EP0600996A1 - Preparation de peptides par une synthese en phase solide, et intermediaires pour la preparation - Google Patents

Preparation de peptides par une synthese en phase solide, et intermediaires pour la preparation

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Publication number
EP0600996A1
EP0600996A1 EP92918056A EP92918056A EP0600996A1 EP 0600996 A1 EP0600996 A1 EP 0600996A1 EP 92918056 A EP92918056 A EP 92918056A EP 92918056 A EP92918056 A EP 92918056A EP 0600996 A1 EP0600996 A1 EP 0600996A1
Authority
EP
European Patent Office
Prior art keywords
compound
general formula
group
solid support
amino acid
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.)
Withdrawn
Application number
EP92918056A
Other languages
German (de)
English (en)
Inventor
Ram Prakash Sharma
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.)
University of Southampton
Original Assignee
University of Southampton
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by University of Southampton filed Critical University of Southampton
Publication of EP0600996A1 publication Critical patent/EP0600996A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/04—Esters of silicic acids
    • 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
    • C07K1/061—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 protecting groups
    • C07K1/062—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 protecting groups for alpha- or omega-carboxy functions
    • 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
    • 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

Definitions

  • the present invention relates to the preparation of
  • polypeptides by solid phase synthesis, in particular synthesis on a solid support, to methods for preparing polypeptides under mild conditions and to protected carbonyl group intermediates for use in such preparations and methods.
  • a tertiary butyl oxycarbonyl group is a typical amine protecting group, for example tert-butyl oxycarbonyl (tBoc), which may be removed either by hydrochloric acid gas in organic solvents or by using trifluoroacetic acid (TFA).
  • tBoc tert-butyl oxycarbonyl
  • Solid phase polypeptide synthesis methods were devised principally by Merri field & Sheppard. Their methods involved conducting the reactions using solid phase conditions by covalently bonding the N-protected peptide via a terminal carboxyl group to a polymeric support, typically a resin, whilst the peptide is being synthesised.
  • tBoc is a preferred protective group, for Merri field's method.
  • a base labile group such as fluorenylmethoxycarbonyl (Fmoc) is used in Sheppard' s method.
  • WO90/057308 discloses a procedure for solid phase polypeptide synthesis which involves protecting the carboxyl group of a first amino acid by forming the corresponding trialkylsilyl ester, binding the trialkylsilyl ester of the first amino acid to a solid support via its amino group, removing the trialkylsilyl group to leave the support-bound amino acid, and activating the carboxyl group of the bound amino acid, for example by the use of DCC/HOBt (dicyclohexylcarbodiimide- hydroxybenzotriazole). Then the trialkylsilyl ester of a second amino acid is used to produce a peptide bond by nucleophilic attack on the activated carboxyl group ⁇ f the bound first amino acid.
  • DCC/HOBt dicyclohexylcarbodiimide- hydroxybenzotriazole
  • trialkylsilyl protecting groups disclosed in this prior art document include trimethylsilyl- (TMS) and
  • tert-butyldimethylsilyl- (TBDMS) esters of amino acids it is essential to employ a temporary N-protecting group, for example tBoc, to prevent N-silyl derivatisation.
  • tBoc temporary N-protecting group
  • the subsequent removal of the N-protection with acid yields the amino acid trialkylsilyl ester acid salt, which must be neutralised before the amino group can participate in peptide bond formation.
  • the preparation of these amino acid trialkylsilyl esters is thus a three-step process.
  • An example for the production of an amino acid TMS-ester is shown below: tBoc. NH . CHR. COOH + TMSC1 + Base tBoc . NH . CHR. COOTMS . + Base-HC1
  • the amino acid TMS-ester hydrochlorides produced are unstable and decompose even when stored under an inert atmosphere (e.g. N 2 ). If the hydrochloride is neutralised with base, for example pyridine, the resulting 'free' amino acid TMS-esters form a translucent "gel" in most solvents commonly used in peptide synthesis (for example in dichloromethane or dimethylformamide). The severe instability and poor solubility of the amino acid TMS-ester hydrochlorides renders them almost useless in solid-phase peptide synthesis.
  • base for example pyridine
  • Amino acid TBDMS-esters may be used in place of amino acid TMS-esters, but although the amino acid TBDMS-esters have been shown to be more stable and more soluble than the corresponding TMS-esters, they too have their drawbacks;
  • TBDMS-esters are used in solid-phase peptide synthesis.
  • Tri-t-butoxysilyl esters of aspartic and glutamic acids were also N-protected by the use of a t-Boc substituent.
  • the Gruszecki paper discloses the use of an aspartic acid and a glutamic acid tri-t-butoxysilyl ester in a liquid phase coupling reaction with an amino acid to produce a dipeptide.
  • the tri-t-butoxysilyl carboxy-protecting group is disclosed as being unstable in acidic conditions and the ester is said to be split in seconds in the present of 50% trifluoroacetic acid (TFA) in dichloromethane (DCM).
  • WO 90/05738 discloses amino acids or peptide derivatives linked to an insoluable resin support via a terminal amino group.
  • An example of such a resin which can bond to the amino group is a polystyrene resin bearing a -CH 2 OCOCl substituent.
