EP0444068A1 - Trialkylsilyle esters d'acides amines et leur emploi dans la synthese de peptides - Google Patents

Trialkylsilyle esters d'acides amines et leur emploi dans la synthese de peptides

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Publication number
EP0444068A1
EP0444068A1 EP89912478A EP89912478A EP0444068A1 EP 0444068 A1 EP0444068 A1 EP 0444068A1 EP 89912478 A EP89912478 A EP 89912478A EP 89912478 A EP89912478 A EP 89912478A EP 0444068 A1 EP0444068 A1 EP 0444068A1
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European Patent Office
Prior art keywords
protected
amino
group
amino acid
formula
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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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EP89912478A
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German (de)
English (en)
Inventor
Anthony Atkinson
Michael Roger Calder
Ram Prakash Sharma
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Public Health Laboratory Service Board
University of Southampton
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Public Health Laboratory Service Board
University of Southampton
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C07K1/088—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents containing other elements, e.g. B, Si, As
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers

Definitions

  • This invention relates to novel procedures for synthesising peptides and to novel reagents for use in these procedures.
  • the invention also provides kits of reagents for use in carrying out the procedures of the invention.
  • Peptides. are linear polymers derived from amino acids and generally have the formula
  • a , A ... A are the residues of the amino acids making up the peptide.
  • Such amino acids may be represented by the formulae
  • Peptides may additionally include sub-units derived from imino acids (also termed "heterocyclic amino acids”) such as, for example, proline. These sub-units may be represented by the formula
  • amino acids that form the sub-units of proteins. These amino acids, which in nature are of the L-configuration, have been described as "naturally occurring amino acids”.
  • the side chain R may be a hydrocarbyl group, for example an an alkyl or aryl group, as in alanine, valine, leucine, isoleucine, methionine and phenylalanine.
  • the residue R may contain polar, but non-ionizable groups, as in. asparagine, glutamine, threonine and serine, or ionizable groups, such as in aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, tryptophan and cysteine.
  • Peptides may be synthesised by two distinct routes, biological and chemical. This invention is concerned with the second route.
  • next amino acid precursor is added and couples with the N-terminal benzyloxycarbonyl-proteced amino acid precursor:
  • any reactive groups (other than the -NH_ group and -COe group) which might enter into undesired side reactions need to be protected from attack by other active species.
  • the Merrifield strategy utilises an N-protected amino acid HOOC.A.NHProt (XI) which is converted to an activated form e.CO.A.NHProt (XII) Traditionally tertiary butoxycarbonyl (tBoc) has been the protective group of choice.
  • the initial (C-terminal) amino acid NH_.A n .C00H is covalently bonded to a resin via its carboxyl group. This may be achieved by reacting a resin having free -CH-C1 groups with a protected amino acid caesium salt
  • Resin.CH 2 0.CO.A n .NH 2 (XIV) and the resin particles, with the nth amino acid attached, are reacted with the next (n-l ' )th activated protected amino acid e.C0.A n_1 .NHProt (XII 1 ) which is to form the (n-l)th amino acid of the desired peptide, i.e.
  • tertiary butoxy oxycarbonyl (tBoc) has been the protective group of choice.
  • this protective group requires the use of strong acids in both deprotection and final cleavage.
  • acid-stable protecting moieties e.g. benzyl groups
  • protecting moieties for protecting the side chains of amino acids such as the basic amino acids (Lys, His and Arg) .
  • the peptide chain is built amino acid-by-amino acid starting with the one intended to be the carboxyl or C-terminal residue of the peptide chain.
  • This C-terminal amino acid is covalently bonded via its -C00H group to an insoluble resin support.
  • a chloromethylated polystyrene resin is most often used because the benzyl ester group formed with the C-teminal amino acid may be readily cleaved.
