EP2403829A1 - Quervernetzungsmittel - Google Patents

Quervernetzungsmittel

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Publication number
EP2403829A1
EP2403829A1 EP10707893A EP10707893A EP2403829A1 EP 2403829 A1 EP2403829 A1 EP 2403829A1 EP 10707893 A EP10707893 A EP 10707893A EP 10707893 A EP10707893 A EP 10707893A EP 2403829 A1 EP2403829 A1 EP 2403829A1
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EP
European Patent Office
Prior art keywords
crosslinking agent
compound
protein
agent according
mol
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.)
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Application number
EP10707893A
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English (en)
French (fr)
Inventor
Jean Martinez
Gilles Subra
Christine Goubet
David Paramelle
Eric Forest
Michaël Heymann
Christophe Geourjon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Universite de Montpellier I
Universite Montpellier 2 Sciences et Techniques
Original Assignee
Centre National de la Recherche Scientifique CNRS
Universite de Montpellier I
Universite Montpellier 2 Sciences et Techniques
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Application filed by Centre National de la Recherche Scientifique CNRS, Universite de Montpellier I, Universite Montpellier 2 Sciences et Techniques filed Critical Centre National de la Recherche Scientifique CNRS
Priority to EP10707893A priority Critical patent/EP2403829A1/de
Publication of EP2403829A1 publication Critical patent/EP2403829A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom

Definitions

  • the invention relates to the field of structural analyzes of proteins by mass spectrometry.
  • the analysis of the three-dimensional structures of proteins and their interactions with their environment e.g. DNA, proteins, membranes
  • their environment e.g. DNA, proteins, membranes
  • NMR and XRD are commonly used analytical methods.
  • these methods are very limited by certain constraints: protein crystallization, use of large amount of material (5 to 10 mg of proteins).
  • This approach consists in carrying out an enzymatic digestion of the proteins of the reaction medium after carrying out the crosslinking reaction in solution.
  • the peptide mixture obtained is then analyzed by mass spectrometry in order to identify the peptides modified by the crosslinking agents. Structural data are then obtained from this identification. This strategy has been used in low-resolution structural studies of proteins or protein-protein interactions.
  • the chemical crosslinking agents have one or two reactive functions connected by a spacer arm. These reactive functions are able to react with the side chains of amino acids of proteins. After identification of the amino acid residues of the proteins modified by the two reactive functions, distance constraints within these proteins can be determined by virtue of the known length of the spacer arm of the chemical crosslinking agent. Homobifunctional agents, such as bis-imido esters or dialkyl halides, were developed and used on proteins as early as the 1950s.
  • the crosslinking experiment is carried out with an equimolar mixture of two crosslinking agents of identical structure but one of which is modified by stable isotopes.
  • the isotopes used are deuterium and oxygen 18. This type of labeling will have the effect of creating a resolution of the crosslinked peptide signal by virtue of the difference in mass between the two types of crosslinking agents, which makes it easier to detect modified peptides.
  • cleavable function within the crosslinking agent makes it possible to facilitate the identification of the crosslinked peptides by comparison between the mass spectrum of all the peptide signals of interest and that obtained after cleavage of the agent. crosslinking. It is thus possible to identify the signals that have been reached by the cleavage and thus to deduce the masses of the products modified by the crosslinking agent. Cleavage can be induced either by chemical treatment or directly in tandem mass spectrometric (MS / MS) analyzes. - Fluorescent marking
  • crosslinking agent labeled with a fluorescent motif is to facilitate the detection of crosslinked peptides during purification by chromatography. - Affinity purification
  • HCCA or HCCA derivatives make it possible to identify by MALDI-TOF mass spectrometry peptides of interest of a protein in a complex peptide mixture by labeling in solution whereas said peptides are not visible on the MALDI-TOF mass spectrum of the protein. This selective effect is further accentuated by the use of the HCCE matrix.
  • the subject of the invention is therefore a crosslinking agent for proteins of formula
  • R 1 is an aryl group optionally substituted one or more times with a group selected from the group consisting of hydroxy, C 1 -C 4 alkyl, OBoc, SO 3 Na, Deu, C 1 -C 4 alkoxy,
  • n and m are identical or different integers between 0 and 10, preferably between 1 and 5 p is an integer between 0 and 5, k is 0, 1, 2 or 3,
  • X and X ' which are identical or different, are a reactive function of the proteins.