  • This activation can be difficult to achieve and an accurate measure of the amount of amino acid bound to the resin is not easy to obtain.
  • this type of linkage is very stable and requires unfavourably harsh acidic conditions, such as liquid HF, for cleavage of the synthesised peptide.
  • N ⁇ -amino groups is known per se in conventional synthetic chemistry (e.g. cf. CARPINO, L.A. & HAN, G.Y. (1972) J. Org. Chem 37 3404) but harsh conditions have precluded the use of such linker compounds in peptide syntheses.
  • the present invention can provide a process for preparation of peptides, especially extended chain polypeptides, by a solid phase synthesis under mild conditions.
  • the mild conditions apply both in the chain extension steps and in the cleavage of the finished desired peptide from the solid phase support.
  • the present invention provides a process for the production of a peptide of general formula (I) (I)
  • n+m(x+1) which comprises the following steps:
  • n is a positive integer
  • n a positive integer
  • x is o or a positive integer
  • Z is a tri-t-alkoxysilyl, alkyl-di-t-alkoxysilyl
  • Y is a linker compound linked to the
  • A represents the residue of an amino acid
  • ii) represents a peptide residue; or iii) the structure NH.A represents the residue N ⁇ A of an imino acid HN ⁇ ACOOH
  • N ⁇ A represents a heterocyclic group
  • step (f) the terminal carboxyl group of the solid support bound peptide is modified by substitution of or addition to the terminal -OH group, resulting in production by step (g) of a modified polypeptide of formula (I) 1.
  • step (I) the terminal carboxyl group of the solid support bound peptide is modified by substitution of or addition to the terminal -OH group, resulting in production by step (g) of a modified polypeptide of formula (I) 1.
  • M is any substituent group other than -OH.
  • the present invention provides a process for the production of a peptide of general formula (I) (I)
  • n,m,x,Z,Y, ⁇ and A are as defined above.
  • the present i nventi on further provides a process for the producti on of a pepti de of general formula (I) (I)
  • n,m,x,Z,Y, ⁇ and A are as defined above
  • unsaturated alkyl or t-alkoxy groups containing 1 to 20 carbon atoms which may be unsubstituted or substituted by one or more groups selected from C 4 -alkoxy, nitro, tri(C 1 -C 4 alkyl)silyl and halogen; and wherein at least one of R 1 , R 2 and R 3 is a t-alkoxy group.
  • Preferred alkoxysilyl groups are tri-t-butoxysilyl
  • step (c) may be carried out in aqueous solution at pH ⁇ 4, for base-labile solid supports.
  • Step (c) is preferably carried out in dilute TFA
  • step (c) is carried out in about 5% TFA in the presence of DCM (dichloromethane).
  • step (c) may be carrried out in aqueous solution at pH>8.
  • step (c) is preferably carried out in the presence of 5% piperidine in N,N-di methyl acetamide.
  • an amino acid or peptide derivative may be linked to these compounds via an "oxycarbonyl" group following
  • linker compounds with phosgene.
  • linkers may also be attached to any solid support W bearing a terminal amino function.
  • A) allows release of a newly synthesised peptide under mild conditions such as 15% piperidine in dimethyl formamide.
  • B) the conditions for peptide release are slightly more rigorous, requiring 90% trifluoroacetic acid (TFA).
  • Cleavage from C) can be achieved under mild basic conditions, such as 0.1N sodium hydroxide.
  • the solid support W is a resin having a terminal amino group, for example commercially available resins MBHA., BHA., or aminomethyl ated resins.
  • W and the linker compound are preferably reacted to form a W-Y bond in the presence of DCC/HOBt.
  • linker group containing solid supports W-Y for example:
  • amino acid or peptide derivative may be linked via an oxycarbonyl bond following functionalisation of the resin hydroxy moiety.
  • the subsequent peptidyl-resin bond may be cleaved with tetrabutyl ammonium fluoride or by hydrazine in chloroform/methanol;
  • Suitable leaving groups ⁇ introduced by carboxyl group activating agents are those of general formula (XI)
  • R 4 -NH-C N-R 5 (XI) wherein R 4 and R 5 , which may be the same or different, represent C 1 -C 10 hydrocarbyl groups. In a well studied example both are cyclohexyl groups.
  • R 4 and R 5 represent C 1 -C 10 hydrocarbyl groups. In a well studied example both are cyclohexyl groups.
  • Other suitable leaving groups include
  • Steps (c) or (d) and (d) or (e), respectively) can take place simultaneously.
  • Any reactive side chains on residues A are protected and subsequently deprotected, as necessary by known procedures.
  • the choice of side-chain protecting groups may be adjusted to suit the method of removal of the protecting group Z.
  • the invention further provides compounds of general formula (II); (II)
  • A is either Asp or Glu
  • Figure 1 is an HPL Chromatograph of Leucine-Enkephalin synthesised using tert-butoxysilyl (TBOS) esters;
  • Figure 2 is a FIB Mass Spec Analysis of Leu-Enkephalin synthesised using L-amino acid-TBOS esters;
  • Figure 3 is an HPL Chromatograph of Leucine Enkephalin Alcohol
  • Figure 4 is a FIB Mass Spec of Leucine Enkephalin Alcohol
  • Figure 5 is a FIB Mass Spec of Leucine Enkephalin Diol.
  • Figure 6 is a FIB Mass Spec of Leucine Enkephalin Chloromethyl Ketone. The invention will now be illustrated by way of example with reference to the following description.
  • the compound of general formula III may be 9-(methoxycarbonyl amino acid t-al koxysi lyl ester)-2-Fluorene acetic aci d:
  • This compound may be produced by a process which comprises the following steps:
  • the carboxy-protected compound 9-(methoxy carbonyl amino acid t-alkoxysilyl ester)-2-Fluorene acetic acid (9) can couple to a solid support resin W via the "free" carboxylic acid group (*). Coupling with a resin carrying a terminal amino group is preferred.
  • a synthesis can be carried out using compound (9) according to the following procedure:
  • Benzyl acetate resin Chioromethylated co-polystyrene-2% divinylbenzene (5g. 1mmol./g.) suspended in 2-methoxyethanol
  • Benzyloxycarbonyl resin L- leucine ethyl ester L- leucine ethyl ester.
  • L-leucine ethyl ester hydrochloride 0.294g.; 1.5mmol.
  • triethylamine 0.21mis.; 3.75mmol.
  • Methylchloroformate resin (500mg.) was prepared as described before.
  • the resin was then swollen in dimethyl acetamide (2 x 10mls.) and treated with L-tryosine methyl ester hydrochloride (1.0mmol.; 0.231g.) and triethylamine (2.0mmol.; 0.112mls.) in
  • the leucine-enkephalin pentapeptide was assembled by four successive cycles of addition and deprotection to yield the desired peptidyl-resin.
  • a typical addition cycle consisted of treating the deprotected resin with the amino acid TBOS ester (1.0mmol.), HOBt (1.0mmol; 0.135g.) and diisoproplycarbodiimide (1.0mmol.; 0.156mls.) in DCM (10mls.) for 4 hours at room temperature. The resin would then be washed thoroughly with DCM before being deprotected by treatment with 25% TFA in DCM (10mls.) for 10 minutes at room temperature followed by washing with DCM (6 x 10mls.) before commencing the subsequent addition cycle.
  • Buffer A 0.1% TFA., H 2 O
  • Buffer B 0.1% TFA., 80% ACN., 30% H 2 O
  • the peptides were compared by injecting individual ly and
  • the preferred carboxy-protected amino acids (II) are the tri-t-butoxysilyl esters. These can easily be produced in a one step process as follows:
  • the reaction is rapid and typically yields 85-90% product.
  • the simplicity of production contrasts favourably with the prior three step process for production of the silyl esters of W090/05738.
  • the tri-t-alkoxysilyl esters, alkyl-di-t-alkoxysilyl esters and dialkyl-t-alkoxysilyl esters can be made from free amino acids without the need for N ⁇ -protection (by groups such as tBoc).
  • the free amino acids are abundantly available and cheap.
  • tri-t-alkoxysilyl esters, alkyl-di-t-alkoxysilyl esters and di alkyl-t-alkoxysilyl esters show good stability when stored under anhydrous conditions, hence permitting long term storage. Large scale preparation of these derivatives may be carried out well in advance of the peptide synthesis.
  • esters are stable between pH 4.0 and pH 8.0 and are stable to alcoholic solvents. They can be readily removed at pH values less than 4 or greater than 8.
  • the procedure for the preparation of several tri-t-butoxysilyl amino acid esters is illustrated in more detail in the example below:
  • the pyridinium hydrochloride is removed by filtration, washing the precipitate with ethyl acetate (50ml). All solvents, (i.e.
  • the product was characterised by means of its infra-red (I.R.) spectrum, thin layer chromatography (t.l.c.) and Mass Spectrum (M.S.).
  • I.R. infra-red
  • t.l.c. thin layer chromatography
  • M.S. Mass Spectrum
  • the tri-t-alkoxysilyl, alkyl-di-t-alkoxysilyl and dialkyl-t-alkoxysilyl amino acid esters are convenient to prepare, they have suitable stabilities, they are soluble in all commonly used organic solvents and they have exhibited no signs of suffering from problems of steric hindrance. Hence, it would appear that most of the problems encountered with the trimethylsilyl and the t-butyldimethylsilyl amino acid esters do no effect these
  • the mild nature of the synthetic strategy proposed may provide a means of synthesising modified peptides, such as phosphopeptides or glycopeptides, that cannot withstand the conditions used at some stages of conventional synthetic procedures.
  • modified peptides such as phosphopeptides or glycopeptides
  • the reversal of the direction of synthesis from the conventional C- to -N terminal also opens up the possibility of carrying out solid-phase fragment coupling for the production of large peptides.
  • a further advantage of synthesising in the N to C direction is that the C-terminal remains free and this allows for modifying the C-terminal of peptides whilst they are bound on the solid support.
  • Examples of C-terminal modified peptides which have been prepared are enkephalin-alcohol, -diol and -chloromethylketone. Data on these analogues is shown in Figures 3-6. The products made
  • t.l.c solvent was chloroform: methanol (9:1).
  • Bz refers to Benzyl.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • 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 se rapporte à un procédé de préparation de polypeptides par synthèse en phase solide. Ledit procédé consiste à fixer des acides aminés protégés par carboxy à l'extrémité carboxyle d'une chaîne peptidique liée à son extrémité N-terminale à un support solide. La protection carboxy est faite par un groupe tertiaire alcoxy silyle. L'invention se rapporte également à de nouveaux esters d'acides aminés du groupe tertiaire alcoxy silyle pouvant être utilisés comme intermédiaires.
EP92918056A 1991-08-30 1992-08-26 Preparation de peptides par une synthese en phase solide, et intermediaires pour la preparation Withdrawn EP0600996A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9118669 1991-08-30
GB919118669A GB9118669D0 (en) 1991-08-30 1991-08-30 Preparation of peptides by a soliphase synthesis and intermediates therefor
PCT/GB1992/001567 WO1993005065A1 (fr) 1991-08-30 1992-08-26 Preparation de peptides par une synthese en phase solide, et intermediaires pour la preparation

