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WO1998017677A1 - Ameliorations apportees a la synthese en phase solide de peptides et de composes apparentes - Google Patents

Ameliorations apportees a la synthese en phase solide de peptides et de composes apparentes Download PDF

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Publication number
WO1998017677A1
WO1998017677A1 PCT/AU1997/000711 AU9700711W WO9817677A1 WO 1998017677 A1 WO1998017677 A1 WO 1998017677A1 AU 9700711 W AU9700711 W AU 9700711W WO 9817677 A1 WO9817677 A1 WO 9817677A1
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Prior art keywords
peptide
spacer
gly
construct
group
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Darren Englebretsen
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University of Queensland UQ
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University of Queensland UQ
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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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier

Definitions

  • This invention relates to spacers for use in synthesis of peptides, especially peptides made by solid- phase methods, and to solid-phase methods for synthesis of "difficult" or poorly soluble peptides.
  • the spacers of the invention are applicable to the synthesis, manipulation and purification of "difficult" or poorly-soluble peptides, especially peptides containing large numbers of hydrophobic amino acids. These spacers are also applicable to synthesis of peptide-related compounds and compounds comprising peptide sequences, such as peptide nucleic acids .
  • SPPS solid phase peptide synthesis
  • 5-15 amino acids are exactly the types of peptides which it is most frequently desired to synthesize.
  • Peptides containing multiple repeating alanine residues are often used to investigate difficulties in synthesis or solublization (see for example Bedford et al , 1992) .
  • Polyalanine peptides synthesized using either Boc or Fmoc frequently show poor coupling yields and inhomogeneous final products (Merrifield et al , 1988; Beyermann et al , 1992) .
  • An accompanying problem with polyalanine and other "difficult sequences" sequences is their poor solubility, which can make purification and analysis by HPLC and electrospray mass spectrometry (ESMS) difficult, if not impossible.
  • ESMS electrospray mass spectrometry
  • RP-HPLC RP-HPLC
  • ESMS electrospray mass spectrometry
  • MALDI-TOF MS matrix-assisted laser desorption- time of flight mass spectrometry
  • a spacer moiety such as a spacer peptide
  • PNAs peptide nucleic acids
  • the invention provides a spacer construct for enhancing the efficiency of synthesis of peptides, or of peptide-related compounds, which is a) able to be linked to a solid support by a chemical linkage, b) linked to, or incorporates at another point, a selectively-cleavable chemical linkage means, and c) can be linked to a desired peptide or peptide-related compound to form a construct, whereby processes can be performed on the construct which cannot be performed on the desired peptide or peptide-related compound per se .
  • the spacer construct of the invention enables the desired peptide or peptide-related compound to be solubilised, where the peptide or peptide-related compound alone would be insoluble or very difficult to solubilise.
  • the spacer of the invention would be a chemical compound which possesses groups which are, or which can be made, strongly positively or negatively charged.
  • the spacer of the invention must be stable to the conditions used solid phase peptide synthesis, including the cleavage and purification steps.
  • l e . m order to serve as a means for modifying the properties of a peptide or peptide- related compound, the compound must be stable to hydrogen fluoride, to base, and to compounds such as trifluoroacetic acid .
  • the invention provides a spacer peptide construct useful m synthesis of peptides or of peptide-related compounds, comprising a spacer peptide and a reversible linker group covalently bound to the N-termmal thereof, and optionally comprising a cleavable coupling group whereby to link the construct to a solid support; and further optionally comprising a solubilismg peptide group, covalently bonded to the spacer peptide either directly or via a reversible linker group.
  • solubilismg peptide can be attached directly to the spacer peptide via an amide bond, to give
  • Solubilismg peptide-spacer peptide which can then eventually be used to give Target peptide-reversible lmker-solubilismg peptide-spacer peptide.
  • the spacer is preferably a soluble peptide, but can otherwise be any easily synthesized sequence of length suitable to ensure that the peptide or peptide-related compound which it is desired to synthesize (the target peptide or peptide-related compound) is at a distance equivalent to at least 5-15 ammo acids, preferably at least 9-10 ammo acids, from the solid support.