  • Another subject of the invention is a process for preparing a crosslinking agent according to the invention.
  • Another object of the invention is the use of a crosslinking agent according to the invention for the analysis of the three-dimensional structure of a protein in mass spectrometry.
  • Another object of the invention is a method of structural analysis of a protein or a protein complex comprising the following steps: a) crosslinking the protein or the protein complex on the crosslinking agent according to one of the following: any of claims 1 to 8 by the X and / or X 'functions, b) enzymatic digestion of the protein or protein complex attached to the crosslinking agent according to any one of claims 1 to 8, c) analysis by mass spectrometry.
  • alkyl Ci -1 O means a saturated hydrocarbon chain, linear or branched, having 1 to 10 carbon atoms, such as a group methyl, ethyl, isopropyl, tert-butyls, pentyl, etc.
  • C 2 -C 4 alkenyl is meant a hydrocarbon chain, linear or branched, comprising at least one unsaturation and containing from 2 to 10 carbon atoms, for example an ethenyl, propenyl or 2,4-hexadienyl group, etc.
  • aryl group is meant an aromatic group, preferably comprising from 5 to 10 carbon atoms, comprising one or more rings and optionally comprising a heteroatom, in particular an oxygen, a nitrogen or a sulfur, for example a grouping phenyl, furan, indole, pyridine, naphthalene, etc.
  • halogen refers to fluorine, bromine, chlorine or iodine.
  • Boc refers to an amine protecting group of the formula t-butyloxycarbonyl.
  • Deu denotes deuterium
  • (Ci-Ce) alkoxy is understood within the meaning of the present invention, a (Ci-C ⁇ 5) alkyl, as defined above, bonded to the molecule via a oxygen atom.
  • a (Ci-C ⁇ 5) alkyl as defined above, bonded to the molecule via a oxygen atom.
  • tBu refers to tert-butyl.
  • the crosslinking agent of the proteins of the invention has the formula (I)
  • R 1 is an aryl group optionally substituted one or more times with a group selected from the group consisting of hydroxy, C 1 -C 4 alkyl, OBoc, SO 3 Na, Deu, Ci-C 4 alkoxy,
  • R 2 is N, , or n and m are identical or different integers between 0 and 10, preferably between 1 and 5, p is an integer between 0 and 5, k is 0, 1, 2 or 3,
  • X and X ' which are identical or different, are a reactive function of the proteins.
  • n m.
  • R 1 is a phenyl group optionally substituted one or more times with a group selected from the group consisting of hydroxy, C 1 -C 4 alkyl, OBoc, SO 3 Na, Deu, C 1 -C 4 alkoxy.
  • R 1 is a phenoxy group, most preferably a para-hydroxyphenyl group.
  • R 2 is N, or
  • crosslinking agent according to the invention has formula (II), (III) or (IV)
  • X and X ' which are identical or different, are chosen from the group consisting of imidoester, N-hydroxysuccinimide ester, isocyanate, isothiocyanate, N-maleimide, disulfide, 1,2-dicarbonyl, benzophenone and arylazide functions.
  • the crosslinking agent of the invention comprises two reactive functions of the X and X 'proteins. It is possible to design a very large variety of crosslinking agents depending on the nature of the reactive functions used which may be identical or not (i.e. homo or heterobifunctional agent).
  • crosslinking of the protein or protein complex on the solid support crosslinking agent of the invention is the reaction of one or more reactive functional groups X and / or X 'of the crosslinking agent A with one or more several groups of the protein or protein complex resulting in the covalent attachment of said protein or protein complex to the crosslinking agent A.
  • the homobifunctional crosslinking agents have two identical reactive functions that can react with the same type of function.
  • one of the two reactive functions reacts on a side chain of an amino acid residue.
  • the second reactive function then reacts either intramolecularly on another nearby side chain, or on a side chain of an amino acid residue belonging to another protein. Since the two reactive functions are identical, the reaction protocol is in a single step.
  • the heterobifunctional crosslinking agents have two reactive functions targeting different amino acids.
  • crosslink proteins are generally used to crosslink proteins in two steps (e.g. crosslinking agents having a nonspecific and specific reactive function). Once the first fixation has been carried out, it is therefore possible to carry out purification of the modified proteins before carrying out the reaction of the second reactive function. This may favor intramolecular reactions and may provide more diverse data than with homobifunctional crosslinking agents.
