40nmol), catalase (30nmol), cytochrome C (30nmol), ovalbumin (50nmol) and bovine serum albumin (20nmol), be dissolved in respectively in the 200 μ L damping fluids, contain 8M urea in this damping fluid, 0.2M ammonium hydrogencarbonate and 0.02M calcium chloride (CaCl 2), pH8.2.In the solution that contains egg white mixture 1,2, add 5 μ mol tributylphosphines (TBP) respectively, place 37 ℃ of thermostat container insulations 1 hour.
With egg white mixture with 1 times of the aqueous solution (pH8.0-8.2) dilution of the ammonium hydrogencarbonate of 0.03M after, use 1 times of pure water redilution again.In the solution that contains egg white mixture 1,2, add 2% trypsin solution, 10 μ l, place 37 ℃ of thermostat container insulations after 8 hours two solution, add 2% trypsin solution, 10 μ l again, 37 ℃ are incubated 15 hours, reserve 10 μ l and carry out μ LC-MS analysis, are labeled as enzymolysis product.
3. what add in the reaction solution of egg white mixture 1 that 10 hydrogen of 5mg replace contains iodine resin (10H), what add in the reaction solution of egg white mixture 2 that 10 deuteriums of 5mg replace contains iodine resin (10D), the concussion reaction is after 2 hours, the beta-mercaptoethanol quencher reaction that in two reaction solutions, adds 2 μ l respectively, suction filtration, collect filtrate, this filtrate is also carried out μLC-MS analyzes, and is labeled as elutriant, and solid water, methyl alcohol, methylene dichloride wash respectively three times, drying.
4. above-mentioned solid is placed acidic solution, the composition of acidic solution is: trifluoroacetic acid-dithioglycol-thioanisole-phenol-water=81.5: 2.5: 5: 5: 6.Concussion reaction 2.5 hours.Suction filtration, filtrate is concentrated into dried under nitrogen gas stream, add the 5ml ether, can see that the adularescent decorating film produces, and adds the aqueous solution (PH8.0-8.2) of the ammonium hydrogencarbonate of 100 μ l 0.03M again, the solution layering of concussion back, after removing most of ether layer, centrifugal, after the water intaking layer concentrates, carry out μ LC-MS and analyze, be labeled as recovery liquid.
5. μIt is the A phase that LC-MS analyzes used condition: the aqueous solution that contains 0.1% formic acid; B phase: the acetonitrile solution that contains 0.1% formic acid.Gradient elution, B phase 3-85% in 80 minutes.
μAfter LC-MS analyzed, the result was as follows:
1. by to enzymolysis product μLC-MS analyzes, and has obtained the signal of each proteic typical enzymatic fragment, but has had a sizable part that signal overlap has taken place.The ownership of peptide section be use ExPASy Molecular Biology Server ( Http:// cn.expasy.org/sprot/) TagIdent instrument on is retrieved and is obtained, and used database is 41.10.
2. by to filtrate μLC-MS analyzes, and finds that the peptide segment signal that contains halfcystine obviously reduces, have in addition blackout, the strength of signal of other peptide section substantially becomes.
3. by to reclaiming liquid μLC-MS analyzes, and has only found to contain the signal of halfcystine peptide section, and the intensity of respective segments is suitable among its intensity and the enzymolysis product result, but signal to noise ratio has clear improvement.
4. reclaiming liquid μIn the LC-MS analytical results, find that a series of total mass numbers differ the group peak of the signal of 10Da, by retrieval, one group of peak promptly corresponding one contain halfcystine peptide section (m/z is peptide segment molecule amount+mark part molecular weight 170/180Da).Each content of organizing corresponding protein in the strength ratio at two peaks and the egg white mixture 1,2 is directly proportional, with this quantitative error less than 5%.

Claims (12)