  • the spacer is a peptide, this may be any easily synthesized peptide sequence of the required length, and may be tailored for specific target peptides m relation to length, hydrophobicity, identity of the ammo acids m the sequence, secondary structure-forming properties, oligomer formation, solubility, and incorporation of additional sequence moieties.
  • the spacer peptide will desirably include cationic residues in order to facilitate ESMS or MALDI-TOF MS analysis.
  • a particularly preferred sequence for use m the Boc method is (Gly-Arg) 4 -Gly .
  • this sequence may cause difficulties, and so for this method either (Gly (Lys-Gly) b Gly or (Arg-Gly-Gly) iGly is preferred.
  • the person skilled in the art will be able to optimise the length of spacer for each desired target peptide using routine trial and error methods.
  • the reversible linker group between the spacer and the target peptide or peptide-related compound should preferably be stable to hydrogen fluoride (HF) , which is the most commonly-used reagent for cleavage from the solid support, and labile to base.
  • HF hydrogen fluoride
  • One suitable linker group is glycolamide ester, which is known to be compatible with most side-chain protecting groups used with Boc (Baleux et al , 1984, 1986) and with Fmoc.
  • glycolamide ester linkage has also been used m Fmoc SPPS (Mendre et al , 1992) .
  • the glycolamide ester linkage is suitable for use with most groups used Boc SPPS for protection of reactive side-chains (Kent, 1988).
  • Trp (CHO) and His (Dnp) must be deprotected m solution after cleavage and purification of the target peptide-glycolamide ester- spacer peptide construct.
  • the glycolamide ester is prepared by reacting the N-termmal amine of the spacer peptide with the anhydride of bro oacetic acid.
  • the cesium salt of the desired C-termmal Boc-ammo acid is added, and nucleophilic displacement of the bromine by the carboxylate group of the ammo acid results formation of a glycolamide ester between the Boc-ammo acid and the spacer peptide .
  • esters of 4-Hmb may also be used as a selectively cleavable reversible chemical link for the processes of the invention.
  • Esters of 4-Hmb have the advantage that they can be used m Fmoc SPPS synthesis as well as in Boc-based SPPS.
  • 4-Hmb esters can be more conveniently formed than glycolamide esters.
  • the 4-Hmb ester linkage is stable to trifluoroacetic acid and is labile to nucleophiles .
  • Esters of 4-Hmb may be formed either by the cesium salt method, or by an anhydride method (Atherton et al , 1991) .
  • Linker systems which may be used include photolabile, thiolytic or reductively cleaved linkers; allyloxycarbonyl linkers ("ALLOC"), which are cleaved in the presence of a palladium complex; linkers, such as phenacyl linkers, which are cleaved m the presence of a metal ion; or "safety-catch” linkers, whose properties are changed by a simple reaction.
  • ALLOC allyloxycarbonyl linkers
  • linkers such as phenacyl linkers, which are cleaved m the presence of a metal ion
  • safety-catch linkers
  • the spacer peptide itself may be designed so as to confer solubilismg properties, for example by incorporating charged ammo acids, especially cationic ammo acids, an additional solubilismg peptide or other solubilizmg moiety may be used.
  • the solubilismg group is linked to the spacer peptide via an amide, using a linker group as described above,
  • a non-peptide solubilizmg moiety is to be used, the nature of the moiety can be selected depending on the nature of the compound which is being synthesized.
  • polyethylene glycol derivatives may assist m solubilismg an attached peptide m both aqueous or non-aqueous solvents.
  • co-carboxyl polyethylene glycol derivatives may be useful, which constructs of the form:
  • n 1-10, may be synthesised.
  • Extremely hydrophilic peptides although not
  • tail analogous to the solubilizmg peptide described above, compensates for this problem.
  • This tail may be a hydrophobic peptide, or may be a hydrophobic compound such as 16-hydroxydecano ⁇ c acid or N-Boc- 6-ammocapro ⁇ c acid.
  • the construct may also optionally comprise a cleavable coupling group whereby to link the construct to the solid support.