  • REACTIVE FUNCTIONS X and X ' are specific reactive functions of proteins capable of reacting with a reactive group of proteins.
  • X and X ' which are identical or different, are chosen from the group consisting of the specific reactive functions of the amines, the specific reactive functions of the carboxylic acids, the specific reactive functions of the thiols, the specific reactive functions of the guanidines and the non-reactive functional groups. specific.
  • the specific reactive functions of the amines react with the primary amines present on the proteins.
  • Three types of functions are commonly used for this purpose: imidoesters, N-hydroxysuccinimide esters ( ⁇ HS), isocyanates (and isothiocyanates). They are all electrophilic activated agents with a good leaving group by nucleophilic substitution.
  • the specific reactive functions of the carboxylic acids are present in the Asp, Glu and C-terminal residues of the proteins. These functions are not reactive per se and require activation.
  • This activation is usually carried out with carbodiimides.
  • the O-acylurea formed by this activation makes it possible to react with a primary amine and form an amide bond.
  • the specific reactive functions of the thiols are the N-maleimide and disulfide functions.
  • the thiol function is the most reactive nucleophilic function within a protein.
  • the side chains of the cysteine residues are often engaged in disulfide bridges which prevents their reaction with chemical crosslinking agents. Therefore, a reduction reaction (with ethanedithiol or EDT) of these bonds is generally necessary in order to recover free thiol functions.
  • EDT ethanedithiol
  • the pKa of the thiols of the cysteine residues are approximately 8.6, their reactivity increases when the thiolate ion is formed at a pH greater than 8.6.
  • the specific reactive functions of guanidines are the 1,2-dicarbonyl compounds which react specifically with the side chains of arginines.
  • Non-specific reactive functions react on molecules by exposure to UV light.
  • An ideal photoreactive agent must have different qualities: - It has a high reactivity
  • reaction mechanism of these functions are generally radical which allows to act indifferently on various residues.
  • chemical crosslinking agents having a nonspecific reactive function also have a specific reactive function. In this way, it is possible to target residues of interest with a first fixing step involving the specific reactive function and then to mark all the residues present in a certain perimeter (defined by the length of the spacer arm linking the two functions reactive) through the action of the non-specific reactive function.
  • the crosslinking agents of the invention are preferably prepared in the following manner.
  • the process for preparing the crosslinking agents of the invention comprises three steps.
  • Step (a) consists of a peptide coupling between the amine RR'NH and the carboxylic acid R 1
  • R 1 having the meaning given above with R and R 'representing: both - (alkyl) -COOY
  • one of the two H's and the other with Y is H or tBu.
  • Z is C1-C8 alkyl
  • Step (a ') comprises steps (i), (ii) and (iii)
  • Step (i) consists of the reaction of RR 'NH with O where HaI is a halogen atom in the presence of a base to give
  • Step (ii) consists of the RR'N reaction
  • Step (iii) consists of the reaction of RR'N with RiCHO in the presence of a base to give
  • Step (b) consists in the optional hydrolysis of the ester obtained in step (a) or (a ') to give R 1
  • Step (c) consists in reacting the compound obtained in the preceding step (b) to obtain a reactive function of the proteins.
  • This reactive function of the proteins is preferably an NHS ester function.
  • Peptide coupling with a carboxylic acid is preferably carried out in the presence of a coupling agent, such as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), hydrochloride of 1- (3-dimethylaminopropyl) -3- ethylcarbodiimide (EDC), carbonyldiimidazole (CDI), 2-H-benzotriazol-1-yl) - 1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2- (1H-benzotriazol-1-yl) 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) or else O- (7-azobenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), optionally combined with a coupling aid such as N-hydroxy succinimide (NHS), N-hydroxy
  • the supported chemical crosslinking agent is placed in the presence of the protein dissolved in a saline buffer in order to approach the physiological conditions. This results in a covalent attachment of the protein to the crosslinking agent via the reactive functions X and / or X '. Enzymatic digestion
  • the crosslinked proteins are digested.
  • the mixture is placed in the presence of an enzyme (e.g., trypsin) solubilized, preferably in a salt buffer.
  • an enzyme e.g., trypsin
  • the mass spectrometric analysis is carried out by MALDI-TOF spectrometry.
  • the samples are deposited either in dried drop or thin layer. Deposition in dried drop is preferred. It is carried out by co-crystallization of a drop of matrix solution and a drop of an analyte solution.