  • the next amino acid to be used in the peptide chain being formed after having its amino group blocked with a t-butyloxycarbonyl (t-Boc) group, is activated with a coupling activator, such as dicyclohexyl- dicarbondiimide (DCC) , and coupled to the deprotected amino group of the amino acid attached to the resin - (H 2 NCHRC00-CH 2 -Resin XVIII' )
  • a coupling activator such as dicyclohexyl- dicarbondiimide (DCC)
  • the t-Boc group is then removed by treatment with trifluoracetic acid, and after neutralisation the growing peptide chain attached to the resin is ready for addition of the next amino acid precursor. This sequence is repeated until a peptide chain having the required structure is synthesized. Having the growing peptide chain attached to resin particles large enough to be separated from a liquid phase by filtration, simplifies removal of excess reagents and washing of the resin particles in the many repetitious steps involved, and makes the procedure more convenient for the synthesis of larger peptides and proteins. When the peptide chain is complete, it is cleaved from its resin support by a reaction that does not affect the peptide linkages. Typically, hydrogen fluoride is utilized for cleaving the peptide from the resin.
  • Fmoc fluorenylmethoxy carbonyl
  • the tBoc and the Fmoc systems require the use of respectively stringent acidic conditions and basic conditions in order to remove the tBoc and Fmoc protective groups.
  • strong acids such as trifluoroacetic acid have to be used and bases such as, for example, piperidine in DMF, are required in the Fmoc system.
  • bases such as, for example, piperidine in DMF.
  • the use of trifluoroacetic acid in order to remove the tBoc protective group is particularly troublesome as the repeated contacting of the resin particles at each cycle with the trifluoroacetic acid reagent causes degradation of the resin.
  • activated protected amino acids symmetrical anhydrides
  • Such systems require the use of reagents which are relatively stable because supplies of all the reagents necessary to synthesise a desired peptide need to be stored for relatively long periods.
  • activated protected amino acids are generally unstable and susceptible to decomposition in the presence of even trace amounts of water and many commercially available activated protected amino acids are prohibitively expensive.
  • the tBoc and Fmoc procedures require the use of an excess of the activated protected amino acid reagent in each cycle in order to ensure that the N-terminal amino acid residues of the resin-bound peptide chain react to completion with said reagent.
  • the reagent needs to be present in a mole ratio of at least 4 moles of reagent per mole of N-terminal amino residues. As each mole of reagent is formed of two moles of amino acid, this represents a requirement for an 8x excess of amino acid starting material.
  • the present invention overcomes the problems associated with prior art solid state peptide synthesis procedures by binding the first amino acid of the desired peptide to a resin via its amino group, activating the carboxyl group of the thus-coupled amino acid and utilising a carboxy-protected amino acid reagent to produce a peptide bond by nucleophilic attack on the activated carboxy group of the bound amino acid. Subsequent amino acids are coupled in a similar manner.
  • reactive side chains of the carboxy- protected amino acid may be protected (if necessary) by known procedures.
  • the carboxy-protective groups utilised in accordance with the invention are O-silyl ester groups.
  • amino acids having O-silyl ester protective groups are novel and form a further aspect of the invention.
  • the present invention in its process aspects, is based on the realisation that distinct advantages ensue from the synthesis of peptides by a procedure in which an initial amino acid forming the N-terminus of the desired peptide forms the starting point for the step-wise synthetic route, the carboxyl group of this amino acid is activated and the activated carboxy group is reacted with the free amino group of a carboxy-protected amino acid.
  • the present invention provides a process for producing a peptide of the formula
  • step (B) reacting the activated solid phase reactant from step (A) with a carboxy-protected amino acid of formula
  • n is a positive integer
  • x is 0 or a positive integer
  • Prot is a silyl group, each A, which may be the same or different, either represents the residue of an amino acid or the structure NH.A is the residue
  • the process of the invention comprises the following steps:
  • H.NH.A.CO.OProt with a support material having groups capable of forming a covalent bond with the amino or imino group of the carboxyprotected amino acid, and removing said protective group Prot.
  • amino acid residues A may be derived from a wide range of amino acids, which is not restricted to the amino acids commonly occuring in natural proteins.