  • the coupling group is attached at the C-terminal end of the spacer peptide, or the solubilismg peptide if present .
  • Any suitable coupling group known m the art for this purpose may be used, for example thiol, thioacid, hydrazme, hydrazide or aldehyde.
  • the invention provides a peptide construct comprising a solubilismg peptide linked via a first, base-labile reversible linking group at its C-termmal to a desired target peptide or peptide-related compound, which m turn is linked via a second reversible linking group as defined for the first aspect of the invention to a spacer, and optionally a cleavable coupling group at the C-termmal of the spacer peptide.
  • this construct is of the form
  • the reversible linking group between the solubilismg peptide and the desired peptide or peptide-related group is different from the reversible linker used m the first aspect of the invention, that the linking group for this aspect must be base-labile only, whereas the linking group for the first aspect of the invention must be both HF stable and base-labile .
  • the constructs of the invention may also comprise special peptide sequences designed for use in specific manipulations of the final desired peptide or peptide-related compound, for example His for use in immobilised metal affinity chromatography purification (Arnold, 1991), or sequences providing epitopes for use in monoclonal antibody affinity chromatography purification or in immun ⁇ assay, eg. FLAGTM.
  • His for use in immobilised metal affinity chromatography purification (Arnold, 1991)
  • sequences providing epitopes for use in monoclonal antibody affinity chromatography purification or in immun ⁇ assay, eg. FLAGTM for analysis of combinatorial libraries, a tag which facilitates mass spectroscopy , ESMS, MALDI-TOF MS and/or sequencing, or a tag for ELISA assay, may be incorporated.
  • Transport sequences such as those susceptible to intracellular esterases, may be incorporated to facilitate penetration into the target cell .
  • delivery sequences may be required in order to enable the peptide to cross the cell membrane and/or an intracellular membrane.
  • These may include non-naturally occurring amino acids incorporating lipophilic side chains, or ester-peptide-linker delivery sequences, which can be designed so as to be cleavable in a time-dependent manner.
  • ester-peptide-linker delivery sequences which can be designed so as to be cleavable in a time-dependent manner.
  • Other such special-purpose sequences will be known to those skilled in the art.
  • the invention provides a method of solid-phase peptide synthesis comprising the steps of: a) coupling a spacer peptide to a solid support , b) adding a reversible linker group at the N-terminal end of the spacer peptide, c) synthesising the target peptide by sequential addition of amino acids, d) removing the product thus produced from the solid support, and e) removing the target peptide from the spacer peptide by cleavage of the reversible linker group.
  • a solubilismg peptide may be incorporated between the spacer peptide and the target peptide, or at the N-terminal end of the target peptide, via a base-labile reversible linker group.
  • the solid support will be a resin, preferably a polystyrene resin.
  • Particularly suitable supports include Boc-Gly-phenylacetamidomethyl (PAM) polystyrene resin and 4-methylbenzhydrylamme (MBHA) resin (Matsueda and Stewart, 1981) .
  • Standard methods used in solid-phase peptide synthesis may be employed, for example as originally described by Merrifield (1986), or any of the many variations of this technique developed subsequently.
  • ammo acids may be protected with Boc or Fmoc
  • activation of ammo acids may be effected with dicyclohexylcarbodiimide or HBTU
  • cleavage of the final ammo acid chain from the resin will usually be with hydrogen fluoride.
  • Cleavage of the linker group may be effected by treatment with mild base, such as aqueous t ⁇ ethylamme .
  • the target peptide once separated from the spacer peptide and solubilismg peptide construct, may be solubilised m an appropriate solvent, such as water, buffer, trifluoroacetic acid, etc.
  • an appropriate solvent such as water, buffer, trifluoroacetic acid, etc.
  • the most suitable solvent for each individual peptide may be empirically determined. In some cases it may be necessary initially to use a solvent such as TFA, whereas the peptide construct may be soluble m water after initial lyophilisation from TFA.
  • PNAs peptide nucleic acids
  • PNAs ammo acid derivatives which instead of the normal ammo acid side chains carry the four nucleotide bases of DNA.