  • MASS SPECTROMETRY DATA ANALYSIS SOFTWARE
  • data from mass spectrometry analysis of peptide mixtures are analyzed by software.
  • the purpose of these software is to quickly provide a list of potential peptides unmodified or modified by the crosslinking agent used, corresponding to each m / z value of the mass spectrum.
  • the experimental values are compared with the theoretical values calculated from an in silico digestion of the studied protein model.
  • MSX-3D a prediction software, called MSX-3D, has been created within the Institute of Biology and Protein Chemistry (IBCP) in Lyon.
  • HCCA is commonly used in MALDI-TOF peptide mass spectrometry analyzes. It is able to absorb UV light from the MALDI source laser. The energy that it absorbs is restored in the form of thermal energy and allows the desorption of the co-crystallized compounds with the matrix.
  • HCCE methyl ⁇ -cyano-4-hydroxycinnamate ester
  • ⁇ -CNME methyl ester of the HCCA matrix. Due to their close structures, they both have similar characteristics: - Molecular weights (189 g / mol for HCCA and 203 g / mol for HCCE)
  • the HCCA matrix has two distinct pKa acid functional groups: the carboxylic acid function of pKai equal to 2 and the phenolic alcohol function of pKa 2 equal to 8.
  • the HCCE matrix has only one acid function: the phenolic alcohol function of pKai equal to 8.
  • the HCCA matrix thus has a carboxylic acid function that makes it possible to act as a proton donor during the ionization process involved in the MALDI source: this is why it is called an acid matrix.
  • Figure 1 Analysis of an equimolar mixture of the five model peptides JMV3346 to JMV3350 with the HCCA matrix.
  • FIG. 2 Mass spectrum of the peptide mixture resulting from tryptic digestion of unmodified cytochrome c (with HCCA matrix).
  • Figure 5 Mass spectrum of the peptide mixture from the trypsic digestion of unmodified apomyoglobin (with HCCA matrix).
  • the following examples illustrate the invention without limiting its scope.
  • the five model peptides are C-terminal amidated decapeptides having the same amino acid sequence in order not to influence their intrinsic ionization capacity in MALDI-TOF mass spectrometry. They were synthesized on solid support in Fmoc strategy. A first peptide JMV3346 is not labeled and serves as a reference for evaluating the effect of spectral discrimination.
  • Model peptides JMV3347, JMV3348 and JMV3349 were synthesized by coupling HCCA and two derivatives at position 4 (ie, ⁇ -cyanocinnamic acid or
  • Spacer arm 6.2 A ( ⁇ ) The direct coupling between the carboxylic acid function of HCCA and the secondary amine of diethyl iminodiacetate ester was first considered in order to synthesize JMV3378.
  • HCCA Unlike basic treatments, HCCA does not appear to be sensitive to TFA.
  • MALDI-TOF and model peptides labeled with HCCA supported the use of concentrated solutions of TFA.
  • Lane No. 1 has three steps.
  • the secondary amine of iminodiacetic diacid is first protected with a benzyloxycarbonyl group in order to obtain compound 1.
  • This reaction is carried out using a solution of benzyl chloroformate in sodium hydroxide in quantitative yield.
  • An esterification reaction of the compound 1 carried out at 55 ° C. in a solution of dimethylacetamide (DMAC) containing benzyltriethylammonium chloride, potassium bicarbonate and tert-butyl bromide afforded compound 2 in 17% yield.
  • DMAC dimethylacetamide
  • deprotection of the amine to give compound 4 was carried out with a yield of 50% by hydrogenolysis performed in methanol with hydrogen bubbling catalysed by palladium on carbon.
  • Lane # 2 has only two steps.
  • Compound 3 is obtained directly by a double nucleophilic substitution of the primary amine of benzylamine on two molecules of tert-butyl 2-bromoacetate. This reaction is carried out with a yield of 99% in a solution of dimethylformamide (DMF) containing potassium bicarbonate at 45 ° C.
  • DMF dimethylformamide
  • the hydrogenolysis of the benzyl group leading to the compound 4 was carried out quantitatively using bubbling hydrogen and palladium on carbon in 95% ethanol.
  • Route No. 2 seems to be more interesting because it makes it possible to obtain the final diester 4 in two stages of very good yield and uses a commercial tert-butyl ester.