  • amino acids may be represented by the general formula
  • N j represents either the group .NHB wherein B represents a
  • R may be defined as being selected from C._. n hydrocarbyl groups optionally substituted by one or more substituents selected from hydroxy, thio, C « _ alkylthio, C._ i . alkoxy, carboxy, acetamido, guandidyl, 3"indolyl and 2-imidazolyl.
  • hydrocarbyl groups include branched and straight chain alkyl groups (which preferably contain from 1 to 6 carbon atoms) and aryl groups, particularly phenyl groups, which may be unsubstituted or substituted by one or more moieties selected from C. alkoxy, nitro and halogen.
  • the groups of the support which are capable of forming covalent bonds with the amino or imino groups of the carboxy protected amino acid are preferably activated carboxy groups of formula -COe or activated oxycarbonyl groups of formula -OCOe (e is as defined above).
  • silyl groups are those having the formula Si(R 1 ,R 2 ,R 3 ) (XXXI) wherein . , R_ and R fur, which may be the same or different represent saturated or unsaturated hydrocarbyl groups containing 1 to 20 carbon atoms, which may be unsubstituted or substituted by one or more groups selected from C j ,-alkoxy, nitro, tri(C, j ,alkyl)silyl and halogen.
  • the aforementioned hydrocarbyl groups include branched and straight chain alkyl and alkenyl groups (which preferably contain from 1 to 6 carbon atoms) and aryl groups, particularly phenyl groups.
  • silyl groups have the formula Si(R R 2 ,R 3 ) (XXXI) wherein 1 , R restroom and R_, which may be the same or different represent C_._ 2n alkyl groups.
  • the alkyl groups may be straight and branched (and a combination of both may be present). Examples include methyl, ethyl, n-propyl, n-butyl, sec-butyl, t-butyl and dodecyl
  • the carboxy protected amino acids XXVII may be formed from the corresponding amino acid or protected amino acid by converting a free carboxyl group to a silyl ester group using known silylating agents.
  • Such agents may generally have the structure X-SitR.R-R- where R., R_ and R_ are as defined above and X is a leaving group.
  • suitable leaving groups X include (i) Cl, Br and I (as in trimethylsilyl chloride) (ii) alkoxy, e.g. ethoxy and methoxy (as in trimethylethoxy silane) (iii) secondary amino, for example di-C.._ i ,-alkylamino.
  • a typical secondary amino group is dimethylamino (as in N-trimethylsilyldimethyl araine) (iv) dlsilazano (as in hexa ethyl disilazane)
  • the leaving group 6 should be selected according to known criteria such that the carbon atom to which it is attached is activated sufficiently that it can undergo nucleophilic attack by a lone electron pair on the free amino or imino group of the carboxy protected amino or imino acid.
  • group e should be what is termed "good leaving group”.
  • Particularly effective leaving groups e are strongly electron withdrawing.
  • reaction(s) which introduce leaving group e minimise racemisation and that also the activated intermediate containing e should not be susceptible to racemisation.
  • Examples of leaving groups e are groups having the structure
  • Compounds having leaving groups (a) may be produced by reacting a compound having a carboxyl-function with a corresponding carbodiimide.
  • Compounds having leaving groups (b) and (c) may be made from compounds containing leaving groups (a) by reaction with pentafluorophenol and 1-hydroxybenzotriazole.
  • the groups -COe represent carboxyl groups activated by known procedures, for example by reaction with a diimide such as for example, dicyclohexylcarbodiimide, by formation of a hydroxybenzotriazole ester or pentafluorophenyl ester or by using benzotriazole-l-yl-oxy-tris-(dimethyl-amino)phosphonium hexafluorophosphate.
  • a diimide such as for example, dicyclohexylcarbodiimide
  • carboxyl activation and addition of the carboxy-protected amino acid may be carried out successively or simultaneously.
  • the carboxyl group can be activated prior to addition of protected amino acid, e.g. carboxyl activated by formation of the hydroxybenzotriazole ester or pentafluorophenyl ester.