  • PNAs peptide nucleic acids
  • These compounds have a pseudopeptide backbone composed of N- (2-ammoethyl) glycme units, in which the nucleotide bases are attached to the nitrogen atom of glycme via carbonyl methylene linkers (reviewed m Neilsen and Haaima (1997).
  • Oligomers of PNAs are able to bind strongly and with high sequence specificity to complementary oligomers of DNA, RNA or other PNAs, and m particular can bind to double-stranded DNA to form a PNA - DNA triplex m a strand displacement complex. They therefore show potential m gene therapy, as anti-sense DNA drugs, and as reagents for genome analysis (Nielsen and Haaima (1997) .
  • PNAs have been synthesised by a process analogous to SPPS, using both Boc and Fmoc chemistries (Thomson et al , 1995; Hyrrup and Neilsen, 1996). For example, essentially standard SPPS protocols were used m the Boc solid phase synthesis of PNA oligomers, with final cleavage of the PNAs using liquid hydrogen fluoride
  • the spacer means, particularly spacer peptides, and other constructs and methods of the invention are applicable to the synthesis of PNAs.
  • the invention includes within its scope the spacer means, preferably tne spacer peptide, and optional solubilizmg peptide construct linked either to a solid support or to a first ammo acid.
  • the invention contemplates a set of [construct] -ammo acid pairs, as well as SPPS kits comprising one or more constructs of the invention.
  • Figure 1 shows a reaction scheme for synthesis of Ala 12 - [glycolamide ester] - [Gly-Arg] 4 -Gly using Boc chemistry with in si tu neutralisation (Schnolzer et al , 1992).
  • Figure 2 shows the results of HPLC chromatography of crude HF-cleaved Ala 12 - [glycolamide ester] - [Gly-Arg] 4 -Gly on a Vydac C 4 column.
  • Figure 3 shows a reconstructed electrospray mass spectrum of crude HF-cleaved Ala J2 - [glycolamide ester] - [Gly-Arg] 4 -Gly .
  • -X represents [glycolamide ester] - [Gly- Arg] 4 -Gly.
  • Figure 4a shows the results of HPLC chromatography of crude cleaved H-(CP-10 4? j5 ) -glycolamide ester- (Gly-Arg-4) 4 -Gly-OH;
  • Figure 4b shows an ES-MS spectrum of H-(CP-10 ⁇ 2 r )-OH after base cleavage.
  • the inset shows a reconstructed electrospray mass spectrum.
  • Figure 5 is a flow chart showing the use of an ester of 4-hydroxymethylbenzoic acid (4-Hmb) as a selectively-cleavable chemical linkage in the synthesis of constructs of the form desired peptide- [4-Hmb ester] -spacer using Fmoc SPPS chemistry
  • Peptide A desired peptide
  • Peptide B spacer peptide.
  • Figure 6 shows base cleavage of di-addition of the 4-Hmb linker to the spacer peptide-resin .
  • Peptide B spacer peptide
  • Figure 7 shows a MALDI-TOF MS spectrum of a sample of Fmoc-Ala- [4-Hmb linker] -Gly (Lys-Gly) b -amide cleaved after attachment of the Fmoc-Ala to the HO-4-Hmb- Gly (Lys-Gly) 6 -Resin using the anhydride method, after
  • Figure 8 shows an HPLC chromatogram of a peptide construct made using the 4-Hmb linkage as the selectively- cleavable chemical linkage, using Fmoc chemistry.
  • Figure 9 shows a HPLC chromatogram of a second peptide construct made using the 4-Hmb linker and Fmoc chemistry .
  • Figure 10 shows the use of 16-hydroxyhexadecanoic acid as modifier of the physicochemical properties of target peptide.
  • Figure 11 shows the use of 6-aminocaproic acid as modifier of the physicochemical properties of target peptide .
  • Figure 12 shows the use of a hydrophilic peptide as modifier of the physicochemical properties of target peptide .