  • the preparation of the tert-butyl ester is the limiting step in the No. 1 route.
  • this channel has one more step and does not make it possible to obtain the diester with as good a yield as the channel N ° 2.
  • SUBSTITUTE SHEET (RULE 26) potentially be involved in intermolecular side reactions and react with an active ester of another molecule.
  • Compound 4 is first acylated with 2-chloroacetyl chloride in a mixture of sodium hydroxide and tetrahydrofuran (THF) quantitatively.
  • the substitution of chlorine with potassium cyanide in dimethylsulfoxide (DMSO) at 65 ° C. makes it possible to obtain compound 6.
  • DMSO dimethylsulfoxide
  • a Knoevenagel reaction between compound 6 and 4-hydroxybenzaldehyde carried out in a mixture of pyridine and piperidine with 5O 0 C provides the compound 7 with a yield of 74%. During this reaction, the aldehyde can be used in large excess in order to optimize the yield of the reaction.
  • JMV3378 is synthesized by activating the two carboxylic acids with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccimmide in THF with a yield of 69%
  • DCC dicyclohexylcarbodiimide
  • N-hydroxysuccimmide in THF with a yield of 69%
  • the chemical crosslinking agent JMV3378 was thus obtained through a seven-step synthetic route with an overall yield of 31%.
  • a second homobifunctional crosslinking agent having two NHS ester functions has been synthesized N-hydroxysuccimmidyl 5- ( ⁇ -cyano-4-hydroxycinnamido) isophthalate (see scheme 8).
  • This crosslinking agent has a spacer arm of similar length. to that of the compound JMV3378, however its structure is rigid because of the isophthalic aromatic ring. This rigidity can make it possible to obtain new structural information during crosslinking experiments on proteins
  • Figure 9 Synthetic route of the homobifunctional agent with 5-aminoisophthalic pattern.
  • the synthesis route is directly initiated by the acylation of 5-aminoisophthalic acid with 2-chloroacetyl chloride in a mixture of sodium hydroxide and THF.
  • Compound 9 is recovered by flash precipitation in petroleum ether with a yield of 96%.
  • Substitution of the chlorine atom of 9 with potassium cyanide in water in the presence of potassium bicarbonate provides compound 10 in 77% yield.
  • the diacid 11 is then obtained in a yield of 99% by means of a Knoevenagel reaction with 4-hydroxybenzaldehyde in a mixture of pyridine and piperidine at 50 ° C.
  • the compound is synthesized by activation of the two carboxylic acids with dicyclohexylcarbodiimide (DCC) and N- hydroxysuccinimide in THF.
  • DCC dicyclohexylcarbodiimide
  • N- hydroxysuccinimide in THF.
  • the purification conditions of this compound are identical to those of JMV3155.
  • a heterobifunctional crosslinking agent having a nonspecific reactive function can provide many more diverse information than a homobifunctional crosslinking agent.
  • a crosslinking agent having both an NHS ester function and a benzophenone photoactivatable function has been synthesized: N-hydroxysuccinimidyl (S) -3- (4-benzoylphenyl) -2- ( ⁇ -cyano-4-hydroxycinnamido) propanoate or JMV3480 (see Figure 10).
  • Scheme 11 Synthetic route of the heterobifunctional agent JMV3480.
  • Example 3 Applications of the crosslinking agent JMV3378 to study model proteins Cytochrome c and horse heart apomyoglobin were used as model proteins.
  • the protein samples were analyzed by MALDI-TOF mass spectrometry using the sinapinic acid matrix as a dried drop deposit. After checking the degree of crosslinking obtained, the samples were digested with trypsin. Each peptide mixture obtained was analyzed by mass spectrometry with the HCCA acid matrix in dried drop type deposition and with the HCCE matrix in thin layer type deposition.
  • Table 1 List of experimental masses and predicted peptides crosslinked by the
  • the detected signals may correspond to different types of peptide crosslinks (type 0, 1 and 2). After verifying the theoretical distances between the side chains of cytochrome c lysines, a total of 8 types of crosslinking reactions of type 1 and 2 were identified (see Figure 12).
  • Table 2 List of experimental masses and predicted peptides crosslinked by JMV3378.
  • LSDGEWQQV 10 LNVWGkVEAD 20 IAGHGQEVLI 30 RLFTGHPETL 40 EKFDkFKHLj- 50
  • the spectrometer is equipped with a SCOUT source and the desorption / ionization is carried out using a nitrogen laser with a wavelength of 337 nm and the frequency used is 50 Hz.