  • the "activation" step (A) and the "addition of amino acid step” - Step (B) can be achieved in one step using e.g. benzotriazole-1-yl-oxy-tris(dimethyl-amino)phosphoniurn hexafluorophosphate (known as BOP or Castro's reagent), base and protected amino acid (stoichimetry 1:2:1).
  • BOP benzotriazole-1-yl-oxy-tris(dimethyl-amino)phosphoniurn hexafluorophosphate
  • base and protected amino acid stoichimetry 1:2:1
  • the invention may also be used to synthesise peptides in the liquid phase, thus according to a further aspect of the invention there is provided a process for producing a peptide of the formula
  • Prof represents an -NH_ protecting group with a carboxy protected amino acid of formula
  • the protective groups Prof are preferably t-butoxycarbonyl (tBoc), fluorenylmethoxycarbonyl (Fmoc) or triphenylmethyl (trityl or Trt) groups.
  • the silyl groups are preferably of the formulae specified above, i.e. of the formula
  • R_. , R_ and R_ which may be the same or different represent hydrocarbyl groups containing 1 to 10 carbon atoms, preferably of the formula
  • silyl esters suitable for use in accordance with the process aspects of the invention are novel. These include the following:
  • N-formyl tryptophan 4-methoxy benzyl cysteine, and dinitrophenyl histidine.
  • H.NH.A.CO.O.SKR ⁇ R-) (XXXIV) wherein R.. , R and R_, which may be the same or different represent alkyl groups having 1-20 carbon atoms, and H.NH.A.CO.0 is the residue of an ⁇ -amino acid selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, histidine, arginine, phenylalanine, tyrosine, tryptophan, cysteine, methionine and proline, with the proviso that where the amino acid is other than lysine, aspartic acid or glutamic acid, at least one of R 1 , R_ and R render is other than methyl.
  • novel silyl esters of the invention posess protective groups of the amino function
  • protective groups of the amino function such groups are preferably t-butoxycarbonyl (tBoc) groups, fluorenylmethoxycarbonyl (Fmoc) groups or triphenylmethyl (Trt) groups.
  • amino-protecting groups are preferably t-butoxycarbonyl, fluorenylmethoxycarbonyl or triphenylmethyl groups.
  • kits comprising such esters, particularly kits of reagents for use in the synthesis of peptides comprising supplies of silyl esters of amino acids, and a supply of a reagent for activating carboxyl groups to convert them to activated carboxyl groups capable of reacting with amino groups to form peptide bonds.
  • the solid phase procedure for the synthesis of a peptide of predetermined structure by the process of the present invention typically involves the following steps:
  • Trialkylsilyl esters of amino acids are most conveniently prepared according to the invention by the reaction of a trialkylsilyl chloride with an N-blocked amino acid.
  • Many N-blocked amino acids may be obtained commercially, however in general they may be prepared in the well known manner from the corresponding amino acid.
  • a t-Boc amino acid may be produced by reacting an amino acid with di-t-butyldicarbonate.
  • the blocking group may be any conventional amine blocking group such as t-butoxycarbonyl, fluorenylmethoxycarbonyl, phenylacetyl, acetoacetyl, N-benzylidine, benzoyl, benzyl, t-amyloxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, choroacetyl, carbamyl triphenylmethyl and the like, which can be readily cleaved, at the appropriate time, by acid or base, by hydrogenation or by enzymatic action.
  • amine blocking group such as t-butoxycarbonyl, fluorenylmethoxycarbonyl, phenylacetyl, acetoacetyl, N-benzylidine, benzoyl, benzyl, t-amyloxycarbonyl, benzyloxycarbonyl, p-toluenesulfonyl, choro
  • t-Boc t-butoxycarbonyl
  • reaction mixture is then acidified to pH 3 with citric acid or IN HCl and extracted 2 x with ethyl acetate.
  • the organic phase is then evaporated to isolate the t-Boc-protected amino acid.