  • Boc-Gly-PAM polystyrene resin (0.8 mmol/g; ABI, Foster City, CA; 1 mmol scale) was used to synthesise
  • Boc- (Gly-Arg) 4 -Gly-PAM-resin Boc-amino acids (2 eq) were activated with HBTU and coupled for 10 minutes. The average coupling yield as measured by ninhydrin assay was 99.81%. Boc removal from 0.33 mmol of this resin followed by neutralisation, and reaction with 1 mmol bromoacetic anhydride gave N-bromoacetyl- (Gly-Arg) 4 -Gly-PAM-polystyrene resin. This was reacted with 1 mmol Boc-Ala, Boc-Ala cesium salt in 5 ml dimethylformamide (DMF) for 14 hours.
  • DMF dimethylformamide
  • Boc-Ala residues were then added (2 mmol, 6 eq) as above.
  • a sample of the Boc-Ala,;- [glycolamide ester] - (Gly- Arg) 4 -Gly-PAM-polystyrene resin was subjected to removal of Boc, cleaved with HF, then the crude Ala b - [glycolamide ester] - (Gly-Arg) 4-Gly was analysed by ESMS.
  • the precipitate from another base cleavage experiment was collected by centrifugation, and was sparingly soluble m 90% aceton ⁇ t ⁇ le-10%water-0.1% TFA (HPLC solvent B) .
  • the resulting suspension was filtered for analysis by ESMS.
  • FIG. 1 A minor impurity (peak 1) was a Des-Ala peptide (1767 Da), while another (peak 3) was a low molecular weight impurity probably generated during HF cleavage.
  • Figure 3 shows a reconstructed ESMS spectrum of the crude Ala i2 - [glycolamide ester- (Gly-Arg) -Gly, showing the expected product at 1838 Da and low levels of alanme deletion peptides
  • the amide peptide construct was easily purified by HPLC, using the method described above, to give Ala _- [glycolamide ester] - (Gly-Arg) 4 -Gly .
  • Homogeneous Ala i2 was obtained by purification of the Ala ⁇ 2 - [glycolamide ester] - (Gly-Arg) 4 Gly construct followed by base cleavage at th glycolamide ester linkage.
  • the Ala ⁇ 2 was separated from the highly soluble HO-CH 2 -CO- (Gly-Arg) 4 Gly by filtration or centrifugation.
  • Figure 4a shows an HPLC chromatogram of crude cleaved H- (CP-10 42 " ) -glycolamide ester- (Gly-Ala) _-Gly-OH .
  • ESMS of the major peak from Figure 4a gave a mass of 2699 Da (calc. 2700 Da).
  • HPLC-pu ⁇ fled H-(CP-10 42 )- glycolamide ester - (Gly-Ala) i-Gly-OH was also cleaved with base.
  • H-Ala ⁇ 2 -glycolamide ester- (Gly-Arg) 4 -Gly-OH and H- (CP-10 42-55 ) -glycolamide ester- (Glyn-Ala) 4 -Gly-OH (2.0 mg in 900 ⁇ l water, a clear solution) were treated with 100 ⁇ l of triethylamine for 10 minutes (pH 12.2) .
  • the resultant peptide precipitates were collected by centrifugation. After washing six times with 1 ml water the precipitates were shaken with a mixture of 20 ⁇ l TFA and 980 ⁇ l acetic acid.
  • the suspension of peptide which resulted was filtered for analysis by ESMS.
  • 4-Hmb esters may be formed using the cesium salt method.
  • the anhydride of 4-bromomethyl benzoic acid can be reacted with the N-terminal amine of a spacer chemical linkage- (Resin) to give Br-CH 2 - [C 6 H 4 ] -CO-spacer chemical linkage- (Resin) .
  • the second linker would clearly be present at a level of 10% or less, as only 1.1 eq of linker were used in the reaction and it had been shown that no unreacted amine was present.
  • the resin was washed with dioxane, and then treated for two minutes with a mixture of 1 M NaOH/MeOH/dioxane 1:4:15.
  • the NaOH cleaves the base-labile ester in the di-4-Hmb addition product to give the single addition material, ie. 4-HO-CH 2 -C 6 H 4 ] -CO spacer chemical linkage- (Resin) .