  • the laser power can be modulated thanks to an attenuator.
  • the calibration of the MALDI - TOF spectrometric analyzes was carried out in external mode. Two calibration kits were used:
  • the external calibration is performed by depositing one of these commercial mixtures with a suitable matrix (eg ⁇ -cyano-4-hydroxycinnamic acid (HCCA) for the “standard calibration peptide II", sinapinic acid (AS) for the “Standard Protein Calibration I”) near the deposit (s) to be analyzed.
  • a suitable matrix eg ⁇ -cyano-4-hydroxycinnamic acid (HCCA) for the “standard calibration peptide II", sinapinic acid (AS) for the “Standard Protein Calibration I
  • Samples are deposited on a Bruker Daltonics MTP 384 target plate in polished steel using two methods.
  • Dried Drop Deposition has been preferred in most assays using the HCCA matrix. It is carried out by co-crystallization of a drop of matrix solution and a drop of an analyte solution. Each deposit can contain between 0.5 and 1 ⁇ L of each solution.
  • HCCA matrix solutions are prepared by saturation in a water / ACN / TFA solution (50: 50: 0.1).
  • HCCE consists of depositing 0.5 ⁇ L of a saturated solution of HCCE acetone in band on two targets marked on the deposit plate.
  • 0.5 ⁇ L of analyte solution is deposited on a target containing the dried matrix deposition. Due to the difficulty of reproducing the deposit aspect by this method, it is preferable to renew the deposit a second time for each analysis.
  • 0.5 ⁇ L (0.5 pmol) of peptide solution at 1 ⁇ M in a water / ACN / TFA mixture (50: 50: 0.1) are co-crystallized on a deposit of 0.25 ⁇ L of saturated neutral HCCE matrix in acetone.
  • the model peptides JMV3346, JMV3347, JMV3348, JMV3349 and JMV3350 were manually synthesized on a solid support according to an Fmoc strategy.
  • Peptide 3 was synthesized on a solid support according to an Fmoc strategy using a microwave synthesizer.
  • the three peptides were synthesized with as coupling agent HBTU in the presence of NMM on a Fmoc-Rink-amide-aminomethyl-polystyrene resin (100-200 mesh, 0.7 mmol / g).
  • the medium is placed in an ice bath to stabilize its temperature at 0 0 C. 2 times
  • 0.800 g of compound 1 are dissolved in the presence of 1.640 g of benzyltriethylammonium chloride in 75 ml of DMAC under argon. 10 g of K 2 CO 3 are introduced into the medium and then 30 ml of tert-butyl bromide are added. The medium is stirred vigorously at 55 ° C. for 30 hours. The medium is cooled in the open air to room temperature.
  • the medium is placed in an ice bath and 400 ml of cold water are added.
  • the aqueous phase is extracted with 5 times 100 ml of AE.
  • Each organic phase is then extracted with 3 times 50 ml of saturated aqueous NaHCO 3 solution and 2 times 50 ml of water.
  • the combined organic phases are dried with MgSO4, filtered on a frit then the solvent is evaporated under reduced pressure.
  • the compound is purified by column chromatography with the following eluent:
  • the balloon is purged with argon.
  • the medium is filtered on celite and rinsed several times with methanol.
  • the solvent is then evaporated off under reduced pressure and the medium is then taken up in 100 ml of EA and extracted with twice 50 ml of a saturated aqueous solution of NaHCO 3 then 2 times 50 ml of water.
  • the organic phase is dried with MgSO 4 and filtered on a frit. The solvent is then evaporated under reduced pressure.
  • the compound is purified by column chromatography with eluent: EP / AE (95: 5).
  • the reaction is stopped after 1 hour by the addition of 400 ml of water.
  • the solvent is then evaporated under reduced pressure.
  • the medium is taken up with 200 mL of water and then extracted with 4 times 200 mL of EA. Each organic phase is washed with 250 mL of brine.
  • the organic phases are combined and then dried with MgSO 4 and filtered on a frit.
  • the solvent is evaporated under reduced pressure.
  • the crude reaction product is purified by column chromatography using an AE / EP mixture (3: 7) as eluent.