  • the individual amino groups generally require different blocking groups so that only the amino group destined to form the peptide bond is subsequently de-blocked.
  • the £-amino group is preferably blocked with a benzyloxycarbonyl group prior to blocking the ⁇ -amino group with t-Boc. This may be achieved by forming a copper complex with the ⁇ -NH_ and C00H groups, reacting with benzyloxycarbonyl chloride (Z-chloride) and then decomplexing and reacting with t-butyl dicarbonate as described above.
  • Amino acids having additional carboxyl groups are preferably in the form of their benzyl esters.
  • hydroxyl groups of serine and threonine can be blocked, if necessary, by formation of benzyl esters and the hydroxyl group of tyrosine by formation of the 2-bromobenzyloxycarbonyl derivatives.
  • the indolyl nitrogen of tryptophan may be blocked by for ylation and the thio group of cysteine by 4-methylbenzylation. Histidine may be protected as a dinitrophenyl derivative.
  • the reaction between the N-protected amino acid and the trialkylsilyl chloride is effected in an inert solvent, preferably an aprotic solvent such an ether, for example diethyl ether or tetrahydrofuran or in dimethyl formamide.
  • an inert solvent preferably an aprotic solvent such an ether, for example diethyl ether or tetrahydrofuran or in dimethyl formamide.
  • the trialkylsilyl chloride neat or dissolved in the solvent, is added dropwise over a period of time to a solution of the N-t-Boc blocked amino acid in a reaction vessel chilled in a cold water or water-ice bath.
  • the reaction medium preferably contains a tertiary amine, e.g. triethyl amine, pyridine or imidazole, to serve as a scavenger for the hydrogen chloride formed during the course of the reaction.
  • the reaction mixture is generally allowed to stand for about an hour to permit the reaction to reach completion. If the reaction mixture had been cooled, it may at this stage be permitted to warm to room temperature.
  • the solid tertiary amine hydrochloride, formed during the course of the reaction, may then be separated, most conveniently by filtration. Removal of the solvent from the filtrate under reduced pressure gives an almost theoretical yield of highly pure trialkylsilyl ester of the N-protected amino acid.
  • the silylation reaction is preferably carried out in an aprotic solvent in the presence of one equivalent of base (triethylamine, pyridine or imidazole).
  • base triethylamine, pyridine or imidazole
  • diethyl ether is the preferred solvent.
  • Dimethylformamide is preferred for production of the t-butyldimethylsilyl esters.
  • the reaction is generally complete in one hour at room temperature for the trimethylsilyl esters and in 2-8 hours for the t-butyldimethyl silyl esters.
  • the intermediate N-t-Boc blocked ester may be dissolved in dry ether and dry hydrogen chloride passed through the solution, preferably chilled to about 0-5°C, for about 30 minutes.
  • the product can be isolated either by filtration or by evaporation of the solvent.
  • Glu(OBz) - trimethylsilyl ester lie - trimethylsilyl ester
  • Trp(Formyl), His(DNP) Arg(Tos) and Lys(2-Cl-Z) trimethyl silyl esters were found to be unstable on prolonged storage at room temperature due to their hygroscopic nature.
  • the trialkylsilyl ester of the intermediate peptide precursor is readily cleaved with methanol or methanol/acetic acid and the liberated carboxylic acid group activated for coupling with a conventional activator such as DCC/HOBT. This sequence is repeated until a peptide chain of the required structure is formed. Then, the trialkylsilyl ester at the C-terminal end of the chain is cleaved and the N-blocking group removed with hydrochloric acid/diethyl ether to liberate the peptide. In order to prepare a pure peptide product, it is generally necessary to isolate and/or separate unreacted reagents from one or more of the intermediate peptide precursors produced during the course of the synthesis.
  • Tyr-Ala-Ala-Phe-Leu-OH and Ala-Ala-OH were prepared using the liquid phase procedure described above.