  • Figure 7 shows a MALDI spectrum of a sample of Fmoc-Ala- [4-Hmb linker] -Gly (Lys-Gly) b -amine cleaved after attachment of the Fmoc-Ala to the HO-4-Hmb-Gly (Lys-Gly) 0 - Resin using the anhydride method, with NaOH/methanol/ dioxane cleavage to remove di-addition of the 4-Hmb linker.
  • the only product seen on MALDI-TOF MS was the desired Fmoc- Ala- [4-Hmb linker] -Gly (Lys-Gly) b -amide (found mass
  • Example 5 Use of 4-Hydroxymethyl Benzoic Acid (4-Hmb) as the Selectively-Cleavable Linker
  • Peptide A represents the desired peptide
  • Peptide B represents the spacer.
  • 1.1 eq 4-Hmb, 1.1 eq HBTU, and 1.3 eq DIEA were reacted in DMF solvent at room temperature overnight . This reaction has been shown to be complete within one-two hours (Step 1 in flow chart) .
  • cleavage reagent for example, a mixture of [TFA 10 ml, thioanisole 0.5 ml, water 0.5 ml, ethanedithiol 0.25 ml, phenol 0.75 g] , was used as the cleavage reagent.
  • the peptide construct was cleaved from the spacer by 0.1 M NaOH two minutes or less (Step 8) .
  • Linkage Figure 8 shows the HPLC chromatogram of a peptide construct made using the 4-Hmb linkage as the selectively- cleavable chemical linkage.
  • the construct was made using an ABI 433 peptide synthesiser with standard ABI Fmoc coupling chemistry.
  • the peptide sequence was:
  • the mass of the major peak elutmg at 32 06 minutes was found to be 2699 Da, while the calculated mass was 2700 Da.
  • Linkage Figure 9 shows a HPLC chromatogram of another construct
  • the mass of the major peak elutmg at 31.381 minutes on the HPLC was 2513.1 Da, and the calculated mass was 2511.8 Da.
  • Peptides The use of a hydrophobic tail is referred to above for the purification of extremely hydrophilic peptides .
  • hydrophobic peptide 16-hydroxyhexa- decanoic acid (CAS number 506-13-8, Aldrich 1996-1997 catalogue p844) can alternatively be used.
  • This hydrophobic compound is able to retard the retention time of an attached hydrophilic peptide on HPLC, and already includes in its structure the basis of a selectively cleavable chemical linkage, l e . the 16-hydroxyl group, which can form an ester linkage to the hydrophilic peptide
  • This compound HO-(CH9) : -COOH is attached to the resm support via its carboxyl group. Then an acid stable, base labile ester is formed between the hydroxyl group and the first ammo acid of the peptide to be synthesised.
  • the construct is cleaved using acid, to give a construct of the form:
  • the ester between the hydrophilic peptide and the -O- (CH 2 ) 1 5-COR portion is stable to acid used m cleavage from the resm, but labile to alkali. Thus once the construct has been purified by HPLC or some other method, the ester is cleaved by aqueous alkali to liberate the hydrophilic peptide.
  • the HO- (CH 2 ) 15-COR may be soluble in alkali if R is OH, for example. Acidification of the solution or addition of Ca 2 " ions would then precipitate the HO- (CH 2 ) 15-CO-OH, thus allowing recovery of the hydrophilic peptide.
  • Hydrophilic Peptides A second related compound which may be used is N-Boc-6-aminocaproic acid, ie. Boc-NH- (CH 2 ) -COOH . This is easily made from 6-aminocaproic acid (CAS number 60-32-2) . In this case the hydrophobic alkyl chain is also used to modify the solubility of an attached hydrophilic peptide.
  • Hydrophilic pep ide-CO-0-CH - [C 6 H Register] -CONH0 (CH ; ) ,-CO-R, which is then cleaved at the 4-Hmb linkage by aqueous alkali to give the hydrophilic peptide and
  • the Boc-ammo acid anhydride can be formed using standard methods (eg. 2 mmole Boc-ammo acid dissolved m 4 ml DCM, add 1 mmole dicyclohexylcarbodimide m 2 ml DCM, stir 20-30 mm, filter off the precipitate of dicyclohexylurea and add the solution to the resm) .