  • 250 mg of compound 7 are dissolved in 50 ml of a TFA / TIS / H 2 O mixture (95: 2.5: 2.5) with magnetic stirring. After 1 hour, the solvent is evaporated under reduced pressure. The medium is taken up in 50 ml of a water / ACN mixture (50:50), frozen with liquid nitrogen and freeze-dried.
  • Synthesis protocol 775 mg of compound 8 are dissolved in 100 mL of THF and 2.115 g of DCC are added. After 10 minutes, the medium is cloudy and 715 mg of HOSu are added.
  • the solvent is evaporated under reduced pressure and the medium is taken up with a minimum of cold DMF and placed in an ice bath for 10 minutes. Dicyclohexylurea precipitates to a white powder. The medium is filtered on a sinter of porosity 4 cold. The process is repeated a second time. The solvent is then evaporated under reduced pressure. The precipitate is dissolved in 300 mL of EA and extracted with 2 times 100 mL of an aqueous solution of 1M KHSO4. The organic phase is dried with MgSO 4, filtered on frit and the solvent is evaporated under reduced pressure.
  • the solvent is evaporated under reduced pressure.
  • the medium is taken up with 200 ml of EA and then extracted with 3 times 80 ml of an aqueous solution of KHSO 4 IM.
  • the organic phase is then dried with MgSO 4 and then filtered on a frit.
  • the solvent is evaporated under reduced pressure.
  • the compound is purified by preparative HPLC with the following gradient:
  • fractions containing the purified compound are pooled, frozen with liquid nitrogen and lyophilized.
  • the dicyclohexylurea precipitated to a white powder is filtered on a sinter of porosity 4 cold.
  • the solvent is then evaporated off under reduced pressure and then the medium is taken up in 150 ml of EA and extracted with 4 times 100 ml of an aqueous solution of 1M KHSO 4 .
  • the organic phase is dried with MgSO4, filtered on frit and the solvent is evaporated under reduced pressure.
  • 3,516 of 5-aminoisophthalic acid are dissolved in 150 ml of THF containing 20 ml of a 10% aqueous sodium hydroxide solution. After 5 minutes with magnetic stirring, the diacid is completely solubilized. 7.729 mL of 2-chloroacetyl chloride are slowly added to the medium and the pH of the solution is maintained at 10 by addition of 1N sodium hydroxide solution under strong magnetic stirring. The reaction is exothermic. After stirring for 5 minutes, the THF is evaporated under reduced pressure. The medium is taken up with 400 ml of EA and then extracted with 2 times 100 ml of an aqueous solution of KHSO 4 IM. The aqueous phases are extracted with 2 times 100 ml of AE.
  • the reaction is stopped and the medium is acidified by adding a few milliliters of a 6N aqueous HCl solution to a pH below 3.
  • the medium is then extracted with 8 times 100 ml of AE.
  • Each organic phase is washed with 100 mL of an aqueous solution of KHSO 4 IM.
  • the organic phases are combined and dried with MgSO 4 and filtered on a frit. The solvent is evaporated under reduced pressure.
  • 500 mg of compound 10 are dissolved in 200 ml of pyridine and 100 ⁇ l of piperidine are added. After 5 minutes, 246 mg of 4-hydroxybenzaldehyde are added and the medium is placed in an oil bath at 50 ° C. for 3 hours. The medium is cooled slowly to room temperature. It is added dropwise to 150 ml of an aqueous solution of KHSO 4 IM. The pH is below 4. A yellow precipitate appears and the solution is triturated with a spatula. The precipitate is filtered on a porosity frit 4 and then dissolved in DMF. The solvent is evaporated under reduced pressure. The solid is taken up in 150 ml of an aqueous solution of KHSO 4 IM and then triturated and filtered as above.

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CN103483223B (zh) * 2013-09-12 2015-12-09 吉林大学 α-氰基-4-羟基肉桂酸正丙酯、制备方法及应用
CN107129455B (zh) * 2016-02-29 2019-11-15 北京大学 一种多功能化学交联剂及其制备方法与应用
CN112979674B (zh) * 2019-12-02 2023-04-07 中国科学院大连化学物理研究所 一种多功能交联剂及其制备方法和应用

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US7368290B2 (en) * 2003-05-12 2008-05-06 Sandia National Laboratories Structural determination of intact proteins using mass spectrometry
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CN111505175B (zh) * 2019-01-30 2022-07-05 上海科技大学 一种质谱用交联剂及其制备与应用

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