Publications (1)

Publication Number Publication Date
EP0600996A1 true EP0600996A1 (fr) 1994-06-15

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP92918056A Withdrawn EP0600996A1 (fr) 1991-08-30 1992-08-26 Preparation de peptides par une synthese en phase solide, et intermediaires pour la preparation

Country Status (4)

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EP (1) EP0600996A1 (fr)
AU (1) AU2464992A (fr)
GB (1) GB9118669D0 (fr)
WO (1) WO1993005065A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5798035A (en) 1996-10-03 1998-08-25 Pharmacopeia, Inc. High throughput solid phase chemical synthesis utilizing thin cylindrical reaction vessels useable for biological assay
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
ATE480561T1 (de) 2004-10-19 2010-09-15 Lonza Ag Verfahren zur festphasen-peptidsynthese
GB0505221D0 (en) * 2005-03-14 2005-04-20 Southampton Polypeptides Process
EP3693377A4 (fr) * 2017-10-03 2021-06-30 Nissan Chemical Corporation Procédé de production de composé peptidique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8827083D0 (en) * 1988-11-19 1988-12-21 Porton Prod Ltd Trialkylsilyl esters of amino acids & their use in synthesis of peptides
DE3924257A1 (de) * 1989-07-19 1991-01-31 Gruszecki Wojciech Silikonester als neue schutzgruppen in der organischen chemie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9305065A1 *

Also Published As

Publication number Publication date
WO1993005065A1 (fr) 1993-03-18
AU2464992A (en) 1993-04-05
GB9118669D0 (en) 1991-10-16

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