  • Dimethylammopy ⁇ dme (DMAP) is added to catalyse the reaction of the hydroxyl group with the Boc-ammo acid anhydride .
  • the ester formed between the Boc-ammo and the 16-hydroxyhexadecano ⁇ c acid is marked * (Step 4) .
  • Boc SPPS is performed using standard methods (Step 5) .
  • the construct is cleaved using strong acid eg. liquid hydrogen fluoride plus scavengers (Step 6) .
  • the ester marked * is stable to these conditions but labile to aqueous alkali.
  • Example 11 Alternative Use of an Organic Compound as Modifier of the Physicochemical Properties of an Attached Peptide This example also uses the MBHA-type peptides synthesis resm and Boc chemistry, but m this case the 4-Hmb linker is used. The process is illustrated in Figures 11 and 12, which respectively show the use of
  • 6-ammocapro ⁇ c acid and hydrophilic peptide to modify the properties of the target peptide.
  • the resm is treated with TFA to remove the Boc group prior to the addition of the 4-Hmb linker (Steps 1-3) .
  • Some di-addition of the 4-Hmb linker may occur, as described m Example 4. Should this occur, treating the resm with aqueous alkali m organic solvents (eg. a mixture of 1 M NaOH/methanol/dioxane 1:4:15) results m cleavage of the second 4-Hmb molecule.
  • Steps 4 and 5 are performed as Example 10.
  • the construct is cleaved using strong acid eg. liquid hydrogen fluoride plus scavengers (Step 6) .
  • strong acid eg. liquid hydrogen fluoride plus scavengers (Step 6) .
  • the 4-Hmb ester marked * is stable to these conditions but labile m aqueous alkali.

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Abstract

L'invention concerne des espaceurs utilisés dans la synthèse en phase solide de peptides et de leurs composés apparentés. On utilise une construction d'espaceur pour faire en sorte que le peptide cible ou le composé apparenté cible soit séparé du support solide par une distance égale ou supérieure à 5-15 acides aminés. On peut notamment utiliser les constructions de la présente invention dans la synthèse d'un peptide fortement hydrophobe, qu'il serait difficile, voire impossible, de purifier et d'analyser. Les constructions et les méthodes faisant l'objet de cette invention peuvent également être appliquées à la synthèse d'acides nucléiques peptidiques.
PCT/AU1997/000711 1996-10-24 1997-10-24 Ameliorations apportees a la synthese en phase solide de peptides et de composes apparentes Ceased WO1998017677A1 (fr)

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AUPO3240A AUPO324096A0 (en) 1996-10-24 1996-10-24 Linker peptide

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8022181B2 (en) 2006-05-03 2011-09-20 Mallinckrodt Llc Composition and method for the release of protected peptides from a resin
WO2017020569A1 (fr) * 2015-07-31 2017-02-09 南京斯拜科生化实业有限公司 Procédé de synthèse-purification par séparation pour un polypeptide indissoluble
WO2024181781A1 (fr) * 2023-02-27 2024-09-06 에스피투티엑스주식회사 Substrat pour la synthèse de polymères biologiques ayant une fonction anti-agrégation et procédé de synthèse de polymère biologique l'utilisant

Citations (3)

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US5369017A (en) * 1994-02-04 1994-11-29 The Scripps Research Institute Process for solid phase glycopeptide synthesis
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US8022181B2 (en) 2006-05-03 2011-09-20 Mallinckrodt Llc Composition and method for the release of protected peptides from a resin
WO2017020569A1 (fr) * 2015-07-31 2017-02-09 南京斯拜科生化实业有限公司 Procédé de synthèse-purification par séparation pour un polypeptide indissoluble
WO2024181781A1 (fr) * 2023-02-27 2024-09-06 에스피투티엑스주식회사 Substrat pour la synthèse de polymères biologiques ayant une fonction anti-agrégation et procédé de synthèse de polymère biologique l'utilisant

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