  • the trialkysilyl ester of the initial amino acid in the peptide chain to be formed is bound via its amino group to the insoluble resin.
  • the resin is a polystyrene resin bearing a -CH DC0C1 substituent, e.g.
  • the carboxyl group is then deprotected by cleaving the trialkylsilyl ester with methanol or methanol/acetic acid, and the thus liberated carboxy group activated with DCC/HOBT.
  • a trialkylsilyl ester of a second amino acid is added, and the amino group of the second amino acid couples with the activated carboxyl group of the first amino acid, which remains bound to the resin. This sequence of reactions is repeated until a peptide chain of the desired structure is formed.
  • the last trialkylsilyl ester is cleaved with methanol or methanol/acetic acid, and the peptide is liberated from the resin under acidic conditions e.g. HBr/acetic acid, trifluoromethanesulphonic acid (TFMSA) in trifluoracetic acid (TFA) and HP.
  • the reagents and reaction condition utilized are milder than those customarily utilized (in the present process, methanol may be used to cleave the protecting group; (c.f. the Merrifield procedure: trifluoroacetic acid is used to remove the N-t-Boc blocking group and Sheppard's Fmoc system: the base piperidine is used to remove the N-terminal blocking group) .
  • a further advantage of the method of the invention is that it is possible to test for completion of the individual reaction stages by measuring the conductivity of the reaction medium.
  • the trimethylsilyl ester of L-alanine was prepared by the following procedure.
  • N-t-Boc-L-alanine (10 m.moles) was dissolved in anhydrous diethyl ether (40 ml) and pyridine (10 m.moles) was added. The solution was cooled to 0°C on an ice bath and stirred.
  • Trimethylsilyl chloride (11 m.moles) was then added dropwise. Immediately a white precipitate began to form (pyridine-HCl) . The mixture was stirred for 1 hour and then allowed to warm to room temperature. The white solid was removed by filtration and the organic layer was evaporated under reduced pressure to give N-t-Boc-L-alaninetrimethylsilyl ester as a liquid in a yield of 94#, which was stored under nitrogen. All operations were carried out under anhydrous conditions.
  • the product was characterised by its infra-red (IR) spectrum, H Nuclear Magnetic Resonance (NMR) spectrum and by thin layer chromatography (tic) analysis.
  • IR infra-red
  • NMR Nuclear Magnetic Resonance
  • tic thin layer chromatography
  • the H NMR spectrum were recorded on a R-1500 Hitachi (60 MHz) instrument using tetremethylsilane (TMS) as the internal standard) .
  • Trimethylsilyl esters of the title amino acids and protected amino acids were prepared by the procedure described in Example 1.1 above, but substituting the following amino acids and protected amino acids for N-t-Boc-L-alanine: t-Boc-glycine, L-valine, L-phenylanine, L-isoleucine, L-leucine, N-2-Cl-Z-L-lysine, L-methionine, L-asparagine, L-proline, S-4-MBzl-L-cysteine, 0-2-Br-Z-L-tyrosine, 0-Bzl-L-threonine, 0-Bzl-L-glutamic acid, 0-Bzl-L-aspartic acid, L-glutamine, N-Tos-L-arginine, N-formyl-L-tryptophan, N-DNP-L-histidine and 0-Bzl-L-serine.
  • Diethylether was used as solvent in all cases except for Met, Asp, Glu, Arg and His for which dichloromethane was used.
  • N-t-Boc-L-alanine trimethyl silyl ester (10 m.moles) prepared as described in Example 1.1 was dissolved in anhydrous diethyl ether (50 ml) . Dry hydrogen chloride gas was then bubbled into the solution until saturation. A white precipitate formed which was filtered, washed with diethyl-ether to remove traces of HCl and was dried under reduced pressure. The solid was then stored tinder inert atmosphere (N_) .
  • the resulting L-alanine-trimethyl silyl ester hydrochloride was characterised by its Infra-red spectrum, and by melting point determination.