1. one kind contains the iodine resin derivative, and it has following structure:
Solid-Link-R ' (H/D)-COCH 2I, wherein,
A) Solid is the solid phase part, and this part is to have the resin of methylamino-as reactive group;
B) Link is the connection portion, and this part is the connection portion that can be used for solid phase synthesis;
C) R ' is a mark part (H/D), and this part is to contain a chain or the catenate amino acid that 8-12 hydrogen/deuterium replaces;
D) active group part, this part is an iodoacetyl.
2. the iodine resin derivative that contains as claimed in claim 1, it has following structure:
Solid-Link-R ' (H/D)-COCH 2I, wherein,
A) Solid is the solid phase part, and this part is to have the resin of methylamino-as reactive group;
B) Link is the connection portion, and this part-structure general formula is as follows, wherein contains the unsettled C-N key to acid,
The unstable C-N key of acid
N=1,2,4,5R is a substituted-phenyl, R4=H or substituted-phenyl
Substituting group on the described substituted-phenyl is single the replacement or polysubstituted electron-donating group;
C) R ' is a mark part (H/D), and this part is to contain a chain or the catenate amino acid that 8-12 hydrogen/deuterium replaces;
D) active group part, this part is an iodoacetyl.
3. the iodine resin derivative that contains as claimed in claim 1 or 2 is characterized in that described amino acid is leucine, Isoleucine, Xie Ansuan, methionine(Met) etc.; The described methylamino-that has is methylamino acrylic resin (Aminomethyl polystyrene resin) as the resin of reactive group.
4. the iodine resin derivative that contains as claimed in claim 1 or 2 is characterized in that described connection portion Link is that general structure is as follows, wherein contains the unsettled C-N key to acid,
The unstable C-N key of acid
N=1,2,4,5R1, R2, the alkoxyl group of R3=H or C1～C4
Figure A2004100176620003C2
The alkoxyl group of R5=C1～C4, m=1～3;
Described mark part R ' (H/D) is
X=H or D in the formula
5. the iodine resin derivative that contains as claimed in claim 1 is characterized in that having electron-donating group in this derivative; B in this derivative), c) part all provides amino, carboxylic group; Described mark part contains the locating information of limiting analysis thing residue.
6. as claim 2 or the 5 described iodine resin derivatives that contain, it is characterized in that described electron-donating group is for being C 1～C 3Alkoxyl group, OH, Me 2NCH 2CH 2O, Et 2NCH 2CH 2O, NH 2, C 1～C 4Alkyl.
7. as claim 1, the 2 or 3 described synthetic methods that contain the iodine resin derivative; it is characterized in that the connection of each several part in this derivative; be amino by fluorenylmethyloxycarbonyl (Fmoc) protection; deprotection under alkaline condition; carry out condensation reaction with carboxyl again, the amino circulation of deprotection is again finished.
8. the synthetic method that contains the iodine resin derivative as claimed in claim 8 is characterized in that the described iodine resin derivative that contains synthesizes by following method:
In non-protonic solvent, the mol ratio that has the solid support resin of aminotoluene base and connection portion, condensing agent BOP/DCC/DIC, condensation catalyst HOBt is 1: 1～5: 1～5: 1～5,10 ℃-50 ℃ reactions 0.5-5 hour, obtain solid through aftertreatment
Above-mentioned solid carries out the end socket reaction with aceticanhydride in non-protonic solvent; the unreacted amino of acetylize; 10-50 ℃ was reacted 0.5-3 hour; through aftertreatment, under alkaline condition, take off the Fmoc protection; enter the condensation of next round after the washing drying; washing, the deprotection process also is connected to the active group ethanamide at last and gets destination agent on the solid.
9. synthetic method as claimed in claim 8 is characterized in that whether each step condensation reaction all detects remaining amino judgement reaction in order to sensitive ninhydrin reaction complete, if find not exclusively, the repetition condensation step is to reacting completely.
10. synthetic method as claimed in claim 8, it is characterized in that washing used solvent is DMF, CH 2Cl 2, CH 3OH etc.; Taking off the used alkaline reagents of Fmoc protection is piperidines/DMF (20-40%).
11. synthetic method as claimed in claim 8 is characterized in that used device is the vierics that have the stop,threeway of polypropylene material, reacts on vibrator.
12. as claim 1, the 2 or 3 described purposes that contain the iodine resin derivative, it is characterized in that containing the peptide of halfcystine as halfcystine, proteinic qualitative, quantitative and comparative analysis.
CN 200410017662 2004-04-14 2004-04-14 New type ramification of resin containing iodine, synthetic method and application in researching protein group based on mass spectrum Expired - Fee Related CN1288177C (en)

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CN106632877A (en) * 2015-08-26 2017-05-10 中国科学院大连化学物理研究所 Preparation of protein solid-phase alkylation reagent, and solid-phase alkylation reagent and applications thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106632877A (en) * 2015-08-26 2017-05-10 中国科学院大连化学物理研究所 Preparation of protein solid-phase alkylation reagent, and solid-phase alkylation reagent and applications thereof
CN106632877B (en) * 2015-08-26 2018-10-26 中国科学院大连化学物理研究所 The preparation of protein solid phase alkylating reagent and solid phase alkylating reagent and application

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