  • L-Phenylalanine (5) L-Isoleucine, (6) L-Leucine, (7) 2-Cl-z-L-Lysine, (8) L-Methionine, (9) L-Asparagine, (10) L-Proline, (11) (4-MBzl)-L-Cysteine, (12) (2-Br-Z)-L-Tyrosine, (13) (Bzl)-L-Threonine,
  • Trimethylsilyl esters of the title amino acids and protected amino acids were prepared by the procedure described in Example 2.1 above, but substituting the corresponding t-Boc amino acid and t-Boc protected amino acid trimethylsilyl esters (2) to (20) referred to in Table 1 for t-Boc L-alanine trimethylsilyl ester.
  • t-Boc-L-alanine-t-butyldimethylsilyl ester was prepared by the following procedure.
  • N-t-Boc-L-alanine (10 m.moles) was dissolved in N,N-dimethylformamide (20 ml) and imidazole (20 m.moles) added to act as base and catalyst.
  • the mixture was diluted with diethyl ether (60 ml) and washed with 10 sodium bicarbonate (1 x 20 ml), water (1 x 20 ml), 0.1 M hydrochloric acid (1 x 20 ml), and water (2 x 20 ml).
  • the organic layer was then dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give N-t-Boc-L-aianine-t-butyldimethyl silyl ester as an oil.
  • N-t-Boc-L-alanine-t-butyldimethyl silyl ester was characte ⁇ rriisseedd bbyy iittss IInnffrraa--rreedd ssppeeccttrruumm,, HH nnuuccll ⁇ ear magnetic resonance spectrum and by thin layer chromatography (tic).
  • t-Boc (Tos)-L-Arginine t-Butyl-dimethylsilyl esters of the title t-Boc amino acids and protected amino acids were prepared by the procedure described in Example 3.1 above, but substituting the following t-Boc amino acids and protected amino acids for N-t-Boc-alanine: t-Boc Phenylalanine t-Boc O-Bzl-L-Aspartic acid, t-Boc L-Leucine, t-Boc 2-Br-z-L-Tyrosine, t-Boc L-proline t-Boc L-Valine, t-Boc DNP-L-Histidine and t-Boc L-Isoleucine, t-Boc L-Glycine, t-Boc Tos-L-Arginine.
  • t-Boc-L-alanine t-butyldimethylsilyl ester (10 m.moles) was treated with 25% trifluoroacetic acid in dichloromethane (25 ml) for 30 minutes at room temperature.
  • the solvents i.e. dichloromethane, trifluoroacetic acid
  • dichloromethane, trifluoroacetic acid were then removed under reduced pressure at room temperature and an oil was obtained in high yield.
  • the resulting L-alanine t-butyldimethylsilyl ester was characterised by its Infra-red spectrum and H n.m.r. spectrum.
  • a resin containing -CH D.C0.C1 groups was prepared from a standard, commercially available "Merrifield” resin (bearing -CH intendC1 groups) by the following procedure (see Scheme 2).
  • the resin as prepared in Example 5 was placed in a reaction vessel and washed with dimethylacetamide (DMA) (3 x 10 ml).
  • DMA dimethylacetamide
  • Leu t-butyl dimethylsilyl ester was added in DMA (10 ml) followed by triethylamine and the reaction vessel was shaken at room temperature for 2 hours. The liquid phase was drained out and the resin washed with DMA (2 x 10 ml) . Estimation of unreacted Leu derivative indicated that 0.61 mmol/g had been bound to the resin. The remaining free chloroformate on the resin was capped with diethylamine in DMA.
  • the t-butyldimethylsilyl Leu resin was treated with warm (4 ⁇ °C.) methanol (10 ml) for 30 minutes to remove the t-butyl dimethysilyl group.
  • the resin was washed with DMA (3 x 10 ml) and was shaken with H-Ala-0Si-(Me) 2 t-Bu in DMA in the presence of DCC/HOBT for 45 minutes.
  • the solvents were drained and the resin washed with DMA (3 x 10 ml). 100 mg resin was withdrawn from the reaction vessel.
  • the resin was then coupled as described above to H-Gly-0Si(Me)_t-Bu and 100 mg of
  • Resin-(C g H ,-CH 2 0-C0-NH-Leu-Ala-Gly-0Si(Me) 2 tBu was withdrawn as usual. It was then coupled to H-Val-0Si(Me) 2 tBu in a similar manner and the desired resin peptide was obtained.
  • the peptides Leu-Ala, Leu-Ala-Gly, and Leu-Ala-Gly-Val were released from the resin by the standard HF cleavage procedure.

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  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un nouveau procédé de synthèse de peptides, dans lequel on fixe des acides aminés successifs à une chaîne de polypeptides naissante par addition d'un acide aminé à protection carboxy à l'extrémité carboxy de ladite chaîne. Le procédé met en oeuvre l'emploi de silyle esters d'acides aminés, dont beaucoup sont nouveaux et constituent un autre aspect de l'invention.
EP89912478A 1988-11-19 1989-11-15 Trialkylsilyle esters d'acides amines et leur emploi dans la synthese de peptides Withdrawn EP0444068A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8827083 1988-11-19
GB888827083A GB8827083D0 (en) 1988-11-19 1988-11-19 Trialkylsilyl esters of amino acids & their use in synthesis of peptides

Publications (1)

Publication Number Publication Date
EP0444068A1 true EP0444068A1 (fr) 1991-09-04

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Application Number Title Priority Date Filing Date
EP89912478A Withdrawn EP0444068A1 (fr) 1988-11-19 1989-11-15 Trialkylsilyle esters d'acides amines et leur emploi dans la synthese de peptides

Country Status (10)

Country Link
EP (1) EP0444068A1 (fr)
JP (1) JPH04502908A (fr)
AU (1) AU4517289A (fr)
CA (1) CA2003308A1 (fr)
FI (1) FI912411A0 (fr)
GB (1) GB8827083D0 (fr)
HU (1) HUT58106A (fr)
IL (1) IL92338A0 (fr)
WO (1) WO1990005738A1 (fr)
ZA (1) ZA898774B (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9118669D0 (en) * 1991-08-30 1991-10-16 Univ Southampton Preparation of peptides by a soliphase synthesis and intermediates therefor
US7244815B2 (en) 2001-05-23 2007-07-17 The Curators Of The University Of Missouri Attachment and elaboration strategies for inverse peptide synthesis
US7214769B2 (en) 2001-05-23 2007-05-08 The Curators Of The University Of Missouri Method for inverse solid phase synthesis of peptides
EP2060580A1 (fr) 2007-11-19 2009-05-20 SOLVAY (Société Anonyme) Procédé de préparation de peptides persilylés
CA2819510A1 (fr) * 2010-12-22 2012-06-28 Onyx Pharmaceuticals, Inc. Couplages peptidiques efficaces et leur utilisation dans la synthese et l'isolement d'un sel trisodique de cyclopenta[g]quinazoline
US9828393B2 (en) * 2013-07-22 2017-11-28 The Regents Of The University Of Colorado, A Body Corporate Silylalkyloxyaryl compounds and methods for treating cancer
EP3693377A4 (fr) 2017-10-03 2021-06-30 Nissan Chemical Corporation Procédé de production de composé peptidique
EP3922638A4 (fr) 2019-02-04 2022-11-30 Nissan Chemical Corporation Procédé de production d'un composé peptidique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9005738A1 *

Also Published As

Publication number Publication date
IL92338A0 (en) 1990-07-26
GB8827083D0 (en) 1988-12-21
CA2003308A1 (fr) 1990-05-19
HU896580D0 (en) 1991-08-28
HUT58106A (en) 1992-01-28
JPH04502908A (ja) 1992-05-28
ZA898774B (en) 1990-08-29
WO1990005738A1 (fr) 1990-05-31
AU4517289A (en) 1990-06-12
FI912411A0 (fi) 1991-05-17

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